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Naive CD8+ T cells differentiate into protective memory-like cells after IL-2–anti–IL-2 complex treatment in vivo
Daisuke Kamimura,
Daisuke Kamimura
Department of Immunology and Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195
Michael J. Bevan
CORRESPONDENCE Michael J. Bevan: mbevan@u.washington.edu
Abbreviations used: C57BL/6, B6; HP, homeostatic pro liferation; LM-OVA, Listeria monocytogenes–expressing soluble OVA.
Received: March 16 2007
Accepted: July 05 2007
J Exp Med (2007) 204 (8): 1803–1812.
https://doi.org/10.1084/jem.20070543
Daisuke Kamimura, Michael J. Bevan; Naive CD8+ T cells differentiate into protective memory-like cells after IL-2–anti–IL-2 complex treatment in vivo . J Exp Med 6 August 2007; 204 (8): 1803–1812. doi: https://doi.org/10.1084/jem.20070543
An optimal CD8+ T cell response requires signals from the T cell receptor (TCR), co-stimulatory molecules, and cytokines. In most cases, the relative contribution of these signals to CD8+ T cell proliferation, accumulation, effector function, and differentiation to memory is unknown. Recent work (Boyman, O., M. Kovar, M.P. Rubinstein, C.D. Surh, and J. Sprent. 2006. Science. 311:1924–1927; Kamimura, D., Y. Sawa, M. Sato, E. Agung, T. Hirano, and M. Murakami. 2006. J. Immunol. 177:306–314) has shown that anti–interleukin (IL) 2 monoclonal antibodies that are neutralizing in vitro enhance the potency of IL-2 in vivo. We investigated the role of IL-2 signals in driving CD8+ T cell proliferation in the absence of TCR stimulation by foreign antigen. IL-2 signals induced rapid activation of signal transducer and activator of transcription 5 in all CD8+ T cells, both naive and memory phenotype, and promoted the differentiation of naive CD8+ T cells into effector cells. IL-2–anti–IL-2 complexes induced proliferation of naive CD8+ T cells in an environment with limited access to self–major histocompatibility complex (MHC) and when competition for self-MHC ligands was severe. After transfer into wild-type animals, IL-2–activated CD8+ T cells attained and maintained a central memory phenotype and protected against lethal bacterial infection. IL-2–anti–IL-2 complex–driven memory-like CD8+ T cells had incomplete cellular fitness compared with antigen-driven memory cells regarding homeostatic turnover and cytokine production. These results suggest that intense IL-2 signals, with limited contribution from the TCR, program the differentiation of protective memory-like CD8+ cells but are insufficient to guarantee overall cellular fitness.
The CD8+ T cell response to an acute systemic infection entails vigorous expansion of antigen-specific cells, followed by a contraction phase in which 90–95% of the cells die by apoptosis. The cells remaining after the contraction phase become memory T cells, which possess properties distinct from the naive population, such as rapid acquisition of effector function on reencounter with pathogen. Accumulating evidence suggests that naive CD8+ T cells are programmed to become memory cells in the early phase of the CD8+ T cell response, when appropriate signals from the TCR, co-stimulatory molecules, and cytokines associated with inflammation are thought to be required (1, 2).
IL-2, which is produced by activated T cells, is one of the cytokines involved in the CD8+ T cell response. IL-2 induces intracellular signals through the IL-2 receptor complex, consisting of CD25 (IL-2Rα), CD122 (IL-2/IL-15Rβ), and CD132 (common γ chain). Stimulation of the receptor complex by IL-2 induces several signal transduction pathways, including the activation of STAT5 (3). Recent studies have demonstrated that IL-2 supports the maintenance of Foxp3+ CD4+ regulatory T cells, which bear the IL-2Rα chain (CD25) (4–6). The requirement for IL-2 signals to maintain regulatory T cells limits the use of cytokine or cytokine receptor knockout mice to study other in vivo roles of IL-2. Instead, the effect of IL-2 signals on CD8+ T cells during an immune response has been investigated by creating situations in which CD25, CD122, or IL-2 are selectively deficient in CD8+ T cells. These studies revealed a modest role for IL-2 in the primary expansion and differentiation of CTL (7–12), whereas IL-2 appears to support expansion of primary CTL in nonlymphoid organs (13). Very recently, another role for IL-2 signals in antigen-stimulated CD8+ T cells was revealed using mixed BM chimeric mice containing both wild-type and CD25−/− cells. In these mice a complete compartment of regulatory T cells is reconstituted, and the mice remain healthy (11, 12). Upon acute infection, effector and memory CD8+ T cells lacking CD25 were generated and normally maintained, but their secondary expansion after pathogen rechallenge was severely compromised compared with that of CD25-sufficient memory cells (11, 12). Intriguingly, replenishment of IL-2 signals to CD25−/− CD8+ T cells during the primary infection, but not during the secondary challenge, restored their ability to expand in a recall response (11). This result clearly indicates a programming effect of IL-2 signaling during the primary response in driving the complete differentiation of memory CD8+ T cells.
Recent data suggest that the anti–IL-2 mAb S4B6, which has been widely used as a neutralizing antibody in vitro, enhances the bioactivity of IL-2 in vivo (14, 15). Administration of rIL-2 mixed with the anti–IL-2 mAb (IL-2–anti–IL-2 complexes) or the concurrent treatment with plasmid DNA expressing mouse IL-2 and the antibody substantially and preferentially increased the proliferation of CD44hi CD122hi memory-phenotype CD8+ T cells and NK cells (14, 15). Injection of anti–IL-2 mAb alone had a similar effect because of the capture of endogenously secreted IL-2 by the mAb, although the efficacy of this treatment is much weaker than the cotreatment with IL-2–anti–IL-2 complexes (14, 15). The precise mechanism of the enhanced potency of the immune complex remains unclear, although the presentation of IL-2–anti–IL-2 complexes via the Fc portion of the mAb has been suggested (14).
We report that the administration of IL-2–anti–IL-2 complexes stimulated all CD8+ T cells, both naive and memory phenotype, in vivo. The naive CD8+ T cells proliferated, became effector cells, and differentiated to memory-phenotype cells capable of providing protection against pathogen challenge. Remarkably, proliferation of naive CD8+ T cells by treatment with IL-2–anti–IL-2 complexes was induced in the absence of foreign antigen and when competition for self-MHC class I ligands was intense. Because most experimental systems used for the study of CD8+ T cell responses are associated with strong TCR stimulation, our approach using IL-2–anti–IL-2 complexes allowed us to dissect and examine the relative contribution of IL-2 and antigen signals to CD8+ T cell responses, revealing that intense IL-2 signals coupled with weak TCR ligation have the potential to program the differen tiation of protective memory-like CD8+ T cells in vivo.
IL-2–anti–IL-2 complexes activate STAT5 in all CD8+ T cells
CD44hi CD122hi memory-phenotype CD8+ T cells and NK cells express high levels of CD122 (IL-2/IL-15Rβ), providing an explanation for the preferential expansion of these populations after treatment with complexes of rIL-2 and anti–IL-2 mAb (IL-2–anti–IL-2 complexes) (14, 15). However, CD44lo naive CD8+ T cells also express detectable levels of CD122, intermediate between those of memory-phenotype CD8+ T cells and naive CD4+ T cells (unpublished data) (16). In addition, after treatment with IL-2–anti–IL-2 complexes, almost the entire CD8+ population adopts a CD44hi CD122hi phenotype (unpublished data) (14). We therefore asked whether CD44lo naive CD8+ T cells could respond to IL-2–anti–IL-2 complexes injected in vivo. IL-2 signal transduction is known to involve the activation of STAT5, which is important for the biological activity of IL-2 (3, 4). Therefore, we used the phosphorylation status of STAT5 as a readout of induction of an IL-2 signal in vivo. C57BL/6 (B6) mice were given a single injection of IL-2–anti–IL-2 complexes, and at the indicated time points, splenocytes were fixed without restimulation in vitro, and activated (tyrosine-phosphorylated) STAT5 was detected by flow cytometry. As shown in Fig. 1, CD44hi memory-phenotype CD8+ T cells responded rapidly after the treatment: the phosphorylation of STAT5 was induced in ∼50% of the population at 15 min and increased to >90% by 60 min. Surprisingly, the rapid activation of STAT5 was also observed in CD44lo naive CD8+ T cells with slightly delayed kinetics. At 60 min, >80% of the CD44lo naive CD8+ population contained the activated form of STAT5. NK cells (NK1.1+ TCRβNEG) also showed STAT5 activation, whereas B cells (B220+ TCRβNEG) showed no detectable activation of STAT5 at 60 min (unpublished data). The rapid activation of STAT5 suggests that CD44lo naive CD8+ T cells, as well as memory-phenotype CD8+ T cells, receive signals directly from IL-2–anti–IL-2 complexes.
IL-2–anti–IL-2 complexes induce proliferation of naive CD8+ T cells
These results prompted us to examine the physiological consequence of treating naive CD8+ T cells with IL-2 signals in vivo. Congenically marked CD44lo CD122lo naive OT-I cells were isolated, labeled with CFSE, and transferred into nonirradiated syngeneic B6 mice (Fig. S1 A). After treatment of the host animals with IL-2–anti–IL-2 complexes, the naive OT-I cells divided extensively (Fig. 2 A) and accumulated (Fig. 2 B) in the absence of any other stimulation. The expansion of naive OT-I cells was almost equivalent to that of the host CD8+ T cell population (Fig. 2 B). Surface markers associated with activation/memory, including CD27, CD44, and CD122, were up-regulated on OT-I cells that received IL-2 signals, whereas CD62L and CD127 levels remained unchanged as compared with OT-I cells in animals receiving control injections (Fig. 2 C). This phenotype resembles a central memory phenotype (17). IL-2–anti–IL-2 complex–mediated proliferation in a lympho-replete environment was not unique to OT-I cells or mediated by a second TCR possibly expressed on TCR-transgenic cells, because other naive TCR-transgenic CD8+ T cells (from P14 or Vβ5 TCR-transgenic mice) and OT-I cells on a RAG-1−/− background also proliferated (Fig. S2). In contrast, proliferation of naive polyclonal CD8+ T cells was not as vigorous (unpublished data), as previously reported (14). Functionally, OT-I cells that had received IL-2 signals were able to produce IFN-γ after a 4-h stimulation with their cognate peptide in vitro (Fig. 2 D) and killed target cells in an antigen-specific manner in vivo (Fig. 2 E), both of which are typical characteristics of effector cells. These results indicate that signals provided by IL-2–anti–IL-2 complexes induce the proliferation and activation of naive antigen-specific CD8+ T cells in nonlymphopenic environments without cognate antigen stimulation.
Fcγ receptors are dispensable for the action of IL-2–anti–IL-2 complexes
Why IL-2–anti–IL-2 complexes are so potent in vivo is still unclear. One possible mechanism that has been proposed is the presentation of IL-2–anti–IL-2 complexes by Fcγ receptor–bearing cells to cells expressing CD122/CD132 (14). Thus, IL-2 complexed to F(ab′)2 fragments of the anti–IL-2 mAb had only a modest effect in vivo (14). To investigate the role of Fcγ receptors, mice deficient in both FcRγ chain and FcγRII (FcRγ−/−FcγRII−/−) were used. Because the FcRγ chain is necessary for surface expression of the activating IgG Fc receptors, including FcγRI, III, and IV, whereas the inhibitory receptor FcγRII does not share the FcRγ chain (18, 19), FcRγ−/−FcγRII−/− mice should be devoid of known functional Fcγ receptors on hematopoietic cells. In fact, FcγRII and FcγRIII expression was undetectable on B cells from FcRγ−/−FcγRII−/− mice by staining with anti-CD16/32 (FcγRIII/II) mAb (Fig. S3 A). In addition, anti-CD3–induced cytokine release in vivo, which requires presentation and cross-linking via Fcγ receptors (20, 21), was severely impaired in FcRγ−/−FcγRII−/− mice, indicating a gross functional defect of Fcγ receptors in these mice (Fig. S3 B). Because FcRγ−/−FcγRII−/− mice are on a mixed genetic background, we tested the efficacy of IL-2–anti–IL-2 complexes by sampling PBLs before and after treatment rather than using an adoptive transfer system. Both control and FcRγ−/−FcγRII−/− mice displayed a similar small population of CD44hi CD122hi memory-phenotype CD8+ T cells before treatment (Fig. 3 A). By day 5 after treatment with IL-2–anti–IL-2 complexes, CD44hi CD122hi memory-phenotype CD8+ T cells increased dramatically in control and FcRγ−/−FcγRII−/− mice (Fig. 3 A). Total numbers of PBLs increased significantly after treatment in both strains and correlated with vigorous expansion of CD8+ T cells and NK cells (Fig. 3 B). These results suggest that Fcγ receptor–mediated presentation is dispensable for the action of IL-2–anti–IL-2 complexes in vivo.
IL-2–anti–IL-2 complexes stimulate naive CD8+ T cells with limited TCR stimulation by self-ligands
IL-2 signals induced the proliferation of naive TCR-transgenic CD8+ T cells in a complete lymphoid compartment and in the absence of cognate antigen stimulation (Fig. 2 A and Fig. S2). However, it was possible that TCR stimulation by self-ligands had an important role, as is the case for homeostatic proliferation (HP) of naive CD8+ T cells in a lymphopenic environment (22–24). To explore the dependency on MHC class I ligands, we chose MHC class I Kb−/−Db−/− mice as host animals, because the homeostasis of IgG is impaired in β2 microglobulin–deficient mice (25), which might affect the half-life of IL-2–anti–IL-2 complexes. Because CD8+ T cells transferred into MHC class Ia–deficient mice were almost completely rejected after IL-2–anti–IL-2 complex treatment (unpublished data), MHC class Ia–deficient mice were irradiated with 10 Gy and transplanted with syngeneic BM cells to prevent rejection. Irradiated, BM-reconstituted mice were injected with CFSE-labeled naive OT-I cells and IL-2–anti–IL-2 complexes. As controls, B6 mice were also irradiated and reconstituted with syngeneic BM. As shown in Fig. 4 A, naive OT-I cells underwent three to four divisions in lethally irradiated WT hosts after rat IgG treatment, because of lymphopenia-induced HP. Proliferation was delayed in MHC class Ia–deficient hosts, confirming that HP of naive CD8+ T cells is MHC class I–dependent (22–24). In contrast, IL-2 signals provided by injected IL-2–anti–IL-2 complexes stimulated extensive division of naive OT-I cells in the MHC class Ia–deficient and –sufficient host animals. The recovery of donor OT-I cells in the spleen after the complex treatment was also similar in both groups (Fig. 4 B).
These results suggest a minimal requirement for TCR ligation by self-MHC for the proliferation of naive CD8+ T cells stimulated by IL-2–anti–IL-2 complexes, but it does not rule out the possibility of stimulation by MHC class I molecules on the donor cells themselves or by irradiation-induced cytokines. In this regard, a recent report has strongly suggested the requirement for TCR recognition of self-MHC ligands to observe the accumulation of IL-2–driven CD8+ cells (26). We took a second approach to examine the dependency on TCR stimulation based on the finding that adoptively transferred TCR-transgenic CD8+ T cells do not undergo HP in hosts with the identical TCR clonotype because of clonal competition for MHC class I/peptide ligands (27, 28). Accordingly, naive OT-I cells were labeled with CFSE and transferred into nonirradiated OT-I/RAG-1−/− hosts. As shown in Fig. 4 C, signals provided by IL-2–anti–IL-2 complexes induced several divisions of naive OT-I cells even in this environment, where competition for self-MHC ligands is intense. This result suggested that robust expansion of naive CD8+ T cells in response to potent IL-2 signals requires only minimal TCR stimulation.
IL-2–anti–IL-2 complexes generate memory cells with a central memory phenotype
We next addressed whether naive CD8+ T cells activated by IL-2–anti–IL-2 complexes could differentiate to memory cells. OT-I mice were injected with IL-2–anti–IL-2 complexes, resulting in the conversion of almost all the CD8+ T cells to a CD44hi CD122hi activated phenotype (Fig. S1 B). 15 × 106 IL-2–activated OT-I cells were then transferred at day 12 to normal B6 hosts. At 1 d after transfer, the OT-I cells exhibited rapid IFN-γ production in response to peptide stimulation in vitro (unpublished data). For comparison, conventional, antigen-driven OT-I cells were generated by transfer of 104 naive OT-I cells into B6 recipients, followed by immunization with Listeria monocytogenes–expressing soluble OVA (LM-OVA; Fig. S1 C). More than 75 d after transfer of the IL-2–anti–IL-2 complex–driven or antigen-driven memory cells, the OT-I cells accounted for ∼3% of the CD8+ population in the spleen (Fig. 5). At this point, the antigen-driven memory OT-I cells were CD44hi CD122hi and contained CD62Lhi central and CD62Llo effector memory subsets. In contrast, all of the IL-2–anti–IL-2 complex–driven cells had the phenotype of central memory cells. These results suggest that naive CD8+ T cells activated by IL-2–anti–IL-2 complex treatment revert to stable central memory-phenotype cells.
IL-2–anti–IL-2 complex memory CD8+ T cells protect against bacterial infection
Functional memory CD8+ T cells rapidly expand in numbers and effectively control a challenge with pathogen (1, 2). We therefore assessed the ability of IL-2–anti–IL-2 complex memory OT-I cells to mount a protective recall response to a lethal dose of LM-OVA. Mice bearing IL-2–anti–IL-2 complex memory OT-I/RAG-1−/− cells (91 d after transfer) were infected with a lethal dose of LM-OVA. As shown in Fig. 6 (A and B), IL-2–anti–IL-2 complex memory OT-I/RAG-1−/− cells expanded over 30-fold within 3 d after the LM-OVA challenge. To examine the protective ability of IL-2–driven memory cells, a control group of mice received 1.5 × 106 untreated OT-I/RAG-1−/− cells to constitute a population of these cells at an equivalent level to that of mice containing IL-2–anti–IL-2 complex memory OT-I/RAG-1−/− cells (132 d after transfer). Before challenge with LM-OVA, the OT-I donor cell populations in peripheral blood were 1.3 ± 0.11% and 1 ± 0.04% of CD8+ T cells in control and IL-2–anti–IL-2 complex memory groups, respectively. At 3 d after challenge, mice containing IL-2–anti–IL-2 complex memory OT-I/RAG-1−/− cells were able to significantly decrease LM-OVA burden in both the spleen and liver compared with mice containing untreated OT-I/RAG-1−/− cells and mice without cell transfer (Fig. 6 C). Furthermore, adoptive transfer of IL-2–anti–IL-2 complex memory OT-I cells provided significant protection to naive animals, whereas an equal number of naive OT-I cells did not (Fig. S4). These results demonstrate that IL-2–anti–IL-2 complex–driven memory CD8+ T cells are able to mount a rapid and robust response to antigen challenge and afford protection.
IL-2–anti–IL-2 complex memory CD8+ T cells display incomplete cellular fitness
We further characterized IL-2–anti–IL-2 complex memory OT-I cells, and compared them to LM-OVA memory OT-I cells and with spontaneously arising memory-phenotype CD8+ T cells. Memory CD8+ T cells proliferate slowly to maintain a stable pool (6, 24), and this homeostatic turnover can be measured by the uptake of BrdU. LM-OVA antigen-driven memory OT-I cells displayed a rate of homeostatic turnover that was similar to that of memory-phenotype CD8+ T cells in the same mouse (Fig. 7 A). In contrast, although IL-2–anti–IL-2 complex memory OT-I cells also exhibited homeostatic turnover, the rate was significantly less than that of antigen-driven memory cells or CD44hi CD122hi memory-phenotype CD8+ T cells in the same mouse (Fig. 7, A and B).
Another cardinal feature of memory CD8+ T cells is the rapid production of cytokines after reencounter with antigen (1, 2). Splenocytes containing IL-2–anti–IL-2 complex memory OT-I cells or LM-OVA memory OT-I cells were stimulated with OVA peptide or PMA plus ionomycin for 4 h in vitro, and cytokine production was assessed by flow cytometry. Upon antigenic stimulation, almost all (>95%) antigen-driven memory cells produced IFN-γ; most of the IFN-γ–producing population also produced TNF-α, and ∼40% of them were IL-2 positive (Fig. 7 C). In comparison, a reduced fraction of memory cells generated by IL-2–anti–IL-2 complexes were positive for the cytokines tested (Fig. 7 C). In addition, the amounts of IFN-γ, TNF-α, and IL-2 produced on a per-cell basis, as judged by the mean fluorescence intensity, were also decreased in IL-2–anti–IL-2 complex memory OT-I cells compared with antigen-driven memory cells (Fig. 7 D). Differences in cytokine production were evident when IL-2 complex–driven or antigen-driven memory cells were stimulated with PMA plus ionomycin (Figs. 7, E and F), implying that alterations in signal transduction proximal to the TCR do not account for the changes. The cytokine production levels of IL-2–anti–IL-2 complex memory OT-I cells resembled those of the host memory-phenotype CD8+ T cells upon PMA/ionomycin stimulation, although a marked difference was observed in IFN-γ levels (Fig. 7 F). Similar results were obtained when activated OT-I cells were prepared from OT-I/RAG-1−/− mice that had been treated with IL-2–anti–IL-2 complexes (Fig. 7 B and not depicted). These results suggest that strong IL-2 signals coupled with weak TCR signals are not sufficient to guarantee the overall cellular fitness of memory CD8+ T cells.
The recent realization that complexing IL-2 with anti–IL-2 mAbs leads to a dramatic increase in the potency of signaling via the intermediate affinity CD122/CD132 receptor in vivo (14, 15) allowed us to examine the consequences of such signals to CD8+ T cells in normal, nonlymphopenic animals in the absence of foreign antigen stimulation. Injected IL-2–anti–IL-2 complexes caused the rapid activation of STAT5 in naive as well as memory-phenotype CD8+ T cells (Fig. 1). This was the case even though CD122 levels are much higher on memory than on naive CD8+ T cells (unpublished data) (16) and the IL-2–anti–IL-2 complex signaling depends on CD122 (29). Under such conditions, potent IL-2 signals stimulated naive CD8+ T cells to proliferate and differentiate into functional memory cells. Such IL-2–anti–IL-2 complex–driven memory cells had a conventional central memory phenotype (Fig. 5) and were able to control a bacterial challenge in both lymphoid and nonlymphoid organs (Fig. 6 C and Fig. S4). The downstream molecular basis for the IL-2–anti–IL-2 complex–driven programming of memory CD8+ T cells awaits further investigation. Target genes of STAT5, an important component of IL-2 signaling (3, 4), are likely to be a key component of this process, because overexpression of STAT5 in T cells results in the selective expansion of memory-phenotype CD8+ T cells (30, 31).
Although IL-2–anti–IL-2 complex–generated memory CD8+ T cells were able to expand, accumulate, and provide protection after challenge with a high dose Listeria infection (Fig. 6 and Fig. S4), we found that they differed from conventional antigen-driven memory cells. In comparison with conventional memory cells, they had a slower rate of homeostatic turnover and reduced cytokine production after short-term restimulation (Fig. 7). This slow turnover may result in dis appearance of the complex-driven memory cells in the long term, although a clear correlation between the reduced BrdU uptake and loss of donor cells was not consistently observed up to 80 d after transfer (unpublished data). The turnover of memory CD8+ T cells is regulated by cytokines such as IL-7 and IL-15 (6). The expression levels of the receptors for these cytokines, namely CD127 (IL-7Rα) and CD122 (IL-2/IL-15Rβ) and CD132, did not noticeably differ between complex-driven memory cells, antigen-driven memory cells, and memory-phenotype cells (Fig. 5 and not depicted). In addition, treatment with PMA plus ionomycin did not override the defect in cytokine production of IL-2–anti–IL-2 complex memory CD8+ T cells (Fig. 7, E and F), implying that the reduced fitness of IL-2–anti–IL-2 complex memory CD8+ T cells might be caused by intracellular differences, such as in the epigenetic remodeling of genes involved in hallmark characteristics of memory cells. We speculate that more potent signals via the TCR may be required to operate in conjunction with IL-2 signals to promote the complete programming of memory CD8+ T cell differentiation. In this context, it is interesting to note that naive CD8+ T cells that receive strong TCR signals after pathogen infection, but weak or no IL-2 signals (i.e., IL-2Rα−/− cells), develop into memory cells that show normal levels of homeostatic turnover and cytokine production but fail to accumulate in response to secondary antigenic challenge (11). Thus, predominant TCR signals without IL-2 give rise to memory CD8+ T cells that fail to accumulate in response to a secondary challenge, whereas predominant IL-2 signals with weak TCR stimulation result in memory cells with reduced turnover and cytokine production.
IL-2–anti–IL-2 complexes dramatically enhance IL-2 activity in vivo (14, 15), but the mechanism of this enhancement is still unclear. Because IL-2–anti–IL-2 complexes lacking the Fc region (i.e., IL-2 plus the F(ab′)2 portion of the anti–IL-2 mAb) had less activity than complexes with the whole IgG, it was suggested that Fcγ receptor–positive cells in lymphoid organs presented the IL-2 to T cells (14). However, we show that IL-2–anti–IL-2 complexes showed similar potent activity in FcRγ−/−FcγRII−/− mice as in B6 control mice (Fig. 3). It is possible that other nonclassical Fc receptor–like molecules (32) or leaky expression of FcγRI without the FcRγ subunit (33) may mediate the activity of IL-2–anti–IL-2 complexes in vivo. However, FcRγ−/−FcγRII−/− mice showed a gross functional defect in the stimulatory presentation of anti-CD3 IgG (Fig. S3 B), leading us to conclude that presentation of IL-2–anti–IL-2 complexes via Fcγ receptors may not be responsible for the enhancing effect. The half-life of IgG in vivo is dependent on the presence of the Fc region (25, 34), and this may explain the weak activity of IL-2–anti–IL-2 immune complexes lacking Fc. Moreover, there are many studies documenting the fact that the in vivo half-life of various cytokines is prolonged by the formation of immune complexes. These include human IL-2–anti–IL-2 (35), IL-4–anti–IL-4 (36), IL-6–anti–IL-6 (37), and soluble IL-15–IL-15Rα-Fc (38) complexes. Therefore, we favor the idea that IL-2–anti–IL-2 complexes extend the half-life of IL-2 in vivo by preventing renal excretion, the major pathway of IL-2 clearance in mice (39).
Other recent reports have demonstrated the development of memory CD8+ T cells from naive cells without cognate antigen stimulation. Stimulation of naive CD8+ T cells with soluble IL-15–IL-15Rα complexes, which act as superagonists (40, 41), induced proliferation and converted the cells to memory-phenotype CD8+ T cells (38). Because both soluble IL-15–IL-15Rα complexes and IL-2–anti–IL-2 immune complexes signal through the CD122/CD132 receptor complex on CD8+ T cells (6, 42), these two types of complex may result in similar consequences, although the function of the memory-phenotype CD8+ T cells generated by soluble IL-15–IL-15Rα complexes was not addressed in detail (38). In another report, Hamilton et al. demonstrated that lymphopenia-induced HP resulted in the generation of protective memory-like CD8+ T cells (43). Similar to IL-2–anti–IL-2 complex–mediated proliferation, HP is induced in the absence of cognate antigen (22–24), and similar phenotypic changes occur after proliferation induced by IL-2 signals (Fig. 2 C and Fig. 5) and by HP (43). HP of naive CD8+ T cells in a lymphopenic environment requires both TCR stimulation through MHC class I–peptide complexes, in addition to cytokines such as IL-7 (22–24). Proliferation of naive CD8+ cells driven by IL-2–anti–IL-2 complexes in irradiated mice may also require TCR signals from self-MHC ligands (26). IL-7–dependent HP is enhanced by IL-12, but no substantial involvement of IL-2 has been reported (44–46). Given that stimulation by foreign antigen plus cytokines (such as pathogen infections), self-antigen plus IL-7 (HP), or intense IL-2 signals (IL-2–anti–IL-2 complexes) all give rise to protective memory CD8+ T cells, key components of programming memory CD8+ T cell differentiation may reside within pathways common to these stimulations.
Mice.
B6 mice were obtained from the Jackson Laboratory. B6.SJL, MHC class I Kb−/−Db−/−, OT-I/RAG-1−/−, and FcRγ−/−FcγRII−/− mice were purchased from Taconic. These animals were backcrossed to a B6 genetic background, except for FcRγ−/−FcγRII−/− mice, which were on a 129 × B6 mixed background. Animals were housed in specific pathogen-free conditions in the animal facilities at the University of Washington. OT-I TCR-transgenic mice (47) congenic for Thy1.1 and CD45.1 were bred and maintained in the same facilities. All experiments were performed in compliance with the University of Washington Institutional Animal Care and Use Committee regulations.
Preparation of IL-2–anti–IL-2 complexes.
Carrier-free recombinant mouse IL-2 was obtained from eBioscience. Hybridoma of anti–IL-2 mAb (S4B6) was grown in HB basal medium with HB101 supplement (Irvine Scientific). The anti–IL-2 mAb was purified from the culture medium with a protein G column. IL-2–anti–IL-2 complexes were prepared by mixing 1.5 μg rIL-2 and 50 μg anti–IL-2 mAb per mouse. Rat IgG (Sigma-Aldrich) plus PBS was used as a control treatment, as previously described (15, 48). The dose of IL-2–anti–IL-2 complexes has been shown to be effective in vivo in previous studies (11, 14).
Detection of phospho-STAT5 in vivo.
Mice were killed at the time points indicated in the figures after a single i.p. injection of IL-2–anti–IL-2 complexes. The intracellular phospho-STAT5 staining was performed as previously described (49, 50). In brief, splenocytes were fixed in 1.6% formaldehyde/PBS for 30 min at room temperature, followed by permeabilization with ice-cold methanol. The fixed and permeabilized cells were stained in 2% FCS/PBS with PECy7-labeled anti-CD44, PerCP-labeled anti-CD8α, and Alexa Fluor 647–labeled anti–phospho-STAT5 (Y694; Phosflow, BD Biosciences) mAbs. Alexa Fluor 647–labeled mouse IgG1 mAb (Phosflow; BD Biosciences) was used as the isotype control.
Adoptive transfer of donor CD8+ T cells.
CD44lo naive CD8+ T cells were purified by magnetic cell sorting using the CD8a+ T cell isolation kit (Miltenyi Biotec) supplemented with 0.003 μg per million cells of biotinylated anti-CD44 mAb (BD Biosciences) to deplete CD44hi CD8+ T cells (46). After sorting, >98% of CD8+ T cells were CD44lo CD122lo naive phenotype. 0.5–106 donor cells per mouse were transferred i.v. into nonirradiated hosts. In some experiments, the donor cells were labeled with 1.6 μM CFSE (Invitrogen) for 10 min at 37°C before transfer. Host animals in Fig. 4 (A and B) received 10 Gy total body irradiation and were reconstituted with syngeneic BM trans plantation. For assays of IL-2–anti–IL-2 complex memory cells, OT-I or OT-I/RAG-1−/− mice were injected with IL-2–anti–IL-2 complexes five to six times every other day until most OT-I CD8+ T cells acquired the CD44hi CD122hi phenotype. These cells were isolated by the magnetic cell sorting system without biotinylated anti-CD44 mAb. 7–17 × 106 of these activated OT-I cells per mouse were injected i.v. into host mice. The analysis of memory OT-I cells was performed at least 50 d after transfer. Engraftment of the donor cells at the time of assay was between 1 and 10% of total CD8+ T cells.
Intracellular cytokine staining.
Splenocytes were incubated with 1 μg/ml OVA257-264 (SIINFEKL) or PMA plus ionomycin in the presence of Brefeldin A (GolgiPlug; BD Biosciences) for 4 h at 37°C. After staining of cell-surface molecules, the cells were fixed and permeabilized with the Cytofix/Cytoperm kit (BD Biosciences). The intracellular cytokine staining was performed with FITC-labeled anti–TNF-α, PE-labeled anti–IL-2, and PECy7-labeled IFN-γ mAbs (eBioscience).
In vivo CTL assay.
To prepare target cells, splenocytes were incubated with or without 10 μg/ml OVA257-264 peptide for 30 min at 37°C. These peptide-pulsed target and nontarget populations were labeled with 1.6 and 0.16 μM CFSE, respectively, mixed at a 1:1 ratio, and injected i.v. into mice. After 20 h, the target cell killing activity was evaluated using the ratio of CFSE/positive populations in the spleen. The CFSE-labeled populations were distinguished from host populations by congenic markers.
BrdU labeling in vivo.
Mice were given 0.8 mg/ml BrdU (Sigma-Aldrich) in their drinking water for 7 d. Intracellular BrdU staining was performed by following the instructions of the BrdU Flow Kit (BD Biosciences).
LM-OVA infection, generation of LM-OVA–induced memory OT-I cells, and protection assay.
An erythromycin-resistant recombinant strain of L. monocytogenes that expresses a secreted form of OVA (LM-OVA) was provided by H. Shen (University of Pennsylvania School of Medicine, Philadelphia, PA) (51). LM-OVA was grown in Brain-Heart Infusion broth (BD Biosciences). At a midlog growth phase, culture samples were measured by OD and diluted in PBS for the desired titers. For preparation of LM-OVA–induced OT-I memory cells, mice that received 104 CD44lo CD122lo naive OT-I cells per mouse 1 or 2 d earlier were infected i.v. with 3,000 CFU of LM-OVA. These mice were used at least 50 d later. Engraftment of the donor cells at the time of assay was between 1 and 10% of total CD8+ T cells. For lethal infection, mice were inoculated i.v. with 105 CFU. For protection assay, organ suspension was prepared in 5 ml PBS, and 10-fold serial dilutions were made in PBS containing 0.1% NP-40. These 100-μl dilutions were plated onto the Brain-Heart Infusion plates containing 5 μg/ml erythromycin. The limit of detection was 50 CFU per organ.
Data with logarithmic presentation were transformed to log10 (43). Statistical differences between groups were examined by a paired, two-tailed (for Fig. 3 B) or an unpaired, two-tailed Student's t test using Prism software (GraphPad). P < 0.05 was considered statistically significant.
Online supplemental material.
Fig. S1 shows the experimental protocols used for studying IL-2–anti–IL-2 complex–induced proliferation and for the generation of IL-2–anti–IL-2 complex memory and antigen-driven memory cells. Fig. S2 shows the proliferation of different kinds of TCR-transgenic CD8+ T cells induced by IL-2–anti–IL-2 complexes. Fig. S3 shows the surface staining and the functional defect in FcRγ−/−FcγRII−/− mice. Fig. S4 shows the protection against bacterial challenge afforded by an equal number of naive OT-I, IL-2–anti–IL-2 complex memory OT-I, or LM-OVA–induced memory OT-I.
We thank Beverly Dere and Xiao-cun Pan for excellent technical support.
This work was supported by the Howard Hughes Medical Institute and National Institutes of Health grant AI19335 (to M.J. Bevan).
The authors have no conflicting financial interests.
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Figure S1
Experimental protocols. (A) Protocol for effector time points. CD44lo CD122lo naive CD8+ T cells were isolated by magnetic bead sorting with biotinylated anti-CD44 mAb. 0.5-1 x 106 donor cells were transferred i.v. into host animals on day -1. IL-2-anti-IL-2 complexes were injected i.p. (1.5 µg rIL-2 mixed with 50 µg anti-IL-2 mAb) on days 0 and 2. Assays were performed on day 5 or 6. (B) Protocol for memory time points. OT-I mice or OT-I/RAG-1-/- mice were treated i.p. five to six times with IL-2-anti-IL-2 complexes until most of the OT-I cells became CD44hi CD122hi memory-phenotype cells. After magnetic bead sorting to purify CD8+ splenic cells, 7-17 x 106 activated OT-I or OT-I/RAG-1-/- cells were transferred i.v. into normal hosts. Assays were performed at least 50 d later. Numbers in A and B represent the percentage of gated cells that are within the boxes. (C) Protocol for generating antigen-driven memory OT-I cells. Mice that had received 104 CD44lo CD122lo naive OT-I cells i.v. 1 or 2 d previously were infected i.v. with 3,000 CFU of LM-OVA and rested for at least 50 d. Engraftment of the donor cells in B and C at the time of the assays was between 1 and 10% of host CD8+ T cells.
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IL-2-anti-IL-2 complexes induce proliferation of OT-I/RAG-1-/-, P14, and Vβ5 TCR-transgenic CD8+ T cells. CFSE-labeled CD44lo CD122lo naive CD8+ T cells from P14 TCR-transgenic mice specific for lymphocytic choriomeningitis virus GP33, Vβ5 TCR-transgenic mice that express the same TCRβ transgene as OT-I and endogenous TCRα chains, or total splenocytes from OT-I/RAG-1-/- mice were transferred into host mice on day -1. IL-2-anti-IL-2 complex treatment was performed on days 0 and 2. The CFSE levels in the donor cells were examined on day 6.
Expression of Fcγ receptors and anti-CD3-induced IL-2 production in FcRγ-/-FcγRII-/- mice. (A) Expression of CD16/32 (FcγRIII/II) on B cells (B220+ CD3NEG) is shown. Numbers represent the percentage of gated B cells that are positive for staining. (B) 10 µg anti-mouse CD3ε mAb (145-2C11; BD Biosciences) was injected i.p. Mice were bled 90 min later, and serum IL-2 levels were measured by ELISA (eBioscience). *, P < 0.05 versus nontreated B6 mice; **, P < 0.01 versus anti-CD3-treated B6 mice.
IL-2-anti-IL-2 memory OT-I cells provided protection better than naive OT-I cells. An equal number (105 cells) of naive OT-I, IL-2-anti-IL-2 memory OT-I (12 d of treatment plus 117 d after transfer), or LM-OVA-generated memory OT-I (55 d after infection) was transferred to naive animals. On the next day, mice were infected with 105 CFU of LM-OVA. CFU in the spleen were examined 3 d later. *, P < 0.05 versus the naive B6 group.
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IL-2–anti–IL-2 complex treatment induces rapid activation of STAT5 in naive CD8+ T cells. Mice were given a single injection of IL-2–anti–IL-2 complexes. At the indicated time points, splenocytes were fixed and permeabilized for intracellular staining of the tyrosine-phosphorylated form of STAT5. Mice for the zero time point received no injection. The data are gated on the CD8+ population, and the numbers represent the percentage of gated cells in each quadrant.
IL-2–anti–IL-2 complexes induce proliferation of naive antigen-specific CD8+ T cells. 0.5 × 106 purified CD44lo CD122lo naive OT-I cells were transferred into nonirradiated mice on day −1. The mice were given IL-2–anti–IL-2 complexes or control (rat IgG) on days 0 and 2, and the assays were performed on day 6. (A) Proliferation of donor cells was evaluated by CFSE dilution. (B) Absolute number of donor cells (left) and host CD8+ T cells (right) in the spleen. The data represent the mean + SEM (n = 3). ***, P < 0.001 versus rat IgG treatment. (C) Phenotypic changes of the donor cells. Rat IgG and IL-2–anti–IL-2 complex treatment are indicated with open and shaded histograms, respectively. (D) IFN-γ production by donor OT-I cells was assessed after 4 h of stimulation in vitro. Numbers represent the percentage of gated OT-I cells making IFN-γ. (E) Equal numbers (1.1 × 106 cells) of naive OT-I cells and IL-2–anti–IL-2 complex–activated OT-I cells were transferred to naive animals. An in vivo CTL assay with OVA peptide–pulsed (CFSEhi) and control (CFSElo) targets was performed the next day. The killing activity was examined 20 h later. Numbers represent the percentage of CFSEhi and CFSElo targets remaining. The donor cells (A–D) and target cells (E) were distinguished from host populations by congenic markers.
Fcγ receptors are dispensable for the action of IL-2–anti–IL-2 complexes. B6 mice and FcRγ−/−FcγRII−/− mice were treated with IL-2–anti–IL-2 complexes on days 0 and 2. The mice were bled before starting the treatment (pretreatment) and on day 5. (A) Phenotype of the CD8+ T cell population of the same animal in each group is shown. The data are gated on CD3+ CD8+ cells. Numbers represent the percentage of gated cells within the box. (B) Absolute numbers of total leukocytes, CD8+ T cells (CD3+ CD8+), and NK cells (NK1.1+ CD3NEG) in the peripheral blood before and after (day 5) the treatment. Each column represents the mean + SEM (n = 3). *, P < 0.05; or **, P < 0.01 versus pretreatment.
IL-2–anti–IL-2 complexes stimulate naive CD8+ T cells with limited TCR stimulation. Host mice that received CFSE-labeled naive OT-I cells 1 d earlier were treated with IL-2–anti–IL-2 complexes on days 0 and 2. CFSE levels of donor cells from the spleen were examined on day 6. The data are gated on CD3+ CD8+ congenically marked donor cells. (A) WT and MHC class I Kb−/−Db−/− mice that were irradiated with 10 Gy and transplanted with syngeneic BM cells were used as host animals. (B) Absolute number of OT-I cells recovered from the spleen of animals in A is shown. The data represent the mean + SEM (n = 2). ***, P < 0.001 versus rat IgG treatment. (C) Nonirradiated OT-I/RAG-1−/− mice were used as hosts.
IL-2–anti–IL-2 complexes generate memory cells with a central memory phenotype. Memory OT-I cells generated by LM-OVA infection (75 d after infection; population A), IL-2–anti–IL-2 complex memory OT-I cells (12 d of treatment plus 76 d after transfer; population B), and host CD8+ T cells (population C) were examined for the expression of cell-surface markers. Contour plots are gated on CD8+ cells. Numbers represent the percentage of CD8+ cells within the box.
IL-2–anti–IL-2 complex memory CD8+ T cells protect against bacterial infection. (A) Enumeration of IL-2–anti–IL-2 complex memory OT-I/RAG-1−/− cells (9 d of treatment and 91 d after transfer; CD45.2+) in the spleen before and 3 d after challenge with 105 CFU of LM-OVA. The data are gated on CD8+ cells. Numbers represent the percentage of CD8+ cells within the box. (B) Absolute numbers of IL-2–anti–IL-2 complex memory OT-I/RAG-1−/− cells in the spleen before and after LM-OVA challenge. Data represent the mean + SEM (n = 3–4). ***, P < 0.001. (c) CFU of LM-OVA in the spleen and liver at 3 d after challenge of mice containing equal numbers of naive OT-I/RAG-1−/− or IL-2–anti–IL-2 memory OT-I/RAG-1−/− cells. Data represent the mean + SEM (n = 4–6). ***, P < 0.001 versus the OT-I/RAG-1−/− group.
IL-2–anti–IL-2 complex memory CD8+ T cells display incomplete cellular fitness. (A) Mice bearing LM-OVA antigen-driven memory OT-I cells at 68 d after infection or IL-2–anti–IL-2 complex memory OT-I cells after 81 d (12 d of treatment plus 69 d after transfer) were given BrdU in drinking water for 7 d. BrdU incorporation by the indicated subsets of memory CD8+ T cells in the spleen is shown. Numbers represent the percentage of gated cells that are BrdU positive. (B) BrdU-positive population of host CD44hi CD122hi CD8+ T cells and IL-2–anti–IL-2 complex memory OT-I/RAG-1−/− cells after 61 d (9 d of treatment plus 52 d after transfer). The data are the mean + SEM (n = 3). *, P < 0.05. (C–F) Splenocytes containing memory OT-I cells generated by LM-OVA infection (62 d after infection) or IL-2 signals (12 d of treatment plus 63 d after transfer) were stimulated in vitro with OVA peptide (C and D) or PMA plus ionomycin (E and F). Intracellular cytokine production was examined 4 h later. Numbers in C and E represent the percentage of gated cells in each quadrant. (D and F) The mean fluorescent intensity (MFI) of cytokine staining is shown (mean + SEM; n = 3). The MFI was calculated within the population positive for the respective cytokine. * and **, P < 0.05 and 0.001, respectively, versus the complex-driven memory cells; ***, P < 0.001 versus LM-OVA memory cells.
Local microbleeding facilitates IL-6– and IL-17–dependent arthritis in the absence of tissue antigen recognition by activated T cells
Autoimmune arthritis associated with mutated interleukin (IL)-6 receptor gp130 is driven by STAT3/IL-7–dependent homeostatic proliferation of CD4+ T cells
ATP spreads inflammation to other limbs through crosstalk between sensory neurons and interneurons


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Redefining Micro-Budget Filmmaking: The $6,000 'Layover' (Guest Blog)
Director, writer and producer Joshua Caldwell reveals the backstory behind his first feature film
Joshua Caldwell Guest Writer
Last Updated: October 14, 2014 @ 8:51 AM
Courtesy White Bear PR
Like most filmmakers, I didn't think my first feature film, Layover, would have a budget of $6,000. Even $100,000 seemed like barely enough, but $6,000? I've made short films and music videos with budgets several times that amount! A camera rental alone could cost me that much. Not only did it feel difficult; it felt impossible.
On Jan. 1, 2013, I started feeling the pressure. I had been living in Los Angeles for seven years. I had won an MTV Movie Award, directed a bunch of well-received short films, music videos and commercials, and spent the last three years producing and directing digital content as an executive for a production company. However, I hadn't made a feature film and my self-imposed deadline of doing so before I turned 30 was quickly approaching.
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It wasn't for lack of trying. I had developed and written scripts with my writing and producing partner Travis Oberlander, but we found that most of the projects we were writing would cost a minimum of six figures. Then, two things happened:
1) I read an article where writer/director/actor Ed Burns discussed how he was producing films for under $10,000, and
2) I saw the film "For Lovers Only," a zero budget black & white feature shot by the Polish Brothers on a Canon 5D.
In an instant, my thinking flipped from "I need a lot of money" to "How little can I get away with?" I had everything I needed to make a film: actors, cameras, locations, editing systems, and so on. I thought back to an idea I had about a young woman stuck in Los Angeles on a layover and thought it might be a concept easily executed for very little money.
So, in February I wrote the script for "Layover." In May, we shot the film over the course of six weekends with a total budget of $6,000. A year after we wrapped, we had our World Premiere at the Seattle International Film Festival, where we were nominated for the New American Cinema Award. And now the film is available via our own self-distribution model on LayoverFilm.com.
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For Travis and I, this whole process of producing and distributing a $6,000 film has been one big experiment, largely born out of the desire to create our own path and our own destiny as filmmakers — where we control the projects we make and distribute in a way that allows us to keep making those films.
After directing with a DIY approach for 14 years, I've had my share of failures, films that were shot but not finished, and a graveyard of ideas and concepts, scripts and shorts. From each of my failures and successes, I learned. I was able to see what worked and what didn't, where and how to cheat, what's possible to get away with, what isn't, and how to tell a compelling story with interesting characters without significant funding.
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As a result, I've managed to figure out a few things that allowed me to shoot a feature film with the scope of Layover on a very, very limited budget.
DEVELOPMENT/WRITING: MODULAR STORYTELLING
Over the past couple years I've found myself in the position of developing and writing treatments and scripts for projects with budgets that had a huge spread ("We could end up with $200,000 or we might get $1M, we don't know yet.") Having no idea what budget we were writing for, I had to craft a story that could easily be scaled up or scaled down depending on what funding came through.
That is essentially what I call "Modular Storytelling." Think of it like a film camera. At its most basic, it's a lens and a camera body. But its design allows you to add all sorts of extras,: a rails system, matte box, follow focus, monitors, and gadgets of all kinds. But adding those things doesn't change the fundamental nature of the camera itself: a body and a lens. Even if you can't afford all those other add-ons, you can still use the camera.
The camera and lens is your story and all the add-ons are the execution-based accessories you can or can't afford. The story is the story whether you have all the bells and whistles or not; you should be able to tell an engaging and interesting version of the story without them.
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For example, in "Layover," Simone (our main character) finds herself at a house party looking for her friend. The story beats at that house would be the same whether we had a massive PROJECT X style party or one with a few random people. As such, I knew we'd never be able to afford to get enough people there for a massive party and it would be obvious if we tried to fake it. So I wrote it as a random house party with a handful of people getting together after their time at the club — I wrote it specifically as that because I knew I could execute it.
PRODUCTION: USING THE APPROPRIATE CAMERA
One of the most important things to successfully making a low or no-budget film is shooting on the camera that is appropriate to your production. No doubt, the ARRI Alexa is an amazing camera and delivers superior images. But you need more than just the camera to get those images.
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You need an experienced camera crew, you need an experienced lighting and grip crew, you need appropriate lighting equipment which then may require generators. You would need to get ALL OF THAT for free to justify using the ALEXA on a no-budget production. And even then, your lighting setups are taking longer, leaving you less time on an already short shoot to get what you need. Is that worth it?
On "Layover," I used the only camera available to me at the time that would allow me to be light and compact, without drawing attention to myself (since we were shooting guerilla style) and didn't require a ton of light to get an acceptable image (since were shooting the whole film at night). I used the Canon 5D mk II, and we lit the whole movie using a 1×1 LED panel light, china balls, some Home Depot can lights, and 250 watt practical bulbs. That's it.
One of the great things about the Canon 5D mk II and mk III is its light sensitivity. You can shoot using much less light than would be required on an Alexa or a RED. It's also small and compact. It's easy to move, to hold for long periods of time, and delivers a beautiful image if used correctly. For the World Premiere of "Layover," we had the final film blown up to 2K for a DCP and projected onto a 50 foot screen, and it looked really good.
And that's the other thing: Your audience doesn't really care what you shot the film on. With everyone talking about camera innovation and resolution and tech and gear, we forget the audience just wants a good story.
What is the best tool to allow you to tell that story? Is it a RED that will take forever to light and give you less than the ideal amount of time to shoot? Or is it the Canon 5D which doesn't deliver the same resolution as the RED but will allow you to light quickly and simply and allow more time for what's important: your actors, in front of the camera, delivering their lines with emotion and not being rushed. You never want to say, "It's good enough."
POST-PRODUCTION: IT'S ALL SOUND
In screening after screening of "Layover," we get asked about and complimented on the sound mix. I think people have a hard time believing that quality sound can be achieved with only $6,000. As most of us have been told during our filmmaking careers, it's really sound, not image, that can make or break your films.
So, how did we pull it off? Did we have a professional production sound mixer with thousands of dollars in equipment on set? We did. For one day. And then we realized we couldn't afford it throughout production. So, one of my producers, Vertel Scott, came to me with a solution: an H4N Zoom recorder, wireless lavs, a boom mic and pole. That's it. Our post sound mixer asked us what we had recorded on. I told him and he responded that our rough tracks sounded better than 90 percent of the professional raw audio he gets in.
We were very fortunate to have an amazing sound team volunteer their time to help me get this film made, and I couldn't have made this film without them. There's no easy trick to getting a good sound mix for very little money — it's all relationships and work history. I've been fortunate to work with my guys on several projects, most of which were paid, so I had bought myself an "ask" and because they liked the film and trusted my talent as a director, they were willing to cut me a deal.
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It's the same reason why composer Bill Brown ("CSI: New York") created our incredible score. We had collaborated to the point where even if he wasn't getting paid, the work load was manageable. (I wasn't coming in and doing 10 rounds of notes on every cue.) He liked the movie and knew that he was working on something quality-driven. I had to earn the right to ask Bill and my sound mixers for a favor.
THE NEW 'INDIE'
Making "Layover" for as little as we did and seeing the results and the opportunities it has created really forced me to rethink how movies can be made. I'm not saying that every movie can or should be made for $6,000, but it has taught me not to wait on someone else to say, "Yes." It's allowed me to forge my own path and tell the stories that I want to tell, not what some gatekeeper deems to be financially viable.
I can get away with making a small, French-language drama with no stars (which some might call indulgent filmmaking) because I spent so little on it. But if you make a movie for $500,000, that is a $500,000 bill that you have to pay back. So, you better be sure the film you're making is capable of making that amount of money back (and then some) and be okay with your job shifting from being a director to being a salesman. Instead of creating content, you'll be off trying to sell it.
As indie filmmakers, we are no longer in a world where we can make any film for any amount of money we get and hope the audience shows up. Your civil war LBGT musical might not have the audience to justify spending $1 million on it, or even $500k. We are burning through investors who are basically financing really expensive calling cards. Instead, I believe that we have to think more like YouTubers. We have to:
1) Cultivate an audience by creating and delivering consistent content. Doesn't have to be every day but say bye-bye to spending four years focused on making and selling only one movie. Your audience won't remember you.
2) Make that content at a responsible budget level so that a ROI is possible through direct-to-customer distribution on a network you've built by building and rewarding your audience.
The current state of indie film is in flux and there are new and every growing opportunities available if you're willing to move beyond the traditional approach and think differently.
Want to see what $6,000 can get you? Head over to LayoverFilm.com to buy "Layover" today and help directly support indie film and filmmakers.
BIO Joshua Caldwell
Joshua Caldwell is an MTV Movie Award winning director, writer, and producer. He has worked with a number of high profile producers, including CSI: creator Anthony E. Zuiker. His award-winning short film Dig, starring Mark Margolis of Breaking Bad, was featured in numerous film festivals and his Superman fan fiction short film Resignation will screened at Comic-Con 2014. Layover had its World Premiere at the 2014 Seattle International Film Festival where it was nominated for the prestigious New American Cinema Award. Follow Joshua on Twitter @Joshua_Caldwell.
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text:
Strike Three for ADHD: How Baseball Helped 'The Killing' Actress Bex Taylor-Klaus
Playing baseball allowed the TV star to channel her ADHD energy and anxiety into building her character – both on and off the screen.
Medically Reviewed by Niya Jones, MD, MPH
Bex Taylor-Klaus firmly believes that if she hadn't played baseball as a kid in Atlanta, she would not be playing Bullet on AMC's murder-mystery drama "The Killing" today. "Without a doubt, sports got me where I am today," said Bex, who has attention deficit hyperactivity disorder (ADHD) and has been living in Los Angeles since she turned 18 about a year ago.
As a kid, she knew something was different about her. "I could never sit down and focus on things," she recalled. "I had to do things my own way."
Her mother, Elaine Taylor-Klaus, who still lives in Atlanta, said her daughter was diagnosed with ADHD when she was 4 or 5 "but I knew it from the time she was a few weeks old because she was a very classic hyper kid, bouncing off the walls and easily distracted." Her mother described young Bex as "extremely bright, but having a hard time getting her work done, even in kindergarten."
As a certified life coach, Bex's mom knew that sports could help with her daughter's attention and anxiety. Exercise can be an effective alternative treatment for ADHD, because it helps kids in two ways. "One is the impact it has on the brain in terms of organization, planning, and structure," said Taylor-Klaus. "The other is in terms of self-esteem and development and finding something they can be good at."
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Like Taylor-Klaus, Jay P. Granat, PhD, a psychotherapist and licensed marriage and family counselor in River Edge, N.J., believes that participating in sports can build kids' self-esteem because it gives them an opportunity to shine. Also, he said, "they can take advantage of their energy on the playing field and use it in a positive way."
ADHD and Exercise: The Power of Parents and Coaches
Taylor-Klaus enrolled Bex in all kinds of sports when she was little: karate, hockey, swimming. Nothing stuck. Then, at age 6, Bex found her sport when she started playing coed baseball. "She played it every year until she left Atlanta," her mom said.
Bex started in the outfield, but that didn't work for her because she didn't see much action. "You could see her standing there, picking daisies," Taylor-Klaus recalled.
But then her coach recognized she needed more action and put her behind the plate as catcher, and that made all the difference. "She just soared," Taylor-Klaus said. "I love that man because he engaged her instead of counting her out and saying she can't pay attention."
Bex's Solution: Baseball as ADHD Treatment
Bex played on the team every year spring and fall because she loved it so much. As catcher, she was able to hone her concentration skills in order to keep track of where the ball and players were. And because she was good at her infield position, it also helped boost Bex's confidence, which allowed her to focus better on other tasks off the baseball diamond.
When Bex wasn't playing baseball, Taylor-Klaus would encourage her to exercise and stay active.
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Even now, "when I hear her starting to get low, the first thing I say is, 'When was the last time you exercised?'" said Taylor-Klaus, who keeps in close touch with her daughter despite the nearly 2,000 miles between them.
Playing sports isn't a cure for every kid with ADHD, Granat said, "but for many kids, it is. I have seen many kids with ADHD who, if they didn't play sports, would have been set back."
Finding Time to Exercise
Eventually Bex got into acting through summer camps, first in Atlanta and later in LA. She and her best friend and roommate both auditioned for the role of Bullet, a tough and scrappy yet big-hearted street kid. Bex knew the role was hers as soon as she saw the script because she could connect the fictional character's life with her own.
Because she's now so busy on the set, Bex doesn't have as much time as she'd like for exercise, but she's hoping to change that. "Exercising is one of the greatest releases or outlets that I have ever found," she said. "Just not running — I'm not a big fan of running."
Regardless of how busy her schedule is, however, Bex will always carry a love for the game that helped her gain control of her ADHD symptoms.
"Some people complain that baseball is like watching paint dry," Bex said. "I totally disagree. There are so many subtle nuances to the whole game. You can't get bored. It's a game of skill and studies — like chess but cooler. When you're playing, it is exhilarating."
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Q: Div Side By Side Background Color Not Filling Height I know a lot of topics have been written about div side by side, but I couldn't find a solution for my specific issue. I managed to get 2 divs side by side, and be mobile friendly (not require sideways scrolling), but there's a background color problem. If 1 div has little text while the div next to it doesn't, then the missing blocks of height just show blank instead of having a background. How can I fix this while staying mobile friendly?
Example page: http://www.gloryhood.com/articles/ztest.html
CSS:
div.table {
max-width: 100%;
}
div.firsthalf {
background-color: #ffffff;
float: left;
max-width: 50%;
text-align: left;
word-wrap: break-word;
}
div.secondhalf {
background-color: #ffffff;
margin-left: 50%;
max-width: 50%;
text-align: left;
word-wrap: break-word;
}

HTML:

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Righvid

A: I think you want something like this,

$(document).ready(function(){
        $('.container').each(function(){
            var firstDiv = $(this).find('.firsthalf');
            var secondDiv = $(this).find('.secondhalf');
            if(firstDiv.height() >= secondDiv.height()){
                secondDiv.css('height',firstDiv.height());
            } else {
                firstDiv.css('height',secondDiv.height());
            }
        });
    });
body{background: #39b1a4;}
    div.table {
    max-width: 100%;
}
div.firsthalf {
    background-color: #ffffff;
    float: left;
    width: 50%;
    text-align: left;
    word-wrap: break-word;

}
div.secondhalf {
    background-color: #ffffff;
    width: 50%;
    text-align: left;
    word-wrap: break-word;
    float: right;

}

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        Righvid

redpajama_set_name: RedPajamaStackExchange


Looking for a super cool Halloween mask? NEVERMORE will you have to search! This moveable-mouth Raven mask is original and fascinating! The movement and sound effects are brilliant and will have everyone mystified and terrified! Legends come to life with this illusive classic Halloween horror story character animated mask with scary sound effects! See VIDEO below.
Detailed raven face is black with beady eyes, cawing beak and shrieking mouth.
The Beak opens when you open your mouth! Mouth moves when you move your mouth, creating the illusion of a real crow! Black Bird mask is made from molded plastic and covered in faux fur feather-inspired fabric with beady eyes. Lightweight, comfortable and easy to wear, this realistic Raven mask is made from molded plastic and covered in fake fur.
Comes with push-button activated loud cawing wild bird sounds that you operate by a push button device you hold in your hand (a cord runs down your sleeve). Creates an authentically terrifying and realistic attacking bird effect!
This Halloween cosplay mask is lightweight, comfortable and easy to wear. Adjustable foam inserts enable a snug fit at the jaw for realistic movement.


Dubois L, et al. Genetic and environmental contributions to weight, height, and BMI from birth to 19 years of age: an international study of over 12,000 twin pairs. PLoS One. 2012;7(2):e30153.
Hubal MJ, et al. Variability in muscle size and strength gain after unilateral resistance training. Med Sci Sports Exerc. 2005 Jun;37(6):964-72.
Davidsen PK, et al. High responders to resistance exercise training demonstrate differential regulation of skeletal muscle microRNA expression. J Appl Physiol (1985). 2011 Feb;110(2):309-17.
Zempo H, et al. Heritability estimates of muscle strength-related phenotypes: A systematic review and meta-analysis. Scand J Med Sci Sports. 2017 Dec;27(12):1537-1546.
Beaven CM, et al. Significant strength gains observed in rugby players after specific resistance exercise protocols based on individual salivary testosterone responses. J Strength Cond Res. 2008 Mar;22(2):419-25.
Yamada M, et al. Age-dependent changes in skeletal muscle mass and visceral fat area in Japanese adults from 40 to 79 years-of-age. Geriatr Gerontol Int. 2014 Feb;14 Suppl 1:8-14.


It was Friday night at the USTA Billie Jean King National Tennis Center. There was 121st-ranked Nicole Gibbs, on Court 10, in the final round of the US Open qualifying versus 128th-ranked Naomi Broady. Gibbs had won the first set 6-4, lost the second by the same score, now led 5-2, 40-love in the third and would soon make an unprecedented statement.
At 5-2, 40-love, Gibbs missed a makeable forehand. It was now 40-15, Gibbs again one point away from making the main draw. This time, Broady missed a forehand.
Here is where Nicole Gibbs entered new territory: She cried. There had been tears in defeat, but never in victory.
The grind it must be. For Gibbs, life has always been about the fight. When Gibbs was a child, her parents thought she'd be a lawyer. Be it with her intellect or her tennis game – a style based less on weaponry and more hinged on point-to-point problem-solving – Nicole Gibbs is arguably (sic) never more content than when she has the chance to make the case for herself.


Its time for the feature of Blogger of the Month!
This March I bring you the non-typical fashion blogger, her name is Ana (I know best name ever right?!). She is from the UK and I love love love her outfits. She is very fashion forwards and out there when it comes to trends. But what I love the most about Ana is that her posts are full of rich arguments about her opinions of things that are going on around the world. Her posts are not the typical experience or tips on trends, although if you really want to know how to mix prints and trends in general, you need to follow her. She enriches her audience with articles that create controversy and arguments among her followers.
If this got you curios about her read the interview below to get to know her more!
Growing up I had always followed blogs but never made the decision to start my own because I was in full time education. Come university graduation, I was finally ready to start my own blog and it has been a whirlwind journey. I have been very ill for a few years and the blog was my way of coping and expressing myself in a way that I could not necessarily do in person and it felt good to share my experiences with others. I am ( at least before blogging) a very private person and often would dismiss and ignore problems but my blog forces me to acknowledge them and recognize that I need to look after myself before I help others. Blogging has opened my eyes to a new world and challenged personal and social boundaries to the extreme but I would not have it any other way. I am proud to call myself a blogger and wanted to challenge social perspectives on the nature of blogging as an occupation. I have been questioned to what exactly I do all day with some people implying that I just sit on my arse and that is not the case. Blogging is hard work and a lot to deal with when you have other responsibilities too. I wanted my blog to be a platform for the voiceless, oppressed by society for being different and in many ways I am an outsider. I don't follow the crowd and am very vocal on important issues like body image, abuse, bullying and more prevalent political issues affecting our society today. I also write about fashion but often my fashion articles are rooted in historical/social/political/economical context or examine wider social issues. Some of my viral posts have been centered around my experiences with bullying and abuse and as someone who has never had counselling the blog has become an outlet for all the worries, insecurities and negative energy I had inside me. It detoxifies me and makes me feel like a more positive person.
Do you consider your blog a full-time job? Or do you have any other job? If so, what is it?
I work insane hours especially since I am early to rise and late to sleep and suffer from insomnia. So while the people I live with are sleeping I am up at 3 am scribbling away or completing blog threads because I can't sleep and it is the perfect time to start blogging when there is noone to distract me. I work three jobs including my blogging which I consider a full time job, and am a freelance journalist, fashion & lifestyle blogger with a passion for advocating social issues and a nanny and primary/secondary school tutor- specifically English. I take the kids to school in the morning and then attend blog events, write future blog posts, reply to emails, write articles for other companies and create meetings with PR/Brands/Photographers and edit photoshoots. During this time I also take time to manage my social media and plan shoot outfits and locations. After picking the kids up I am either working on my blog, working in general or attending blog events and meeting up with friends. I have a very strong work ethic and can sometimes become slightly obsessed because I live, breathe and am 'work'. I love to go out and have fun with my friends too and meet new bloggers. I have even met some of my fans and we have become good friends.
In five years I want to be able to publicly advocate for social minorities and campaign to make the lives of others better. I want to start creating my own blog events and travel the world meeting some of my fans along the way. I am not asking for fame but I do want prestige so that I can help those in need. I want my blog to become a safe haven for those who have been through some dark times and sprinkle some positivity into their lives. I have always been career minded from a young age and I am hoping that my blog will showcase journalistic prowess and work alongside my freelance journalism. I want to expand on my clientele and work for some magazines and sites that I admire. I am also thinking about turning my blog into an online magazine although I may open that up as a seperate venture which would be split into fashion, social issues and debates which is central to my ethos as a brand and blogger. I would love to launch my own clothing range and although I can't draw to save my life I have ideas that would work very well and open up a new sense of style to a mass audience.
Do you have any piece of advice for new bloggers or girls/boys hesitating about opening one?
Be sure that becoming a blogger is your 'everything', this is no 9-5 job and you will face some adversity and hardships but it is worth it if you know this is what you want to do. Some people choose to do blogging as a hobby which is completely fine but for those wanting to go full time I urge you to 'look past the freebies'. Do not accept opportunities that do not fit your brand simply because you get something for free or it is paid because at the end of the day your readers will know when you are being authentic and when you are not. Some people I know tried becoming bloggers because they thought it was the luxurious lifestyle portrayed in the media and its not. It takes time, blood sweat and tears to build up your blog and following but remember it does not matter how many followers you have. All that matters is that you be true to yourself and the readers will soon come pouring in. Use social media groups and interact with other bloggers because blogging can become alienating so remember to keep being social.
If you wish to read Ana's blog Faded Spring click here!
LOVE these outfits! So unique and colorful! I wish I had the confidence to pull off these types of outfits!
I know right?! Her style is one of the reasons why I wanted to feature her! She is so bold when it comes to dressing up!
Awesome choice for blogger of the month. I'm fairly new to Ana's blog and I've enjoyed everything I've seen so far.
Great interview! I love Ana's blog so it is nice to get to learn more about her!
Wonderful interview! So many beautiful photos too, the outfits are just gorgeous!
Your blogger of the month is fab and I actually read her posts! She has incredible personal style and is inspiring 🙂 Thanks for featuring her!
I love when bloggers feature other bloggers, give credit, and display a sense of the community promoting and encouraging everyone in the process! Great post !
Ive become a regular reader of Ana's blog because, well, its just really good. I loved the additional insight you gave into her world with this interview! Thanks for sharing!
I really love this blogger! I have followed her for a long time, she is all sorts of fabulous!
Thank you for all your kind comments it means a lot to me and thank you Ana for featuring me lovely. Love the layout. Hope people like my content as well as my pictures lol!
Wonderful blogger to feature! I've been reading her blog for a few months now. I love her writing style and her fashion sense – refreshingly unique!
I am fairly new to Ana's blog but she always has the most unique and beautiful backgrounds in all of her photography. Can't wait to see who you feature next month!
She does have a great style. We've been able to connect through some groups, and I hope that her story is changing some girls life!
Great interview! I always love to check out fashion bloggers – I'm always inspired by their outfits!