Class II major complex mutant mice PNAS PLUS to study the germ-line bias of T-cell receptors

Daniel Silbermana,b, Sai Harsha Krovib, Kathryn D. Tuttleb, James Crooksc, Richard Reisdorphd, Janice Whitea, James Grossa, Jennifer L. Matsudaa, Laurent Gapinb, Philippa Marracka,b,e,1, and John W. Kapplera,b,e,1

aDepartment of Biomedical Research, National Jewish Health, Denver, CO 80206; bDepartment of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO 80045; cDivision of Biostatistics and Bioinformatics, National Jewish Health, Denver, CO 80206; dPharmaceutical Sciences, University of Colorado School of Medicine, Aurora, CO 80045; and eHoward Hughes Medical Institute, National Jewish Health, Denver, CO 80206

Contributed by Philippa Marrack, July 6, 2016 (sent for review March 2, 2016; reviewed by Erin J. Adams and Martin Flajnik)

The interaction of αβ T-cell antigen receptors (TCRs) with peptides anism termed “negative selection” (13, 14). The remaining T bound to MHC molecules lies at the center of adaptive immunity. cells go on to mature and form the peripheral T-cell repertoire. Whether TCRs have evolved to react with MHC or, instead, pro- The effect of positive and negative thymic selection on limiting cesses in the involving coreceptors and other molecules the T-cell repertoire has made it difficult to test directly whether select MHC-specific TCRs de novo from a random repertoire is a germ-line features of TCRs and MHC molecules have been con- longstanding immunological question. Here, using nuclease-tar- served to promote their interaction. However, some data consistent geted mutagenesis, we address this question in vivo by generating with this notion have accumulated over the past several decades three independent lines of knockin mice with single- through sequencing, X-ray crystallographic, mutational, and de- mutations of conserved class II MHC amino acids that often are velopmental studies. For example, random examination of mouse – T cells before positive selection showed a high frequency of MHC- involved in interactions with the germ-line encoded portions of – TCRs. Although the TCR repertoire generated in these mutants is reactive cells (15 17). In mice constructed to allow positive selec- similar in size and diversity to that in WT mice, the evolutionary tion but incomplete negative selection, an even higher frequency of bias of TCRs for MHC is suggested by a shift and preferential use of generically MHC-reactive T cells was observed (18). Structural and some TCR subfamilies over others in mice expressing the mutant sequencing studies of MHC molecules have shown that the great majority of their polymorphisms are within the peptide-binding

class II MHCs. Furthermore, T cells educated on these mutant MHC

groove, not on the tops of the MHC α-andβ-chain helices that IMMUNOLOGY AND molecules are alloreactive to each other and to WT cells, and vice interact with TCRs (Table 1). The CDR1 and CDR2 loops of versa, suggesting strong functional differences among these rep- TCRsaremuchlessvariableinlengththanthoseofIgs(19).Inthe ertoires. Taken together, these results highlight both the flexibility dozens of structures of peptide–MHC/TCR complexes that have of thymic selection and the evolutionary bias of TCRs for MHC. been solved, a diagonal orientation of the TCR is nearly always seen. This orientation usually causes the somatically generated T-cell | MHC | evolution | mutation | variable region CDR3s to be focused on the peptide and the germ-line–encoded CDR1 or CDR2 amino acids, especially those of CDR2, to be he genes for immunoglobulins (Igs), αβ T-cell receptors docked on the conserved portions of the MHC helices (9). T(TCRs), and antigen-presenting MHC proteins appeared at Mutation of these TCR amino acids impairs T-cell recognition least 450 million years ago in the cartilaginous fish and are present of the ligand and affects thymic development of the T cells in in all modern vertebrates (1–3). The more primitive hagfish and vivo (8, 20–22). Some of these germ-line TCR amino acids can lampreys lack these genes and have an adaptive be traced back to the TCRs of fish, and, despite their overall comprised of unrelated proteins (4). The main ligands for αβ TCRs are short peptides derived from self and foreign proteins, Significance captured in a specialized groove of MHC class I (MHCI) and class II (MHCII) molecules and presented to T cells (5, 6). Functional The evolutionary hypothesis for T-cell antigen receptor–pep- Igs and TCRs are created by very similar recombination mecha- tide major histocompatibility complex (TCR–pMHC) interaction nisms involving fusion of V, J, and sometimes D gene segments posits the existence of germ-line–encoded rules by which the with additional variations at the junctions to create an enormous TCR is biased toward recognition of the MHC. Understanding potential repertoire of Igs and TCRs, suggesting a common, un- these rules is important for our knowledge of how to manip- known evolutionary origin for these loci. ulate this important interaction at the center of adaptive im- These observations have raised several unanswered questions. munity. In this study, we highlight the flexibility of thymic For example,why did a separate TCR-rearranging gene system – selection as well as the existence of these rules by generating develop for recognizing peptide MHC ligands? How knockin mutant MHC mice and extensively studying the TCR did the extraordinarily polymorphic MHC genes stay functionally repertoires of T cells selected on the mutant MHC molecules. connected to TCR genes throughout 450 million years of evolu- Identifying novel TCR subfamilies that are most evolutionarily tion? One long-standing hypothesis has been that certain features conserved to recognize specific areas of the MHC is the first of TCRs and MHC molecules are evolutionarily conserved to step in advancing our knowledge of this central interaction. promote their interaction (7–10). Like Igs, the antigen-recogni-

tion portions of TCRs are partially encoded in the comple- Author contributions: D.S., S.H.K., L.G., P.M., and J.W.K. designed research; D.S., S.H.K., mentary determining region (CDR) CDR1 and CDR2 loops of K.D.T., R.R., and J.W. performed research; D.S., S.H.K., J.G., J.L.M., and J.W.K. contributed germ-line TCR Vα (TRAV) and Vβ (TRBV) genes and are new reagents/analytic tools; D.S., J.C., and L.G. analyzed data; and D.S., S.H.K., L.G., P.M., partially generated by somatic recombination processes that and J.W.K. wrote the paper. form the CDR3 loops. This initial repertoire is culled dramati- Reviewers: E.J.A., University of Chicago; and M.F., University of Maryland. cally during T-cell development in the thymus. First, only those T The authors declare no conflict of interest. cells whose TCRs have at least some minimal affinity for the self- Freely available online through the PNAS open access option. – peptide MHC molecules expressed in the thymus are positively 1To whom correspondence may be addressed. Email: [email protected] or kapplerj@ selected for further development (11, 12). The T cells in this njhealth.org. population whose TCRs have too high an affinity for these self- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. peptide–MHC molecules are eliminated by an apoptotic mech- 1073/pnas.1609717113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1609717113 PNAS Early Edition | 1of10 Downloaded by guest on September 29, 2021 Table 1. Alignment of I-A haplotype helix residues with alanine (A), and alanines were replaced with glutamine (Q). Alanine was chosen as a neutral, frequently used mutational re- placement, and glutamine was chosen because it is already present at the other positions on the helix and thus would not greatly alter the chemistry at the surface of the protein. Genes encoding either the mutant I-Ab α-orβ-chain, paired with the corresponding WT I-Ab α or β gene, were transduced into an MHCII-deficient B-cell lymphoma, M12.C3 (27, 28), to create APCs expressing the mu- tant I-Ab molecules. M12.C3 cells, derived from an H-2d mouse, lack an I-Ad β-chain but express a functional I-Ad α-chain from the original M12 BALB/c lymphoma. This I-Ad α-chain can some- times pair with some other introduced I-A β-chains, including that of I-Ab. For this reason we prepared M12.C3 cells transduced with only the WT I-Ab β-chain to control for the possible activity of the I-Ad/b mixed molecule. M12.C3 cells transduced with both of the WT I-Ab genes served as a positive control, and M12.C3 cells with b α *The solvent-exposed residues of I-Aα or I-Aβ mutated in this study are only the WT I-A -gene were also used as a negative control. numbered. All the M12.C3 transductants were cloned at limiting dilution, † b Consensus sequence. and surface expression of I-A was confirmed by flow cytometry. ‡Differences from consensus sequence. Because each mutation might have affected the recog- nized by individual mAb differently, we stained the cells using a variety of anti–I-Ab–specific mAbs. Fig. 1B shows data for the weak sequence homology, substitution of fish V segments for the 227 mAb, the least affected by the mutations. With this mouse V segments preserves antigen recognition of the mouse peptide–MHC complex (23). Finally, although RAG-mediated rearrangement makes the CDR3 more diverse, the CDR1 and CDR2 loops in TCRs, unlike those in Igs, do not undergo an- tigen-selected somatic mutation; thus they keep their germ-line sequence and antigen-driven responses throughout develop- ment, suggesting a conserved function (24, 25). Another model for the MHC restriction of TCRs has been put forth. According to the selection model, MHC restriction is not intrinsic to TCR structure but imposed is by the CD4 and CD8 coreceptors that promote signaling by delivering the tyrosine Lck to TCR–MHC complexes through coreceptor binding to MHC during positive selection (26). In the current study we assessed the importance of several MHCII conserved docking sites for TCRs by introducing specific point mutations into mouse I-Ab MHCII α or β genes. In vitro these mutations had little effect on the collection of self-peptides bound by the mutant I-Ab but often disrupted the recognition of peptide plus MHC by T cells specific for a variety of foreign or self- peptides. In vivo, mice carrying these MHC point mutations de- veloped TCR repertoires that were similar in size to those of WT mice but with altered TRAV or TRBV gene use. Furthermore, in vitro in mixed reactions, T cells from each of the WT and mutant mice responded strongly to antigen-presenting cells (APCs) from the other mice but not to their own cells. We discuss these results in relation to the current ideas and data about the role of evolution vs. somatic selection in framing the T-cell repertoire. Results b Mutations of the I-A Conserved Amino Acids Affect the Presentation Fig. 1. Screening and selection of I-Ab mutants that affect mature T-cell of Foreign Peptides to Antigen-Specific T-Cell Hybridomas. Although responses. The WT and mutant MHC molecules are expressed at similar levels MHC genes are extremely polymorphic, the amino acids on the α in cell lines and present peptides. (A) The solvent-exposed residues that were and β helices of MHCII that are frequently engaged by the TCR targeted for mutations are indicated for the α-(cyan)andβ- (magenta) chains of CDR1 and CDR2 loops are usually conserved, sometimes even H-2 I-Ab. Residues were chosen for mutational analysis based on their conservation across species (9). Ten of these amino acids tend to be almost between H2 molecules and their predicted interaction with TCRs. (B) M12.C3 cells monomorphic in the mouse I-A alleles found in the majority of expressing the mutant constructs were stainedwiththe17/227mAb,andthe expression of I-Ab was determined by flow cytometry. (C)Hybridomaswere laboratory strains (Table 1). A number are conserved as well in b mouse I-E molecules and in the MHCII alleles of humans and stimulated with cognate antigen presentedbyAPCsexpressingWTormutantI-A . other species (Table S1). These residues are located on the tops Activation was determined by flow cytometry and was defined as the MFI of CD69. α The ability of each mutant to stimulate thehybridomasisdisplayedafternor- of the MHCII -helices, in positions where they are less likely to malization to WT responses. Data are representative of three or four biological affect peptide binding and are more likely to affect interactions < A replicates per group; *P 0.05 by a one-sample t test with a true value of 100. (D) of the MHCII protein with TCRs (Fig. 1 ). Thus, we hypothe- Peptides were eluted from WT I-Ab and were analyzed by MS. Peptides with size that these amino acids may have been conserved during identical HPLC retention times that were present in three separate WT samples evolution to promote interactions with TCRs. were identified. Data indicate the percentage of peptides that were also identified To study the relative importance of these amino acids in TCR in duplicate runs isolated from βT77A, βR70A, and αA64Q cells. (E)Peptidespre- b recognition of peptide–I-A complexes, we mutated each of these sent in duplicate runs from each of the mutants were identified and compared 10 residues separately. Nonalanine amino acids were replaced with WT. Data are representative of multiple MS and MS-MS runs.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1609717113 Silberman et al. Downloaded by guest on September 29, 2021 mAb, the WT and mutant I-Ab cells all stained with a mean somal peptide loading into MHC (30), and in TCR/MHC struc- PNAS PLUS fluorescence intensity (MFI) 10- to 30-fold higher than that of the tures often makes a surface-exposed H-bond to the peptide negative controls. The MFI for the cells expressing the I-Ab/d mixed backbone. Therefore we decided not to mutate this amino acid in molecule was much lower. Thus, all the mutants were expressed at our experiments. βR70 is nearly monomorphic in all mouse I-A aboutthesamelevel. alleles (Table 1) but is not conserved in mouse I-E alleles or in the Next, we devised a system in which the responses of many dif- MHCII alleles of other species. In nearly all published TCR/I-A ferent T cells to antigen bound to the mutant MHCIIs could be structures it lies in the central region of the TCR footprint assessed simultaneously. C57BL/6 mice were immunized separately interacting with the TCR CDR3s and therefore might be expected with five different (Table 2). Seven days later, T cells from to influence somatic CDR3 selection during thymic selection but the draining lymph nodes of the immunized mice were restimulated perhaps not to have as strong an influence on germ-line Vα and with their cognate antigens, expandedinvitro,andfusedinbulkto Vβ use. Therefore we choose αA64Q and βT77A as the primary − the TCR αβ BW5147 thymoma cell line to create T-cell hybrid- mutations to test our hypothesis and βR70A as a potential control. omas. The preparations were named for their target MHC-II allele, I-Ab, and antigen (Table 2). Effects of the βT77A, βR70A, and αA64Q Mutations on the Peptides The bulk T-cell hybridoma preparations were cultured with Bound to I-Ab. Before proceeding to in vivo experiments with M12.C3 cells expressing WT I-Ab, each of the mutant I-Abs, or these mutants, we considered the possibility that, despite the WT I-Ad/b with or without the immunizing antigen. Activation of predicted lack of a direct role for these I-Ab amino acids in the T-cell hybridomas was assessed by up-regulation of CD69 on peptide binding, they might indirectly change the spectrum of the cells, as measured by flow cytometry (Fig. 1C). On average, I-Ab–presented self-peptides. Such changes would confound our about 50-fold more of the T-cell hybridomas in the bulk pop- experiments because positive selection involves reaction of the ulations responded to their immunizing antigen plus M12.C3 TCRs with both MHC and peptide, and we intended to examine cells bearing WTαβ I-Ab than to control M12.C3 cells with only the effects of MHC mutations independent of changes in the I-Ab WTβ. Nearly all the responses of the peptide or hen egg ly- bound peptide. To determine whether the MHCII mutations we sozyme (HEL)-specific bulk T-cell hybridomas were significantly created altered the spectrum of bound peptides, we compared reduced when the mutant APCs were used instead of WT APCs, the repertoire of peptides bound to WT I-Ab with those of the again with their immunizing antigen. The responses by bulk key- three I-Ab mutants expressed in our M12.C3 transfectants. One hole limpet hemocyanin (KLH)-specific T-cell hybridomas were caveat of these experiments is the thymus presents a different set also reduced 1.5- to 16-fold in various β-chain mutants but not in of peptides in a cathepsin L-dependent fashion (31) and thus α

-chain mutants, perhaps because the large KLH protein may have may behave differently than the transfected M12.C3 cells. INFLAMMATION many potential I-Ab–binding epitopes, and therefore, as group, T The WT and mutant I-Ab proteins were immunoprecipitated IMMUNOLOGY AND cells specific for KLH may be less sensitive to any one MHCII from lysates of the transduced M12.C3 cells. Peptides were mutation. Consistent with this relative lack of sensitivity to α-chain eluted from these preparations and subjected to MS or MS-MS mutants, some of the bulk KLH-specific cells were also cross-re- analysis as previously reported (32) and as described in Materials active to KLH presented by APCs bearing the mixed I-A molecule and Methods. A preliminary MS-MS analysis of the peptides in which the I-Ad α-chain replaced the I-Ab α-chain. isolated from WT and mutant I-Ab showed that they had I-Ab– These results confirmed and extended our previous studies binding motifs (Table S2) (33, 34). This finding served to vali- (29) because they showed that the conserved amino acids on the date our method of peptide isolation and suggested that the MHCII helices are not required for MHCII surface expression. I-Ab mutations did not affect the I-Ab peptide-binding motif. However, in agreement with previous work, they are often impor- To compare the peptides bound to WT vs. mutant I-Ab pro- + tant for TCR docking during CD4 T-cell responses, leaving open teins, immunoprecipitations and elutions for each sample were the possibility that their conservation might be required to ensure performed and analyzed with duplicate runs by MS. Limited MS- germ-line–encoded favorable MHCII docking sites for TCRs. MS again confirmed the presence of the I-Ab–binding motif We selected mutants from this group for in vivo studies to find in the peptides. A list of the peptides with identical HPLC re- out if they also affected T-cell thymic development. We con- tention times and calculated masses that were present in three sidered the four mutations that most consistently inhibited T-cell separate WT I-Ab samples was compared with those in duplicate activation: A64Q on the α-chain and R70A, T77A, and H81A on runs of mutant samples (Fig. 1D). Nearly all the total peptide the β-chain. αA64 is invariant in mouse and human MHCII and intensities found in the WT I-Ab samples were also identified in creates a docking “cup” for TCR Vβs that contain a tyrosine (Y) all the mutant I-Ab samples. To determine if unique peptides at position 48 of CDR2 (9). βT77 and βH81 are adjacent on the appeared only in the mutants, we first created a list of peptides I-Ab β-chain α-helix (Fig. 1A). βT77 is invariant in common that were found in duplicate MS runs of the same mutant sam- mouse I-A and I-E alleles and in human HLA-DR and HLA-DQ ple. Any peptide in this list that also appeared in any of the three alleles. In TCR/MHC structures, βT77 and βH81 are often WT samples was also called present. Once again, most of the contacted by the TRAV CDR1 loop (9). However, the highly peptides and nearly all the intensity from the mutant samples conserved βH81 has been implicated in the activity of mouse were found in the WT runs (Fig. 1E). Less stringent criteria, e.g., H-2DM and human HLA-DM, the proteins that catalyze endo- requiring that a peptide be present in only two of the three WT or mutant I-Ab samples, identified even more peptides shared among the samples. This analysis does not identify peptides Table 2. Bulk hybridomas and their immunizing antigen belonging to nested sets; therefore the similarity between sam- ples may be underestimated, because differently trimmed pep- Protein or peptide tides were considered to be different by our analyses, although Bulk hybridoma antigen Sequence the peptide they present to T cells is identical. Taken together, BB5 Vaccinia B5 peptide FTCDQGYHSSDPNAV these experiments support the notion that βT77A, βR70A, and BNP LCMV NP peptide SGEGWPYIACRTSIVGRA αA64Q MHC mutations do not notably alter the repertoire of b BHEL Hen egg lysozyme Whole protein self-peptides bound to I-A . We therefore proceeded to test the BKLH Keyhole limpet Whole protein effects of these mutations on thymic selection in vivo. hemocyanin β β α BMOG Myelin oligodendrocyte MEVGWYRSPFSRWHLYRNGK Creation of Mice Bearing the T77A, R70A, and A64Q Mutations. We produced mice expressing only the WT or mutant forms of glycoprotein peptide I-Ab. To ensure that the mutant and WT genes were expressed Bulk hybridomas are named for the haplotype of the mouse (H-2b) and on the proper cells at the appropriate levels, we changed the the immunized antigen shown in this table. coding sequences of the genes in situ, using zinc finger nuclease

Silberman et al. PNAS Early Edition | 3of10 Downloaded by guest on September 29, 2021 (ZFN) technology to generate knockin point mutations directly only in comparisons within TRAV families and to be just semi- in fertilized C57BL/six eggs (35, 36). quantitative in comparisons between TRAV families. Never- Custom ZFNs were designed (Sigma Aldrich) for both H2-Ab1 theless, the biases in analysis between each TRAV family should and H2-Ab2, the genes that encode the α- and β-chains of the be common to the different types of mice, so we believed com- only MHCII molecule expressed in C57BL/6N mice. To reduce parisons of TRAV use between mouse genotypes were justified. off-target effects, the ZFNs were designed to ensure that no other We compared the use of the TRAV family and the TRAV region of the mouse genome had fewer than five DNA base subfamily among the mice. First, we looked at the average use of mismatches to the sequence targeted by the ZFNs. A template for the 20 TRAV families present in the mice (Fig. 3D) in the WT vs. homology-directed repair (HDR) was used to introduce our mu- mutant mice. Use of the DESeq2 package (38), which is often tations into mice. This template had four components: (i)the implemented in comparing mRNA expression in different cell mutation of interest; (ii) a silent mutation to create a new re- populations, revealed no significant differences in the frequency of striction enzyme site for screening of progeny; (iii) a silent mu- TRAV use by the T cells in WT and R70A mice. However, there tation to disrupt the ZFN binding so that a subsequent insertion or were reproducible and statistically significant differences in TRAV deletion event caused by nonhomologous end joining (NHEJ) use by the T cells in T77A and WT mice. The TRAV 3, 6, and 11 would not occur after our mutation of interest had been in- families were used more frequently, and the TRAV 5, 7, and 14 troduced; and (iv) roughly 1,000 bp of homology on either side of families were used less frequently by T cells in T77A mice than by the target of the ZFN (Fig. 2A)(37). T cells in WT mice; the latter finding confirms our mAb-staining DNA from the resultant mice was analyzed to identify chro- results (Fig. 3 A and B). mosomes bearing the desired mutation. The method was sur- However, TRAV family analysis does not compare the use of prisingly robust, with NHEJ events identified in nearly all the mice TRAV subfamily members. In C57BL/6 mice, a portion of the and at least one chromosome with the correct mutation found in TRAV locus has been triplicated. Herein genes in the supposed >10% of the mice overall. Mutant mice were crossed to WT mice “original” [ImMunoGeneTics (IMGT) designation] mouse TRAV and then intercrossed to create mice homozygous for each of the locus are designated “A.” Genes in the second of the triplications three mutations. All mice showed equivalent levels of I-Ab cell- are designated “D,” and genes in the third triplication are desig- surface expression on peripheral cells (Fig. 2B). nated “N.” Distinction between these subfamily genes is important because often, but not always, the various subfamily members differ Phenotypic Analysis of Thymic T Cells in the Mutant Mice. To de- in nucleotide and consequently in protein sequence, particularly termine whether any of our MHC mutations affected the de- in their CDR1 and CDR2 sequences, which are of interest for our + velopment of CD4 T cells, the thymus of each mouse strain was experiments (9, 19). Thus, each family is designated by a number analyzed by flow cytometry. No significant difference in the (e.g., TRAV1, TRAV6, and so forth), and a second number and number of thymocytes in the double-negative (DN), double- letter are used to designate the particular subfamily member (e.g., positive (DP), and single-positive (SP) populations was detected TRAV6-5A, TRAV6-5D, and TRAV6-5N). between the thymi of the mutant mice vs. WT mice (Fig. 2 C–E). We compared TRAV subfamily use for all the 88 TRAVs we + Analysis of CD5 and CD69 expression, markers of DP thymocyte could distinguish with our sequencing by the CD4 T cells in the activation during positive selection, showed that the size of the WT, βT77A, and βR70A mice. The data for all the individual expressing population was not changed in the βR70A and βT77A TRAV subfamily genes are contained in Fig. 3 E and F. TRAVs mutant mice but was significantly reduced in the αA64Q mutant underrepresented in the βT77A mice were 7-6A, 7-6D, 7-6N, mice (Fig. 2 C, D,andF), suggesting that at least the αA64Q 8-1AD, 8-2D, 12-1AN, 13-2AN. and 14-3A. TRAVs overrepresented mutation reduced positive selection and MHCII reaction by TCRs. in the βT77A mice were 3-3A, 4-4D, 6-2A, 6-3ADN, 6-4A, 6-4D, 6-6N, 6-7A, 9-2D, and 11-1AD. Differences between WT and T77A Effect of the βT77A and βR70A Mutations on the Use of TRAVs. We cells for all the subfamilies and their significance scores can be next examined the TCR repertoire of peripheral T cells selected found in Table S3. Many of these subfamily differences account in WT and mutated mice. Because the germ-line portions of the for the differences in overall family use shown in Fig. 3D. TCRs interacting with I-Ab βT77 were predicted to be those of TRAV CDR1s, we predicted that the TRAVs used in the mutant Comparison of the TCRα Repertoire Used by Naive CD4 T Cells from mice would be more affected by these mutations than the TRBVs. WT and I-Abβ Mutant Mice. Sequencing identified not only the Therefore, we compared TRAV use in the βT77A mice with that TRAV families and subfamilies used in the WT and mutant mice in the WT mice, using the βR70A mutant mice as a possible but also the complete sequences of the TCRα domains, including control, because this amino acid most often interacts with randomly the TRAVs, TRAJs, and the somatically generated CDR3α re- generated CDR3 regions rather than the germ-line encoded DR1 gions. Thus, we analyzed the diversity of the entire TCRα se- and CDR2 regions. Anti-TRAV staining with the four available quences among the naive splenic CD4 T cells in WT and mutant anti-TRAV mAbs (TRAV14, TRAV9, TRAV12, and TRAV4) mice in several different ways. First, we examined the properties of revealed a significant reduction in TRAV14 use in the mutant mice the overall TRAV–CDR3–TRAJ repertoires. Initially, to measure (Fig. 3 A–C). As expected, this reduction was seen only in CD4 T the richness and diversity in the population, we used a species cells, not in CD8 T cells. This TRAV14 shift was the first indication accumulation curve (39) in which a random sampling of our of an altered TCR repertoire in the T77A mice. population along the x axis is shown on the y axis if each included This analysis was limited by the small number of anti-TRAV– sequence adds a unique sequence to the total number of unique specific mAbs available. Moreover, the TRAV that sequences (Fig. 4A). This curve should plateau as the data ap- are available might not distinguish between subfamily members in proach the saturation of all sequences present in the cDNA sample. each TRAV family (see below). To overcome this reagent limi- Sequences of the TCRαs from the three types of mice have similar tation, we examined the TRAV repertoires of the MHCβ mutant curves and do not plateau even after analyzing 500,000 randomized mice in greater detail using deep sequencing. We used a set of sequences. Therefore, the naive CD4 T cells in WT, βR70A, and forward primers specific for the TRAV families and common Cα βT77A mice all express similarly large, diverse TCRα repertoires. reverse primers to generate a diverse PCR product that encoded However, a different type of accumulation curve shows that + the TRAVs present in the naive CD4 T cells from each strain of this large repertoire is not randomly dispersed, i.e., the frequency mice. These fragments were sequenced with high-throughput of each sequence is not determined by a simple Poisson distri- methods. Using software developed in house, we filtered out bution (Fig. 4B). Despite the lack of saturation, the average short sequences and determined the TRAV, TRAJ, and CDR3 number of repeats of any given unique sequence in the samples used by each sequence. Although we designed our TRAV pri- was about five but ranged from 1 to more than 10,000. Using this mers to be family specific, of similar length, and with similar frequency, we constructed a Poisson-predicted accumulation melting temperatures, we expected our results to be quantitative curve that predicts the proportion of total sequences that should

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Fig. 2. Generation of MHCII mutant mice and characterization of the effects of mutation on thy- mic selection. (A) The schematic depicts the ZFN- targeting strategy used to generate MHC mutant mice. The DNA for HDR was designed to target exon 3 of either H2-AB (R70A, T77A) or H2-AA (A64Q); the positions of screening primers are indicated. The structure of the DNA for HDR included 1,000 bp of homology flanking at either end the ZFN recogni- tion site. Mutations in H2-AB are indicated in red, and mutations in H2-AA are shown in teal. The re- striction sites introduced to allow screening are in- INFLAMMATION

dicated in green. The locations of ZFN recognition IMMUNOLOGY AND sites are also indicated; these sites were disrupted by the introduction of a silent mutation in the vector. (B) Splenocytes were stained for MHCII and markers of lymphocyte lineages and were analyzed by flow cytometry. Histograms depict the level of expression − of MHCII on TCRβ cells in βR70A and βT77A (ma- genta traces), αA64Q (teal trace), and WT (dark gray shaded area) mice. (C and D) Thymocyte composition is shown for WT, βR70A and βT77A (C), and for WT and αA64Q mice (D) as determined by expression of CD4 and CD8. Thymocytes undergoing selection are identified by the expression of CD5 and CD69. (E) Frequency of mature SP4 and SP8 thymocytes in the indicated strains. (F) Frequency of thymocytes undergoing selection in the different mice. Data in C–F are representative of three or four independent experiments containing 7–10 mice per group. Error bars represent SEM; *P < 0.05.

accumulate as we added sequences that occur from 1 to 20 times. (the principal components) are defined by the variability in the This curve predicts that, if TCRα use is Poissonian, we should data. By construction, the first principal component is the linear account for nearly all the sequences by the time we include those combination of TCRs that yields the highest variance in expression that occur 15 times or less, but the experimental accumulation levels between samples. The second principal component is then curve generated from the sequencing data shows that these se- the linear combination of TCRs that yields the highest variance in quences account for only ∼50% of the total sequences. Likewise, expression levels subject to being perpendicular to the first prin- more sequences were found fewer than three times than pre- cipal component, and so forth. Often the first several principal dicted by the Poisson curve. Similar results were seen with the components explain the majority of the variance in the data. One data from the mutant mice. Thus, despite the great diversity of then can plot the samples along the first few principal component sequences, their frequency was not as predicted by a Poisson axes to visualize high-dimensional expression data in terms of a set distribution, a feature shared with previous repertoire analyses of of simpler axes that represent the most important features of these different human T-cell populations (40). Some of these results data. In these plots, clear separation between and clustering might be attributable to uneven efficiencies during the PCRs within genotype groups indicates that the genotype is driving with the cDNA templates, but it is likely that both thymic and repertoire-wide differences in expression patterns. The WT, βT77A, peripheral selective pressures also contributed. and βR70A mice clustered well and were separated from each other Finally, we examined the differences in overall sequences for for two of the three components. Particularly well separated were the three types of mice. Two types of analyses were done on the the WT and βT77A data. As a second analysis we directly compared total TCRα (combining the TRAV subfamily, TRAJ, and CDR3) the TRAV–CDR3–TRAJ combinations in the nine mice. Given the sequences. First, in Fig. 4C theoveralldatafromtheninemiceare very large number of comparisons being made, the bar for sig- represented as a three-component principal component analysis nificance differences was set very high. To reduce the number of (PCA). PCA is a transformation of the data (in this case expression comparisons, we set a threshold of TCRαs sequenced at least 10 values for all samples) into a new coordinate system whose axes times combined in all nine runs. As shown in the heat maps in

Silberman et al. PNAS Early Edition | 5of10 Downloaded by guest on September 29, 2021 Fig. 3. Differential use of TRAV families and subfamilies in T77A mutant mice. (A) Representative analysis via flow cytometry of TRAV14 expression in spleen + + + + CD4 T cells from WT, βR70A, or βT77A mice. (B and C) Frequency of TRAV14 cells in CD8 (B) and CD4 (C) T cells in the spleen of the indicated mice. Data are representative of three or four independent experiments containing 7–10 mice per group. Error bars indicate SEM. (C) Next-generation sequencing of the + entire TCRα repertoire expressed in naive CD4 T cells sorted from the spleen of WT, βR70A, or βT77A mice. (D) Frequencies indicate the proportion of any given TRAV family out of all valid sequences. (E and F) Heat maps representing the positive (E) or negative (F) fold change in the use of the individual TRAV + subfamily genes in CD4 cells from the indicated strains. Green, yellow, and red indicate high, medium, and low use, respectively. Statistical significance (P < 0.05) is indicated by an asterisk in C and by blue squares in D–F.

+ Fig. 4 D and E, 84 combinations were found to be significantly elements. We analyzed CD4 SP thymocytes and splenic CD4 different between WT and T77A mice. The figure gives a “ge- T cells from WT and αA64Q mice with a mAb that discriminates stalt” view of the data; the complete data for these sequences, TRBV13-2 from TRBV13-3 (Fig. 5 A and B). Flow cytometric including the TRAV, TRAJ, and CDR3 sequences and signifi- data showed a substantial, significant shift in use from TRBV13-2 cance scores are contained in Table S4. to TRBV13-3 in both populations in the A64Q mutant mice as In summary, although both WT and mutant mice develop compared with WT mice. large diverse repertoires, significant changes have occurred in Next, with a strategy similar to that used in our analysis of the TRAV family and subfamily and in TCRα CDR3 sequences to TCRα repertoire in the βT77A and βR70A mice, we deep se- + accommodate the mutations. quenced the TRBV13 domains present in naive CD4 T cells in three WT and αA64Q mice. We created a PCR fragment with a Effect of the αA64Q Mutation on the T-Cell TRBV13 Repertoire. Our 5′-primer common to all three members of the TRBV13 family and a analysis of the thymus in the αA64Q mice showed apparently 3′-primer within Cβ.Fig.5C shows that the sequence data confirmed reduced activation from positive selection in DP thymocytes the significant shift from TRBV13-2 to TRBV13-3 in the αA64Q based on CD5/CD69 expression (Fig. 2 D and F). Because sub- mice, but there was no change in the use of the third family member, stantial biological and structural data have shown that the site TRBV13-1. The heat map in Fig. 5D shows all the TRBV13/TRBJ that includes αA64Q is often used as a docking site for βY48 of combinations with an increased frequency in WT samples compared the CDR2 loop of the TRBV13-2 Vβ element and perhaps also with A64Q, and Fig. 5E shows the combinations used more fre- the TRBV13-3 Vβ element (21), we focused our analysis of the quently in the mutant. The blue squares in Fig. 5 D and E indicate effects of this mutation on the repertoire of T cells using these statistical significance (Table S5), and these combinations group

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1609717113 Silberman et al. Downloaded by guest on September 29, 2021 previously demonstrated influence of the varying peptide rep- PNAS PLUS ertoire among MHC haplotypes (41), these results provide evi- dence for the previously suggested (42) role of the germ-line bias of TCRs for MHC in alloreactivity. Thus, it is likely that both the peptide repertoire and the germ-line–encoded bias contribute to alloreactivity to some extent. Discussion The roots of our current thinking on the evolutionary conser- vation of interactions between TCR and MHC amino acids came + from our studies of CD4 T cells in mice expressing a single fixed peptide–MHCII complex (18). These mice had impaired negative selection because of the absence of a diverse set of self-peptides bound to their MHCII. The T cells thus created reacted strongly to self-MHC occupied by the normal complement of self-peptides and also, surprisingly, to many different allo-MHCII alleles. We concluded that, although negative selection functions to remove high-affinity self-specific T cells, in so doing it also eliminates a large population of highly MHCII cross-reactive T cells. Based on our subsequent functional, mutational, and struc- tural studies, we concluded that the high cross-reactivity of these T cells was caused by dominant interactions of certain conserved amino acids in their TRAV and TRBV CDR1 or CDR2 loops with conserved sites on the MHCII helices. In complexes be- tween the TCRs of T cells from normal mice and their activating peptide–MHCII ligands, reported by ourselves (20) and others (8), these conserved interactions were seen often, but they usu- ally were not so dominant. These findings have led us to our

current hypothesis that random combinations of germ-line TCR INFLAMMATION

α β IMMUNOLOGY AND Fig. 4. Differential use of complete TCRα in T77A mutant mice. (A) Species ac- - and -genes create, with high frequency, T cells reactive to cumulation curves for the WT, βR70A, and βT77A mice. Each curve represents the MHCII, regardless of allele, and that, to escape negative selec- average of the accumulation curves of three mice of the indicated genotype. (B) tion and contribute to the functional peripheral repertoire, A Poisson-predicted distribution based on the average number of repeats in any T cells must bear TCRs whose somatically generated CDR3s sample is compared with the accumulationofsequencesbasedonitsfrequencyin have modulated this tendency away from generic MHC reactivity the sample. (C) PCA showing that each individual run clusters by genotype on the and toward peptide dependence. The results of direct mutagenesis first three components. (D and E)TCRα sequences differentially expressed in T77A experiments by us and others are consistent with this idea. and WT cells are expressed in heat maps ordered by positive (D) or negative Our purpose in this present study was to determine how mu- (E) fold-change. Green, yellow, and red indicate high, medium, and low use, tations in MHCII I-Ab amino acids affect T-cell development and respectively. Statistical significance (P < 0.05) is indicated by blue squares. the peripheral T-cell TCR repertoire. We chose I-Ab βT77 and αA64 for this study for several reasons: They are highly conserved among MHCII molecules; they have been seen repeatedly as sites almost perfectly with TRBV13 subfamily. Furthermore, looking of interaction with certain germ-line TRAV CDR1 and TRBV at the more commonly found TCRs, DESeq2 identifies indi- CDR2 amino acids; their mutation often disrupts the activation of vidual TCRβs that are differentially expressed in the WT and E peripheral antigen-specific T cells in response to antigen; and A64Q mice (Fig. 5 and Table S6). Thus, these data support the they do not participate directly in peptide binding. We choose I-Ab previous findings on a more global scale, with particular Rβ70 as a control because it is less conserved and usually interacts – TRBV13-2 containing domains associating the TRBV13-2 with the TCR CDR3 loops. β CDR2 loop with docking on the portion of the MHCII 1 helix Our results show that none of the mutations prevented the α containing with an evolutionary preference for MHC A64. development of large, diverse peripheral CD4 T-cell populations.

b However, depending on the mutation, there were significant Reciprocal T-Cell Recognition of the I-A Mutations. Our data clearly changes in thymocyte subpopulations and changes in the pe- point to adjustments in the use of particular germ-line TRAV ripheral CD4 T-cell TCR repertoire. The subtlest changes were and TRBV elements driven by the mutations in the conserved β b seen with the T77A mutation. There were no changes with this amino acids on the α1 and β1 helices of I-A , but they do not mutation in thymic cellularity or in the size of the thymic pop- + + reveal the functional consequences of the overall change in the ulation undergoing selection (CD4 CD69 ). However, com- TCR repertoire. To begin to address this question, we tested how pared with WT mice, the βT77A mutation led to significant shifts b + “foreign” the WT and mutant I-A molecules appeared to CD4 in TRAV family and subfamily use. In addition, this mutation led T cells from the various mice. We set up one-way mixed lym- – – + to changes in the TRAV CDR3 TRAJ repertoire, demonstrated phocyte reactions using all combinations of purified CD4 T by PCA that clearly separated the unique sequences in the mutant cells and APCs from the WT and mutant mice. T cells and APCs mice from WT mice and from each other. Our analysis of the from an I-Af mouse (B10.M) were used as a control. The results α + A64Q mice also showed normal thymic cellularity, but in this case (Fig. 6) show that the CD4 T cells did not respond to APCs there was a significant reduction in the activation in thymocytes from the same mouse but did respond to APCs from all the other undergoing selection. In a more abbreviated peripheral repertoire mice, as measured by IL-2 production. The T-cell responses from analysis, we compared the use of TRBV13-2 with the other two the WT and mutant I-Ab mice were on the same order of mag- members of this family. The importance of the intimate interaction nitude as the alloresponses seen with the I-Af T cells and APCs. of evolutionarily conserved TRBV-Y48 in the CDR2 of TRBV13-2 These results predict that differences in the TCR repertoires with the portion of MHCII α-chain helix containing A64 has been among the WT and mutant I-Ab mice should be similar to those documented in numerous structural, functional, and thymic de- among mice of different MHCII haplotypes, indicating that the velopmental studies (1, 20, 21). The importance of this amino acid changes in TCR repertoire had dramatic effects on the specificity in the other family members is not as clear: It is present, but there and alloreactivity of the T cells. Furthermore, in addition to the are other differences between the family members in their CDR1

Silberman et al. PNAS Early Edition | 7of10 Downloaded by guest on September 29, 2021 Fig. 5. Differential use of TRBV13 subfamilies in A64Q mutant mice. (A and B) Representative staining with mAb MR5-2 distinguishing TRBV13-2 and TRBV13-3 (A) and frequency, represented as a ratio of TRBV13-2 to TRBV13-3 in thymic and splenic CD4+ T cells (B). Data are representative of three in- dependent experiments containing seven mice per group. Error bars indicate SEM. (C) The frequency of the three genes that are members of the TRBV13 family as determined by next-generation sequencing. Data are averages of three independent runs. Error bars indicate SEM. (D and E) Heat maps for the TRBV–TRBJ combinations are ordered by positive (D) or negative (E) fold change. (F) Individual TCRβ sequences that are differentially expressed in A64Q and WT cells. Statistical significance (P < 0.05) is indicated by asterisks in B and C and by blue squares in D–F.

and CDR2 regions. Our analysis showed that TRBV13-2 use by its CDR2 loop unfailingly react with related sites on the MHCII both thymic and peripheral CD4 T cells is reduced in the mutant α1 helix, even in the face of various docking angles of the TCRs. mice, with a concomitant rise in TRBV13-3 but no change in The set of structures available for analysis involving other TRAV TRBV13-1 compared with the WT mice. and TRBV elements has not been as extensive, so analyses with The results of the present study clearly show that mutation of the other TRAVs and TRBVs are not currently possible. How- either βT77 or αA64 alters the repertoire of developing CD4 T ever, because much of the tops of the MHC helices are con- cells. However, the magnitude of these effects was less than served, it is possible that individual TRAV or TRBV elements those we saw on the response of antigen-primed WT peripheral prefer docking to different conserved sites or can use alternatives CD4 T cells to antigenic peptides presented by the mutant to the preferred site. MHCII proteins. Likewise, mutation of conserved amino acids in A recent study consistent with this idea comes from the Garcia the CDR2 loop of TRBV13-2 had a much more profound effect laboratory (43). They analyzed the structures of the same TCR on T-cell development than did the αA64Q mutation (21). These bound to the MHCI allele, H2-Ld (Ld), engaged by many peptides. results suggest that, during the development of the TCR reper- The results showed that, although the TRAV CDR1 and CDR2 toire, adjustments not only in TRAV use but likely also in αβ locations on the Ld α2 helix were very similar in the structures, the pairing and somatically generated CDR3 sequences can largely TRBV13-1 CDR1 and CDR2 loops had more than one docking compensate for the loss of a single conserved docking site on site on the Ld α1 helix, altering the angle of engagement of the MHCII. However, once a T-cell has been selected by WT MHCII, TCR with Ld. Interestingly there were discrete docking positions, it no longer can make these adjustments to the loss of the docking not a continuous series. These results establish multiple discrete, site. It also is worth noting that our previous results with mutations conserved sites for TRBV13-1 docking on MHCI, the choice of in TRBV13-2 CDR2 were done with a transgenic TCR β-chain which is determined by the peptide. Therefore, the single amino with a fixed CDR3, thus limiting the possible adjustments in acid mutational approach used here may make it difficult to repertoire to changes only in α-chain pairing. With the advent of establish completely the TRAV or TRBV partners for a par- paired TCRαβ sequencing from single cells, a future direction ticular conserved site on the MHC helices. could be to explore the entire TCRαβ pairs and elucidate any The results of the current study are not inconsistent with any of potential compensation on the opposite TCR chain. the recent reports that have shown highly unusual MHC docking It has been suggested that the great deal of latitude seen in the modes by some TCRs and non-MHC ligands for some TCRs. For docking angle of TCRs binding to MHC argues against the idea example, natural killer T cells (NKT cells) and mucosal-associated of evolutionarily conserved amino acids in TCR–MHC inter- invariant T cells (MAIT cells) have nonconventional MHC ligands actions. However, the many structures of TCRs that include that lack the conserved MHC dockingsites(44,45).Thein- TRBV13-2 bound to MHC show that conserved amino acids in variant NKT and MAIT TCRs dock on their ligands in very

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1609717113 Silberman et al. Downloaded by guest on September 29, 2021 capacity of the thymus to generate an enormous number of unique PNAS PLUS TRAV and TRBV CDR3 loops, their existence is inevitable. However, if the initial, unselected TCR repertoire is random, the frequency of T cells specific for any particular protein, such as CD155 or MHC proteins, will be very low. Subsequent culling of this scarce MHC-specific repertoire during the nonproliferative phase of T-cell development to make it both self-MHC restricted and self-MHC tolerant will further reduce its size greatly, making the generation of the well-established, very large peripheral T-cell repertoire very difficult. However, predisposing the preselection TCR repertoire toward MHC recognition via embedded con- served amino acids in MHC and TCR proteins to promote their interaction should separate the “wheat” from the “chaff” during selection much more efficiently. Evidence presented here and in previous papers suggests that this idea is, to some extent, correct and that the preselected TCR repertoire is already skewed toward MHC reactivity (9, 15–17, 21, 23). Materials and Methods Mutant MHC I-Ab α- and β-Chains. Plasmids encoding MHCII I-Ab α-and β-chains were previously used (29). MHC mutations were cloned by over- lapping primers using engineered restriction sites. The I-Ab α-chain was cloned into a murine stem cell virus (MSCV)-based retroviral plasmid with an internal ribosome entry site plus Thy1.1 as a reporter. The I-Ab β-chain was cloned into a similar MSCV vector with a GFP reporter. These MSCV vectors were also available in the J.W.K./P.M. laboratory.

Retroviral Packaging. Retroviral plasmids were cotransfected into Phoenix cells with pCLEco accessory plasmid using Lipofectamine 2000 (Invitrogen) ’ INFLAMMATION according to the manufacturer s instructions. Retrovirus-containing super- IMMUNOLOGY AND natants were collected 48–72 h after transfection and were filtered through a 0.45-μm filter to remove cell debris. Fig. 6. Functional TCR repertoire differences identified by reciprocal T-cell recognition of the I-Ab mutations. One-way mixed lymphocyte reactions using MHC-Expressing Cell Lines. MHC constructs were expressed by retroviral + all combinations of purified CD4 T cells and APCs from WT and mutant mice. transduction of an APC line, M12.C3. M12.C3 cells are derived from a BALB/C T cells and APCs from an H-2f haplotype mouse (B10.M) were used as a control. B-cell lymphoma that was selected for loss of I-A expression (27), although Data shown are from three independent experiments. Error bars indicate SEM. they contain a functional I-Ad α-chain. For retroviral infection of M12.C3 cells, 105 cells were spin-infected with retroviral supernatants containing 8 μg/mL of Polybrene (Sigma-Aldrich) for 90 min at 37 °C. Cells were ex- nonconventional ways. These specialized T cells and their ligands panded in culture and subsequently were cloned by limiting dilution; clones arose evolutionarily after the development the conventional TCR– of equal MHC expression were chosen. MHC system. One could consider that they have “hijacked” apart of system for another purpose, much as certain MHC-like mole- Preparation and Stimulation of Bulk T-Cell Hybridomas. Antigen-specific T-cell cules no longer function as ligands for T cells but have taken on new hybridomas were generated by immunizing mice with the desired antigen functions over evolutionary time. emulsified in complete Freund’s adjuvant. The para-aortic lymph node cells The set of conventional TCRs that deviate most in the orienta- were isolated 7 d later, expanded in culture for 3 d with the same antigen with which the mice had been immunized, and cultured in IL-2 for 5 d. After tions and location with which they interact with conventional pep- α−β− tide–MHC complexes comes primarily from autoreactive T cells. this in vitro culture, activated T cells were fused to BW , a variant of the fusion partner BW5147 generated to lack both TCR α- and β-chains (50). Their footprints on MHC can drift dramatically away from those 4 5 – For stimulations, 5 × 10 to 1 × 10 hybridomas were cultured with different seen with foreign peptide MHC complexes and, in one case, even stimuli for 4–24hin200μL culture medium in 96-well microtiter plates. Hy- reverse the orientation of the TCR on the ligand (46). These T cells bridoma responses were measured by CD69 expression and IL-2 production. IL-2 are the survivors of thymic negative selection and as such may need ELISAs were done using the anti–IL-2 antibody JES6-1A12 (eBioscience) to cap- to venture into these unusual docking modes, not found in the ture and the biotinylated antibody clone JES6-5A4 (eBioscience) with streptavi- thymus, to improve their affinity to achieve T-cell activation. din conjugated to HRP (Jackson ImmunoResearch) to detect the bound antibody. Experiments aimed at the discovery of T cells that use non- MHC ligands have turned up T cells that recognize other mole- MS. WT and mutant I-Ab proteins were immunoprecipitated from lysates of cules. Most dramatically, one laboratory constructed a mouse roughly 109 of the transduced M12.C3 cells using antibody clone Y3P. Pep- lacking MHCI, MHCII, CD4, and CD8 and introduced mutations tides were eluted in 2.5-M acetic acid and were separated from beads, to uncouple essential downstream TCR-signaling molecules from antibodies, and MHCII molecules by passage through a 10,000-Da cutoff essential interactions (47–49). The mice developed a peripheral ultrafiltration unit (Millipore) and were subjected to MS or MS-MS analysis T-cell repertoire that contains T cells reactive to the surface protein as previously reported (32). Peptides were analyzed via LC/MS-MS or LC/MS CD155. The authors conclude that these experiments show that the on an Agilent Q-TOF instrument (model 6520) as described in detail in SI TCR repertoire need not be MHC dependent and that the usual Materials and Methods. specificity for MHC is not inherent in the germ-line sequences of Flow Cytometry. Cells, either ex vivo or hybridomas, were preincubated with the MHC and TRAV/TRBV elements. Rather, they suggest that in supernatant from the anti-CD16/CD32 producing hybridoma, 2.4G2. Cells normal mice MHC specificity arises by selection from a somatically were stained under saturating conditions with antibodies to mouse TCRβ generated random repertoire of TCRs, yielding TCRs that can (clone H57-597), CD4 (clone GK1.5), CD8 (clone 53-6.7), CD25 (clone PC61), satisfy the MHC-dependent geometry of the many components of CD44 (clone IM7), CD5 (clone 53-7.3), CD69 (clone H1.2F3), CD24 (clone M1/ the large TCR/coreceptor signaling complex. 69), B220 (clone RA3-6B2), CD11b (clone M1/70), γδ TCR (clone GL3), CD62L Our experiments do not argue against the generation of T cells (clone MEL-14), Vβ8.x (clone F23.1), Vβ8.2 (clone F23.2), Vβ8.3 (clone 1B3.3), of these non-MHC specificities. In fact, given the recombinational Vβ8.1/2 (clone MR5-2), and Vα2 (B20.1), purchased from eBioscience or BD

Silberman et al. PNAS Early Edition | 9of10 Downloaded by guest on September 29, 2021 Pharmingen or generated in house. Cells were analyzed by flow cytometry SuperScript VILO Kit (Invitrogen). Details on the PCRs used to generate the on a FACScan, LSR II, or LSRFortessa system (BD Biosciences). sequencing library and the sequences of the primers can be found in SI Materials and Methods. Generation of Knockin MHC Mutant Mice. As described in detail in the SI Materials and Methods, embryos were isolated from superovulated female Statistical Analysis of TCR Repertoires. Differential expression analyses were mice (51) and pronuclear injections performed with ZFN mRNA (Sigma- performed using the DESeq2 package (v1.8.1) (38) in the R language (v3.2.2) Aldrich) to introduce point mutations in the genes encoding IAb. All animals (52). Details of these analyses can be found in SI Materials and Methods. were housed and maintained in the Biological Resource Center within NJH in accordance with the research guidelines of the National Jewish Health In- ACKNOWLEDGMENTS. We thank Francis Crawford and Ella Kushner, Randy stitutional Animal Care and Use Committee. Anselment and Thomas Danhorn of the National Jewish Health Center for Genes, Environment and Health, Josh Loomis and Shirley Sobus of the Na- tional Jewish Health Cytometry Core for technical assistance, and Dr. Greg Sequencing of TCR Repertoire. Naive CD4 T cells were stained as described Kirchenbaum for assistance with some of the experiments in this paper. This above and sorted at the National Jewish Health Flow Cytometry Core Facility. work was supported by NIH Grants AI-18785 (to P.M.), AI092108 (to L.G.), RNA was isolated using the RNeasy Kit (Invitrogen). cDNA was made using the AI103736 (to L.G.), and T32 AI007405.

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