T cell immunodominance is dictated by the positively selecting self-peptide
An individual pathogen encodes thousands of potentially immunogenic epitopes , yet during the course of an infection , generally only a fraction of epitopes actually stimulate T cell response ( Sercarz et al . , 1993; Sant et al . , 2007; Weaver and Sant , 2009; Jenkins et al . , 2010 ) . This fundamental feature of T cell responses is known as immunodominance ( Sercarz et al . , 1993; Chen et al . , 2001; Yewdell and Del Val , 2004; Yewdell , 2006; Sant et al . , 2007; Weaver and Sant , 2009; Jenkins and Moon , 2012 ) . Most of the effort in immunodominance studies has focused on the antigen presentation and processing pathways ( Deng et al . , 1993; van Ham et al . , 1996; Nanda and Sant , 2000; Chen et al . , 2001; Crowe et al . , 2003; Chen and McCluskey , 2006; Lazarski et al . , 2006; Yewdell , 2006; Weaver and Sant , 2009 ) . Yet immunodominance is also defined by the frequencies of naive T cells specific for individual antigens ( Kotturi et al . , 2008; Jenkins and Moon , 2012; Kwok et al . , 2012 ) . Given that thymic positive selection serves as a major developmental process to generate mature T cell repertoires ( Jameson et al . , 1995; Morris and Allen , 2012 ) , one may reason positive selection would play a role in determining naive T cell precursor frequency to dictate immunodominance . However , this possibility has been difficult to address directly , due to several technological issues . First , the frequency of T cells specific for individual peptide–MHC ligands is at most about 20–100 cells per million naive T cells ( Jenkins and Moon , 2012 ) , making it difficult to identify such an infrequent population . Second , to directly test the potential role of positive selection in immunodominance requires the knowledge of what T cell population specific for a defined antigen a positively selecting ligand is capable of selecting . Therefore , the low frequency of naive T cell precursor and the ignorance of essential information have limited the examination of the roles of positive selecting ligands in dictating naive T cell precursor frequency and immunodominance . The murine CD4+ T cell response against cytochrome c is an outstanding model system to investigate the impact of positively selecting ligands on immunodominance ( Solinger et al . , 1979; Schwartz , 1985; Engel and Hedrick , 1988 ) . Only one single dominant epitope emerges from the immunization of H−2k mice with the whole protein of moth cytochrome c ( MCC ) or closely related pigeon cytochrome c ( PCC ) ( Hedrick et al . , 1982; Winoto et al . , 1986; Hedrick et al . , 1988; McHeyzer-Williams and Davis , 1995 ) . Also , the MCC- or PCC-stimulated CD4+ T cell response shows highly organized immunodominance hierarches . The MCC- or PCC-specific responses are highly dominated by Vα11+ TCR , and exhibit several conserved CDR3 features ( Winoto et al . , 1986; Hedrick et al . , 1988; McHeyzer-Williams and Davis , 1995; McHeyzer-Williams et al . , 1999; Mikszta et al . , 1999; Newell et al . , 2011 ) . During MCC-specific responses , the Vα11+Vβ3+ CD4+ T cells are the most dominant responders , while Vα11+ TCRs pairing with Vβ6+ , Vβ8+ , or Vβ14+ are the subdominant responders ( Miyazaki et al . , 1996; Malherbe et al . , 2004 ) . Based on the structural data , certain positions at CDR3α and CDR3β regions , where TCR make contact with MCC peptide , present highly conserved amino acid usages ( McHeyzer-Williams et al . , 1999; Newell et al . , 2011 ) . These features constitute the strength of utilizing cytochrome c as a model antigen to study CD4+ immunodominance . Moreover , the MCC/I-Ek tetramers have been shown to be able to detect most primary MCC-specific T cells ( Savage et al . , 1999 ) . Importantly , our laboratory had previously identified a naturally occurring positively selecting self-peptide , termed gp250 , for its ability to positively select the MCC-specific TCR: AND ( Lo et al . , 2009 ) . In this study , we have generated a transgenic mouse line , the gp250 single chain ( SC ) mouse , in which the gp250/I-Ek was the only MHC class II ligand presented . Combining MCC tetramer analysis and our gp250 SC mice permitted us to elucidate the relationship between positively selecting ligands and antigen specificities of post-selection CD4+ T cell repertoires . Several studies have attempted to investigate the antigen specificities of the post-selection T cell repertoire by limiting the diversity of positively selecting self-peptides ( Kouskoff et al . , 1993; Ignatowicz et al . , 1996; Miyazaki et al . , 1996; Fukui et al . , 1997; Grubin et al . , 1997; Ignatowicz et al . , 1997; Nakano et al . , 1997; Surh et al . , 1997; Tourne et al . , 1997; Gapin et al . , 1998; Barton and Rudensky , 1999; Chmielowski et al . , 2000; Barton et al . , 2002; Huseby et al . , 2005 ) . Studies that limit the diversity of positively selecting self-peptides to a single peptide have involved the introduction of a transgene that encoded a defined peptide covalently linked to MHC class II ( Ignatowicz et al . , 1996 , 1997; Liu et al . , 1997; Huseby et al . , 2005 ) , disruption of the peptide exchange molecules H-2M ( Miyazaki et al . , 1996; Grubin et al . , 1997; Surh et al . , 1997; Tourne et al . , 1997 ) , expression of a human invariant chain transgene in which CLIP peptide was replaced with other self-peptides ( Barton and Rudensky , 1999; Barton et al . , 2002 ) , or viral expression of altered peptide ligands in the thymus ( Kouskoff et al . , 1993; Nakano et al . , 1997 ) . Altogether these studies concluded that a single peptide could select a large repertoire of T cells and that the recognition of positively selecting ligands is the driving force behind determining the antigen specificities of post-selection T cell repertoire ( Ignatowicz et al . , 1996; Fukui et al . , 1997; Grubin et al . , 1997; Ignatowicz et al . , 1997; Surh et al . , 1997; Fukui et al . , 1998; Gapin et al . , 1998; Barton and Rudensky , 1999; Chmielowski et al . , 2000; Barton et al . , 2002; Huseby et al . , 2005 ) . However , these studies were unable to examine immunodominance because they did not utilize a naturally occurring positively selecting ligand for a defined foreign antigen . The selection of Vα11+Vβ3+ TCRs was greatly enhanced in our gp250 SC mice . CDR3 sequencing revealed that gp250 skewed the positive selection toward MCC-reactive conserved CDR3 features , especially those CDR3s with serine at α91 and asparagine at β97 . Our hypothesis that gp250 favors the positive selection of MCC-reactive T cells was further supported by the greatly expanded MCC-tetramer+ population in gp250 SC mice . Our data provide direct evidence that positive selection plays a central role in determining the post-selection T cell repertoire and is a major determinant in immunodominance .
To explore the relationship between positive selection and CD4+ T cell immunodominance , we utilized the MCC model antigen system . The MCC88–103 peptide ( referred to MCC hereafter ) is an immunodominant peptide following immunization with the MCC protein , and the MCC primary responders and memory cells are exclusively dominated by Vα11+ T cells ( Winoto et al . , 1986; Hedrick et al . , 1988; McHeyzer-Williams and Davis , 1995; McHeyzer-Williams et al . , 1999; Mikszta et al . , 1999; Malherbe et al . , 2004; Newell et al . , 2011 ) . The basis for the Vα11+ dominant response has not been established . The AND TCR is one of the dominant MCC-responsive TCRs and consists of Vα11 and Vβ3 gene segments ( Hedrick et al . , 1988; Kaye et al . , 1989; McHeyzer-Williams and Davis , 1995; McHeyzer-Williams et al . , 1999; Mikszta et al . , 1999 ) . Essential for these studies was our previous identification of the naturally occurring positively selecting ligand , gp250 , for AND T cells ( Lo et al . , 2009 ) . To examine gp250’s effect on the post-selection MCC-specific T cell repertoire , we constructed a transgenic mouse line using the MHC class II promoter in which all of the MHC class II molecules contained the gp250 self-peptide via a covalent linkage of the peptide to the MHC class II β chain . We obtained one B6 founder in which the gp250 covalently linked I-Ek β chain had successfully integrated and was expressed . The I-Ek α chain was introduced into the founder by crossing to previously generated I-Ekα transgenic mice ( Griffiths et al . , 1994 ) . The mice were subsequently bred onto B6 MHC class II−/− to eliminate other class II molecules and CD74−/− background to assure the gp250/I-Ek is the only MHC class II ligand ( Ignatowicz et al . , 1996 , 1997 ) . The following experiments were all executed with the transgenic mice on the B6 MHC class II−/− and CD74−/− background ( termed gp250 SC mice hereafter ) . In gp250 SC mice , the transgenic covalently linked gp250/I-Ekβ successfully paired with transgenic I-Ekα , restoring the surface expression of I-Ek in splenic B cells and CD11c+ dendritic cells ( Figure 1A–D ) . The level of expression of the gp250/I-Ek was readily detectable , but reconstituted to approximately a 10% level on B cells and 40% on dendritic cells ( Figure 1A–D ) . Immunofluorescence staining of thymic frozen sections of gp250 SC mice showed surface expression of I-Ek in the cortex and medulla of the thymus ( data not shown ) . This level of expression was advantageous for our study in that we had detectable expression , but not supra-physiological levels of a single peptide-MHC and provided us with a suitable window to examine the positive selection mediated by gp250 self-peptide . 10 . 7554/eLife . 01457 . 003Figure 1 . The I-Ekα and covalently-linked gp250/I-Ekβ transgenes restore surface expression of I-Ek in gp250 SC mice class II-deficient mice . The surface expression of I-Ek on B cells ( A and B ) and dendritic cells ( C and D ) from the spleens of gp250 SC mice . The B cells and dendritic cells from MHCII−/−CD74−/− mice were used as negative controls . The plots are representative of three independent experiments ( A and C ) and summarized in the bar graph ( B and D; mean ± SD; n = 3; two-tailed Mann–Whitney test ) . DOI: http://dx . doi . org/10 . 7554/eLife . 01457 . 003 The covalently linked gp250/I-Ek positively selected a large number of CD4+ T cells in the thymus with an average of 1 . 6 × 108 total thymocytes and 6 . 8 × 107 CD4SP T cells ( Figure 2A–C ) . The positive selection efficiency for CD4SP T cells in the gp250 SC mouse was approximately 40% of that seen in the B6 . K wild type ( Figure 2B , C ) , in line with other published single chain mice . The size of CD8SP T cells in gp250 SC mouse was comparable to that in B6 . K ( Figure 2D ) , as anticipated given that the gp250 SC mice expressed normal class I molecules . The higher percentage of CD8SP T cells is a reflection of the less efficient CD4SP selection ( Figure 2B–D ) . 10 . 7554/eLife . 01457 . 004Figure 2 . The single gp250/I-Ek ligand positively selects a significant population of CD4+ T cells in gp250 SC mice . ( A ) Total number of thymocytes in B6 . K or gp250 SC mice . ( B ) Plot of thymocytes from B6 . K or gp250 SC mice . ( C and D ) Quantification by percentage of CD4SP ( C ) or CD8SP ( D ) thymocytes , and of total number of CD4SP ( C ) or CD8SP ( D ) in B6 . K or gp250 SC mice are shown in the bar graph . ( E ) Total number of splenocytes in B6 . K or gp250 SC mice . ( F ) Plot of splenocytes from B6 . K or gp250 SC mice . ( G and H ) Quantification by percentage of CD4+ ( G ) or CD8+ ( H ) T cells and of total CD4+ ( G ) or CD8+ ( H ) T cell numbers in the spleens of B6 . K or gp250 SC mice are shown in the bar graph . ( A–H ) The data are representative of three experiments ( B6 . K , n = 7; gp250 SC , n = 6; mean ± SD; two-tailed Mann-Whitney test ) . DOI: http://dx . doi . org/10 . 7554/eLife . 01457 . 00410 . 7554/eLife . 01457 . 005Figure 2—figure supplement 1 . TCR Vβ usages of peripheral T cells in gp250 SC mice . ( A and B ) Vβs expression of peripheral CD4+ T cells in B6 . K or gp250 SC mice . The frequencies of three Vβs were decreased ( A ) and three were increased ( B ) . n = 9; data are representative of three experiments . n = 4; mean ± SD; two-tailed Mann–Whitney test . ( C and D ) B6 . K HSC cells were used to reconstitute lethally irradiated gp250 SC or B6 . K mice . After a 12-week reconstitution , Vβx expression repertoire in CD4+ or CD8+ T cells were examined ( D ) , and the frequencies of Vβs that were increased in gp250 SC mice ( B ) were shown in ( C ) . n = 5; the data are representative of three experiments . n = 4; mean ± SD; two-tailed Mann–Whitney test . DOI: http://dx . doi . org/10 . 7554/eLife . 01457 . 005 In the spleen , the peripheral CD4+ T cell population in gp250 SC mice was readily detectable , being around 40% of the B6 . K , yet still averaged 1 . 7 × 106 CD4+ T cells per spleen ( Figure 2E–G ) . The single gp250/I-Ek ligand selected a full TCR Vβ repertoire , in that we did not observe any complete absence of Vβs ( Figure 2—figure supplement 1 ) . However , the frequencies of some TCR Vβs were decreased , including Vβ2+ , Vβ4+ , Vβ8 . 1/8 . 2+ , suggesting gp250/I-Ek did not positively select individual Vβs with a uniform selection capability ( Figure 2—figure supplement 1A ) . The usages of Vβ5+ , Vβ11+ , and Vβ12+ were also increased in peripheral CD4+ T cells in gp250 SC mice ( Figure 2—figure supplement 1B ) . Given gp250/I-Ek was the only ligand presented by MHC class II molecules in gp250 SC mice , it was possible that the increased Vβs were caused by the lack of negative selection . We therefore used B6 . K hematopoietic stem cells ( HSCs ) to reconstitute lethally irradiated gp250 SC mice ( B6 . K > gp250 SC; Figure 2—figure supplement 1C ) . In B6 . K > gp250 chimeras , radiation-resistant epithelial cells presented gp250 as the only ligand to promote positive selection , while B6 . K bone marrow-derived antigen presenting cells presented a full peptide repertoire to restore negative selection . After a 12-week reconstitution , in B6 . K > gp250 chimeras , the frequencies of Vβ5+ , Vβ11+ and Vβ12+ were indistinguishable when compared with B6 . K > B6 . K chimeras ( Figure 2—figure supplement 1C , D ) . The CD8+ T cell population in gp250 SC mice was similar to the size in B6 . K mice ( Figure 2H ) , and the Vβ usages of CD8+ T cells in B6 . K > gp250 chimeras were comparable to the frequencies in B6 . K > B6 . K mice ( Figure 2—figure supplement 1D ) . The data reflected MHC class I-mediated positive selection was relatively intact , given no alternation was made on MHC class I-restricted peptide presentation . Together , the data show that the single gp250/I-Ek ligand was capable of positively selecting a large population of CD4+ T cells , which then could be further analyzed in detail . We used MCC-tetramers to identify MCC-specific population in CD4SP thymocytes and peripheral CD4+ T cells . The gp250-mediated positive selection dramatically expanded the MCC-specific CD4+ T cell response ( Figure 3 ) . In gp250 SC mice , the MCC tetramer+ CD4+ population showed a threefold increase in the periphery and a twofold increase in the thymus , compared with the population in B6 . K mice ( Figure 3A , B ) . Considering that the size of the CD4+ T cell population in gp250 SC mice was around 40% of the CD4+ T cell population in B6 . K mice ( Figure 2 ) , the augmented MCC-tetramer+ frequency was even more impressive . While T cell populations specific for other foreign antigens very likely became smaller , T cells specific for MCC increased in percentage , or at least stayed comparable to T cells selected by diverse self-peptide repertoire in terms of the absolute number . Indeed , the MCC tetramer+ population was uniquely expanded in the gp250-selected CD4+ T cell repertoire , as the frequency of the hemoglobin antigen ( Hb ) tetramer-bound CD4+ T cell population was equivalent , if not slightly decreased ( Figure 3C , D ) . To show that the increased MCC tetramer+ population was the product of gp250-mediated positive selection not due to the lack of negative selection , we generated radiation bone marrow chimeras ( Figure 3E , F ) . We used B6 . K hematopoietic stem cells ( HSCs ) to reconstitute lethally irradiated gp250 SC mice ( B6 . K > gp250 SC ) . The B6 . K HSC-reconstituted B6 . K bone marrow chimeras served as the controls ( B6 . K > B6 . K ) . After a 12-week reconstitution , the frequency of MCC tetramer+ CD4+ T cells was 1 . 5% in B6 . K HSC-reconstituted gp250 SC mice ( Figure 3E , F ) . Compared with the frequency of MCC tetramer+ population in B6 . K HSC-reconstituted B6 . K mice , which was about 0 . 18% , gp250-mediated positive selection enriched MCC tetramer+ cells more than eightfold ( Figure 3E , F ) . The B6 . K HSC-reconstituted gp250 chimeras suggested the augmented MCC tetramer+ population in gp250 SC mice was not due to the lack of negative selection . Taken together , our data showed that gp250-mediated positive selection enriched for MCC-specific CD4+ T cells . 10 . 7554/eLife . 01457 . 006Figure 3 . gp250-mediated positive selection greatly expands MCC-tetramer+ CD4+ T cells . ( A and B ) The frequency of MCC-tetramer+ CD4SP thymocytes or peripheral CD4+ T cells in B6 . K or gp250 SC mice . The plot was gated on the live CD4+CD3+CD8− population . The data are representative of at least five experiments . Bar graph shows the summary ( B; thymus , n = 12; spleen , n = 17; mean ± SD; two-tailed Mann–Whitney test ) . ( C and D ) The frequency of Hb-tetramer+ CD4SP thymocytes or peripheral CD4+ T cells in B6 . K or gp250 SC mice . The plot was gated on live CD4+CD3+CD8− population . The data are representative of at least three experiments . ( D ) : n = 3; mean ± SD; two-tailed Mann–Whitney test . ( E and F ) The frequency of MCC-tetramer+ peripheral CD4+ T cells in B6 . K or gp250 SC bone marrow chimeras reconstituted with B6 . K bone marrows . ( F ) : n = 4; mean ± SD; two-tailed Mann–Whitney test . DOI: http://dx . doi . org/10 . 7554/eLife . 01457 . 006 The polyclonal MCC-specific T cell memory responses are strongly dominated by Vα11+ CD4+ T cells; however , the basis of this is not known ( Winoto et al . , 1986; Engel and Hedrick , 1988; McHeyzer-Williams and Davis , 1995; McHeyzer-Williams et al . , 1999; Mikszta et al . , 1999; Malherbe et al . , 2004; Newell et al . , 2011 ) . We hypothesized that recognition of a positively selecting ligand may influence a specific antigen response through optimizing the selection of dominant TCRs . We therefore included the TCR Vα11 staining in MCC-tetramer experiments to examine the Vα11+ TCR expression in gp250-selected MCC-specific CD4+ T cells ( Figure 4A , B ) . In B6 . K CD4+ T cells , the MCC tetramer+ populations that used Vα11− vs Vα11+ were about equal , whereas in gp250 SC mice , the vast majority of MCC tetramer+ population expressed Vα11+ ( Figure 4A , B ) . It was the increased Vα11+ MCC tetramer+ cells that specifically contributed to the enlarged MCC tetramer+ population in gp250 SC mice , since the frequency of Vα11− MCC tetramer+ population in gp250 SC mice was comparable with the frequency in B6 . K mice ( Figure 4B ) . The exclusively enlarged Vα11+ MCC tetramer+ population in gp250 SC mice suggested that gp250/I-Ek specifically favored the positive selection of Vα11+ TCR , thus resulting in a T cell repertoire profile with the enhanced MCC tetramer-bound population . The MCC tetramer+ T cells were responsive to MCC , as the sorted MCC tetramer+ CD4+ T cells upregulated CD69 while stimulated overnight with MCC-pulsed APC in vitro ( Figure 4C , D ) . 10 . 7554/eLife . 01457 . 007Figure 4 . The majority of gp250-selected MCC-specific CD4+ T cells express Vα11+ TCR . ( A and B ) The Vα11 expression of MCC-tetramer+ peripheral CD4+ T cells in B6 . K or gp250 SC mice . ( B ) n = 6; mean ± SD; two-tailed Mann–Whitney test . ( C and D ) The CD69 upregulation of sorted MCC-tetramer− or MCC-tetramer+ CD4+ T cells from B6 . K or gp250 SC splenocytes . Data are representative of three experiments . ( D ) : n = 3 , paired t tests . DOI: http://dx . doi . org/10 . 7554/eLife . 01457 . 007 The TCR Vβ usages in Vα11+-driven MCC-specific response exhibit a highly organized immunodominance hierarchy . On day 7 post immunization , about 50% of MCC-reactive T cells were Vα11+Vβ3+ , and Vα11+Vβ6+ , Vα11+Vβ8+ , Vα11+Vβ14+ constituted the remaining 50% MCC-reactive T cells . Observing gp250-mediated selection promoted the MCC-specific CD4+ T cells toward the usage of Vα11+ , we next examined the selection of MCC-preferred dominant and subdominant TCR pairs in gp250 SC mice ( Figure 5 ) . The Vα11+-expressing CD4+ T cells significantly increased in the thymus and periphery in gp250 SC mice ( Figure 5A , B ) , suggesting gp250/I-Ek actively skewed the post-selection TCR usages to influence antigen specificities . While examining the TCR Vβ expression among gp250-selected Vα11+ CD4+ T cells , MCC-preferred dominant pair Vα11+Vβ3+ increased about 1 . 4-fold in thymic CD4SP and at least twofold in periphery ( Figure 5C–E ) . Certain subdominant MCC-preferred TCRs also increased ( Figure 5C–E ) . Interestingly , for some Vβs , found commonly to pair with Vα11 in normal mice ( such as Vβ2 and Vβ4 ) , the frequencies were unchanged ( Figure 5C ) . This observation argued that gp250 specifically promoted the selection of MCC-specific TCR usages , and was not merely functioning as a good selecting ligand for all Vα11+ . The increased frequency of Vα11+Vβ3+ did not result from the absence of negative selection in gp250 SC mice ( Figure 5F ) . While reconstituted lethally irradiated gp250 SC mice with B6 . K HSC cells , the negative selection in gp250 SC mice was restored due to the presence of B6 . K HSC-derived antigen presenting cells in the thymus . After a 12-week reconstitution , Vα11+ CD4+ peripheral T cells in B6 . K > gp250 SC bone marrow chimeras were about 2 . 5-fold larger than the size in B6 . K > B6 . K chimeras ( Figure 5F ) . The Vα11+Vβ3+ population increased to approximately threefold in B6 . K > gp250 SC chimeras , compared with the population in B6 . K > B6 . K chimera mice as well ( Figure 5F ) . The bone marrow reconstitution data clearly supported gp250-mediated positive selection was the driving force to cause the enlarged MCC-specific CD4+ population . 10 . 7554/eLife . 01457 . 008Figure 5 . The gp250 self-peptide skews the selection of MCC-reactive preferred TCR pairs . ( A ) The Vα11 and Vβ3 expression of thymic CD4SP or peripheral CD4+ T cells in B6 . K or gp250 SC mice . The data are representative of three experiments . ( B ) The quantification by percentage of Vα11+ CD4SP thymocytes or CD4+ peripheral T cells in B6 . K or gp250 SC mice . n = 7; mean ± SD; two-tailed Mann–Whitney test . ( C ) The distribution of Vβ usages among Vα11+ CD4SP thymocytes or CD4+ peripheral T cells in B6 . K or gp250 SC mice . The pie chart was plotted with the mean of three experiments ( n = 7 ) . ( D and E ) The quantification of increased dominant and subdominant MCC-reactive TCR pairs of CD4SP thymocytes or CD4+ peripheral T cells in B6 . K or gp250 SC mice . n = 7; mean ± SD; two-tailed Mann–Whitney test . ( F ) The Vα11 and Vβ3 expression of thymic CD4SP or peripheral CD4+ T cells in B6 . K or gp250 SC bone marrow chimeras reconstituted with B6 . K HSC cells . The data are representative of three experiments . n = 4; mean ± SD; two-tailed Mann–Whitney test . DOI: http://dx . doi . org/10 . 7554/eLife . 01457 . 008 In addition to the preferred TCR Vα and Vβ usages , MCC-responsive CD4+ T cells also showed strikingly conserved CDR3 features , including the preferred four CDR3 residues: glutamic acid at CDR3 α89 ( α89E ) , serine at α91 ( α91S ) , asparagine at CDR3 β97 ( β97N ) , and alanine/glycine at β99 ( β99A/G ) ( McHeyzer-Williams et al . , 1999; Newell et al . , 2011 ) . The crystal structures of TCR:pMHC complexes have provided the structural explanation for these conserved CDR3 residues for each may form salt bridge or hydrogen bond with MCC TCR contact residues at P3 ( tyrosine ) , P5 ( lysine ) , and P8 ( threonine ) ( Newell et al . , 2011 ) . To elucidate the amino acid residues at these specific CDR3 positions , we performed single cell repertoire analysis and large-scale bulk CDR3 sequence studies ( Figure 6 ) . 10 . 7554/eLife . 01457 . 009Figure 6 . The gp250 self-peptide skews the selection of TCRs exhibiting conserved MCC-reactive features . ( A ) The pie chart of amino acid usages at CDR3 α89 and α91 of bulk-sorted peripheral Vα11+Vβ3+ CD4+ T cells in B6 . K or gp250 SC mice . The gp250 SC data were obtained by single cell repertoire analysis . ( B ) The frequency of MCC-reactive preferred CDR3α amino acids , α89N and α91E from gp250-selected peripheral Vα11+Vβ3+ CD4+ T cells in single cell analysis ( left ) or bulk population analysis ( right ) . ( C ) The pie chart of amino acid usages at CDR3 β97 and β99 from bulk-sorted peripheral Vα11+Vβ3+ CD4+ T cells in B6 . K or gp250 SC mice . The data were representative of two independent experiments . ( D ) The frequencies of CDR3 β97N , β99A , and β99E of Vα11+Vβ3+ CD4+ T cells in B6 . K or gp250 SC mice . ( B and D ) The single cell B10 . A data were plotted based on McHeyzer-Williams et al . ( 1999 ) published data . DOI: http://dx . doi . org/10 . 7554/eLife . 01457 . 009 We single cell sorted Vα11+Vβ3+ peripheral CD4+ T cells from a gp250 SC mouse for single cell TCR analysis . We successfully recovered 38 sequences of TCR Vα11 ( Table 1 ) , among which five also had the co-expressed Vβ3 sequences identified ( Table 2 ) . The Vα11+Vβ3+ populations were also sent for large-scale bulk CDR3α and CDR3β sequencing to extensively explore CDR3 features . We obtained about 2900 unique CDR3α and 1500 CDR3β sequences in gp250-selected Vα11+Vβ3+ CD4+ T cells ( Table 3 ) . We found AND TCR and its closely related TCR: 5cc7 and 226 TCRs ( Tables 1 and 3 ) . In bulk population sequence analysis , the majority of published MCC-reactive TCR CDR3α sequences were identified in our dataset , and we found six of the published CDR3β sequences ( Table 3 ) ( Hedrick et al . , 1988; McHeyzer-Williams et al . , 1999 ) . 10 . 7554/eLife . 01457 . 010Table 1 . Examples of CDR3α sequences of Vα11+Vβ3+ CD4+ T cells from gp250 SC mice in the single cell repertoire analysisDOI: http://dx . doi . org/10 . 7554/eLife . 01457 . 010CDR3α sequencesJα usagesFrequencyNotesCAAEASSGSWQLIFTCRA1721% ( 8/38 ) Also published in McHeyzer-Williams et al . ( 1999 ) CAAEANSGTYQRFTCRA117 . 8% ( 3/38 ) CAAEAGGGSGGKLTLTCRA365 . 2% ( 2/38 ) CAAEASSGQKLVFTCRA135 . 2% ( 2/38 ) AND TCR CDR3α; also published in McHeyzer-Williams et al . ( 1999 ) CAAEPPHANTGANTGKLTFTCRA445 . 2% ( 2/38 ) CAANTGNYKYVFTCRA335 . 2% ( 2/38 ) CAADRSNNRIFFTCRA245 . 2% ( 2/38 ) CAAEPSSFSKLVFTCRA242 . 6% ( 1/38 ) CAAESSNMGYKLTFTCRA82 . 6% ( 1/38 ) CAARSSNTNKVVFTCRA82 . 6% ( 1/38 ) 10 . 7554/eLife . 01457 . 011Table 2 . The single cell repertoire analysis of Vα11+Vβ3+ CD4+ T cells from gp250 SC miceDOI: http://dx . doi . org/10 . 7554/eLife . 01457 . 011CDR3α sequencesJα usagesCDR3β sequencesJβ usagesCAADRSNNRIFFTCRA17CASSKQANSYNSPLYFJβ2 . 4CAAESSNMGYKLTFTCRA24CASSGDGRGNTLYFJβ1 . 8CAARSSNTNKVVFTCRA27CASSLWANTGQLYFJβ2 . 2CAAEANSGTYQRFTCRA11CASSLLHKQYFJβ2 . 1CAAEASSGSWQLIFTCRA17CASSPGTQNTLYFJβ2 . 410 . 7554/eLife . 01457 . 012Table 3 . The CDR3 sequences of bulk-sorted Vα11+Vβ3+ CD4+ T cells from gp250 SC mice that overlap with the published data from normal mice ( Hedrick et al . , 1988; McHeyzer-Williams et al . , 1999 ) DOI: http://dx . doi . org/10 . 7554/eLife . 01457 . 012Published CDR3α ( Hedrick et al . , 1988; McHeyzer-Williams et al . , 1999 ) Current studyNotesB6 . KSC36232906Unique sequencesCAAEASSGQKLVFG++AND TCRCAAEPSSGQKLVFG++226 TCRCAAEASGSWQLIFG++CAAEASNTNKVVFG++5c . c7 TCRCAAEGSNTNKVVFG+CAAEASAGNKLTFG++CAAASSGSWQLIFG++CAAEAGSNAKLTFG++CAAEASNNNAPRFG+CAAEAASLGKLQFG++CAAEASSGSWQLIFG++CAAEASNYNVLYFG++CAAEASSSFSKLVFG++CAAEASNMGYKLTFG++CAAETGGYKVVFG++CAAEANYNQGKLIFG++CAAEAGSGTYQRFG++CAGLSGSFNKLTFG++CAAEGNTGNYKYVFG+CAAEEGNMGYKLTFG++CAATSSGQKLVFG+Published CDR3β ( Hedrick et al . , 1988; McHeyzer-Williams et al . , 1999 ) Current studyNotesB6 . KSC28211583Unique sequences ( mean ) CASSLNSANSDY+AND , 5c . c7 , 226 TCRsCASSLNNANSDY++CASSLSTSQNTLYF+CASSLQGTNTEVFF++CASRLGQNTLYF+CASSLGASAETLYF+ Analysis of CDR3α residues in the bulk populations revealed that gp250-mediated positive selection significantly enriched the usage of serine at CDR3 α91 in Vα11+Vβ3+ CD4+ T cells ( Figure 6A , B ) , which hydrogen bonds to the MCC peptide tyrosine residue at P328 . In B6 . K mice , about 20% of Vα11+Vβ3+ CD4+ T cells expressed serine at CDR3 α91 , whereas in gp250 SC mice , the frequency was increased 1 . 5-fold to become about 30% ( Figure 6A , B ) . We observed a similar 1 . 5-fold increase of serine at α91 in single cell repertoire analysis compared to the published data ( McHeyzer-Williams et al . , 1999 ) ( Figure 6B ) . However , gp250-mediated selection had little impact on skewing the amino acid residue at CDR3 α89 , as α89E usages of Vα11+Vβ3+ CD4+ T cells were comparable with the frequency seen in B6 . K ( Figure 6A , B ) . The large-scale bulk CDR3β sequencing showed gp250-mediated positive selection dramatically escalated the usages of β97N , slightly increased the frequency of β99A , and had no effect on the frequency of β99G ( Figure 6C , D ) . Nevertheless , B6 . K CDR3β features were highly similar to the published CDR3β’s ( McHeyzer-Williams et al . , 1999; Figure 6C , D ) . The CDR3 β97N residue was hypothesized to hydrogen bond with MCC P8 backbone carbonyl and MCC P8 threonine side-chain hydroxyl groups ( Newell et al . , 2011 ) . The significantly increased usages of CDR3 α91S and β97N in gp250-selected Vα11+Vβ3+ T cells may also help explain the augmented MCC-tetramer bound population in gp250 SC mice . Altogether , our data changed our current understanding of the relationship between positively selecting self-peptides and post-selection T cell repertoire ( Figure 7 ) . With the well-defined MCC’s highly organized immunodominance hierarchy , we discovered gp250’s active role in promoting the positive selection of good MCC responders . The self-peptide gp250 enhanced the selection of Vα11+Vβ3+ T cells , which were the dominant TCR pair against MCC . These gp250-selected Vα11+Vβ3+ T cells also exhibited conserved MCC-reactive features , including the specific amino acid usages at CDR3 α91 and β97 . Our studies showed an ideal positively selecting self-peptide may be a key determinant to optimize immune response against a specific antigen , by enhancing the positive selection of good responders . 10 . 7554/eLife . 01457 . 013Figure 7 . The evolving concepts of the relationship between positively selecting self-peptide and post-selection T cell repertoire . The figures show the progress of our understanding about the relationship between thymic self-peptides and post-selection CD4+ T cell repertoire , and are arranged in chorological order . The first finding from late 90′ revealed that one positively selecting self-peptide was capable of selecting many different TCRs ( Ignatowicz et al . , 1996; Ignatowicz et al . , 1997; Liu et al . , 1997; Huseby et al . , 2005 ) . A few years later , Barton and Rudensky proposed that even though one self-peptide may select many TCRs , a diverse low abundant self-peptide repertoire is required to generate a full TCR repertoire ( Barton and Rudensky , 1999 ) . Subsequently , with the publication of a series of single chain mice , the model was further refined because even though the TCR repertoire selected by one single ligand was not reduced significantly enough to allow an visualization of a repertoire ‘hole’ , the postselection T cells in each single chain mouse showed different degrees of self-peptide reactivity in mixed lymphocyte culture ( Barton et al . , 2002 ) . These studies showed that a diverse collection of positively selecting self-peptides were necessary to generate a full T cell repertoire ( Barton and Rudensky , 1999; Barton et al . , 2002 ) . This present study involving the gp250 self-peptide showed increased frequency of Vα11+Vβ3+ CD4+ T cells with MCC-preferred CDR3 features in gp250 SC mice . The Vα11+Vβ3+ CD4+ T cells with specific CDR3 features were the dominant responders to promote MCC primary and memory responses . Therefore , gp250’s selecting capability provided a possible explanation to elucidate Vα11+ TCR-driven MCC immunodominance: a positively selecting self-peptide may favor the selection of TCR pairs and CDR3 features that were specific for MCC responses . DOI: http://dx . doi . org/10 . 7554/eLife . 01457 . 013
Immunodominance describes a common phenomenon of T cell immune responses . Yet the role of positive selection in immunodominance has not been established . In this study , we reported that CD4+ T cells specific for a defined antigen are positively selected by a common self-peptide . The self-peptide gp250 positively selected AND TCR , supporting our previous in vitro study ( Lo and Allen , 2013 ) , and also selected many other TCRs that have been reported as good MCC responders ( Hedrick et al . , 1988; McHeyzer-Williams et al . , 1999 ) . Our data reveal for the first time that positive selection may influence the TCR V ( D ) J preference and specific CDR3 junction sequences , and thus shape T cell response to a specific antigen . Our studies therefore offer direct evidence and a new perspective on how to generate a post-selection CD4+ T cell repertoire with desired antigen specificities , through engineering the positively selecting self-peptide . The gp250-selected CD4+ T cell repertoire analysis uncovered several very interesting results regarding the specificity of recognizing a positively selecting self-peptide , as well as the relationship between a positively selecting self-peptide and post-selection T cells . In the bulk population TCR repertoire analysis , the CDR3α was found for the well-characterized AND , 5c . c7 and 226 TCRs and also for the majority of published MCC-preferred clones . These findings suggested that one selecting ligand can select as many CDR3s with conserved features for specific antigen recognition as a full self-peptide repertoire could select . Previously , the Davis laboratory and we have shown in vitro that the self-peptide for AND and 5c . c7 TCRs were mutually exclusive with each other ( Ebert et al . , 2009; Lo and Allen , 2013 ) . The basis for why 5c . c7 was selected in vivo but not in vitro by gp250 probably reflects the relative inefficiency of the in vitro positively selecting assays . Our data suggest these strong MCC-reactive TCR clones may share a common positively selecting ligand , and support our hypothesis that positive selection may be the foundation of T cell immunodominance . Our finding that gp250 was sufficient to generate a reduced but still a large number of mature CD4+ T cells , with a full but skewed range of TCR usages , was similar to the observations in other single peptide mice , such as Eα- , CD22- , or Rab-mediated positive selection ( Ignatowicz et al . , 1996 , 1997; Barton and Rudensky , 1999; Barton et al . , 2002 ) ( Figure 7 ) . However , in those previously published studies , the specificity of post-selection repertoire was only examined at a cursorily level by stimulating the CD4+ T cells with random known antigens . The gp250 SC mice , however , successfully provided some key insights toward the relationship between positively selecting self-peptide and post-selection CD4+ T cells . Thanks to the well-studied MCC polyclonal T cell responses , we were thus able to discover some unique features of positive selection ( McHeyzer-Williams and Davis , 1995; McHeyzer-Williams et al . , 1999; Mikszta et al . , 1999; Malherbe et al . , 2004 ) . We found positively selecting self-peptide may favor some unique CDR3 features , which may serve as a good primary and memory responder toward a specific antigen in the periphery . Consistent with previous studies , the gp250 peptide was able to select a large number of CD4+ T cells . This observation makes sense when one considers that the number of selecting self-peptides presented in cortical thymic epithelial cells is smaller than the size of mature CD4+ T cell repertoire ( Lo and Allen , 2013 ) . Therefore , one positively selecting self-peptide has to select a large number of CD4+ T cell with diverse , unrelated antigen specificities , to be able to generate a full CD4+ T cell repertoire . The thymic development of regulatory T cells is also dependent upon self-peptides , and requires the strong interaction with these self-peptide MHC ligands ( Gottschalk et al . , 2010; Ohkura and Sakaguchi , 2010; Corse et al . , 2011; Ohkura et al . , 2013 ) . However , our preliminary analysis of CD25 expression of CD4SP in the gp250 SC mice did not reveal any significant differences from B6 . K mice . The comparable regulatory T cell frequencies in gp250 SC mice and B6 . K mice suggested that merely altering the self-peptide diversity was not sufficient to manipulate the affinity threshold for regulatory T cell selection . The finding was perhaps not surprising , given the potential TCR diversity through CDR3 recombination mechanism is nearly infinite , and both the TCR and the self-peptide determine the strength of interaction . Our data showed gp250 could select many different Vα11 TCRs . How could a peptide influence the selection of many different Vα chains , with a range of CDR3α sequences ? The general binding mode of TCR to pMHC has the CDR1 and CDR2 contacting the MHC , and the CDR3 contacting the peptide . One explanation comes from the crystal structure of the 226 TCR bound to MCC/I-Ek28 . The side chain of the Arg29 residue of the Vα11 CDR1 makes a hydrogen bond with the backbone carbonyl oxygen of the P3 residue ( Newell et al . , 2011 ) . Thus gp250 could position the P3 carbonyl oxygen ideally to form this hydrogen bond , and thus favoring Vα11 TCRs . Further structural studies will be needed to reveal the molecular basis of how a single peptide can select defined TCRs .
B6 . K ( H-2k ) ( JAX 001148 ) and B6 MHC class II−/− ( JAX 003584 ) mice were obtained from the Jackson Laboratory . The B6 CD75−/− mouse line was a gift from the Kappler/Marrack laboratory ( Bikoff et al . , 1995 ) . All mice were bred and housed in specific pathogen-free conditions of the animal facility at the Washington University Medical Center . All the use of laboratory animals was approved and done in accordance with the Washington University Division of Comparative Medicine guidelines . Fluorescence-conjugated antibodies from commercial sources were as follows: fluorescein isothiocyanate- , allophycocyanin- or allophycocyanin–indodicarbocyanine-conjugated anti-CD4 ( GK1 . 5; BioLegend , San Diego , CA ) ; phycoerythrin–indodicarbocyanine-conjugated anti-CD8 ( 53-6 . 7; BioLegend ) ; fluorescein isothiocyanate- , or phycoerythrin-conjugated I-Ek ( 17-3-3; BioLegend ) ; allophycocyanin- or phycoerythrin–cy7-conjugated anti-CD69 ( 1-11 . 2F3; BioLegend ) ; LIVE/DEAD Fixable Blue Dead Cell Stain ( Invitrogen , Carlsbad , CA ) ; or fluorescein isothiocyanate-conjugated or biotinylated anti-Vα11 ( RR8-1; BD Pharmingen , Franklin Lakes , NJ ) . Antibodies against the panel of TCR Vβ chains were purchased from BD Pharmingen ( 557004 ) . All samples were analyzed on a FACSCalibur , FACSLSRII , or FACSAria ( BD ) and data were analyzed with FlowJo software ( TreeStar , Ashland , OR ) . We used a construct based on the hemoglobin peptide ( Hb64–76 ) covalently linked to the I-Ek β chain with a flexible linker ( a generous gift of J Kappler ) . We replaced the hemoglobin DNA sequences with the gp250 peptide by PCR to create the gp250/I-Ekβ construct . The gp250/I-Ek construct was subsequently subcloned into the EcoRI sites of pDOI-5 vector . A BglI linearized fragment was isolated and used to inject the pronuclei of fertilized B6 oocytes to obtain the gp250/I-Ek mouse . We screened 374 mice and obtained one founder . The founder was crossed to I-Ek α chain transgenic mice ( Griffiths et al . , 1994 ) , and subsequently bred onto the MHCII−/− and CD74−/− background . All of the experiments reported here used the gp250 single chain mice on MHCII−/− and CD74−/− background . Thymocytes or splenocytes from gp250 SC mice or B6 . K mice were stained with a panel of FITC-conjugated antibodies against a panel of antibodies against mouse TCR Vβ chains ( 557004; BD Pharmingen ) , along with APC-CD4 , PE-Vα11 , and APC-Cy7-CD8 . APC-labeled I-Ek tetramers in complex with MCC , or CLIP were obtained from the NIH facility . All washes and incubations were done in modified RPMI-1640 containing 2% FCS and 0 . 075% sodium bicarbonate . For tetramer staining , peripheral CD4+ T cells were enriched by CD4+ MACS beads ( Miltenyi Biotech , Germany ) , and CD4SP thymocytes were enriched by negative selection of CD8+ MACS beads ( Miltenyi Biotech ) . Enriched CD4+ peripheral T cells or CD4SP thymocytes were stained with APC-conjugated MCC tetramer or PE-conjugated Hb tetramer on ice for about 1 . 5–2 hr . The APC-conjugated or PE-conjugated CLIP tetramers were used as a negative control . In the case of MCC tetramer staining , the cells were also stained for APC/Cy7-conjugated CD4 , PE/Cy7-conjugated CD8 , and Pacific blue-conjugated CD3 antibodies . Cells positive for LIVE/DEAD Fixable Blue Dead Cell Stain were gated out of the analysis . B6 . K or gp250 SC hematopoietic stem cells were enriched by c-kit MicroBeads ( #130-091-224; Miltenyi Biotech ) , stained for c-kit and Sca-1 , and sorted out c-kit+Sca-1+ population on an AriaII . Hematopoietic stem cells were resuspended at 104 cells per 50 µl PBS and were adoptively transferred into lethally irradiated B6 . K or gp250 SC mice by retro-orbital injection on day 0 ( at least 104 hematopoietic stem cells per mouse ) . After a 12-week of reconstitution , cells were recovered from thymus and spleen of each recipient mouse , and analyzed by flow cytometry . The single cell repertoire analysis was conducted by following the protocol developed by McHezyer-William’s lab ( McHeyzer-Williams et al . , 1999 ) . Single peripheral Vα11+Vβ3+CD3+CD4+CD8− cells from gp250 SC mice were sorted into a 96-well plate on Aria II . Each single cell was sorted into the center 60 wells with the first and last well of each row serving as a negative control . Each single cell was directly sorted into a well containing 5 μl of reaction mixture: 1× Cell Direct Reaction Buffer ( 11753-100; Invitrogen ) , 0 . 1 μl SuperScript-III RT/Platinum Taq Mix ( 11753-100; Invitrogen ) , and 2 . 5 nM external primers ( Va11 . L2: AATCTGCAGTGGGTGCAGATTTGCTGG; Ca . 2: AATCTGCAGCGGCACATTGATTTGGGA; Vβ3 . L2: ATGGCTACAAGGCTCCTCTGGTA; Cb . 2: CACGTGGTCAGGGAAGAA ) . After the single cell sort , the plate was immediately held at 50°C for 15 min , followed by 95°C for 2 min , and then 22 cycles of 95°C for 15 s and 60°C for 4 min for cDNA reaction . The cDNA products were diluted to 20 μl with molecular-grade water , and 2 μl of the first round product was used for further 10 μl amplification for TCR Vα11 and Vβ3 as previously described with the Internal Primers . For Vα11 , the Internal Primers are Vα11 . L3: AGATTTGCTGGGTGAGAGGAG and Cα . ext: GAGTCAAAGTCGGTGAACAGG . For Vβ3 , the Internal Primers are Vβ3 . 1: AATCTGCAGAATTCAAAAGTCATTCAG and Cβ . 3: AATCTGCAGCACGAGGGTAGCCTTTTG . The first round and second round PCR reaction were prepared and conducted at a separate bench in the lab where no AND transgenic TCR-related experiments had been previously done . After the PCR amplication , 4 μl PCR products were run on 2% and screened for the single positive bands of the right sizes . PCR products were then TOPO cloned to pCR2 . 1 vectors , and prepared for sequencing . The sequencing primers were as previously described ( McHeyzer-Williams and Davis , 1995; McHeyzer-Williams et al . , 1999 ) . The peripheral Vα11+Vβ3+ , Vα11+ Vβ6+ , Vα11+ Vβ14+ CD4+ cells from B6 . K or gp250 SC mice were stained and sorted on an Aria II . For TCR CDR3α analysis , RNA was isolated and used to generate cDNA as described ( Lathrop et al . , 2011 ) . A bulk Vα11 cDNA library was generated by PCR using primers derived from published studies ( Dash et al . , 2011 ) and then sequenced using Illumina MiSeq technology at the Washington University Genome Sequencing Center . For TCR CDR3β repertoire , the bulk-sorted populations were pelleted at 400×g for 10 min and snap-frozen on dry ice . The prepared cell pellets were sent to Adaptive Biotechnologies for conducting bulk CDR3β analysis . The CDR3 amino acid usages were analyzed in Excel . All data were analyzed nonparametrically by the Mann-Whitney U-test or paired t tests with Prism 6 software ( Graph Pad ) . p values of less than 0 . 05 were considered significant .
Naive T cell precursor frequency determines the magnitude of immunodominance . While a broad T cell repertoire requires diverse positively selecting self-peptides , how a single positively selecting ligand influences naive T cell precursor frequency remains undefined . We generated a transgenic mouse expressing a naturally occurring self-peptide , gp250 , that positively selects an MCC-specific TCR , AND , as the only MHC class II I-Ek ligand to study the MCC highly organized immunodominance hierarchy . The single gp250/I-Ek ligand greatly enhanced MCC-tetramer+ CD4+ T cells , and skewed MCC-tetramer+ population toward V11α+Vβ3+ , a major TCR pair in MCC-specific immunodominance . The gp250-selected V11α+Vβ3+ CD4+ T cells had a significantly increased frequency of conserved MCC-preferred CDR3 features . Our studies establish a direct and causal relationship between a selecting self-peptide and the specificity of the selected TCRs . Thus , an immunodominant T cell response can be due to a dominant positively selecting self-peptide .
The immune system protects against disease by recognizing invading pathogens , such as bacteria and viruses , and launching various responses to eliminate them . In vertebrates , such as mice and humans , this response often involves both an ‘innate’ and an ‘adaptive’ component . The innate immune response is fast but it is not targeted at the specific pathogen that needs to be eliminated . The adaptive immune response is slower , but it is also stronger and tailored to combat the specific pathogen that is attacking the host . In general , both the innate and adaptive immune responses work together to combat the infection . The innate immune system is activated when host cells are damaged or stressed , or when ‘foreign’ molecules that are tell-tale signs of microbes and pathogens are detected . It enlists white blood cells that engulf and digest infected host cells or pathogens , and then display the digested fragments via proteins called major histocompatibility complexes . The adaptive immune response relies on other white blood cells called B cells ( which make antibodies ) and T cells ( which carry out a variety of roles ) . Each T cell will respond to a very specific fragment of protein displayed by a major histocompatibility complex . Although a bacteria or virus can generate thousands of protein fragments , only a select few will stimulate an immune response . This phenomenon is known as immunodominance . As T cells mature in the thymus—a specialized organ located in the chest—they undergo both positive and negative selection . To survive positive selection , the T cells must be able to recognize fragments from the host’s own proteins . Negative selection removes T cells that interact too strongly with these ‘self-peptides’ because such interactions will cause the T cells to destroy the host’s tissues and cause auto-immune diseases . Positive selection is thought to have an important role in immunodominance because it shapes the population of T cells released from the thymus , but it has been difficult to test this hypothesis . Now Lo et al . have explored this question by engineering transgenic mice in which gp250—a self peptide that positively selects T cells that respond to a foreign protein called MCC—was the only self peptide to be presented during positive selection . Subsequent experiments showed that the mature T cells released by the thymus increased the fraction of T cells that were capable of responding to the dominant fraction of possible MCC fragments . By establishing a clear connection between positive selection and immunodominance , the work of Lo et al . could assist the development of more effective vaccines .