H-Y T cells in men are 1/3 as frequent as in women but have similar functional avidity

It has long been thought that clonal deletion efficiently removes almost all self-specific T cells from the peripheral repertoire. We found that self-peptide MHC-specific CD8 + T cells in the blood of healthy humans were present in frequencies similar to those specific for non-self antigens. For the Y chromosome-encoded SMCY antigen, self-specific T cells exhibited only a 3-fold lower average frequency in males versus females and were anergic with respect to peptide activation, although this inhibition could be overcome by a stronger stimulus. We conclude that clonal deletion prunes but does not eliminate self-specific T cells and suggest that to do so would create holes in the repertoire that pathogens could readily exploit. In support of this hypothesis, we detected T cells specific for all 20 amino acid variants at the p5 position of a hepatitis C virus epitope in a random group of blood donors.

These results indicate that clonal deletion is a factor, but not the most critical one, in mechanisms that establish tolerance. Instead, it might be that self-specific CD8T cells are imprinted with a less harmful genetic program, either in the thymus or in the periphery, and that this, together with other peripheral tolerance mechanisms such as CD4regulatory T cells (), is the principal bulwark against autoimmunity. We also suggest that the wholesale removal of T cell specificities is avoided because infectious pathogens are a much greater threat to evolutionary fitness than autoimmunity and that it is therefore imperative that T cells cover every possible peptide-MHC variant. In support of this model, we show that there are CD8T cells specific for every natural amino acid at position 5 of a hepatitis C virus (HCV) epitope presented by HLA-A0201, including one peptide variant reported to represent a blind spot in the TCR repertoire caused by clonal deletion ().

These results strongly suggest that clonal deletion does not play an absolute role in shaping the peripheral repertoire, but because they do not directly compare the same specificity in both self and non-self situations, we surveyed the frequency of SMCY specific T cells in males (who express this Y chromosome encoded antigen) and females (who do not), and found only a 3-fold reduction in males. A parallel experiment in mice produced a similar result. We then derived male versus female human T clones specific for SMCY and found that they have overlapping functional avidities. To explore whether a genetic program within self-specific CD8 + T cells might contribute to their apparent tolerance, we performed microfluidics-based single-cell quantitative PCR (qPCR) of SMCY specific T cells from women and men and found distinct patterns of gene expression that suggested impaired expansion in self-specific (male) cells. We then confirmed this observation functionally by showing that the activation of primary, self-specific CD8 + T cells from blood is impaired after in vitro stimulation.

In the case of human αβ T cells, assessing the effect of clonal deletion has been more difficult, although there are sporadic reports mentioning the peripheral survival of self-specific T cells (). In this study, we further explore the fate of self-specific CD8αβ T cells using the unique resource of healthy blood donors. We used specific peptide HLA-A0201 tetramers and a modification of the enrichment scheme of Jenkins and colleagues () to directly measure the frequency of particular CD8T cells and found that the frequency of CD8T cells recognizing endogenous peptides was roughly equivalent to that of naive CD8T cells that recognize foreign epitopes. This is also consistent with what we have found previously with CD4T cells in healthy human volunteers ().

Because of their relatedness in evolution and as components of the immune system, it is of interest to compare the escape of self-specific αβ T cells to other lymphocyte lineages. Up to 20% of human mature circulating B cells are self-reactive and might contribute to natural antibody production (). In the case of mouse γδ T cells, Jensen et al. find that γδ T cells specific for the non-classical class I molecule T10 and the closely related T22, are not appreciably deleted in the thymi of non-transgenic mice expressing these antigens, despite previous results showing the extensive deletion of γδ TCR transgenic T cells having that specificity ().

As a result of these studies in mice, it became generally accepted that the deletion of self-specific αβ T cells is a very efficient mechanism for reducing the threat of autoimmunity (). This paradigm implies that peripheral tolerance regulates only a small number of escaping T lymphocytes that bind to self-antigen with low affinity. A further implication is that the efficient deletion of self-specific T cells will result in gaps in the universe of ligands recognizable by the TCR repertoire (). As a consequence, pathogens could make use of these immunologic blind spots to escape detection.

The clonal selection theory, associated most closely with the work of F. Macfarlane Burnet, provides a model for immunologic tolerance to self: lymphocytes only express antigen receptors of one specificity, and those lymphocytes specific for self are clonally deleted (). With respect to the control of self-specific helper and cytotoxic αβ T cells, mice have been the main experimental animal model used in support of this theory. Classic experiments by Kappler and Marrack showed that specific Vβ-expressing thymocytes were efficiently deleted in mouse strains, which expressed particular endogenous superantigens (). This was followed by a series of TCR transgenic studies in which it was shown that the presence of the relevant peptide-major histocompatibility complex (MHC) ligand of the TCR in the thymus led to massive thymocyte death by apoptosis at the double-positive stage (). Similar results were obtained in studies of TCR transgenics by other laboratories, including ours, where we found extensive thymic deletion of TCR-β-expressing transgenic thymocytes in a CD4system (). More recently, identification of the Aire gene has demonstrated how otherwise tissue-specific genes might be expressed in the thymus to precipitate the deletion of self-specific thymocytes ().

Projection of an immunological self shadow within the thymus by the aire protein.

To create a diverse repertoire of antigen receptors, maturing B and T lymphocytes bring together V, J, and, in some loci, D gene segments to form functional genes to express a very large number of immunoglobulin or T cell receptors (TCRs), respectively (). The semi-random process of V(D)J recombination not only generates antigen receptors with the ability to recognize foreign epitopes but also endogenously expressed self epitopes as well. The potential to mount an immune response against self must therefore be controlled in order to avoid autoimmune disease, an issue raised over 100 years ago by Paul Ehrlich ().

To address whether the CD8T cells specific for HCV variants detected by tetramer staining are able to mount a functional response, we generated T cell clones specific for the wild-type and L5M HCV epitopes, and also the L5N epitope, which was detected at lower frequency in comparison to other variants ( Figure 6 A and Table S7 ). When comparing within the same blood donor, we observed a similar sensitivity to a dose titration of the variant peptides by the CD107 mobilization assay ( Figures S6 A and S6B). We then generated JY target cells expressing the entire HCV NS3 protein containing the wild-type, L5M, or L5N epitope. We were again able to generate CD8T cell clones that preferentially killed the appropriate target cell for all three cases ( Figure 6 B).

Interestingly, at position 5, amino acid residues with ring structures tend to be recognized at a higher frequency than residues with charged or polar side chains. Because amino acids that have aromatic or ring-containing side chains are well known from structural studies to be able to form a wide variety of bonds in protein-protein interactions, this result suggests that they also have a greater propensity for TCR cross-reactivity, resulting in a higher frequency of T cells that recognize them (). In addition, a consistent pattern of frequency variation depending on the type of amino acid side-chain substitution is an additional validation of the accuracy of the tetramer enrichment method.

Tetramer enrichment of HLA-A0201PBMCs from blood bank donors demonstrated that CD8T cells recognizing the L5M variant are present at a comparable frequency to those recognizing the wild-type epitope, showing that previous difficulties in detecting T cells specific for this variant are likely due to a defect in clonal expansion during in vitro priming rather than a physical hole in the repertoire ( Figure 6 A). Furthermore, in the majority of blood samples tested, all the peptide variants are detectable, suggesting that the T cell repertoire can cover all or almost all possible peptide MHC combinations.

(B) Cytotoxicity assay (based on relative target cell survival): CD8T cell clones recognize endogenously processed and presented HCV WT or variant antigen. Preferential killing of JY target cells expressing HCV NS3 protein containing the target HCV variant peptide versus a negative control HCV variant peptide after 18 hr incubation with a 20-fold excess of CD8T cell clone. 95% confidence intervals shown for experiments performed in triplicate. WT indicates wild-type HCV epitope. The dotted line indicates upper 95% confidence interval of a negative control T cell clone (label: non HCV) that does not bind any of the HCV variant tetramers. (p < 0.025: indicates significant difference in killing from negative control.) See also Figure S6 for CD107 mobilization assay with HCV peptide titration curves and Table S7

(A) Frequency of CD8 + T cells recognizing position 5 amino acid substitutions of the HLA-A ∗ 0201 restricted peptide KLVALGINAV. Frequency of tetramer + CD8 + T cells per total CD8 + T cells calculated by tetramer enrichment. Each point represents one sample. Error bar indicates mean ( ∗ Tetramer + T cells not detected in one sample).

Our finding of an extensive pool of self-specific CD8T cells led us to reevaluate the effect of clonal deletion on ligand coverage by the TCR population as a whole. Gaps in the repertoire of epitopes recognized by T cells have been long been proposed to exist due to the effect of clonal deletion during thymic maturation, but it has not been possible previously to determine whether T cells of a particular specificity were actually deleted or just less able to respond to stimuli (). To investigate this further, we chose to focus on the HLA-A0201 restricted epitope KLVALGINAV from the HCV protein NS3. A leucine to methionine escape mutation (L5M) at position 5 seen in chronic HCV infection has been proposed to represent a hole in the T cell ligand repertoire (). We therefore made HLA-A0201 tetramers for this epitope and also for all possible amino acid variants at position 5, in order to systematically examine T cell epitope coverage. Because the aliphatic anchor residues for HLA-A0201 are at the ends of the peptide, these mutations are not expected to affect peptide binding to the MHC.

In each case, the foreign-specific pool of CD8T cells showed increased numbers of tetramercells and upregulation of IL2RA and IL21R ( Figures 5 C and S5 E). In contrast, the self-specific CD8T cells decreased in number after stimulation and showed no increase in IL2RA or IL21R staining. Samples from three of the five blood donors were also stained with carboxyfluorescein succinimidyl ester (CFSE): for all three, the foreign-specific CD8T cells lost CFSE staining, indicating that they had undergone proliferation, whereas the self-specific T cells retained high-intensity CFSE staining ( Figure 5 C). This experiment was repeated using a nonoverlapping set of self and foreign peptides ( Table S6 , bottom). The same result was seen in three more blood samples ( Figure S5 F). To determine whether the expanding cells were representative of the input mixture, we used combinatorial tetramer staining () of an eight peptide-MHC pool post stimulation to determine that at least five of eight foreign specificities were represented in the proliferating T cells in two individuals (data not shown). Thus self-specific human CD8T cells are anergic with respect to peptide stimulation, but foreign-specific CD8T cells are not.

We then compared the response of self- versus foreign-specific primary human CD8T cells to a longer period of in vitro stimulation with peptide antigen and anti-CD28 antibody. PBMCs from five human blood donors were each stained with two pools of HLA-A0201 tetramers: one pool loaded with six self peptides, the other with six foreign peptides to which the blood donors should be naive ( Table S6 , top). We used tetramer pools so that there would be a sufficient number of antigen-specific cells for a robust readout. In addition, we examined the protein expression of IL2RA and IL21R, because these molecules are easily detected on the cell surface by fluorescent antibody. An equal number of self- versus foreign-specific CD8T cells from each donor was sorted into separate wells containing autologous PBMCs. The sorted cells were then stimulated with the same pool of peptides used for tetramer sorting as well as anti-CD28 antibody, and analyzed by flow cytometry after either 4.5 or 7.5 days.

Twelve genes showed a difference in the likelihood that they would be expressed between women and men with p values < 0.05 ( Figure 5 A). With the exception of IL10RA, each gene was detected in a greater proportion of naive CD8T cells versus self-specific cells. We then clustered these genes based on correlated coexpression at the single cell level. In women, but not in men, a statistically significant group of four genes was found to be expressed together (p = 0.025, Figures 5 B and S5 B and S5C). Three of these genes, IL2RA, IL21R, and BCL2L1 (also known as BCLXL), are associated with T cell proliferation and survival (). IL2 transcripts were not detected at the single cell level, most likely due to the inefficiency of the PCR primers, as transcripts were only reliably detected in stimulated CMV-specific memory CD8T cells after increasing the initial amount of cell lysate (data not shown). To control for the influence of gender, we compared CD8T cells from men and women stimulated with either a self peptide (PPI) or foreign peptide (HIV) and observed no consistent statistically significant gender-based differences ( Figure S5 D). Details of these experiments are in the Supplemental Experimental Procedures

(C) Cell expansion after peptide + anti-CD28 antibody stimulation of foreign-, but not self-specific CD8T cells. Equal numbers of tetramerCD8T cells, labeled by pooled tetramers loaded with either self or foreign peptides ( Table S6 , top), were sorted from a single blood sample into separate wells. Approximately 267 sorted cells for each group (self or foreign) were then stimulated in the presence of autologous feeder PBMCs with the same peptides with which they were tetramer selected at 1.5 μg/ml and with anti-CD28 antibody at 5 μg/ml. After 4.5 days, each sample was analyzed by flow cytometry. Panels gated on live CD8T cells. Sample in this figure is representative of five experiments, analyzed at 4.5 and/or 7.5 days; three of the five experiments included CFSE staining. See also Figures S5 E and S5F and Table S6

(B) Coordinated mRNA expression of genes associated with proliferation and survival in female SMCY specific CD8 + T cells but not in males. Two-way clustered heatmaps of the correlation matrices for differentially expressed genes (p < 0.05 and q < 0.15) between male (top) and female (bottom) cells stimulated 14 hr with SMCY antigen and anti-CD28 antibody. Approximate value of correlation coefficient (R) indicated by color; 1.0 (yellow) indicates perfect correlation in the expression of two different genes on a per cell basis.

(A) Microfluidics based qPCR was performed individually on single CD8 + T cells binding the SMCY:HLA-A ∗ 0201 tetramer. Top shows 152 cells from four males and 154 cells from four females (no history of pregnancy) that were analyzed. Cells were stimulated 14 hr with SMCY peptide and anti-CD28 antibody. Only genes differentially expressed between women and men with p < 0.05 and q < 0.15 are shown. Bottom shows fold change in gene expression after stimulation. qPCR was performed on unstimulated single CD8 + T cells binding the SMCY:HLA-A ∗ 0201 tetramer. 41 cells from one man and 38 cells from one woman were used in comparison to calculate fold difference in gene expression after 14 hr stimulation. Fold change could not be calculated for genes that were not expressed in unstimulated cells. Error bars represent SD.

Given their similarities in ligand sensitivity, we hypothesized that self-specific CD8T cells might be kept in check by tolerance mechanisms characterized by distinct patterns of gene expression. A precedent for this has been found in the case in γδ T cells, where self- and non-self-specific cells express different cytokines (). Therefore, we examined the single cell gene expression of 96 genes in male and female SMCY-specific T cells using a microfluidics-based qPCR ( Table S5 ) (). We primarily chose cytokines, cytokine receptors, and genes involved with cell survival because we reasoned that these might be affected in a tolerant cell. After a 14 hr ex vivo stimulation with peptide and anti-CD28 antibody, 152 CD8T cells from four men and 154 cells from four women (with no history of pregnancy) were examined (example qPCR run, Figure S5 A). Among those genes for which for transcript was detectable, the cycle threshold value was similar between cells (data not shown). Therefore, genes were considered expressed or not in a given cell depending on whether detectable transcript levels were measured, and only cells expressing detectable levels of the housekeeping gene GAPDH were considered for analysis. ICOS, an activation inducible gene, was expressed in 52% to 62% of cells; a similar heterogeneity in ICOS expression (∼60%) was seen in CMV:HLA-A0201 tetramer positive memory CD8T cells stimulated similarly (data not shown).

The CD8 coreceptor is known to contribute to peptide MHC-TCR binding (). To determine whether CD8 differentially contributes to SMCY binding by T cells in males versus females, we tested the ability of 9 female (donor ID 38) and 14 male T cell clones (donor ID 61) to bind SMCY HLA-A0201 wild-type tetramer versus a D227K/T228A CD8 binding mutant of the same MHC molecule. Tetramer binding was dependent on CD8 in both females and males ( Figure S4 A). In addition, a large and similar degree of CD8 dependence between self and non-self was observed when performing peptide-MHC tetramer enrichment with pools of 11 or more self or foreign peptides ( Table S4 and Figures S4 B and S4C).

We then performed a CD107 mobilization assay on SMCY-specific CD8T cell clones from five women and five men. CD107a, or lysosomal associated membrane protein-1, is transiently expressed on the cell surface of CD8T cells during the release of cytotoxic granules () and thus represents a functional response. The sensitivity to SMCY antigen ranged from 10M to 10M in both men and women ( Figures 4 A, S2 A and S2B, Table S3 ). To determine whether the SMCY tetramer binding clones are able to react to endogenously processed and presented antigen, we incubated male and female derived T cell clones with either male (JY) or female (OH) HLA-A 0201, EBV-transformed B lymphoblastoid target cells. SMCY specific T cells clones from both genders preferentially killed male target cells with similar efficiency in two different assay types ( Figures 4 B and S3 A–S3E). We conclude that SMCY tetramer binding CD8T cells from men express TCRs with a ligand sensitivity that overlaps with females and are able to recognize endogenously expressed antigen.

(B) Propidium iodide (PI) cytotoxicity assay. Graphs show the percentage of PIJY (male) target cells after incubation with SMCY-specific CD8T cell clones at the indicated effector to target (E/T) ratios (symbols with black lines). Representative clones are from females ID 38 and 23 (bottom), and male ID 61 (top). A nonspecific CD8T cell clone derived from the same individuals is shown in each panel (gray “x” symbols). The gray dotted line indicates the background level of JY cell death in the absence of T cell clones. Performed in duplicate and representative of three to six experiments. The bottom panel combines data from two experiments; for that panel, values for the non HY T cell clone control and background target cell death were averaged. See also Figures S3 C–S3E for additional clone and controls. An alternate cytotoxicity assay based on relative target cell survival is shown in Figures S3 A and S3B.

(A) CD107 mobilization assay performed on SMCY specific CD8T cell clones pulsed with 10M SMCY peptide followed by tenfold dilutions. Each line represents one distinct T cell clone. None of the clones responded to 10M negative control peptide (PPI) by this assay. T cell clones derived from female ID 38 (bottom) and male ID 61 (top). See also Figures S2 A and S2B and Table S3 for more clones.

We found that, ex vivo, CD8T cells from men tended to have an intensity of SMCY tetramer staining that overlapped extensively with that of women, but this intensity distribution was depleted of cells in males, consistent with clonal deletion ( Figures 2 D and 3 A and 3B ). After in vitro expansion, we reassayed T cell clones for SMCY tetramer staining and found a similar difference in the distribution of intensities between women and men, indicating that we had recovered a representative sample of the original population ( Figure 3 C). We also measured the distribution of TCR Vβ gene segment families in SMCY specific T cell clones by antibody staining and found a comparable diversity of Vβ usage in women and men, indicating that the high frequency of self-specific CD8T cells in humans is not due to the peripheral expansion of a few escaping clones ( Figure 3 D). In addition, the Vβ usage of SMCY T cells was skewed in comparison with bulk peripheral blood T cells, consistent with the former being a subpopulation of antigen-specific T cells ().

(D) Pie chart showing TCR Vβ family expression of in vitro expanded SMCY:HLA-A0201 binding CD8T cell clones from four men (IDs 61, 69, 74, 390) and four women (IDs 38, 67, 84, 86). Vβ antibody panel from Beckman Coulter. See also Table S3

(C) MFI of SMCY CD8T cell clones indicates that they are representative of the original population. Plot of MFI for SMCY:HLA-A0201 multimer binding CD8T cell clones after in vitro expansion from one woman with no history of pregnancy (bottom, ID 38) and one man (top, ID 61). Each point represents one distinct T cell clone. Compare with relative fluorescence intensity of primary male or female derived CD8T cells in Figures 3 A and 3B.

(A) FACS plots gated for CD8 + T cells from one woman with no history of pregnancy (bottom, ID 38) and one man (top, ID 61) after tetramer enrichment. Gates shown for single cell FACS of SMCY:HLA-A ∗ 0201 binding CD8 + T cells used for in vitro expansion of T cells clones.

To test whether CD8T cells from men express TCR of sufficient affinity for SMCY to be functionally competent, we used single cell sorting and in vitro expansion with anti-CD3/CD28 or phytohemagglutinin (PHA) stimulation—both strong stimuli—to generate SMCY-specific T clones from five women and five men, and then compared their functional avidity using the CD107 mobilization assay ( Table S3 ) ().

To determine whether this is also true in non-transgenic mice, we analyzed H-Y specific CD8T cells, in which the peptide KCSRNRQYL, derived from the Y chromosome encoded protein SMCY3, is presented by H2-D). The frequency of these CD8T cells was determined using the same tetramer enrichment method for C57BL/6 male and female mice (five each). There was a trend toward a higher frequency of SMCY3-specific CD8T cells in females in comparison to males, but this difference was not statistically significant ( Figure 2 C). This result corroborates the human results in that a significant fraction of self-specific T cells escape clonal deletion. Thus this is not a species-specific difference.

To directly test the effects of deletional tolerance in humans, we measured the frequency of CD8T cells recognizing a Y chromosome-specific SMCY (the H-Y equivalent) epitope in male and female HLA-A0201blood donors ( Table 1 and Figure 2 C) (). In nine men, the frequency of SMCY-specific CD8T cells ranged from ∼1:80k to ∼1:800k with a mean frequency of 1 in 2 × 10CD8T cells ( Figure 2 C). This falls well within the range of frequencies seen for both self and foreign antigens ( Figure 2 A) and represents a substantial number of T cells with this specificity in an average adult male (∼10). Among eight women, the frequency of SMCY-specific T cells ranged from 1 in 5 × 10to 1 × 10with the exception of one individual who had recently given birth to a son and had a frequency of 1 in 3.5 × 10 Figure 2 C and data not shown). In this latter case, 80% of these cells were CD45RA, suggesting that the exceptionally high frequency of these cells was due to the expansion of these T cells after exposure to SMCY antigen from the male fetus. In the remaining seven women, for whom we do not have a complete reproductive history, the average frequency of CD8T cells recognizing the SMCY epitope was approximately 1 in 6.8 × 10, approximately 3-fold greater than in men. This indicates that a large fraction (1/3) of SMCY-specific T cells escape clonal deletion in males.

Of all epitopes analyzed, preproinsulin was recognized by CD8T cells at the highest frequency, up to 1:10in healthy blood bank donors. Preproinsulin is of particular interest because it is associated with type 1 diabetes mellitus (T1D) (). To determine whether there is expansion of PPI-specific CD8T cells in T1D, we measured their frequency in the peripheral blood of five HLA-A0201individuals with T1D and five age-matched controls. We found a 2.66-fold increase in CD8T cells specific for the PPI epitope in T1D individuals (p = 0.0079), consistent with results seen by other groups ( Figure 2 B and Table S2 ) (). It is tempting to speculate that in those susceptible to diabetes, even a partial failure of peripheral tolerance might permit the activation and expansion of some cells in this pool, making this a possible risk factor in this disease.

To explore the differentiation status of these antigen-specific cells, we assessed the surface expression of the molecules CCR7, CD27, CD28, and CD45RA, which are reported to be expressed by naive CD8T cells (). Some degree of lowered expression in at least one of these four surface molecules was seen in 57% of the antigen-specific CD8T cell populations recognizing one of the four foreign epitopes and 47% of those recognizing endogenous antigens ( Table S1 and Figure S1 C). This result suggests that, similarly to recent work on human CD4T cells, there might be a significant degree of TCR crossreactivity to some other antigen or antigens that the subjects have been exposed to ().

Next we examined the frequency of CD8T cells recognizing self, as opposed to foreign, peptides bound to HLA-A0201 tetramers. We chose two endogenous epitopes derived from fructose bisphosphate aldolase (FBA) and keratin (KER) (), as well as two autoimmune disease-associated epitopes derived from preproinsulin (PPI) and glutamic acid decarboxylase (GAD) 65 ( Table 1 and Figure 1 B) (). In a few cases, self-specific CD8T cells were not detectable. Unexpectedly, however, in the majority of cases the frequency of CD8T cells recognizing these four endogenously expressed epitopes was 1:10to 1:10—in the same range as CD8T cells recognizing the foreign antigens ( Figure 2 A). In addition, the intensity of tetramer staining for the self epitopes was robust and comparable to that for foreign epitopes ( Figure 1 B) and therefore most consistent with agonist level TCR affinities ().

Several controls were performed to confirm the accuracy of the tetramer enrichment method: (1) Minimal double labeling was observed when PBMCs were incubated with two HLA-A0201 tetramers loaded with distinct peptide epitopes ( Figure 1 ); (2) Spiking experiments demonstrated that when present at a starting frequency of 1 in 5,400,000 total CD8T cells, 80% of antigen specific cells could be recovered by tetramer enrichment ( Figure S1 A); and (3) Tetramer-enriched T cells were also sorted as single cells by fluorescence activated cell sorter (FACS) and expanded in vitro. 46% to 60% of the clones that grew bound tetramer upon reanalysis ( Figure S1 B).

In order to establish a benchmark comparison, we first determined the frequency of naive antigen-specific CD8T cells in healthy adults. We performed peptide-MHC tetramer enrichment using HLA-A0201 tetramers containing peptides derived from cytomegalovirus (CMV), human immunodeficiency virus (HIV), hepatitis C virus, and the avian influenza A (H5N1) virus on peripheral blood mononuclear cells (PBMCs) obtained from HLA-A0201blood bank donors (seronegative for antibodies against HIV, HCV, and CMV and very unlikely to have been infected by the H5N1 avian influenza virus; Figures 1 A and 1B , Table 1 ) (). The frequency of the CD8T cells recognizing the foreign antigens ranged from 1:10to 1:10in comparison to total CD8T cells ( Figure 2 A). This agrees roughly with the naive T cell frequencies measured in mice (1:10–1:10) and correlates well with the values reported recently in humans ().

(D) Histograms showing the intensity distribution of SMCY:HLA-A ∗ 0201 tetramer fluorescence on CD8 + T cells in men (gray) and women (blue). Histogram areas are normalized for the relative frequency of H-Y + CD8 + T cells per total CD8 + T cells for each blood donor.

(C) Frequency of SMCY peptide-specific CD8 + T cells in males versus females. Tetramer enrichment was used to calculate the frequency of tetramer + CD8 + T cells per total CD8 + T cells. Each point represents one human blood sample or one mouse. Error bar indicates mean. p value calculated using Mann-Whitney test. Left shows frequency of SMCY:HLA-A ∗ 0201 binding CD8 + T cells in men versus women ( ∗ One blood sample from a female with a frequency of 1 in 3.5 × 10 2 was not included; see main text). Right shows frequency of SMCY3:H2-D b binding CD8 + T cells in male mice versus female mice.

(B) Frequency of PPI peptide-specific CD8T cells in HLA-A0201individuals with type 1 diabetes mellitus (T1D) versus controls. HLA-A0201 tetramer enrichment was used to calculate the frequency of tetramerCD8T cells per total CD8T cells in whole blood. Each point represents one human blood sample. Error bar indicates mean. p value calculated using Mann-Whitney test. See also Table S2

(A) Frequency of CD8T cells binding foreign versus self peptide HLA-A0201 tetramers (human blood). The frequency of tetramerCD8T cells per total CD8T cells was determined using tetramer enrichment. Each point represents one sample from a separate individual. Error bar indicates mean (GAD tetramerT cells not detected in two samples). See also Table 1

List of epitopes used for peptide-MHC tetramer enrichment of CD8T cells from human blood bank donor PBMCs in Figure 2 . See text for references.

(A) Flow cytometry gating scheme. PBMCs from a HLA-A ∗ 0201 + blood donor were concentrated for CD8 + T cells by depletion, followed by HIV:HLA-A ∗ 0201 tetramer enrichment over a magnetized column before flow cytometric analysis. Dump channel includes cells labeled with antibodies against CD4, CD14, CD16, CD19, and γδ TCR. In this case, the PE-Cy5 peptide HLA-A ∗ 0201 tetramer was only used as control for peptide MHC specific binding.

The Frequencies of CD8 + T Cells Specific for Self and Non-Self Are Similar in Healthy Adults

Discussion

+ T cells are so abundant in the peripheral blood of healthy adult people. We make this conclusion after making over 40 separate measurements from blood bank donors to determine the frequency of CD8+ T cells recognizing four self and four foreign epitopes (not including SMCY). These self-specific T cells stained robustly with tetramers at an intensity generally consistent with agonist level affinities (Kd ∼100 to 1 μM) ( Savage et al., 1999 Savage P.A.

Boniface J.J.

Davis M.M. A kinetic basis for T cell receptor repertoire selection during an immune response. + T cells overlap broadly with female-derived SMCY-specific clones in their functional avidity to both pulsed and endogenously presented antigen. This is in addition to published data showing that even CD8+ T cells with lower affinity for endogenous antigens can be drawn into immune responses in the setting of inflammation ( Zehn et al., 2009 Zehn D.

Lee S.Y.

Bevan M.J. Complete but curtailed T-cell response to very low-affinity antigen. Enouz et al., 2012 Enouz S.

Carrié L.

Merkler D.

Bevan M.J.

Zehn D. Autoreactive T cells bypass negative selection and respond to self-antigen stimulation during infection. + T cells are found at a lower frequency and have a pattern of gene transcript expression that is distinct in comparison to the equivalent cells in females. In addition, we found that self-specific, primary CD8+ T cells are resistant to activation ex vivo in comparison to foreign-specific cells. Perhaps the most surprising result in our study is that self-specific CD8T cells are so abundant in the peripheral blood of healthy adult people. We make this conclusion after making over 40 separate measurements from blood bank donors to determine the frequency of CD8T cells recognizing four self and four foreign epitopes (not including SMCY). These self-specific T cells stained robustly with tetramers at an intensity generally consistent with agonist level affinities (Kd ∼100 to 1 μM) (). We surveyed 9 additional self epitopes and 11 foreign epitopes with comparable results (See Supplemental Information ). Using the H-Y system, we showed that clones derived from male SMCY (and therefore self-) specific CD8T cells overlap broadly with female-derived SMCY-specific clones in their functional avidity to both pulsed and endogenously presented antigen. This is in addition to published data showing that even CD8T cells with lower affinity for endogenous antigens can be drawn into immune responses in the setting of inflammation (). We wish to emphasize that our results are not consistent with immunologic ignorance. Specifically, we showed that male-derived SMCY-specific CD8T cells are found at a lower frequency and have a pattern of gene transcript expression that is distinct in comparison to the equivalent cells in females. In addition, we found that self-specific, primary CD8T cells are resistant to activation ex vivo in comparison to foreign-specific cells.

Bouneaud et al., 2000 Bouneaud C.

Kourilsky P.

Bousso P. Impact of negative selection on the T cell repertoire reactive to a self-peptide: a large fraction of T cell clones escapes clonal deletion. Zehn and Bevan, 2006 Zehn D.

Bevan M.J. T cells with low avidity for a tissue-restricted antigen routinely evade central and peripheral tolerance and cause autoimmunity. + T cells, from unmanipulated humans, that stain with tetramers at an intensity consistent with TCR ligand agonists ( Savage et al., 1999 Savage P.A.

Boniface J.J.

Davis M.M. A kinetic basis for T cell receptor repertoire selection during an immune response. + T cell clones from both men and women exhibited very similar functional responsiveness to antigen. It is relevant to note two reports by Bousso and Bevan and their colleagues, in which they analyzed two different TCR-β chain transgenic mouse systems that bias T cells toward the recognition of an endogenous antigen (SMCY3 and ovalbumin, respectively) (). In both systems, T cells specific for the endogenous antigen persist, yet have an approximately 100-fold reduction in functional avidity. Our experimental system differs in that we directly observed multiple self-antigen-specific CD8T cells, from unmanipulated humans, that stain with tetramers at an intensity consistent with TCR ligand agonists (). In addition, in the case of the SMCY epitope, CD8T cell clones from both men and women exhibited very similar functional responsiveness to antigen.

von Boehmer and Kisielow, 2006 von Boehmer H.

Kisielow P. Negative selection of the T-cell repertoire: where and when does it occur?. McCaughtry et al., 2008 McCaughtry T.M.

Baldwin T.A.

Wilken M.S.

Hogquist K.A. Clonal deletion of thymocytes can occur in the cortex with no involvement of the medulla. + regulatory T cell lineage has been reported for particular TCR transgenes when their proportion in the thymus exceeds 5% or 1%, respectively ( Huesmann et al., 1991 Huesmann M.

Scott B.

Kisielow P.

von Boehmer H. Kinetics and efficacy of positive selection in the thymus of normal and T cell receptor transgenic mice. Bautista et al., 2009 Bautista J.L.

Lio C.W.

Lathrop S.K.

Forbush K.

Liang Y.

Luo J.

Rudensky A.Y.

Hsieh C.S. Intraclonal competition limits the fate determination of regulatory T cells in the thymus. + T cells might similarly depend on a finite number of niches. One explanation for why so many of the early mouse studies on negative selection in the thymus showed massive deletion could be that TCR transgenes almost always originated from T cell clones that were the best responders to a given antigen, which might have predisposed them to clonal deletion. In addition, in comparison to unmanipulated mice, the introduction of α and β TCR transgenes is known to increase the surface density of TCRs and also results in the early expression of mature TCR at the double-negative (DN) thymocyte stage (). This shift in timing can precipitate lineage diversion and impair proliferation between the DN and double-positive thymocyte stages. Such differences might impact negative selection, although in a H-Y TCR transgenic mouse model with appropriately timed expression of the TCR-α chain, comparable clonal deletion was seen (). A final consideration is the effect of repertoire skewing in transgenic mice. A reduced efficiency in positive selection or diversion into the CD4regulatory T cell lineage has been reported for particular TCR transgenes when their proportion in the thymus exceeds 5% or 1%, respectively (). A limiting number of environmental niches has been proposed as a basis for this phenomenon. The survival and fate of self-specific CD8T cells might similarly depend on a finite number of niches.

Wardemann et al., 2003 Wardemann H.

Yurasov S.

Schaefer A.

Young J.W.

Meffre E.

Nussenzweig M.C. Predominant autoantibody production by early human B cell precursors. Jensen et al., 2008 Jensen K.D.

Su X.

Shin S.

Li L.

Youssef S.

Yamasaki S.

Steinman L.

Saito T.

Locksley R.M.

Davis M.M.

et al. Thymic selection determines gammadelta T cell effector fate: antigen-naive cells make interleukin-17 and antigen-experienced cells make interferon gamma. + T cells ( Mallone et al., 2005 Mallone R.

Kochik S.A.

Reijonen H.

Carson B.

Ziegler S.F.

Kwok W.W.

Nepom G.T. Functional avidity directs T-cell fate in autoreactive CD4+ T cells. + T cells that recognize an antigen expressed endogenously by a transgene in the mouse ( Moon et al., 2011 Moon J.J.

Dash P.

Oguin 3rd, T.H.

McClaren J.L.

Chu H.H.

Thomas P.G.

Jenkins M.K. Quantitative impact of thymic selection on Foxp3+ and Foxp3- subsets of self-peptide/MHC class II-specific CD4+ T cells. + T cells in healthy people, similar to a population previously reported by Romero and colleagues ( Pittet et al., 1999 Pittet M.J.

Valmori D.

Dunbar P.R.

Speiser D.E.

Liénard D.

Lejeune F.

Fleischhauer K.

Cerundolo V.

Cerottini J.C.

Romero P. High frequencies of naive Melan-A/MART-1-specific CD8(+) T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals. Maeda et al., 2014 Maeda Y.

Nishikawa H.

Sugiyama D.

Ha D.

Hamaguchi M.

Saito T.

Nishioka M.

Wing J.B.

Adeegbe D.

Katayama I.

Sakaguchi S. Detection of self-reactive CD8+ T cells with an anergic phenotype in healthy individuals. Our results parallel the developmental observations made in other lymphocyte lineages. In the B cell lineage, 20% of mature B cells are reported to recognize self antigen in the periphery in humans (). Jensen et al. demonstrate that self-specific γδ T cells are not deleted in the thymus of normal non-transgenic mice, in contrast to earlier work using TCR transgenic γδ T cells (). Our results are also consistent with the work of Nepom and colleagues in CD4T cells (), as well as the work of Jenkins and colleagues, who show that the clonal deletion of CD4T cells that recognize an antigen expressed endogenously by a transgene in the mouse () is incomplete, with approximately one third of the specific T cells migrating to the periphery. Recently, Sakaguchi and colleagues reported the presence of anergic MART-1 specific CD8T cells in healthy people, similar to a population previously reported by Romero and colleagues ().

+ T cells, how are they kept in check? Our results showed that self-specific CD8+ T cells are significantly anergic compared to foreign-specific cells, although this can be overcome in vitro by anti-CD3 and anti-CD28 crosslinking combined with interleukin-2 (IL-2). The resistance of self-specific CD8+ T cells to activation was indicated on the transcript level with single cell qPCR in the H-Y system, in which we found the preferential and correlated expression of IL2RA, IL21R, and BCL2L1 (i.e., BCLXL)—genes associated with survival and proliferation—in ex vivo stimulated T cells from women as compared to men. This phenotype was then confirmed functionally by following stimulated, antigen-specific CD8+ T cells that had been sorted using pooled tetramers over several days, and observing proliferation and the protein expression of IL2RA and IL21R in foreign-specific T cells, but not in self-specific cells. Interestingly, IL-2 is required for T cell proliferation, and lack of signaling through the IL-2 receptor has been associated with T cell tolerance, whereas the expression of BCLXL is associated with increased cell survival ( Smith, 1988 Smith K.A. Interleukin-2: inception, impact, and implications. Boise et al., 1995 Boise L.H.

Minn A.J.

Noel P.J.

June C.H.

Accavitti M.A.

Lindsten T.

Thompson C.B. CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-XL. Choi and Schwartz, 2007 Choi S.

Schwartz R.H. Molecular mechanisms for adaptive tolerance and other T cell anergy models. Zeng et al., 2005 Zeng R.

Spolski R.

Finkelstein S.E.

Oh S.

Kovanen P.E.

Hinrichs C.S.

Pise-Masison C.A.

Radonovich M.F.

Brady J.N.

Restifo N.P.

et al. Synergy of IL-21 and IL-15 in regulating CD8+ T cell expansion and function. McGuire et al., 2009 McGuire H.M.

Vogelzang A.

Hill N.

Flodström-Tullberg M.

Sprent J.

King C. Loss of parity between IL-2 and IL-21 in the NOD Idd3 locus. + T cells might be maintained by a protective gene program induced through interactions with any number of cell types in the periphery, including regulatory T cells. Alternatively, such a gene program might be imprinted centrally in the thymus, as both γδ T cells and regulatory CD4+ T cells seem subject to thymic imprinting as well ( Jensen et al., 2008 Jensen K.D.

Su X.

Shin S.

Li L.

Youssef S.

Yamasaki S.

Steinman L.

Saito T.

Locksley R.M.

Davis M.M.

et al. Thymic selection determines gammadelta T cell effector fate: antigen-naive cells make interleukin-17 and antigen-experienced cells make interferon gamma. Wing and Sakaguchi, 2010 Wing K.

Sakaguchi S. Regulatory T cells exert checks and balances on self tolerance and autoimmunity. Given that we detect such a large pool of self-specific CD8T cells, how are they kept in check? Our results showed that self-specific CD8T cells are significantly anergic compared to foreign-specific cells, although this can be overcome in vitro by anti-CD3 and anti-CD28 crosslinking combined with interleukin-2 (IL-2). The resistance of self-specific CD8T cells to activation was indicated on the transcript level with single cell qPCR in the H-Y system, in which we found the preferential and correlated expression of IL2RA, IL21R, and BCL2L1 (i.e., BCLXL)—genes associated with survival and proliferation—in ex vivo stimulated T cells from women as compared to men. This phenotype was then confirmed functionally by following stimulated, antigen-specific CD8T cells that had been sorted using pooled tetramers over several days, and observing proliferation and the protein expression of IL2RA and IL21R in foreign-specific T cells, but not in self-specific cells. Interestingly, IL-2 is required for T cell proliferation, and lack of signaling through the IL-2 receptor has been associated with T cell tolerance, whereas the expression of BCLXL is associated with increased cell survival (). The IL-21 pathway in turn contributes to cytotoxic T cell expansion and has been linked to the pathogenesis of diabetes mellitus in the NOD mouse (). It is tempting to speculate that the inertia against activation in self-specific CD8T cells might be maintained by a protective gene program induced through interactions with any number of cell types in the periphery, including regulatory T cells. Alternatively, such a gene program might be imprinted centrally in the thymus, as both γδ T cells and regulatory CD4T cells seem subject to thymic imprinting as well ().

+ T cells can be found at a relatively high frequency in healthy individuals and especially so in type 1 diabetics. In mice, proinsulin, the proteolytic product of preproinsulin, is proposed as an early target in the epitope spreading hierarchy of NOD diabetes ( Nakayama et al., 2005 Nakayama M.

Abiru N.

Moriyama H.

Babaya N.

Liu E.

Miao D.

Yu L.

Wegmann D.R.

Hutton J.C.

Elliott J.F.

Eisenbarth G.S. Prime role for an insulin epitope in the development of type 1 diabetes in NOD mice. Kent et al., 2005 Kent S.C.

Chen Y.

Bregoli L.

Clemmings S.M.

Kenyon N.S.

Ricordi C.

Hering B.J.

Hafler D.A. Expanded T cells from pancreatic lymph nodes of type 1 diabetic subjects recognize an insulin epitope. Martinuzzi et al., 2008 Martinuzzi E.

Novelli G.

Scotto M.

Blancou P.

Bach J.M.

Chaillous L.

Bruno G.

Chatenoud L.

van Endert P.

Mallone R. The frequency and immunodominance of islet-specific CD8+ T-cell responses change after type 1 diabetes diagnosis and treatment. Skowera et al., 2008 Skowera A.

Ellis R.J.

Varela-Calviño R.

Arif S.

Huang G.C.

Van-Krinks C.

Zaremba A.

Rackham C.

Allen J.S.

Tree T.I.

et al. CTLs are targeted to kill beta cells in patients with type 1 diabetes through recognition of a glucose-regulated preproinsulin epitope. The presence of an abundant pool of self-specific peripheral T cells—as opposed to their elimination by clonal deletion—is important because it further shifts burden of maintaining tolerance to other mechanisms that must function for the life of the individual. For this reason, we find it significant that preproinsulin reactive CD8T cells can be found at a relatively high frequency in healthy individuals and especially so in type 1 diabetics. In mice, proinsulin, the proteolytic product of preproinsulin, is proposed as an early target in the epitope spreading hierarchy of NOD diabetes (). In humans, insulin and preproinsulin is proposed to be an immunologic target associated with T1D patients (). It is possible that the presence of a substantial pool of peripheral insulin reactive T cells contributes to an increased risk of developing T1D when other mechanisms of tolerance break down.