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Therapeutic targeting of the BCR-associated protein CD79b in a TCR-based approach is hampered by aberrant expression of CD79b
Lorenz Jahn, Pleun Hombrink, Chopie Hassan, Michel G. D. Kester, Dirk M. van der Steen, Renate S. Hagedoorn, J. H. Frederik Falkenburg, Peter A. van Veelen and Mirjam H. M. Heemskerk
Blood 2015 125:949-958; doi: https://doi.org/10.1182/blood-2014-07-587840
Lorenz Jahn
Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands;
Pleun Hombrink
Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands; Department of Hematopoiesis, Sanquin Research, Amsterdam, The Netherlands; and
Chopie Hassan
Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands
Michel G. D. Kester
Dirk M. van der Steen
Renate S. Hagedoorn
J. H. Frederik Falkenburg
Peter A. van Veelen
B-cell malignancies were efficiently recognized by T cells expressing high-affinity alloHLA-restricted TCRs specific for CD79b.
Aberrant expression of CD79b in non–B cells caused unwanted reactivity, rendering CD79b unsuitable for TCR-based immunotherapies.
Immunotherapy of B-cell malignancies using CD19-targeted chimeric antigen receptor–transduced T cells or CD20-targeted therapeutic monoclonal antibodies has shown clinical efficacy. However, refractory disease and the emergence of antigen-loss tumor escape variants after treatment demonstrate the need to target additional antigens. Here we aimed to target the B-cell receptor–associated protein CD79b by a T-cell receptor (TCR)-based approach. Because thymic selection depletes high-avidity T cells recognizing CD79b-derived peptides presented in self-HLA molecules, we aimed to isolate T cells recognizing these peptides presented in allogeneic HLA. Peptide-HLA tetramers composed of CD79b peptides bound to either HLA-A2 or HLA-B7 were used to isolate T-cell clones from HLA-A*0201 and B*0702-negative individuals. For 3 distinct T-cell clones, CD79b specificity was confirmed through CD79b gene transduction and CD79b-specific shRNA knockdown. The CD79b-specific T-cell clones were highly reactive against CD79b-expressing primary B-cell malignancies, whereas no recognition of nonhematopoietic cells was observed. Although lacking CD79b-cell surface expression, intermediate reactivity toward monocytes, hematopoietic progenitor cells, and T-cells was observed. Quantitative reverse transcriptase polymerase chain reaction revealed low CD79b gene expression in these cell types. Therefore, aberrant gene expression must be taken into consideration when selecting common, apparently lineage-specific self-antigens as targets for TCR-based immunotherapies.
Clinical studies using chimeric antigen receptor (CAR)-modified T-cells or targeted monoclonal antibodies have shown the efficacy of immunotherapy to treat different hematologic malignancies. The adoptive transfer of T cells modified to express CD19-targeted CARs have led to complete remission in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL).1⇓⇓-4 Because of its high expression on malignant cells, CD19 can be efficiently targeted by CAR-transduced T cells. Similarly, CD20-targeting therapeutic monoclonal antibodies such as rituximab have caused complete remissions in patients treated for follicular lymphoma5,6 and diffuse large B-cell lymphoma7,8 in combination with chemotherapy. In addition to their expression on malignant B cells, CD19 and CD20 are also highly expressed on healthy B cells, causing depletion of the B-cell compartment in patients treated with CD19-targeted or CD20-targeted immunotherapies.1,3⇓-5,9 However, side effects of long-term B-cell depletion are manageable, which indicates that B-cell aplasia can be tolerated.9⇓⇓⇓-13
Although successful in several patients, malignancies refractory to CD20-targeted therapy and the emergence of CD19-negative tumor escape variants after treatment with CD19-targeting CAR-transduced T-cells4 demonstrate the need to identify additional targets for the treatment of B-lineage malignancies. We hypothesized that the B-cell receptor (BCR)-associated protein CD79b presents a suitable target for immunotherapy because CD79b membrane expression is restricted to the B-cell compartment and can be found on many B-cell malignancies.14⇓-16 The BCR-associated heterodimer CD79a/b is critically involved in BCR transport17 and functionality by providing signaling capacity18⇓⇓⇓-22 to regulate processes such as allelic exclusion, proliferation, differentiation, anergy, and apoptosis.23⇓-25 CD79b is encoded by gene B2926 and through the event of alternative splicing can produce different transcripts.27 The truncated variant of CD79b, missing almost entirely its extracellular domain, has been implicated to render CLL unresponsive to BCR-induced apoptosis.28 Furthermore, a high percentage of malignant cells expressing cell-surface CD79b in combination with other phenotype markers has been associated with shortened overall survival in B-CLL patients.29,30
An alternative strategy for the treatment of cancer is the use of T-cell receptor (TCR) gene-transfer therapy.31,32 T cells engineered to express TCRs with defined antigen specificity have already successfully been used in the treatment of solid tumors.33 Furthermore, only very low quantities of agonistic peptide–HLA complex are required for TCR triggering and T-cell activation,34⇓-36 whereas CAR and antibody-based approaches necessitate a greater abundance of the antigen.37 Therefore, T cells modified with CD79b-targeting TCRs can be a valuable addition to current immunotherapies.
T cells expressing high-affinity TCRs recognizing self-antigens such as CD79b in the context of self-HLA molecules are deleted from the naïve T-cell repertoire during thymic development by negative selection to prevent autoreactivity. In contrast, presentation of such self-antigens in the context of allogeneic HLA (alloHLA) can result in strong T-cell responses as was observed in the induction of PRAME-specific T cells after HLA-A*0201–mismatched hematopoietic stem cell transplantation.38 In an additional study we demonstrated that the high-affinity TCRs of these alloHLA-restricted T cells are single peptide–specific.39 Using the concept of self-antigen presented in alloHLA, Wilms tumor antigen (WT1)-reactive T cells have been generated by stimulating T cells from HLA-A2–negative donors with autologous B-lymphocytes coated with HLA-A2 monomers loaded with WT1-derived peptide.40 In addition, antigen-specific T cells were obtained by stimulating T cells from HLA-A2–negative donors with autologous dendritic cells (DCs) pulsed with RNA to express HLA-A2 in parallel with a variety of targeted antigens.41⇓-43
In this study, from HLA-A*0201/B*0702–negative healthy individuals, we isolated 3 T-cell clones targeting 3 distinct CD79b peptides presented in HLA-A*0201 or HLA-B*0702. CD79b-positive primary CLL, ALL, and ALL cell lines were recognized by these T-cell clones, whereas no reactivity toward nonhematopoietic tissue was detected. However, low to intermediate recognition of CD14+ monocytes, T cells, and CD34+ hematopoietic precursor cells (HPCs) could be observed. Although no cell-surface CD79b was present on these cells, aberrant mRNA expression of CD79b could be detected in these cells.
Culture conditions and cells
Peripheral blood mononuclear cells (PBMCs) were obtained from different individuals, after their informed consent, by isolation using Ficoll-gradient centrifugation and were then cryopreserved. T cells were cultured in T-cell medium consisting of Iscove's modified Dulbecco's medium (Lonza, Basel, Switzerland) supplemented with 100 IU/mL IL-2 (Proleukine; Novartis Pharma, Arnhem, The Netherlands), 5% fetal bovine serum (Gibco, Life Technologies, Carlsbad, CA), and 5% human serum. Isolation, generation, and culture of various hematopoietic and nonhematopoietic cell subsets and lines are described in supplemental Methods, available on the Blood Web site. The Leiden University Medical Center review board approved the use of human material, and research was conducted in accordance with the Declaration of Helsinki.
Generation of peptide-MHC complexes
All peptides were synthesized in-house using standard Fmoc chemistry. Recombinant HLA-A2 and HLA-B7 heavy-chain and human β2m light-chain were in-house produced in Escherichia coli. Major histocompatibility complex (MHC) class I refolding was performed as previously described, with minor modifications.44 MHC class I complexes were purified by gel filtration using high-performance liquid chromatography. pMHC tetramers were generated by labeling biotinylated pMHC monomers with streptavidin-coupled phycoerythrin (PE; Invitrogen, Carlsbad, CA) or Quantum dots (Qdots; Invitrogen) Q585, Q605, Q655, and Q705. Complexes were stored at 4°C and spun at 17 000g for 2 minutes before use.
Isolation of CD79b-specific T-cell clones using pMHC tetramers
T cells binding to pMHC tetramers containing CD79b peptides were isolated from cryopreserved PBMCs from healthy HLA-A*02:01 and HLA-B*07:02–negative individuals following a previously described protocol.45 PBMCs were incubated with PE-labeled pMHC tetramers for 1 hour at 4°C. Cells were washed twice and incubated with anti-PE magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). PE-labeled cells were isolated by magnetic-activated cell sorting (MACS) on an LS column (Miltenyi Biotec) according to the manufacturer's instruction. Positive fractions were cultured at 5000 cells per well in 96-well round-bottom culture plates containing 5 × 104 irradiated (35 Gy) autologous PBMCs and 11 000 anti-CD3/CD28 beads (Invitrogen) in 100 µL T-cell medium. After 2 weeks of expansion, CD79b-reactive T-cell clones were obtained from pMHC tetramer–enriched T-cell populations by sorting CD8+ T cells stained with PE-labeled pMHC tetramers containing CD79b peptides and an antibody against CD8 (Invitrogen/Caltag, Buckingham, United Kingdom) combined with antibodies against CD4, CD14, and CD19 (BD Pharmingen, San Jose, CA). Cells were first stained with PE-labeled pMHC tetramers for 1 hour at 4°C before antibodies were added for an additional 15 minutes at 4°C. Single pMHC tetramer+ CD8+ T cells were sorted into 96-well round-bottom culture plates containing 5 × 104 irradiated allogeneic PBMCs in 100 µL T-cell medium supplemented with 0.8 µg/mL phytohemagglutinin (PHA) (Remel, Lenexa, KS). Cell sorting was performed on a FACSAria III (BD Biosciences).
Fluorescence-activated cell sorting (FACS) was performed on an LSRII (BD Biosciences) and analyzed using Diva Software (BD Biosciences) or on a FACS Calibur (BD Biosciences) and analyzed using FlowJo Software (TreeStar, Ashland, OR). T-cell lines generated by MACS enrichment using pMHC tetramers and T-cell clones were analyzed for pMHC tetramer+ CD8+ T cells by staining with pMHC tetramers CD79b173:A2 labeled with PE or Q705, CD79b20:A2 labeled with Q705 or Q655, CD79b9:B7 labeled with Q605 or Q585, and an Alexa700-conjugated antibody against CD8 (Invitrogen/Caltag) combined with fluorescein isothiocyanate–labeled antibodies against CD4, CD14, and CD19 (BD Pharmingen). Cells were first incubated with a mixture of pMHC tetramers (2 µg/mL per pMHC tetramer) for 15 minutes at 37°C before antibodies were added and incubated for an additional 15 minutes at 4°C. PBMCs, purified hematopoietic cell subsets, or activated cells were stained with antibodies against CD79b (clone 3A2-2E7; BD Pharmingen) or isotype control in combination with CD3, CD4, CD14, CD19, CD34 (BD Pharmingen), or CD271 (NGF-R; Sanbio, Uden, The Netherlands) at 4°C for 15 minutes.
Stimulator cells were peptide-pulsed at various peptide concentrations for 30 minutes at 37°C. Responder T cells and peptide-pulsed or unloaded stimulator cells were coincubated at various stimulator to responder (S:R) ratios. After 18 hours of coincubation, supernatants were harvested and interferon (IFN)-γ production was measured by enzyme-linked immunosorbent assay (ELISA; Sanquin Reagents, Amsterdam, The Netherlands).
Adapted from Jedema et al,46 20 000 PKH26GL-labeled (Sigma-Aldrich/Merck, St. Louis, MO) target cells were coincubated with T cells at various effector:target ratios in 100 µL T-cell medium for 17 hours. After coincubation, cells were stained with Sytox Blue dead-cell stain (Invitrogen/Caltag) in a final concentration of 1 µM for 5 minutes. 10 µL Flow-count fluorospheres (Beckman Coulter, Indianapolis, IN) were added and samples were analyzed using FACS. For each sample, 2500 Flow-count fluorospheres were acquired and the percent surviving cells was calculated as follows: (PKH26GL-labeled targets in the presence of effector cells)/(PKH26GL-labeled targets in absence of effector cells) × 100.
Transduction with CD79b retroviral vector and CD79b-specific shRNA
CD79b (accession number #NM_000626.2_variant1) was codon-optimized (GeneArt, Life Technologies) and expressed on a MP71 retroviral backbone in combination with NGF-R. CD79b-targeting short-hairpin RNAs (shRNA) TRC 057 648 and TRC 057 651 (Sigma-Aldrich) were a kind gift of Prof Rob Hoeben (Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands). shRNAs were expressed on retroviral backbone pLKO.1 or pGreenPuro (Systembiosciences, Mountain View, CA), both containing a puromycin resistance gene (see supplemental Methods for production of retroviral supernatant).
For transduction, retroviral supernatant was loaded on 24-well nontissue culture–treated plates that had been coated with 30 µg/mL retronectine (Takara, Shiga, Japan) and blocked with 2% human serum albumin (Sanquin Reagents). Viral supernatant was spun down at 2000g for 20 minutes at 4°C. 1 × 105 K562-A2 or K562-B7 cells were added to CD79b retroviral supernatant and incubated for 18 hours. High-purity bulk CD79b-transduced K562-A2 and K562-B7 cell populations were obtained by sorting cells stained with an antibody against NGF-R. 1 × 105 cells of Epstein-Barr virus–transformed lymphoblastic cell lines (LCLs) or 3 × 105 CD40L-activated B cells were added to retroviral supernatant encoding for CD79b-targeting shRNA and incubated for 18 hours. Transduced cells were selected by supplementing medium with 2 to 10 µg/mL puromycin (Calbiochem, San Diego, CA) for 2 days. Optimal puromycin concentration for each cell type was determined by escalating dose experiments.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was isolated using the RNAqueous Micro-Kit and Small Scale Kit (Ambion, Life Technologies) for a maximum of 0.5 × 106 and 10 × 106 cells, respectively, following the manufacturer's instructions. Total RNA was converted to cDNA using Moloney murine leukemia virus reverse transcriptase (Invitrogen). CD79b expression was measured on the Roche Lightcycler 480 (Roche) using Fast Start TaqDNA Polymerase (Roche) and EvaGreen (Biotium, Hayward, CA) with forward primer 5′-CCAGGCTGGCGTTGTCTCCTG-3′ and reverse primer 5′-GGTACCGGTCCTCCGATCTGGC-3′.
Identification of naturally processed and presented CD79b peptides
To identify MHC-presented CD79b peptides, we adapted a previously described algorithm.45 From a peptide elution library that was generated by eluting and sequencing MHC-bound peptides from human HLA-A2/B7–positive B-LCLs,47 three peptides of CD79b were identified with mascot ion scores >35 and netMHC48,49 binding scores <250 nM (Table 1). Peptides CD79b9 and CD79b173 were newly identified. Peptide CD79b20 had previously been eluted.50,51 All 3 peptides are encoded by each isoform of CD79b. Matching mass spectrometry fragmentation patterns of eluted and newly synthesized CD79b peptides verified correct identification (data not shown). Stable pMHC tetramers of CD79b20 and CD79b173 bound to HLA-A2 (CD79b20:A2 and CD79b173:A2, respectively) and CD79b9 bound to HLA-B7 (CD79b9:B7) were generated by conjugating conventionally refolded pMHC monomers to streptavidin-labeled fluorophores.
Sequences and properties of CD79b-derived peptides
Generation of pMHC tetramer+ CD8+ T-cell lines by tetramer-guided enrichment
T cells binding to pMHC tetramers containing CD79b peptides presented in HLA-A2 or HLA-B7 were isolated from PBMCs of 13 HLA-A2/B7–negative healthy individuals using PE-labeled pMHC tetramers and MACS isolation. From 6 × 107 to 3 × 108 total PBMCs between 10 000 and 160 000 total cells were recovered in the pMHC tetramer–labeled fraction. Among the isolated cells, 7% to 21% were CD8+ T cells, of which only a fraction stained positive for the pMHC tetramers (data not shown). Other cells included CD4+ T cells, monocytes, and B-cells. After 2 weeks of expansion, the generated T-cell lines were subjected to a second enrichment using the same pMHC tetramers. After 2 weeks of expansion, the presence of CD79b-reactive T cells were determined by pMHC-tetramer staining (supplemental Table 1 and supplemental Figure 1). In 11 of 13 individuals CD8+ T cells positive for pMHC tetramer CD79b173:A2 were detected with frequencies of 0.02% to 23.8% of CD8+ T cells. CD8+ T cells staining positive for CD79b20:A2 were present in 3 individuals with frequencies of 0.1% to 31.2%. CD8+ T cells staining positive for CD79b9:B7 were detected in 3 individuals with frequencies of 0.03% to 0.7% of the CD8+ T-cell compartment.
In summary, CD8+ T cells binding to pMHC tetramers presenting CD79b peptides in HLA-A2 or HLA-B7 could be isolated from the allo-repertoire of HLA-A2/B7–negative individuals.
Identification of 3 distinct T-cell clones recognizing CD79b peptides presented in alloHLA
To analyze the functional activity of the CD8+ T-cell populations binding to CD79b pMHC tetramers, pMHC tetramer+ CD8+ T cells were clonally expanded using single-cell sorting. We sorted CD8+ T cells binding to CD79b173:A2 from 4 individuals. CD8+ T-cell clones binding to CD79b20:A2 were obtained from 2 individuals. CD8+ T-cell clones binding to CD79b9:B7 were sorted from one. T-cell clones were analyzed for CD79b peptide-specificity and avidity by loading CD79b peptides on the CD79b-negative cell line K562 expressing HLA-A2 or HLA-B7 (K562-A2 or K562-B7, respectively). Recognition of endogenously processed and MHC-presented CD79b peptides was assessed on 3 HLA-A2/B7–positive B-LCLs, which naturally express CD79b.
CD8+ T-cell clones binding to pMHC tetramer CD79b173:A2 demonstrated varying degrees of peptide specificity and levels of avidity. Figure 1A-C shows the tetramer staining and functional results of 5 representative clones. T-cell clone K110 showed recognition of all 3 HLA-A2+ B-LCLs, as well as unloaded K562-A2 cells, illustrating non-CD79b specificity of the T-cell clone. Clone K64 demonstrated no recognition of peptide-pulsed K562-A2 cells and no reactivity toward any CD79b-expressing B-LCL (Figure 1B-C, respectively). To exclude that absence of recognition could be attributed to failure to produce IFN-γ, granulocyte macrophage colony stimulating factor (GM-CSF) secretion was assessed, but no GM-CSF production was observed (data not shown), suggesting an insufficient sensitivity of clone K64 to recognize MHC-bound CD79b173 peptide. In contrast, T-cell clones K68, K116, and K308 showed CD79b173 peptide–dependent recognition of peptide-pulsed K562-A2 cells. Clone K308 demonstrated the highest peptide sensitivity (Figure 1B). Corresponding with this high peptide sensitivity, clone K308 demonstrated reactivity toward all 3 B-LCLs, whereas the recognition of 3 B-LCLs was low or absent for the low avidity clones K116 and K68 (Figure 1C). These results indicate that clone K308 exhibits sufficient avidity for CD79b173 peptide to be able to efficiently recognize endogenously processed and presented CD79b in the context of HLA-A2 (Figure 1C,G).
pMHC tetramer+ CD8+ T cells exhibit different degrees of CD79b peptide sensitivity and specificity. T cells were clonally expanded by single-cell sorting of CD8+ T cells binding to pMHC tetramers containing CD79b peptides. (A) Shown are pMHC-tetramer staining of 5 representative clones (K110, K64, K68, K116, and K308) isolated using pMHC tetramer CD79b173:A2. Control includes clone HSS12 specific for USP11-derived peptide FTWEGLYNV bound to HLA-A2. Shown are CD8+ cells. (B-C) The same T-cell clones as in (A) were coincubated with K562-A2 cells loaded with decreasing concentrations of CD79b173 peptide (B) or were cocultured with 3 HLA-A2/B7–positive B-LCLs endogenously expressing CD79b (C). Controls included CD79b-negative K562-A2 cells. (D-E) K562-A2 cells peptide-pulsed with decreasing concentration of peptide CD79b173 or CD79b20 as indicated were coincubated with clone K308 isolated using pMHC tetramer CD79b173:A2 (D) or clone S100 isolated using pMHC tetramer CD79b20:A2 (E). (F) K562 cells retrovirally transduced with HLA-B7 (K562-B7) were peptide-pulsed with decreasing concentration of peptide CD79b9 and coincubated with clone A23 isolated with pMHC tetramer CD79b9:B7. (G-I) Clones K308 (G), S100 (H), and A23 (I) were cocultured with HLA-A2 and HLA-B7–positive B-LCLs. CD79b-negative K562-A2 or K562-B7 cells were included as controls. IFN-γ secretion was measured using standard ELISA. Axes were adjusted to maximum cytokine release measured. (J-L) K308 was stained with pMHC tetramer CD79b173:A2 (J), S100 with CD79b20:A2 (K), and A23 with CD79b9:B7 (L). Staining with an irrelevant pMHC tetramer USP11:A2 was used as control. Dot plots are shown with biexponential axis.
Among the T-cell clones isolated using pMHC tetramers CD79b20:A2 or CD79b9:B7, 2 T-cell clones exhibited peptide-specific recognition of peptide-pulsed K562 cells. T-cell clones S100 and A23 recognized peptide CD79b20 and CD79b9 when loaded on K562-A2 and K562-B7 cells, respectively (Figure 1E-F, respectively). Both T-cell clones efficiently recognized all 3 B-LCLs (Figure 1H-I).
Specificity for their cognate pMHC tetramers was confirmed for clones K308, S100, and A23 by FACS analysis (Figure 1J-L). None of the T-cell clones stained with the control pMHC tetramer composed of HLA-A2 presenting peptide FTWEGLYNV derived from USP11.
These data demonstrate that we have isolated high-avidity peptide-specific T-cell clones targeting CD79b peptides presented by alloHLA molecules. T-cell clones K308, S100, and A23 were identified as the most avid clones targeting peptides CD79b173, CD79b20, and CD79b9, respectively.
CD79b transduction and CD79b knockdown verified CD79b specificity of the T-cell clones
To verify CD79b-dependent reactivity by T-cell clones K308, S100, and A23, K562-A2 and K562-B7 cells were retrovirally transduced with CD79b. Clone K308 and S100 specific for HLA-A2–presented peptides CD79b173 and CD79b20, respectively, demonstrated efficient recognition of K562-A2 engineered to express CD79b, whereas reactivity toward nontransduced K562-A2 or K562 carrying the wrong HLA-restriction molecule was absent (Figure 2A). CD79b9-specific clone A23 efficiently recognized K562 simultaneously expressing HLA-B7 and CD79b, whereas no recognition was observed of nontransduced K562-B7. Recognition was also absent when HLA-A2 was expressed in CD79b-transduced K562 instead of HLA-B7.
CD79b expression and shRNA knockdown confirm CD79b specificity of isolated T-cell clones. (A) CD79b-negative K562 cells expressing either HLA-A2 (K562-A2) or HLA-B7 (K562-B7) were retrovirally transduced with codon-optimized CD79b (K562-A2 + CD79b or K562-B7 + CD79b) and sorted to high purity. CD79b-reactive T-cell clones K308 (CD79b173:A2), S100 (CD79b20:A2), and A23 (CD79b9-17:B7) were coincubated with transductants. Control T-cell clone HSS12 recognizes a peptide of the ubiquitously expressed gene USP11 in the context of HLA-A2. (B) CD40L-activated CD19+ B cells of 2 HLA-A2–positive healthy individuals (SRA CD19+ and UHK CD19+) were transduced with CD79b-specific shRNA (shRNA) and selected by puromycin treatment or were left untreated (nt) and coincubated with T-cell clones K308 or S100 or control clone HSS12. Clones K308 and S100 were cocultured at a S:R ratio of 2:1. Control clone HSS was cocultured at an S:R ration of 10:1. IFN-γ production was determined using standard ELISA. Antigen-presenting capacity of transduced and nontransduced target cells was controlled by clone HSS12.
Furthermore, downregulation of CD79b with specific shRNA abrogated recognition of CD40L-stimulated CD19+ B cells from HLA-A2+ healthy individuals by clones K308 and S100, whereas the reactivity of control clone HSS12 specific for a peptide derived from the ubiquitously expressed gene USP11 presented in HLA-A2 was unaffected (Figure 2B).39 Similar results were obtained for clone A23, where CD79b-specific knockdown abrogated recognition of LCL-JY (supplemental Figure 2). In summary, CD79b-dependent and specific recognition were confirmed for T-cell clones K308, S100, and A23.
CD79b-specific T-cell clones recognize B-cell malignancies
Next, CD79b-specific T-cell clones K308 and S100 were assessed for their potency to recognize a variety of primary B-cell malignancies and malignant B-cell lines. Clones K308 and S100 efficiently recognized HLA-A2–positive ALL cell lines (Figure 3A). Clone S100 also recognized 4 HLA-A2–positive primary CLL samples (Figure 3B). In addition, stimulation with primary HLA-A2–positive CLL and ALL samples resulted in strong IFN-γ production of clone K308 (Figure 3C-D). No recognition of the HLA-A2–negative CLL sample ERV 8300 was observed (Figure 3D). Coincubation of the ALL cell lines or primary HLA-A2–positive ALL and CLL samples with clone K308 resulted in efficient lysis of the malignant cells (Figure 3E-F), indicating a cytotoxic effect of clone K308. No lysis of the HLA-A2–negative CLL sample ERV 8300 (Figure 3F) after coincubation with clone K308 was detected. Killing of the malignant cells was not observed for a cytomegalovirus (CMV)-specific control clone recognizing the irrelevant peptide NLVPMVATV bound to HLA-A2 (Figure 3E-F).These results indicate efficient recognition of CD79b-expressing HLA-A2–positive B-cell malignancies by the CD79b-specific clones K308 and S100.
CD79b-specific T-cell clones recognize malignant B-cell lines and primary B-cell malignancies. (A) T-cell clones K308 and S100 recognizing CD79b173 and CD79b20 peptides in the context of HLA-A2, respectively, were stimulated with 3 HLA-A2–positive ALL cell lines ALL-BV, ALL-CM, and ALL-RL at an S:R ratio of 10:1. Controls included CD79b-negative K562-A2 cells and CD79b-positive LCL-JY. (B) T-cell clone S100 was stimulated with 4 primary HLA-A2–positive primary CLL samples at an S:R ratio 10:1. (C-D) Clone K308 was tested against primary CLL (C) and primary ALL (D) samples at the indicated S:R ratios. The HLA-A2 genotype of primary samples is indicated in parentheses. Controls included samples that were exogenously loaded with 100 nM CD79b173 peptide (3:1 + peptide). IFN-γ production was assessed using standard ELISA. (E-F) Cytotoxicity of K308 was determined for ALL cell lines and primary CLL and ALL samples. PKH26GL-labeled ALL cell lines (E) or primary CLL and ALL samples (F) were coincubated with K308 (K308) at different effector-to-target ratios for 17 hours. Living PKH26GL-labeled target cells were counted and analyzed by FACS, and the percent surviving cells was calculated (see Material and methods). Controls included a CMV-specific T-cell clone (CMV clone) recognizing irrelevant pp65-derived peptide NLVPMVATV presented in HLA-A2, and CD79b-negative K562-A2 cells. The experiment was carried out in triplicate showing mean ± standard deviation.
No reactivity against nonhematopoietic tissue but unexpected reactivity against different hematopoietic cell subsets
To further validate that the recognition pattern of the CD79b-specific T-cell clones is restricted to the B-cell compartment, their reactivity toward healthy nonhematopoietic and hematopoietic cells was assessed. No IFN-γ production was measured for K308 and S100 after overnight coculture with HLA-A2–positive fibroblasts or proximal tubular epithelial cells (PTECs) (Figure 4A). Recognition was also absent when fibroblast and PTECs were pretreated with IFN-γ for 4 days to simulate inflamed conditions. Control-clone HSS12 recognized fibroblasts and PTECs under all conditions, indicating the stimulatory capacity of the target cells. In addition, we assessed clone K308's reactivity toward hematopoietic cell populations by isolating different cell subsets from the peripheral blood of HLA-A2–positive healthy individuals. As shown in Figure 4B, clone K308 did not recognize monocyte-derived immature and mature DCs. However, clone K308 exhibited intermediate reactivity against either activated or nonactivated CD4+ T cells, nonactivated CD8+ T cells, and CD34+ HPCs, as well as low reactivity against CD14+ monocytes (Figure 4B-D). Furthermore, coincubation of nonactivated and activated B cells and T cells, and CD34+ HPCs with clone K308, also led to their lysis, which was not observed when target cells were coincubated with a CMV-specific T-cell clone (Figure 4E-F). Similarly, clone S100 also recognized activated CD4+ T cells (Figure 4G).
CD79b-specific T-cell clones spare healthy nonhematopoietic tissue but demonstrate intermediate and low recognition of T cells, HPCs, and monocytes. CD79b-specific T-cell clones were assessed for their reactivity toward healthy nonhematopoietic tissue and hematopoietic cell subsets. (A) Recognition of HLA-A2–positive fibroblasts (FB) and proximal tubular epithelial cells (PTECs) untreated (–) or pretreated with IFN-γ (+) for 4 days was assessed after coincubation with CD79b-reactive T-cell clones K308 and S100 for 18 hours at an S:R ratio of 5:1. Controls included CD79b-negative K562-A2 cells, CD79b-positive LCL-JY, and T-cell clone HSS12 recognizing a peptide of the ubiquitously expressed USP11 presented in HLA-A2. (B-C) Reactivity toward HLA-A2–positive healthy hematopoietic cells was assessed by coculturing clone K308 with cultured (B) and nonactivated (C) cell subsets at the indicated S:R ratios for 18 hours. Controls included peptide-pulsing of stimulators at a concentration of 100 nM peptide (2:1 + peptide or 6:1 + peptide). CD19+ B cells were cocultured with irradiated CD40L-expressing mouse fibroblasts for 6 days before the experiment (CD19 [CD40L]). Immature dendritic cells (imDCs) and mature DCs (mDCs) were CD14+ monocyte–derived (see supplemental Data). Activated CD4+ cells were generated from CD4+ cells that were treated with 0.8 μg/mL PHA and cultured for 10 days. Nonactivated CD19+, CD14+, CD4+, and CD34+ cells were isolated from peripheral blood using MACS isolation. (D) Clone K308 was stimulated with primary CD4+ or CD8+ cells in an S:R ration of 6:1. (E-F) Cytotoxicity of clone K308 was determined for nonactivated CD4+ and CD8+ T cells and activated T cells (containing CD4+ and CD8+ T cells), activated and nonactivated CD19+ B cells (E), and CD34+ HPCs (F). PKH26GL-labeled target cells were coincubated with K308 (K308) at different effector to target ratios for 17 hours. Living PKH26GL-labeled target cells were counted and analyzed by FACS and the percent surviving cells was calculated (see Material and methods). Controls included a CMV-specific T-cell clone (CMV clone) recognizing irrelevant pp65-derived peptide NLVPMVATV presented in HLA-A2. The experiment was carried out in triplicate showing mean ± standard deviation. (G) T-cell clone S100 recognizing peptide CD79b20 in HLA-A2 was stimulated with activated CD4+ T cells or CD40L-activated CD19+ B cells from 2 HLA-A2–positive individuals in an S:R ratio of 10:1. IFN-γ secretion was assessed by standard ELISA.
Aberrant CD79b expression in T cells, HPCs, and monocytes is responsible for reactivity of CD79b-specific T-cell clones
To resolve the unexpected recognition of T cells, HPCs, and monocytes, we examined whether these cell populations demonstrated unreported CD79b cell-surface expression. Staining of PBMCs with anti-CD79b antibody revealed no cell-surface expression of CD79b on CD3+ T cells and CD14+ monocytes (Figure 5A). In addition, activated CD4+ T cells and purified CD34+ HPCs did not show detectable levels of cell-surface CD79b, whereas CD79b was demonstrated on CD19+ B cells (Figure 5A). To investigate whether gene expression of CD79b in cells outside of the B-cell compartment occurred, we performed qRT-PCR. As expected, high expression of CD79b in CLL, ALL, B-LCL, and healthy CD19+ B-cell samples was demonstrated by qRT-PCR (Figure 5B), whereas no expression could be detected for nonhematopoietic cells such as fibroblasts, keratinocytes, and PTECs under normal and IFN-γ–treated conditions. In addition, mature and immature DCs showed no or very low gene expression of CD79b (100-1000–fold lower expression compared with CD19+ B cells). In activated CD4+ T cells and CD34+ HPCs, the expression of CD79b was approximately only tenfold lower than in CD19+ B cells correlating with intermediate recognition of these cell subsets. In weakly recognized CD14+ monocytes and nonactivated CD4+ T cells, the expression of CD79b was approximately ten- to 100-fold lower compared with CD19+ B cells. These results demonstrate a correlation between CD79b expression and the reactivity of the CD79b-specific clones, exhibiting strong reactivity against CD19+ B cells, no recognition of fibroblasts and PTECs, and immature and mature DCs but intermediate to low reactivity against T cells, HPCs, and monocytes.
Aberrant CD79b gene expression in T cells, HPCs, and monocytes in the absence of cell-surface expression. (A) PBMCs were stained with CD79b-specific antibody (αCD79b) or isotype control (Isotype) in combination with lineage-specific markers as indicated. Histograms show CD79b expression after gating on B cells (CD19+), T cells (CD3+), and monocytes (CD14+). CD34+ HPCs were isolated from granulocyte colony-stimulating factor–mobilized blood. Activated CD4+ T cells were stimulated with PHA and irradiated autologous feeders and cultured for 10 days. (B) CD79b mRNA expression was determined for indicated cell subsets by qRT-PCR. Expression was normalized to average CD79b expression in primary CD19+ B cells, which was set to 100. Dots indicate individual samples.
In summary, aberrant gene expression of CD79b in CD4+ T cells, HPCs, and monocytes, although undetectable at the cell surface, caused unexpected reactivity of the CD79b-specific T-cell clones against these cell subsets.
In this study, we aimed to isolate T cells expressing TCRs specific for BCR-associated protein CD79b that is expressed on normal and malignant B cells.14⇓-16 MHC-presented peptides of CD79b were identified from peptide-elution studies.47,50,51 By exploiting the immunogenicity of alloHLA molecules, 3 distinct T-cell clones derived from different HLA-A*0201/B*0702– healthy individuals were isolated targeting distinct peptides of CD79b presented in HLA-A*0201 or HLA-B*0702. The CD79b-specific clones efficiently recognized primary CLL, ALL, and ALL cell lines, whereas no reactivity toward nonhematopoietic cells and professional antigen-presenting cells was detected. However, unexpected reactivity toward monocytes, HPCs, and T cells was observed. Aberrant intracellular CD79b expression in those cell subsets was detected in the absence of cell-surface CD79b.
Although all T-cell clones were isolated by binding to pMHC tetramers containing CD79b peptides, CD79b specificity was not observed in all cases. Various T-cell clones lacked specificity for CD79b peptides, indicated by recognition of CD79b-negative K562-A2 or K562-B7 cells. This reactivity is probably the result of crossreactivity with other alloHLA-bound peptides and resembles the reactivity previously observed for WT1-specific T cells derived from the allo-repertoire.52 Other isolated T-cell clones showed no reactivity toward peptide-loaded stimulator cells and CD79b-expressing B-LCLs, suggesting insufficient T-cell avidity. In contrast, we also isolated pMHC tetramer+ T-cell clones with variable sensitivity for the CD79b peptide-loaded stimulators. Among these T-cell clones, only the most avid clones were capable of recognizing endogenously-processed peptide. We have previously illustrated53 that not pMHC-tetramer binding but TCR-ligand koff-rate as measured by decay of pMHC:TCR interaction correlates with functional characteristics. Assessment of sensitivity and specificity for MHC-bound CD79b peptides on a clonal level was therefore imperative to select for the most avid and CD79b-specific clones.
For 3 high-avidity T-cell clones, we confirmed CD79b specificity by CD79b transduction into K562 cells and abrogation of recognition of B-LCLs and CD19+ B cells after shRNA-mediated CD79b-specific knockdown. Furthermore, absence of reactivity toward CD79b-negative cells such as nonhematopoietic cell lines pretreated with IFN-γ and professional antigen-presenting cells indicated that CD79b-independent activation of our CD79b-specific T-cell clones does not easily occur. However, recognition of T cells, HPCs, and monocytes was observed, which correlated with low aberrant intracellular CD79b expression in these cells. Although we cannot formally exclude the recognition of peptide lookalikes or mimitopes in these cells, the strong CD79b dependency of our CD79b-specific T-cell clones argues otherwise.
Recognition of primary B-cell malignancies by our T-cell clones suggested that the expressed CD79b-specific HLA-A*0201 or B*0702-restricted TCRs could be used for TCR gene transfer approaches to treat HLA-A*0201 or B*0702-positive patients with B-cell malignancies. However, we also observed recognition of monocytes, T cells, and HPCs. In contrast to B-cell depletion, long-term monocytopenia and T-cell and HPC ablation is not tolerable from a clinical perspective.
The high-avidity CD79b-specific T cells recognizing a self-antigen such as CD79b when presented in HLA-A2 or HLA-B7 were isolated from HLA-A2/B7–negative individuals. Negative selection during thymic development does not deplete these high-avidity T cells in these individuals because the HLA-A2 and HLA-B7 restriction molecules are not present. In HLA-A2 and HLA-B7–positive individuals, however, the elimination of such high-avidity CD79b-specific T cells is crucial to prevent loss of B cells, monocytes, and HPCs as the result of autoreactivity. Furthermore, the CD79b expression in HLA-A2/B7–positive T cells would also cause fratricide41 among T cells; T cells would simultaneously express the correct HLA restriction molecule as well as the CD79b antigen recognized by their TCR. In concordance, we have previously shown that self-HLA–restricted PRAME-specific T cells possess insufficient avidity to recognize endogenously expressed PRAME.38 Therefore, it is very unlikely that such high-avidity CD79b-specific T cells as demonstrated in this study could be isolated from the autologous repertoire of HLA-A2 and HLA-B7 individuals.
In conclusion, exploiting the immunogenicity of alloHLA molecules is an effective and attractive strategy to overcome self-tolerance toward self-antigens. TCRs raised from alloreactive T cells can meet the requirements for high affinity interaction with self-antigens while preserving peptide specificity and HLA-restriction. Their identification can be readily achieved by pMHC tetramer–based isolation but requires functional assessment on a clonal level. Aberrant expression of CD79b in monocytes, T cells, and HPCs eliminated CD79b as a suitable target for TCR-based immunotherapy to treat B-cell malignancies. However, CD79b may still be an attractive target for CAR or antibody-based immunotherapies because no cell-surface expression on monocytes, T cells, and HPCs was observed.
Contribution: L.J. designed, performed and analyzed research, and wrote the manuscript; P.H. and M.G.D.K. designed, performed, and analyzed research; C.H., D.M.v.d.S., and R.S.H. performed and analyzed research; P.A.v.V designed research; J.H.F.F. designed research and wrote the manuscript; M.H.M.H. designed and analyzed research, and wrote the manuscript; and all authors reviewed and edited the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Lorenz Jahn, Department of Hematology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands; e-mail: l.jahn{at}lumc.nl.
The authors thank Prof Cees van Kooten (Department of Nephrology, Leiden University Medical Center, Leiden, The Netherlands) for providing PTECs. CD79b-specific shRNA constructs were a kind gift of Prof. Rob Hoeben (Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands). Guido de Roo and Menno van der Hoorn provided technical assistance in flow cytometric cell sorting.
This research was supported by the financial assistance of the Dutch Cancer Society (2010-4832) and the Landsteiner Foundation for Blood Transfusion Research (LSBR0713).
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
Submitted July 10, 2014.
© 2015 by The American Society of Hematology
Porter DL,
Levine BL,
Kalos M,
Bagg A,
June CH
. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med 2011;365(8):725-733.
. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med 2011;3(95):95ra73.
Kochenderfer JN,
Dudley ME,
Feldman SA,
. B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood 2012;119(12):2709-2720.
Grupp SA,
Barrett D,
. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med 2013;368(16):1509-1518.
McLaughlin P,
Grillo-López AJ,
Link BK,
. Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program. J Clin Oncol 1998;16(8):2825-2833.
Witzig TE,
Vukov AM,
Habermann TM,
. Rituximab therapy for patients with newly diagnosed, advanced-stage, follicular grade I non-Hodgkin's lymphoma: a phase II trial in the North Central Cancer Treatment Group. J Clin Oncol 2005;23(6):1103-1108.
Vose JM,
Grossbard ML,
. Phase II study of rituximab in combination with chop chemotherapy in patients with previously untreated, aggressive non-Hodgkin's lymphoma. J Clin Oncol 2001;19(2):389-397.
Coiffier B,
Lepage E,
Briere J,
. CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N Engl J Med 2002;346(4):235-242.
Wilson WH,
Janik JE,
. Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19. Blood 2010;116(20):4099-4102.
Shortt J,
Spencer A
. Adjuvant rituximab causes prolonged hypogammaglobulinaemia following autologous stem cell transplant for non-Hodgkin's lymphoma. Bone Marrow Transplant 2006;38(6):433-436.
Zheng Z,
Levine B,
June C
. Chimeric Antigen Receptor Therapy for B-cell Malignancies. J Cancer 2011;2:331-332.
Wolverton W,
McClenathan B
. Prolonged hypogammaglobulinemia after adjuvant rituximab for Hodgkin's lymphoma presenting with refractory urticaria. Ann Allergy Asthma Immunol 2012;109(6):473-474.
Casulo C,
Maragulia J,
Zelenetz AD
. Incidence of hypogammaglobulinemia in patients receiving rituximab and the use of intravenous immunoglobulin for recurrent infections. Clin Lymphoma Myeloma Leuk 2013;13(2):106-111.
D'Arena G,
Musto P,
Cascavilla N,
Dell'Olio M,
Di Renzo N,
Carotenuto M
. Quantitative flow cytometry for the differential diagnosis of leukemic B-cell chronic lymphoproliferative disorders. Am J Hematol 2000;64(4):275-281.
Cabezudo E,
Carrara P,
Morilla R,
Matutes E
. Quantitative analysis of CD79b, CD5 and CD19 in mature B-cell lymphoproliferative disorders. Haematologica 1999;84(5):413-418.
Olejniczak SH,
Stewart CC,
Donohue K,
Czuczman MS
. A quantitative exploration of surface antigen expression in common B-cell malignancies using flow cytometry. Immunol Invest 2006;35(1):93-114.
Venkitaraman AR,
Williams GT,
Dariavach P,
Neuberger MS
. The B-cell antigen receptor of the five immunoglobulin classes. Nature 1991;352(6338):777-781.
Clark MR,
Campbell KS,
Kazlauskas A,
. The B cell antigen receptor complex: association of Ig-alpha and Ig-beta with distinct cytoplasmic effectors. Science 1992;258(5079):123-126.
Reth M
. Antigen receptors on B lymphocytes. Annu Rev Immunol 1992;10:97-121.
Pleiman CM,
D'Ambrosio D,
Cambier JC
. The B-cell antigen receptor complex: structure and signal transduction. Immunol Today 1994;15(9):393-399.
Lin J,
Justement LB
. The MB-1/B29 heterodimer couples the B cell antigen receptor to multiple src family protein tyrosine kinases. J Immunol 1992;149(5):1548-1555.
Sanchez M,
Misulovin Z,
Burkhardt AL,
. Signal transduction by immunoglobulin is mediated through Ig alpha and Ig beta. J Exp Med 1993;178(3):1049-1055.
Hsueh RC,
Scheuermann RH
. Tyrosine kinase activation in the decision between growth, differentiation, and death responses initiated from the B cell antigen receptor. Adv Immunol 2000;75:283-316.
Tseng J,
Eisfelder BJ,
Clark MR
. B-cell antigen receptor-induced apoptosis requires both Ig alpha and Ig beta. Blood 1997;89(5):1513-1520.
Healy JI,
Dolmetsch RE,
Lewis RS,
Goodnow CC
. Quantitative and qualitative control of antigen receptor signalling in tolerant B lymphocytes. Novartis Found Symp. Vol. 215. 1998. p. 137-144; discussion 144-135, 186-190.
Hashimoto S,
Chiorazzi N,
Gregersen PK
. The complete sequence of the human CD79b (Ig beta/B29) gene: identification of a conserved exon/intron organization, immunoglobulin-like regulatory regions, and allelic polymorphism. Immunogenetics 1994;40(2):145-149.
Koyama M,
Nakamura T,
Higashihara M,
. The novel variants of mb-1 and B29 transcripts generated by alternative mRNA splicing. Immunol Lett 1995;47(3):151-156.
Cragg MS,
Chan HT,
Fox MD,
. The alternative transcript of CD79b is overexpressed in B-CLL and inhibits signaling for apoptosis. Blood 2002;100(9):3068-3076.
Zucchetto A,
Sonego P,
Degan M,
. Signature of B-CLL with different prognosis by Shrunken centroids of surface antigen expression profiling. J Cell Physiol 2005;204(1):113-123.
Bomben R,
Dal Bo M,
. A scoring system based on the expression of six surface molecules allows the identification of three prognostic risk groups in B-cell chronic lymphocytic leukemia. J Cell Physiol 2006;207(2):354-363.
Heemskerk MH
. T-cell receptor gene transfer for the treatment of leukemia and other tumors. Haematologica 2010;95(1):15-19.
van Loenen MM,
de Boer R,
Hagedoorn RS,
van Egmond EH,
Falkenburg JH,
. Optimization of the HA-1-specific T-cell receptor for gene therapy of hematologic malignancies. Haematologica 2011;96(3):477-481.
Robbins PF,
Morgan RA,
. Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol 2011;29(7):917-924.
Sykulev Y,
Joo M,
Vturina I,
Tsomides TJ,
Eisen HN
. Evidence that a single peptide-MHC complex on a target cell can elicit a cytolytic T cell response. Immunity 1996;4(6):565-571.
Irvine DJ,
Purbhoo MA,
Krogsgaard M,
Davis MM
. Direct observation of ligand recognition by T cells. Nature 2002;419(6909):845-849.
Huang J,
Brameshuber M,
Zeng X,
. A single peptide-major histocompatibility complex ligand triggers digital cytokine secretion in CD4(+) T cells. Immunity 2013;39(5):846-857.
Stone JD,
Aggen DH,
Schietinger A,
Schreiber H,
Kranz DM
. A sensitivity scale for targeting T cells with chimeric antigen receptors (CARs) and bispecific T-cell Engagers (BiTEs). OncoImmunology 2012;1(6):863-873.
Amir AL,
van der Steen DM,
. PRAME-specific Allo-HLA-restricted T cells with potent antitumor reactivity useful for therapeutic T-cell receptor gene transfer. Clin Cancer Res 2011;17(17):5615-5625.
. Allo-HLA-reactive T cells inducing graft-versus-host disease are single peptide specific. Blood 2011;118(26):6733-6742.
Savage P,
Gao L,
Vento K,
. Use of B cell-bound HLA-A2 class I monomers to generate high-avidity, allo-restricted CTLs against the leukemia-associated protein Wilms tumor antigen. Blood 2004;103(12):4613-4615.
Leisegang M,
Wilde S,
Spranger S,
. MHC-restricted fratricide of human lymphocytes expressing survivin-specific transgenic T cell receptors. J Clin Invest 2010;120(11):3869-3877.
Abrahamsen IW,
Stronen E,
Wälchli S,
. Targeting B cell leukemia with highly specific allogeneic T cells with a public recognition motif. Leukemia 2010;24(11):1901-1909.
Jeremias I,
. TCR-transgenic lymphocytes specific for HMMR/Rhamm limit tumor outgrowth in vivo. Blood 2012;119(15):3440-3449.
Garboczi DN,
Hung DT,
Wiley DC
. HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proc Natl Acad Sci USA 1992;89(8):3429-3433.
Hombrink P,
Hassan C,
Kester MG,
. Discovery of T cell epitopes implementing HLA-peptidomics into a reverse immunology approach. J Immunol 2013;190(8):3869-3877.
Jedema I,
van der Werff NM,
Barge RM,
Falkenburg JH
. New CFSE-based assay to determine susceptibility to lysis by cytotoxic T cells of leukemic precursor cells within a heterogeneous target cell population. Blood 2004;103(7):2677-2682.
de Ru AH,
. The human leukocyte antigen-presented ligandome of B lymphocytes. Mol Cell Proteomics 2013;12(7):1829-1843.
Nielsen M,
Lundegaard C,
Worning P,
. Reliable prediction of T-cell epitopes using neural networks with novel sequence representations. Protein Sci 2003;12(5):1007-1017.
Lamberth K,
Harndahl M,
Buus S,
Lund O,
Nielsen M
. NetMHC-3.0: accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8-11. Nucleic Acids Res. Vol. 36.(Web Server issue) 2008. p. W509-W512.
Kageyama G,
Kawano S,
Kanagawa S,
. Effect of mutated transporters associated with antigen-processing 2 on characteristic major histocompatibility complex binding peptides: analysis using electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 2004;18(9):995-1000.
Weinzierl AO,
Rudolf D,
Hillen N,
. Features of TAP-independent MHC class I ligands revealed by quantitative mass spectrometry. Eur J Immunol 2008;38(6):1503-1510.
Falkenburg WJ,
Melenhorst JJ,
van de Meent M,
. Allogeneic HLA-A*02-restricted WT1-specific T cells from mismatched donors are highly reactive but show off-target promiscuity. J Immunol 2011;187(5):2824-2833.
Raz Y,
. Mixed functional characteristics correlating with TCR-ligand koff -rate of MHC-tetramer reactive T cells within the naive T-cell repertoire. Eur J Immunol 2013;43(11):3038-3050.
DOI: https://doi.org/10.1182/blood-2014-07-587840
Immunobiology and Immunotherapy
Lymphoid Neoplasia
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You are going to email the following Therapeutic targeting of the BCR-associated protein CD79b in a TCR-based approach is hampered by aberrant expression of CD79b
Lorenz Jahn, Pleun Hombrink, Chopie Hassan, Michel G. D. Kester, Dirk M. van der Steen, Renate S. Hagedoorn, J. H. Frederik Falkenburg, Peter A. van Veelen, Mirjam H. M. Heemskerk
Blood Feb 2015, 125 (6) 949-958; DOI: 10.1182/blood-2014-07-587840
Jahn, L., Hombrink, P., Hassan, C., Kester, M. G., van der Steen, D. M., Hagedoorn, R. S., Falkenburg, J. H., van Veelen, P. A., & Heemskerk, M. H. (2015). Therapeutic targeting of the BCR-associated protein CD79b in a TCR-based approach is hampered by aberrant expression of CD79b. Blood, 125(6), 949-958. Accessed July 14, 2019. https://doi.org/10.1182/blood-2014-07-587840.


WARNING: Do not eat more than ten cookies in one sitting. This is pure cholesterol in a neat and tidy package.
four sticks (one pound) of salted butter. Real butter. Do not profane this recipe with margarine or any other substitute, or suffer like the infidel you are.
four cups of flour. That bleached white stuff.
one and a half cups of brown sugar. Make sure to break up any big Lumps in here. Of course, you could just leave them in, but don't blame me when you chip a tooth on that lump of carmelized sugar.
various flavorings. My favorite is vanilla, but you can use other kinds just as well. Make sure you have at least two tablespoons worth around.
about three to four hours of spare time.
First, the butter. Unwrap it, then chuck all four sticks into a large plastic mixing bowl. Nuke them for about thirty seconds on high so that they're slightly runny, but not melted.
Now, take the brown sugar and dump about half of it in with the butter. If you followed directions, there shouldn't be any lumps to make the next part more difficult than it has to be. Mix the butter and sugar like a madman until they're well blended, then toss in the flavoring and the rest of the sugar. You guessed it, keep mixing.
Now, once you have that sloshed into a nicely amorphous mass, add in about a third of the flour. Fold it into the mix until everything turns the same color. Follow up with the remaining two thirds of the flour and treat them in a similar fashion.
At this point you should preheat the oven to 350 degrees fahrenheit and get out a few cookie sheets (or pans- see the following paragraph).
say, "to hell with this," roll out the dough on a cookie sheet and just cut apart whatever comes out of the oven.
be a good little androgynous figure, roll it out to about a quarter of an inch thick and use all sorts of pretty cookie cutters to dress up your artery cloggers.
or you can do what I always do: grab a glass, roll it out to the aformentioned quarter inch, and start making circles.
Once you have a sufficient number cut out- say, around twenty- arrange them on a cookie sheet so none of the edges are touching, then pop them in the now-hot oven for around fifteen minutes. When they come out, they should be a light golden brown and smell like buttery heaven.
I've never been able to figure out an exact number of how many come out per batch due to wild dogs and hungry older brothers, but it should make at least forty to sixty cookies. Feel free to post variations.
This is my family's recipe. More Christmas cookies!
Cream the butter and sugar together. Add the flavoring and flour. Roll into balls, flatten with your fingers, and prick with a fork (I don't know why that last step is important, but TRUST ME, it is. Put about four rows of fork-holes in).
Bake at 300oF for 20 to 25 minutes. They should still be fairly pale when they come out of the oven.
Blend the butter and sugar. Add the flour. Place on a cookie sheet, perforate, and refrigerate for about 30 minutes while you pre-heat your oven to 350 F. Bake until the edges start to brown (about 15 minutes).
You may have to play with the amount of flour to get the right consistency (neither too oily nor too crumbly).
British Isles Recipes Cornmeal shortbread Chocolate pecan cookies with gooey caramel centers Don't! Buy! Thai!


Welsh No.2 Peter Creed overcame European Junior Champion Patrick Rooney to triumph in the final of the LOC Charing Cross Classic, PSA M5 tournament held in London - lifting his sixth PSA World Tour title.
The World No.75, known on the Tour for his entertaining, all-action displays, progressed to the final courtesy of wins over Portuguese qualifier Claudio Pinto and English duo Lyell Fuller and Joe Green.
19-year-old Rooney, who was unseeded for the tournament, downed a trio of seeds on his way through to the final, putting third seed Chris Fuller, number eight seed Robert Downer and top seed Nathan Lake to the sword, with the latter of those fixtures going all the way to five games.
Rooney, the World No.133, kept up his impressive form to go a game ahead in the final, only to take just a single point in game two after a superb response from Caerphilly-born Creed.
The final two games were more closely-contested affairs, but it was the Welshman who came out on top again in both and he closed out a 5-11, 11-1, 11-5, 11-6 victory to lift his first Tour crown in 16 months.
Teenage duo Patrick Rooney (right) and Kyle Finch moved through to the quarter-final stage of the 2016 LOC Charing Cross Classic after overcoming the Fuller brothers, Chris and Mark, respectively, in their first round fixtures at the Charing Cross Sports Club in London.
Rooney, the reigning European Junior Champion, is appearing at his first PSA World Tour tournament in two months and enjoyed a strong summer after capturing his first Tour crown at the Madeira International back in June.
The 19-year-old wasted no time in establishing himself on his fixture against Chris and surged to an 11-8, 11-6, 11-4 victory to set up a last eight meeting with fellow Englishman Robert Downer, who defeated Michael Harris in straight games.
Elsewhere, Finch, a 17-year-old qualifier, will aim to reach the semi-finals of a PSA World Tour event for the first time since his maiden tournament back in August of last year after a comfortable 3-0 win over Mark earned him a place in the next round, where he will take on top seed Nathan Lake in what looks set to be a fascinating battle between the two Englishmen.
Number two seed Peter Creed is the only non-Englishman to secure a place in the quarter-finals after the Welsh No.2 dispatched Portugal's Claudio Pinto to set up a next round fixture with Lyell Fuller.
Meanwhile, tournament wildcard James Peach caused the day's biggest upset after bridging a 146-place World Rankings gap to shock New Zealand's Lance Beddoes and his reward is a meeting with fifth seed Joe Green for a place in the semi-finals of the PSA M5 tournament.


text:
La vallée de Jezreel (en hébreu :  - "", aussi appelée la plaine d'Esdraelon) est une large vallée dans le nord d'Israël, nommée en référence à la ville de Jezraël située sur une colline dans le sud de la vallée. La vallée de Jezreel fait partie de la Galilée dans le district nord d'Israël.

La vallée de Jezreel est le lieu d'importantes implantations humaines, anciennes et modernes. L'ancienne ville de Megiddo se situe dans le sud-ouest de la vallée. La plus grande ville moderne est la ville de Afoula aussi appelée « Capitale de la vallée ».

La vallée de Jezreel est, pour les chrétiens, le site de la future bataille de l'Armageddon.

Étymologie
Le nom de la vallée provient de celui de la ville antique de Jezraël située sur une colline dominant le sud de la vallée. Ce nom, en , Yizre'el, se traduit par « semence de Dieu ». Le toponyme vallée de Jezreel est parfois uniquement appliqué à la partie centrale de la vallée, autour de la ville de Jezraël tandis que la partie sud-ouest de la vallée est dénommée « vallée de Megiddo » d'après la cité antique de Megiddo qui se situait à cet endroit. L'ensemble a aussi été appelé plaine d'Esdraelon, ce qui correspond à l'équivalent de Jezreel en koinè.

Géographie
Au sens large, on définit parfois la vallée de Jezreel comme une trouée séparant les monts de Galilée de ceux de Samarie situés dans les territoires palestiniens, reliant ainsi la mer Méditerranée et la mer Morte. Dans cette acception, cet espace comprend d'ouest en est la baie de Haïfa, les vallées de Zebulon, de Jezreel proprement dite, de Harod et de Bëit Shéan.

La vallée de Jezreel comprise au sens strict est de forme grossièrement triangulaire. Orientée sud-est - nord-ouest, elle est entourée au nord-est par les collines de la Basse-Galilée, au sud par le mont Guilboa et les collines de Samarie, à l'ouest et la chaîne du mont Carmel au nord-ouest. La vallée est traversée par le Kishon. Elle est au contact avec la mer Méditerranée via la vallée de Zebulon et avec le rift du Jourdain via la vallée d'Harod. Le centre de la vallée se situe à environ  au-dessus du niveau de la mer.

La vallée de Jezreel s'est formée au début du Pliocène. Il s'agit d'un graben dont la topologie est assez plate, constitué d'une couche alluvionnaire très épaisse. Les sédiments de la vallée sont d'origine alluviale et partiellement colluviale. Ils sont sombres et épais, riches en matières organiques. Ces caractéristiques sont dues au fait que la vallée a abrité de nombreuses zones humides jusqu'à une époque récente. Les sols sont en conséquence fertiles et retiennent bien l'eau. Cependant, en raison de l'excès d'eau, certains de ces sols sont hydromorphiques.

La vallée était peut-être à une époque un chenal par lequel la mer Morte, située au sud-est de la vallée, était reliée à la mer Méditerranée. Il y a environ deux millions d'années, alors que les terres situées entre la mer Méditerranée et la vallée du rift du Jourdain connaissaient un phénomène de surrection, le lien entre les deux mers a disparu et le déversement périodique des eaux de la Méditerranée dans la vallée a cessé. La mer Morte désormais complètement fermée est peu à peu devenue extrêmement saline.

Histoire

Antiquité
La vallée était traversée par la via Maris, un axe de communication datant de l'âge du bronze reliant l'Égypte au Levant.

Période ottomane
L'écrivain américain Bayard Taylor dresse une description de la vallée, qu'il a eu l'occasion de traverser, dans un livre dénommé  publié en 1854. Il la désigne comme « l'une des régions les plus riches du monde ». Laurence Oliphant, qui se rend en 1887 dans le sandjak d'Akko, qui fait alors partie de la wilaya de Beyrouth, décrit la vallée comme « un immense lac de blé ondoyant avec ses villages collinaires émergeant pareillement à des îles, elle présente un tableau de fertilité luxuriante des plus frappants qu'il se puisse concevoir. ».

Dans les , la  basée à Beyrouth achète auprès du gouvernement ottoman les terres de la vallée pour une somme de . Entre 1912 et 1925, cette famille vend les  () qu'elle détient dans la vallée au  pour près de . Le but de l'organisation est de fournir des terres aux Juifs établis en Palestine ainsi qu'à ceux souhaitant s'y installer.

Palestine mandataire
Le , nouveau propriétaire des terres de la vallée souhaitait promouvoir le travail Juif, l'un des principes fondamentaux du sionisme travailliste alors prédominant au sein du Yichouv. En conséquence, les métayers arabes travaillant pour les anciens propriétaires terriens se sont retrouvés sans travail. Certains, tels ceux d'Afula (El-Ful) ont refusé de partir de leur terres et ont été expulsés par la police britannique. Les paysans arabes expulsés sont partis vers la côte à la recherche de travail et ont le plus souvent échoué dans des bidonvilles en périphérie de Jaffa et Haïfa.

Les Juifs du Yichouv créent après l'achat des terres les premiers noyaux de peuplement sionistes de la vallée. Ils fondent la ville d'Afoula et drainent les zones marécageuses afin d'accroître les surfaces agricoles. Le premier moshav d'Israël, Nahalal est créé là-bas en 1921.

La  mise en place par les Britanniques après les émeutes arabes de 1929 pour déterminer les causes de l'instabilité du territoire mandataire conclut en ce qui concerne la situation des fellah de la plaine de Jezreel que l'attitude des autorités juives n'est pas à mettre en cause. En effet l'organisation responsable de l'achat des terres les a payées au prix fort et les métayers ont reçu une compensation qu'il n'était légalement pas obligatoire de leur octroyer. La commission impute la responsabilité de leur situation aux autorités coloniales britanniques qui ont failli à leur devoir de protection des populations arabes.

Voir aussi
 Ein el-Jarba

Références

Jezreel
Lieu de la Bible
Graben dans la vallée du Grand Rift
District nord (Israël)

redpajama_set_name: RedPajamaWikipedia


Young Hungarian photographer Bence Bakonyi first came to our attention via his excellent Dignity series, a surrealistic exploration of self-identity captured in arid mountain landscapes. Cognition, his latest collection of images, picks up where the earlier series left off. The Shanghai-based photographer traveled to Dunhuang and Zhangye Danxia in northwestern China, along the Silk Road, where ancient ruins emerge from giant sand dunes. There he captured images of tiny figures milling about the immense desert landscape, putting the viewer in the position of a distant, outside spectator.
From the perspective of these photographs we experience that kind of comprehension which is possible only through considerable withdrawal. Due to its viewpoint, the series represents the spirit of the group. It is not the person, but the community which appears as an individual entity: we see it moving in unity, the trace of its path, and the process through which it discovers the unknown.
View the whole series on Bakonyi's website.


In American Exurbia I photographed children in their homes in newly developed "exurbia" in New Jersey. These communities are isolated, secure, and homogeneous. My interest in photographing these children lies in my fascination with the development of identity in children within newly created environments. Both the girls and boys in my photographs are represented in moments where their individuality confronts the limitations of their transforming culture. The work speaks to my perspective on these children and their futures. My photographs exist as an exploration of identity creation, and globalization.


Just like the title says- looking to transfer my Jeep lease. PM with any questions. Looking to move quickly on this. 10k/yr mileage but I'm so under miles you can drive 1447 miles per month for the remaining 17 months.
Seems like a decent price with the mileage…What is bank and what's the transfer fee?
I'm interested but have never done a lease takeover. How would that work?
Hello I may be interested in this also. Does the bank allow out of state transfers? Also the engine, is it the six or eight cylinder? Thanks.


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WITH the release of the final stage of land, this year should see the completion of Investa Property Group's award-winning Bellflower.
The final piece in the puzzle is the recent release of Bellflower's last allocation of available land, 30% of which is already sold.
The final sale of the lots will mark the completion of the community, which will have more than 400 homes and 800 residents.
Queensland Investa Land General Manager Bruce Harper said this year will see the team finally realising their initial goal of providing a truly sustainable residential development on the Sunshine Coast.
"Bellflower was never a project that simply focused on selling off house and land packages," Mr Harper said. "We always wanted to create a new community that offered buyers a naturally sustainable, active, affordable and friendly place to live.
"Bellflower has always been a popular choice for residents within the local catchment area," Mr Harper said. "Most of our residents have come from all over the Coast – even as far as Beerwah – and even within the Bellflower community."
The residential community, located at Sippy Downs, has been an attractive option for young families and young professional couples on the Sunshine Coast since the development began in 2005.
"Many of our residents have quoted the educational facilities, the close proximity to the rest of the Coast and the value for money as reasons they chose to live in Bellflower," Mr Harper said.
"Bellflower demonstrated two things to Sunshine Coast buyers: that value for money still exists in the property market, and the difference between developing house-and-land packages and developing a sustainable community."

redpajama_set_name: RedPajamaCommonCrawl


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If you were looking for the exploration team in the Mystery Dungeon series, see Team Skull (Mystery Dungeon).
Team Skull (Japanese: スカル団 Skull-dan, literally Skull Gang) is a villainous team based in the Alola region.
Team Skull appears in Sun and Moon. As revealed by some of their members, Team Skull is composed of misfits who failed the island challenge. They steal Pokémon from people, but unlike previous villainous teams, they do not have a specific goal other than to cause trouble and acquire enough money to support themselves. In a stark contrast to other villainous teams, they do not pose a serious threat to the wellbeing of Alola and its residents, the majority of whom consider them thuggish nuisances at worst.
Team Skull originated from a group that coalesced around one of Alola's kahunas. This group provoked the wrath of the four island deities and was struck down. The survivors became the first members of Team Skull. At some point, Team Skull took over Po Town on Ula'ula Island, driving away the original residents, vandalising the buildings with graffiti and structural damage along with generally preventing others from entering the town and hindering the flow of electricity.
The group is known to highly value kinship - many of the grunts are close friends with each other, the team's admin Plumeria treats her underlings like a caring older sibling and in return is respected by them for it, and several grunts mock Gladion for lacking friends in the team and technically not being a true member of Team Skull as hired work.
At some point, Lusamine became the secret benefactor of Team Skull, and they, in turn, started to carry out her orders. Not all of the members were aware of her influence - while Plumeria knew that Guzma respected Lusamine as she was the only older figure to acknowledge his strength, Lusamine's estranged son Gladion was only aware that Team Skull was after Cosmog without knowing of their ties to the Aether Foundation president. Following Team Skull's kidnapping of Lillie and Nebby, Gladion immediately severed his ties with them and joined with Hau and the player to infiltrate and rescue them from Aether Paradise.
After Guzma is rescued from Ultra Space by the player, he disbands Team Skull and its members go their separate ways. Guzma returns to his parents' house on Melemele Island, while Plumeria takes up residence in a camper on Route 13 and later challenges the Alola Pokémon League. One Grunt becomes an apprentice chef under Mallow in Konikoni City, while two others in the Po Town Pokémon Center offer a Skull Tank for sale in order for them to make enough money to leave. Others are taken by Hala to train in Iki Town, deciding to become a rescue team and dubbing themselves "Team Reskull" (Japanese: タスカル団 Tasukaru-dan, literally Rescue Gang). Guzma is one of the former members taken under Hala's wing, but rather to help hone his potential and help him overcome his anger issues.
Team Skull debuted in Alola to New Adventure!, where three Team Skull Grunts, Tupp, Rapp, and Zipp, were seen harassing Kiawe in front of the Pokémon School. Though the three Grunts outnumbered Kiawe and Ash, the two Trainers emerged victorious through the combined efforts of Ash's Pikachu and Kiawe's Turtonator. This particular trio have become recurring characters, often appearing to cause trouble only to be stopped by Ash and Pikachu's Gigavolt Havoc.
More Grunts appeared in a flashback in Yo, Ho, Ho! Go, Popplio!, where they were harassing a Popplio on the beach until Lana intervened by freezing them solid with her Ride Lapras.
Team Skull appeared on Akala Island in Young Kiawe Had a Farm!, and later back on Melemele Island in Alolan Open House!.
Team Skull fought Team Rocket in A Team-on-Team Tussle! over possession of a Darkinium Z, which multiple Alolan Rattata, Raticate, and a Totem Raticate were protecting. They lost after a wild Bewear showed up, scaring the Totem and Ally Pokémon away while Team Rocket unearthed the Darkinium Z from the hill it was embedded in.
Gladion made his physical debut in A Glaring Rivalry!. Unlike the games, however, he is not associated with Team Skull in the anime; and is even shown defeating Tupp, Rapp, and Zipp in a battle.
Plumeria debuted in That's Some Spicy Island Research!.
In the Sun, Moon, Ultra Sun & Ultra Moon chapter, a pair of Grunts are berated by Guzma because their attempt to skip out on paying Sun for his part-time job ended in their defeat. They are ordered to find Sun so that they can be taught a lesson.
Following the conclusion of the Iki Town festival, Guzma follows Gladion to the Ruins of Conflict and praises his strength, asking for him to join Team Skull as its enforcer. To Guzma's surprise, Gladion quickly accepts.
Team Skull is the first villainous team to make their debut in the first episode of an anime series.
The lyrics to the Team Skull encounter theme contain the word "dokuro" (ドクロ), meaning "skull" in Japanese.
In Super Smash Bros. Ultimate, Pichu can wear the Team Skull cap as one of its palette swaps.


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Zelowan larva  è un ragno appartenente alla famiglia Gnaphosidae.

Etimologia
Il nome della specie deriva dal latino larva, nel senso di mascheramento deforme, in riferimento all'aspetto dell'epigino femminile visto ventralmente.

Caratteristiche
L'olotipo femminile rinvenuto ha lunghezza totale è di 3,75mm; la lunghezza del cefalotorace è di 1,38mm; e la larghezza è di 1,00mm.

Distribuzione
La specie è stata reperita nel Congo orientale: l'olotipo femminile è stato rinvenuto sul monte Lubwe, a sudest di Butembo, appartenente alla provincia del Kivu Nord.

Tassonomia
Al 2016 non sono note sottospecie e dal 2010 non sono stati esaminati nuovi esemplari.

Note

Bibliografia
 Murphy, J.A. & Russell-Smith, A., 2010 - Zelowan, a new genus of African zelotine ground spiders (Araneae: Gnaphosidae). Journal of Afrotropical Zoology vol.6, pp.59-82 PDF (pp.65-66)

Collegamenti esterni

Zelowan


For this winter collection, New Man takes inspiration from the heritage of automotive world. The racing style is diverted from its traditional use in the service of technical and comfortable clothing. Worked in a blend of wick and nylon, the sleeves and stitches feature the logo and yellow / navy contrast of the iconic color code. A new look emerges, more modern with its mix of streetwear athleisure. The future is now!


text:
This article is more than 7 years old
Cycling helmet law may not reduce hospital admissions, researchers say
BMJ study cites evidence from Canada that shows 'minimal' fall in head injury admissions in regions where helmets are required
Better infrastructure for cyclists was one reason cited for a fall in hospital admissions for cycling-related head injuries in Canada. Photograph: Tim Ireland/PA
Wed 15 May 2013 04.11 EDT
Introducing a law to force cyclists to wear helmets may not reduce the number of hospital admissions for cycling-related head injuries, a study has suggested.
Researchers said that while helmets reduce head injuries and should be encouraged, the decrease in hospital admissions in Canada, where the law is in place in some regions, seems to have been "minimal".
The authors examined data concerning all 66,000 cycling-related injuries in Canada between 1994 and 2008 – 30% of which were head injuries.
Writing in the British Medical Journal, the authors noted a substantial fall in the rate of hospital admissions among young people, particularly in regions where helmet legislation was in place. But they said that the fall was not found to be statistically significant.
The researchers said admission rates were falling before the implementation of helmet legislation thanks to safety campaigns, improvements to cycling infrastructure and existing use of helmets.
"When baseline trends in cycling-related injury rates were considered, the overall rates of head injuries were not appreciably altered by helmet legislation," they said.
They concluded: "While helmets reduce the risk of head injuries and we encourage their use, in the Canadian context of existing safety campaigns, improvements to the cycling infrastructure, and the passive uptake of helmets, the incremental contribution of provincial helmet legislation to reduce hospital admissions for head injuries seems to have been minimal."
The road safety minister, Stephen Hammond, said: "We encourage cyclists – especially children – to wear helmets to protect them if they have a crash. However, we believe this should remain a matter of individual choice rather than imposing additional regulations which would be difficult to enforce.
"To improve cycle safety we want to see more innovative measures being put in place to help prevent collisions. For example, we have made it easier for local authorities to introduce 20mph zones in built-up areas and authorised a trial of trixi mirrors in London which, if successful, could be used elsewhere in the country to make cyclists more visible to drivers."


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Posts Tagged 'los angeles county jail'
Earlier this week Los Angeles County Sheriff Lee Baca announced plans to shut down L.A.'s notorious Men's Central Jail. This is big news: L.A. County's jails comprise not just the largest and most violent jail system in the nation, but also, by default, one of the nation's largest mental health care providers. Over the years I have been writing this blog, I've often noted stories of violence and other problems in the L.A. County jails. So, planning to shutter the largest of those troubled facilities — Men's Central, which houses as many as 5,000 inmates on any given day — is a noteworthy reform. (Of course, questions remain about whether/how the plans will be implemented.)
How, you might ask, can L.A. County do this — especially at a time when California's realignment policy is shifting more responsibility to the county jails? The ACLU of Southern California, which has been suing L.A. County over its dismal jail conditions for years, explains:
[A] report [PDF here], by nationally-renowned corrections expert James Austin and based on data provided by Los Angeles County Sheriff Lee Baca, finds that Men's Central Jail can be shuttered by safely releasing 3,000 low-risk, non-violent pre-trial and sentenced inmates into community-based supervision and education programs that will curb recidivism, and by increasing the capacity of the county-wide jail system by 2,000 beds through a repurposing of existing facilities.
James Austin may be familiar to readers of this blog, because he also provided the data crunching needed for Mississippi to shut down its horrific solitary confinement wing, "Unit 32". I noted previously that he was also working with New Orleans to downsize its jails, though it appears his recommendations there have not been implemented. His firm has also consulted for a number of states and the federal Bureau of Justice Assistance. Consultants, advisers, policy analysts don't have the flashiest jobs, and unlike celebrity activists and high-profile lawyers rarely become household names, but work like Austin's is what will make it possible for local and state governments to dismantle mass incarceration — and, ideally, to do so in a way that avoids the Pyrrhic victories that Bob Weisberg and Joan Petersilia have warned of.
Tagged with aclu, california, jail, james austin, lee baca, los angeles, los angeles county jail, mass incarceration, mental health, pretrial detention
Los Angeles County Sued Over Violence, Abuse in Jails
"Los Angeles County Sheriff Lee Baca and his top commanders condoned a longstanding, widespread pattern of violence by deputies against inmates in the county jails," said the ACLU yesterday, announcing a federal class-action lawsuit. The named plaintiffs, Alex Rosas and Jonathan Goodwin, claim that they were severely beaten by sheriff's deputies while they were awaiting trial in the jail.
The ACLU of Southern California has long been litigating L.A. jail conditions and has served as court-appointed monitor of the jail — the nation's largest — since 1985. The new lawsuit, however, includes new first-hand eyewitness accounts from chaplains and other observers of violence. The ACLU has put together a timeline of alleged incidents of abuse; you can also read the full complaint here.
The L.A. Times reports:
"Like members of street gangs, these deputies sport tattoos to signal their gang membership," the ACLU alleges. "They beat up inmates to gain prestige among their peers, and 'earn their ink' by breaking inmates' bones."
In an interview with The Times, a recently retired jails commander also said that deputies had formed cliques inside Men's Central Jail and that some guards earned respect from veteran members of those cliques by using excessive force.
Tagged with aclu, aclu of southern california, california, jail, lee baca, los angeles, los angeles county jail, pretrial detention, reform litigation
Fearmongering about Realignment in L.A. County
To follow up on my post about realignment from yesterday, here's an article from the Los Angeles Times quoting various supervisors predicting doom and gloom:
"It's a system that's meant to fail," [Supervisor Michael] Antonovich said, "and who is it going to fail? Every neighborhood, every community where these people are going to be running around….It's a Pandora's box. It's the bar scene — a violent bar scene that you saw in 'Star Wars' — except they're all crazy and nuts."
Antonovich said it is likely that Los Angeles County will run out of jail beds unless it "uses other models of supervisions such as electronic monitoring, work furloughs, weekenders and GPS tracking."
"It's irresponsible for us to turn around and dump these [prisoners] into our communities with an ankle bracelet and hope they don't re-offend," Antonovich said. Without finding a way to increase prison time, Antonovich said, "I believe we'll have a spike in crime." Read the rest of this entry »
Tagged with ab 109, aclu, california, los angeles, los angeles county jail, michael antonovich, overcrowding, plata/coleman, prison, realignment
More Notable Plata News and Commentary
This roundup will be sort of haphazard, but I just wanted to flag a few things that have come across the transom worth your attention:
Here's an informative article by Jeanine Sharrock at New America Media that puts into perspective Gov. Jerry Brown's "realignment" proposal, which would comply with Plata by shifting responsibility for low-level offenders down to the county level. About a third of California prisoners come from Los Angeles County, where the county jails have their own overcrowding problems, not to mention their own ongoing unconstitutional conditions litigation.
Linda Greenhouse of the New York Times has now weighed in. She characterizes the Kennedy opinion as a blast from the past to the 1970s era of sweeping structural reform injunctions. (As, of course, does the Scalia dissent, though Scalia comes to bury, not to praise.) Overall Greenhouse seems to approve, given the uniquely dire state of affairs in California's prisons: "if the court can't solve such problems, it still has the power to illuminate them and to summon our better selves. The court uses that power rarely these days, but in this one decision, it found a nearly forgotten voice from long ago."
And here's a detailed analysis of the opinion from Stuart Taylor, who has some sympathy for both the majority opinion and the Alito dissent but describes the Scalia dissent as "overheated."
EDITED TO ADD: I meant to include one more:
Dan Morain of the Sacramento Bee gives some additional context to the Justice Kennedy opinion. J. Clark Kelso, the court-appointed federal receiver in charge of CDCR, was a Kennedy clerk back in Kennedy's Ninth Circuit days.
Posted in General News, Supreme Court Watch
Tagged with california, cdcr, los angeles, los angeles county jail, overcrowding, plata/coleman, prison, reform litigation, supreme court
ACLU Files Motion for Protective Order for L.A. County Jail Inmates
The ACLU of Southern California, which has long been involved in litigation over conditions in the Los Angeles County Jail, filed a motion earlier this week in federal court seeking a protective order for inmates who report grievances. The motion alleges a pattern of violent retaliation by prison guards against these inmates, ranging from beatings and stompings to broken bones.
L.A. sheriff's spokesman Steve Whitmore says the ACLU is exaggerating:
"What the ACLU is characterizing does not occur in men's central jail," Whitmore said. "The judge that oversees it toured the jail recently without condemnation."
Whitmore said "regrettably from time to time there are physical altercations," but added that every use of force is thoroughly investigated and said the Office of Independent Review, which monitors the department, called the jail system the most transparent in the nation.
The L.A. County jail system is the largest in the nation, with some 20,000 inmates. Men's Central Jail, which is the facility at issue in the protective order motion, holds about 4,500 men, of which about 80% are pretrial detainees. Last week two inmates committed suicide in the facility.
Related news: L.A. Sheriff Lee Baca is calling for an expansion of the ICE Secure Communities program.
Tagged with aclu, california, ICE, immigration, jail, lee baca, los angeles, los angeles county jail, mental health, pretrial detention
Growing Trend: Jails Around the Country Adopting Postcard-only Policies
I've noted a couple of lawsuits against jails that have adopted postcard-only policies for inmate correspondence. The Los Angeles Times reports that this is a nationwide trend that's now spread from Joe Arpaio's Maricopa County jails to at least seven states, including most recently, California's Ventura County jail:
[Ventura County jail official Brent] Morris said that jail officials followed the emerging policy elsewhere through professional associations. They saw it as a way of both cutting security risks and freeing up staff. Two employees now spend most of their shifts sorting through mail flowing to and from 1,500 inmates.
"When you balance it with the challenge of budget and staffing, it seemed like a prudent thing to institute," he said.
But for Los Angeles County, the tradeoff isn't worth it, said Steve Whitmore, a spokesman for the Sheriff's Department.
"We believe the mail coming to inmates is as important as their phone calls," he said. "If we were to limit the mail, we believe we would see a rise in mental challenges, maybe even violence."
UPDATE: Via Twitter, here's a response from Just Detention International, which advocates for prison rape victims: "this could be problematic for organizations like JDI. We send important packets 2 survivors daily."
Tagged with california, first amendment, jail, joe arpaio, los angeles county jail, maricopa county, pretrial detention, restrictions on prisoner correspondence, ventura county jail
"They Come to Expect Sub-Human Treatment"
The ACLU of Southern California wrote an open letter to Lindsay Lohan, Los Angeles County's most famous inmate, that's been making its way around the blogosphere. Worth highlighting is the letter's summary of the important work done by that particular ACLU branch as the court-appointed monitor for the L.A. County system:
Based on the ACLU's decades of experience as an official court-appointed monitor of the jails, and the stories of countless women with whom we've spoken, the facility where you are staying is an overcrowded detention facility where women are needlessly humiliated for so long that they come to expect sub-human treatment.
It's a place where an eight-months-pregnant woman was forced to sleep on the floor because she could not access the top bunk to which she was assigned. A place where women have said they are made to stand naked while menstruating, as they waited for jail-issued clothes. And a place where women routinely tell us they cannot get access to the same medications they took in the community (though we doubt that you will face this same problem.)
Group punishments and degrading group strip searches are routine, as are reports of deputies calling women "bitches" and other derogatory names. And while you get private visits with your family and friends, every other woman's visitors must wait in long lines on the weekends.
We know from the more than 4,500 complaints we receive annually that the women's facility is a lot nicer than the Men's Central Jail – where rats roam the tiers, and violence is as routine as sunshine in California. We've seen men with broken legs and black eyes. It's not uncommon for a prisoner to be thrown up against the wall or punched, simply for asking a deputy a question.
Tagged with aclu, california, incarcerated women, jail, lindsay lohan, los angeles county jail, overcrowding
The New York Times Swings at the L.A. County Justice System—and Whiffs
I get it, even the New York Times needs page views now and then; and I know, Friday evenings aren't the height of the news cycle. But even still, this is front page news (ok, well, homepage news)?
Meanwhile, I clicked on the NYT topic archive for "Los Angeles, Calif." going back as far as October 2009 and found not a single article in that time span on the Los Angeles County criminal justice system's actual problems. From a May 2010 ACLU report: Read the rest of this entry »
Posted in General News, Random Asides
Tagged with california, lindsay lohan, los angeles county jail, mental health, new york times, overcrowding, pretrial detention, prison health care, prisoner grievances
Michael Lohan's Case for Criminal Justice Reform (???)
Thanks to the intrepid journalists over at TMZ.com, here's an excerpt from a letter sent by Michael Lohan's lawyers to the Los Angeles judge who's overseeing his daughter Lindsay's probation for a DUI conviction:
Your Honor is aware that because of the current overcrowding in Los Angeles jails, misdemeanor, non-violent offenders like Lindsay serve only a fraction of their terms. In Lindsay's case, her last term of incarceration lasted only 84 minutes. This not only made a laughingstock of our criminal justice system in the eyes of the world, … but taught her that jail is a revolving door and poses no real threat or deterrent. Overcrowding has not diminished in the last few years since this first incident, and thus we'd expect a similar very brief term of actual confinement.
In the event Lindsay is found to be in violation, Michael renews his request that the Court order Lindsay into an inpatient drug and alcohol rehabilitation program. We have previously written to the Court regarding a very private facility in New York — so private, that the public is unaware rehab takes place there.
Of course, the letter is only intended to help one particular celebrity defendant but I don't see why it wouldn't apply to, well, almost everyone else facing L.A. County jail time on a drug or alcohol probation violation. Too bad they don't all have access to super top secret rehab facilities — what is this place — the Bat Cave? In any event, apparently Michael's pleas were unavailing as the Los Angeles Times is now reporting that Lindsay's been sentenced to jail for 90 days.
Tagged with california, celebrity defendants, jail, lindsay lohan, los angeles county jail, mental health, overcrowding


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Sweven group breaks ground on new Pooler development
Katie Nussbaum
knussbaum@savannahnow.com
Development continued in Pooler this week with the groundbreaking for the construction of the first building in the Kansa Center, located on Pooler Parkway, between St. Joseph's/Candler Pooler campus and Interstate 16.
The 13,000 square-foot shopping center will be the first retail space of the Kansa Center and is currently in the initial leasing stage for its three retail units. The shopping center parcel covers 3.10 acres.
The project is being developed by Sweven Investment Group and Dewitt Tilton Group has been tapped to complete the construction.
Several local leaders also joined Sweven and Dewitt Tilton Group on Tuesday for the ground breaking, including City of Pooler Mayor Rebecca Benton, Pooler Councilman Aaron Higgins, Councilwoman Karen Williams and Councilman Tom Hutcherson; Jabron Webster, President of Pooler Chamber's Board of Directors, and Pam Southard, Pooler Chamber's Executive Director.
"This groundbreaking ceremony marks a milestone for us in the development of Kansa Center," said Sweven owner and CEO Darshil Patel.
"We are excited to become part of the Pooler community and we're confident the center will offer an abundance of resources to the local community."
Future projects for the Kansa Center development include two restaurants, a hotel, a doctor's office and additional retail space. In total, the development will cover a little over 18 acres.
The Dewitt Tilton Group is also working with the Sweven Investment Group on several other projects including office space in Pooler and a truck stop near the Georgia Tech Savannah campus.
"It's an honor to have been chosen by Sweven Investment Group for this project," said Chris Tilton, co-principal of the Dewitt Tilton Group.
"I was first introduced to Darshil during a project our dads worked on years ago in South Carolina. When Sweven planned to build in the Pooler area, Darshil knew we were the team to get the job done right, in budget and on time."


Romantic ⚜ With great sensitivity to the feminine world, the name defines the taste of this collection: romantic.
A New Classic is coming | The atmosphere of the 1950s is the first source of inspiration for the broadway collection, characterized by round shapes and linings in warm colored fabrics with central buckles and cotton embroideries.
The Forever collection enriches its own personality and elegance with new environments and hues. The pastel version enhances also the spaces dedicated to children.
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Inside the University of Maryland's Virtual and Augmented Reality Laboratory earlier this month, 15 middle-school girls showed off the virtual reality games they created during this summer's Computer Science Connect (CompSciConnect) program.
Zoe Smith, Sayeemah Ahmed and Erica Lopez-Haz created a game for the Oculus Rift virtual reality headset where players shoot oncoming asteroids to earn points.
CompSciConnect is a two-week summer day camp that offers middle school girls the chance to explore activities in computing, including Web development, computer programming, robotics and cryptology. They also take field trips to places where they can see the applications in computing, such as the Newseum and research institutions.
Designed as a three-year program, each cohort returns the following summer to learn more advanced skills and meets once a month during the school year to maintain their skills. This year's three cohorts total 82 students. CompSciConnect also provides an opportunity for female undergraduate students in computing-related majors to teach younger students about computing, while sharing their own excitement about and interest in the field.
Founded by Jandelyn Plane, a senior lecturer in computer science at UMD and director of the Maryland Center for Women in Computing, CompSciConnect is entering its fourth year. The "connect" in the program's name has a two-fold purpose, according to Plane. The first goal is to show the students how computing connects to everything in the world. The second goal is to connect the girls to one another, with the goal of them forming long-term relationships.
Back in the Virtual and Augmented Reality Laboratory—also called the "Augmentarium"—Isha Santhosh and Kayla Newby demonstrated their virtual reality game, a driving simulator where players earn points by collecting cubes floating throughout the environment. One of the most interesting parts of the project was designing the environment, said Santhosh and Newby, which they created from scratch. Santhosh will be an eighth-grader this fall at Murray Hill Middle School in Laurel, Md., and Newby will enter ninth grade this fall at Northwood High School in Rockville, Md.
Newby, who joined the program last year after hearing about it at some workshops, said it has been incredibly fun to learn a bunch of new things, like developing a game using Unity 3D this year and creating a website during her first year in the program.
In addition to showcasing their games, the students learned about virtual and augmented reality technologies and applications UMD researchers are working on from Amitabh Varshney, a professor of computer science at UMD, director of the University of Maryland Institute for Advanced Computer Studies, and director of the Virtual and Augmented Reality Laboratory. Varshney also described to the students ways that virtual and augmented reality could change the world in the future.
Initial funding for the UMD Augmentarium came in part from the National Science Foundation (NSF) through a $600,000 Major Research Instrumentation grant used to purchase equipment and develop outreach programs.
"We believe that virtual and augmented reality will play a large role in making STEM education more accessible—and more fun—for a wide group of students. Completely new pathways of discovery, both for science and education, will come from these innovative technologies," said Rita Rodriguez, the NSF program director who manages the UMD Augmentarium award.
The CompSciConnect program has been supported by the Armed Forces Communications and Electronics Association (AFCEA) Bethesda Chapter since its inception. A donation from Brendan Iribe, UMD alumnus and founder and CEO of Oculus VR, allowed the camp to grow this year.