Primary CTL response magnitude in mice is determined by the extent of naive T cell recruitment and subsequent clonal expansion (original) (raw)
Naive CTLp frequencies and the immunodominance hierarchy. The characteristic influenza-specific immunodominance hierarchy in virus-infected B6 mice is shown in Figure 1A for the DbPA224-, DbNP366-, DbPB1-F262–, and KbNS2114-specific CD8+ CTL populations. As described previously (23, 24), the DbPA224-specific T cells are at highest prevalence early (days 7 and 8), with the DbNP366-specific CTLs becoming more prominent by day 10 after infection and the DbPB1-F262- and KbNS2114-specific sets remaining subdominant throughout (ref. 25 and Figure 1A). Importantly, all 4 tetramers detect responding CTLs at frequencies equivalent to those determined for peptide-stimulated T cell populations by the intracellular cytokine staining (ICS) assay (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI41538DS1). There is thus no substantial divergence in binding efficiency or sensitivity that might be thought to skew the results for the different tetramers.
Influenza epitope–specific immune magnitudes do not correlate with naive precursor frequencies. (A) Naive B6 mice were infected i.n. with influenza virus and spleens harvested 7, 8, or 10 days later for analysis of CD8+ DbNP366-, DbPA224-, DbPB1-F262–, and KbNS2114-specific T cell responses. Shown are the mean total splenic numbers of CD8+tetramer+ cells for 4–5 mice ± SD. Results are representative of 2 independent experiments. (B) Representative dot plots of all DbNP366-, DbPA224-, DbPB1-F262–, and KbNS2114-specific CD8+ T cells detected from spleen and all major LNs of naive mice using a magnetic enrichment and staining procedure (as described in Methods). Values indicate the number of tetramer+CD62Lhi cells within the gate shown. (C) Total numbers of epitope-specific CD8+ cells identified from spleen and all major LNs in naive B6 mice or OTI TCR Tg mice. Symbols represent data from individual mice obtained in 4 independent experiments. Horizontal bars indicate mean values.
The tetramer enrichment protocol (14) was used to detect the 4 naive (CD62Lhi) CTLp populations in pooled spleen and LNs from individual, uninfected B6 mice. Shown are the total numbers of epitope-specific cells identified from spleen and all major LNs (Figure 1, B and C), with similar relative values being obtained when these numbers were calculated as frequencies per 106 CD8+ T cells (Supplemental Figure 2). Uninfected B6 mice expressing a Tg TCR (OTI) specific for the ovalbumin 257–264 epitope were used as controls. Minimal evidence of tetramer binding was found for 3 separate OTI TCR Tg samples, with the mean counts being less than 4 for all the pMHCI epitopes (Figure 1C). The average number of naive CD8+DbPA224+ CTLps was 2-fold larger (P < 0.005) than the CD8+DbNP366+ set (68 ± 18 compared with 36 ± 21) (Figure 1C), confirming previous, indirect estimates of naive DbNP366- and DbPA224-specific precursor prevalence (10, 26, 27). Thus, we would conclude that the relative magnitudes and kinetics of the immune CD8+DbNP366+ and CD8+DbPA224+ CTL responses (Figure 1A) are, at least in the initial phase after virus challenge, indeed determined by the naive CTLp frequency (Figure 1C). However, no comparable correlation was apparent for the 2 subdominant epitopes, DbPB1-F262 and KbNS2114. The mean numbers of DbPB1-F262- (225 ± 110) and KbNS2114-specific (282 ± 46) CTLps were significantly higher than those found for either DbNP366 or DbPA224 (P < 0.0001) (Figure 1, B and C), establishing for the first time that there can be a substantial disconnect between naive CTLp frequency and immune magnitude after infection.
Evaluation of CTLp recruitment by TCR Vβ phenotyping. A possible explanation for this discrepancy between precursor frequency and immune magnitude for immunodominant and subdominant CTL populations is that there are differential profiles of recruitment from the various naive CTLp pools, which may be illuminated by the analysis of TCR usage in naive and immune T cell repertoires. The TCR is an αβ-heterodimer with each chain encoded by somatic gene segments that recombine during T cell development. Multiple variable (V), junctional (J), and constant (C) gene segments can combine to form the TCR-α chains, while multiple V, diversity (D), J, and C segments encode for the TCR-β chains. The TCR-α and -β chains have 3 regions of hypervariability, complementarity determining regions (CDR) 1, 2, and 3, which form the antigen-binding site. The CDR1 and CDR2 loops are encoded by the germline V gene segment, while the CDR3 loop is positioned at the junction of the V(D)J segments (28). Diversity within the TCR repertoire is generated via (a) different VJ or VDJ gene segment combinations; (b) pairing of different TCR-α and -β chains; (c) imprecise joining of gene segments; and (d) the addition of nontemplate encoded nucleotides at V(D)J junctions. Given that most of the observed TCR diversity results from imprecise joining of gene segments and the addition of nontemplate encoded nucleotides (29), TCR diversity is heavily focused toward the CDR3 regions. These loops also mediate significant contacts with the peptide, so the analysis of CDR3 sequences can be thought to reflect the fine specificity of T cells (25, 30, 31). Considerable efforts have been made previously to define profiles of TCR Vβ (TRBV) usage in the DbNP366-, DbPA224-, and DbPB1-F262-specific immune repertoires, first by mAb staining, then by CDR3β sequencing (10, 25). The TRBVs (IMGT nomenclature) (32) that are most prominent in these antigen-driven responses (TRBV13-1 for DbNP366, ref. 33; TRBV29 for DbPA224, ref. 34; TRBV19 for DbPB1-F262, ref. 25; and TRBV29 for KbNS2114; Figure 2B), were also detected at high prevalence in the naive TCR sets (Figure 2, A and B). The substantial finding from this analysis, however, was that usage of the dominant TRBV in the DbNP366-, DbPA224-, and KbNS2114-specific repertoires was significantly increased (P < 0.0005) in the immune relative to the naive populations, while this was not seen for DbPB1-F262 (Figure 2B). These data indicate that DbNP366, DbPA224, and KbNS2114 are selectively recruiting or expanding T cell clonotypes from the TRBV13-1+ and TRBV29+ populations, respectively.
Preferred TRBV usage in naive and immune epitope–specific CD8+ T cell populations. (A) Representative dot plots of tetramer versus specific TRBV for CD8+ cells isolated from spleen and all major LNs of naive B6 mice after enrichment with the indicated tetramers. (B) Proportion of tetramer+CD8+ cells expressing the dominant TRBV in naive or immune B6 mice, as identified by flow cytometry (circles) or by single-cell RT-PCR for CD8 and TRBV (diamonds). Symbols represent data from individual mice obtained in at least 2 independent experiments.
Clonotype usage in the naive and immune TCR repertoires. We next sequenced the TCR CDR3β regions from individually sorted, naive CD8+ tetramer+ T cells within the dominant TRBV sets for DbNP366-, DbPA224-, and DbPB1-F262 and compared them with those found previously in immune CTLs (refs. 25, 30, and 31; Table 1 and Supplemental Tables 1 and 2). Prior to developing the capacity to isolate naive CTLps, we used the number of TCR-β aa clonotypes within epitope-specific CTL populations after antigen-driven expansion to infer naive CTLp frequency (10). We thus reasoned that comparison of TCR-β clonotypic usage within the naive and immune repertoires would provide a reliable measure of naive CTLp recruitment into the immune response. Indeed, the same modal CDR3β length (data not shown) and preferred TRBJ (Supplemental Figure 3) gene segment usage was found in both the naive and immune repertoires (25, 30, 31). This consistency of TCR usage for naive and immune T cells was further confirmed by analysis of the CDR3β aa sequences (Table 1 and Supplemental Tables 1 and 2). The relatively “public” TRBV13-1+ DbNP366–specific immune repertoire is characterized by the use of a limited number of clonotypes that tend to be shared among different individuals (30, 35). In contrast, the “private” TRBV29+ DbPA224- and TRBV19+ DbPB1-F262–specific sets are more diverse and demonstrate little overlap (25, 31). These profiles were also found for the naive TCRs, with the DbNP366-specific CTLps exhibiting a degree of TCR clonotype sharing similar to that seen following antigen challenge (Table 1), while neither the naive DbPA224- or DbPB1-F262–specific TRBV repertoires showed significant overlap among individuals (Supplemental Tables 1 and 2). Thus, the public or private nature of any given antigen-driven immune repertoire is likely to reflect CTLp availability in the naive pool.
TCR-β repertoire of naive DbNP366+TRBV13-1+CD8+ T cells in B6 mice
To further ascertain whether the relatively high frequency and “public” nature of particular CDR3β-defined clonotypes in immune individuals may, indeed, be partly explained by their availability in naive populations (36), we analyzed both the immune and the naive repertoires to determine the prevalence of commonly shared DbNP366-, DbPA224-, and DbPB1-F262–specific TCR-β sequences (defined as being present in 33% or more of immune repertoires) within the dominant TRBV populations. The majority of the “shared” TCR clonotypes found in the immune, antigen-specific CTL sets were also detected at least once in uninfected mice (Supplemental Table 3). However, the most abundant clonotypes in the DbNP366, DbPA224, and DbPB1-F262 immune repertoires contributed more substantially to the overall population than the most abundant clonotypes in the naive repertoire (Table 2). Thus, when considering dominant, public clonotypes within a particular immune response, at least some of that profile appears to reflect that multiple T cells bearing the same TCR-β (by aa sequence) are present prior to any encounter with antigen. Additionally, our data suggest that the ultimate, antigen-driven clone size is also likely to reflect the preferential selection of those TCRs with optimal pMHCI-binding characteristics into the immune repertoire (37).
Prevalence of the 1 or 2 most dominant clonotypes in naive and immune repertoires
Comparison of detected and estimated CTLp frequencies. Using single-cell PCR (31), we compared the number of TCR-β aa signatures found in individual naive and immune antigen-specific CTL repertoires (Table 3). Surprisingly, for the dominant DbNP366- and DbPA224-specific populations, more CDR3β clonotypes were found in the immune versus the naive repertoire (Table 3). Clearly, while the sampling procedure (14) provides a “best estimate” of naive CTLp diversity, analyzing the relatively small numbers that can be recovered significantly underestimates (by a factor of at least 1.6) the actual precursor diversity. This likely reflects the cell loss that is the inevitable consequence of the demanding enrichment protocol, together with the exclusion of minor LNs and tissue-associated lymphoid elements from the analysis. Aligning the naive and immune repertoires thus gives the sense that, when antigen is present, these immunodominant responses utilize every available CTLp in the naive pool.
Number of TCR CDR3β clonotypes per mouse
Conversely, for the subdominant DbPB1-F262–specific repertoire, the numbers of different clonotypes found in naive populations were significantly larger (Table 3) than those recovered from the immune repertoire (43.6 and 32.0, respectively; P = 0.03). Given the equivalent capacity of each tetramer to bind its specific population (Figure 1B and Supplemental Figure 1) and assuming the greater than or equal to 1.6 sampling underestimate of the naive T cell pool that we arrived at for the DbNP366- and DbPA224-specific sets, it seems that only about a third of the naive DbPB1-F262–specific population made the transition to immune CTL status. This in turn suggests that the subdominant character of the DbPB1-F262–specific CTL response reflects the incomplete recruitment of naive T cells into the mature response, while exactly the opposite is the case for the immunodominant DbNP366- and DbPA224-specific populations. As related in the supplemental data (Supplemental Figure 4), this analysis was confirmed by Chao1 nonparametric statistical analysis (38).
Immunodominance as a function of CTLp recruitment and expansion. The extent of naive CTLp recruitment into the primary influenza virus–specific CTL response was further analyzed by providing BrdU in the drinking water over 2-day intervals (days 3–4 or 5–6), then sampling on the third day (days 5, 7) to analyze for BrdU incorporation as a measure of CTL cycling (Figure 3). By day 5, the majority of the DbNP366- and DbPA224-specific CTLs were CD44hiBrdU+, indicating that the corresponding naive CTLp pools were completely recruited to proliferate and become differentiated CTLs (Figure 3, A and B). However, only 35% of the DbPB1-F262–specific set was CD44hiBrdU+ at this time point (Figure 3C), with approximately 50% still showing the CD44loBrdU– phenotype of naive, undivided CTLps. Clearly, as predicted from the clonotypic analysis (Table 3 and Supplemental Figure 4), there is incomplete recruitment of the naive DbPB1-F262–specific precursors into this early phase of the immune response. Kinetic analysis of DbPB1-F262 and DbNP366 epitope presentation over time after i.n. influenza infection reveals that the overall level of DbPB1-F262 was substantially less than DbNP366 and peaked earlier, providing a likely mechanism for incomplete CTLp recruitment (Supplemental Figure 5). Looking at the other subdominant response (to KbNS2114), the vast majority of tetramer+ T cells were CD44hiBrdU+ (Figure 3D) by day 5 after infection, indicating that, as far as the recruitment of naive CTLps is concerned, there is no difference between the subdominant KbNS2114-specific and the immunodominant DbNP366- and DbPA224-specific populations. The same conclusions can be drawn from the analysis of CTL cycling and differentiation profiles over the 5 to 6 day interval after respiratory exposure (Figure 3, E–H). Thus, differential mobilization from the naive CTLp pool (Figure 3, A–C) is not the sole explanation for the observed variations in primary CTL response magnitude. Importantly, these data also demonstrate, at least for DbNP366, DbPA224, and KbNS2114, that the CTLps detected within naive animals are true precursors in that they are virtually all capable of being drawn into the immune response.
Poor recruitment of CTLp specific for 1 subdominant epitope. B6 mice were infected i.n. with influenza A virus and given BrdU in the drinking water at days 3 and 4 or days 5 and 6 after infection. Cells from spleen and major LNs were harvested the next day (day 5, panels A–D; day 7, panels E–H), enriched with specific tetramers, and analyzed for BrdU incorporation and CD44 expression. Shown are representative dot plots from a group of 5 individual mice with the proportion of CD8+CD3+CD4–B220–IAb-F4/80–tetramer+ cells shown for each quadrant. Data are representative of 2 independent experiments.
It thus seems that, while the incomplete recruitment of naive CTLps may explain the subdominant status of the DbPB1-F262–specific response, it does not account for the comparably minor status of the CD8+KbNS2114+ set. Given that we can now enumerate naive CTLp numbers, it is also possible to estimate the overall extent of antigen-driven clonal expansion for the different CTL sets in this T cell immunodominance hierarchy. We measured total tetramer+CD8+ T cell numbers in pooled secondary lymphoid tissue taken at days 5, 7, and 9 after primary infection and calculated the fold expansion (Figure 4A) by relating the CD8+DbNP366+, CD8+DbPA224+, CD8+DbPB1-F262+, and CD8+KbNS2114+ T cell counts to naive CTLp prevalence (Figure 1C). Despite evidence of proliferation at day 5 after infection, CTLp numbers in secondary lymphoid organs were minimally increased, likely reflecting the migration of early effector CTL to the infected lung. By day 7, however, the magnitude of the CD8+DbNP366+ and CD8+DbPA224+ sets had increased 30 times or more while the comparable values for the subdominant KbNS2114- and DbPB1-F262–specific responses were ×8 and ×2 respectively (Figure 4A). Over the subsequent 2 days, the immunodominant DbNP366- and DbPA224-specific CTLs expanded a further 120–130 times, while, despite the minimal early proliferation, the CD8+DbPB1-F262+ set increased 50 times, being 10-fold higher than the ×5 value for the KbNS2114-specific response. Overall, the levels of expansion for the subdominant responses were approximately ×40 (KbNS2114) and ×100 (DbPB1-F262) compared with the approximate ×3,500 increase for the immunodominant DbNP366- and DbPA224-specific CTL sets.
Reduced proliferation of subdominant epitope–specific CTLs late in infection. (A) B6 mice were infected i.n. with influenza A virus and total cell tetramer cell numbers determined at days 5, 7, and 9 after infection. The fold expansion was determined by dividing the total number of CD8+tetramer+ CTL at each time point by the average number of naive CTLps identified from Figure 1. Data represent 5 mice per tetramer, per time point. Shown is the fold difference ± SD on a log10 scale. (B) B6 mice were infected i.n. with influenza A virus and fed BrdU in their drinking water on days 3–5 after infection. Cells from spleen and major LNs were harvested at the end of the BrdU treatment on day 6, day 7, or day 8, enriched with specific tetramers, and analyzed for BrdU incorporation and CD44 expression. Shown is the mean proportion ± SD (n = 5 mice per group) of CD44+tetramer+BrdU+ CTL present for each epitope specificity. Data are representative of 2 independent experiments.
An alternative approach for measuring continued CTL expansion is to “pulse” with BrdU, then “chase” to see how this DNA label is diluted with elapsed time (39). Mice were fed BrdU through days 3 to 5 after infection (pulse) and individuals were sampled on day 6 to confirm the extent of BrdU uptake or on day 7 or day 8 (chase) to estimate the rate of division-dependent loss (Figure 4B). The proportion of BrdU+tetramer+ CTLs looked fairly similar for the 4 epitopes at the end of the pulse though, as might be expected from the values in Figure 4A, the extent of BrdU incorporation may have been a little lower for the KbNS2114-specific set (day 6; Figure 4B), indicative of reduced expansion. Thereafter, BrdU+tetramer+ CTL frequencies remained highest for the subdominant CD8+DbPB1-F262+ and CD8+KbNS2114+ populations, further establishing that any continued cycling is at a lower level than that found for the immunodominant CD8+DbNP366+ and CD8+DbPA224+ T cells (Figure 4B). Interestingly, considering the slightly poorer initial labeling of KbNS2114-specific cells, the retention of a larger proportion of BrdU+ cells at day 8 suggests that this population proliferates the least of all the 4 epitope-specific CTL populations (Figure 4B), likely explaining its subdominant status despite comprehensive CTLp recruitment.
Analysis of CD44 expression by naive CTLps. A recent study showed that a significant proportion (10%–30%) of epitope-specific CTLps recovered from naive animals were CD44hi, a phenotype typical of antigen-experienced cells or cells that have undergone homeostatic proliferation (40). As might be expected, these CD44hi cells showed enhanced functional responsiveness to antigen stimulation when compared with the CD44lo subset. Therefore, it was formally possible that the differences in CTLp recruitment and expansion we observed here arose as a consequence of differential expression of CD44 among the epitope-specific populations. This is particularly relevant given that our gating strategy would certainly have included both the CD44hi and CD44lo subsets (Supplemental Figure 6). Analysis of CD44 expression on each of the 4 influenza epitope–specific CTLp populations identified from naive mice showed that all groups had similar proportions of CD44hi cells (Figure 5), with the only significant difference being that the naive DbNP366-specific population (one of the most comprehensively recruited specificities) was least likely to be CD44hi. Thus, the differential expression of CD44 on naive CTLps is unlikely to account for the observed differences in recruitment and/or expansion among these epitope-specific populations.
Expression of CD44 on epitope-specific CTLps from naive mice. Epitope-specific CTLps were identified from naive mice as described in legend to Figure 1. The proportion of CD44hi or CD44lo epitope–specific CTLps are shown. Symbols represent data from individual mice. *P < 0.05 using Student’s t test with Bonferroni’s correction.