Tania Cukalac

Primary CTL response magnitude in mice is determined by the extent of naive T cell recruitment and subsequent clonal expansion

CD8+ T cell responses to viral infection are characterized by the emergence of dominant and subdominant CTL populations. The immunodominance hierarchies of these populations are highly reproducible for any given spectrum of virus-induced peptide-MHCI complexes and are likely determined by multiple factors. Recent studies demonstrate a direct correlation between naive epitope-specific CD8+ T cell precursor (CTLp) frequency and the magnitude of the response after antigen challenge. Thus, the number of available precursors in the naive pool has emerged as a key predictor of immunodominance. In contrast to this, we report here no consistent relationship between CTLp frequency and the subsequent magnitude of the immune response for 4 influenza virus-derived epitopes following intranasal infection of mice with influenza A virus. Rather, the characteristic, antigen-driven T cell immunodominance hierarchy was determined by the extent of recruitment from the available pool of epitope-specific precursors and the duration of their continued expansion over the course of the infection. These findings suggest possibilities for enhancing protective immune memory by maximizing both the size and diversity of typically subdominant T cell responses through rational vaccine design.

Epitope-specific TCRbeta repertoire diversity imparts no functional advantage on the CD8+ T cell response to cognate viral peptides

TCR repertoire diversity has been convincingly shown to facilitate responsiveness of CD8+ T cell populations to mutant virus peptides, thereby safeguarding against viral escape. However, the impact of repertoire diversity on the functionality of the CD8+ T cell response to cognate peptide-MHC class I complex (pMHC) recognition remains unclear. Here, we have compared TCRβ chain repertoires of three influenza A epitope-specific CD8+ T cell responses in C57BL/6 (B6) mice: DbNP366–374, DbPA224–233, and a recently described epitope derived from the +1 reading frame of the influenza viral polymerase B subunit (residues 62–70) (DbPB1-F262). Corresponding to the relative antigenicity of the respective pMHCs, and irrespective of the location of prominent residues, the DbPA224- and DbPB1-F262-specific repertoires were similarly diverse, whereas the DbNP366 population was substantially narrower. Importantly, parallel analysis of response magnitude, cytotoxicity, TCR avidity, and cytokine production for the three epitope-specific responses revealed no obvious functional advantage conferred by increased T cell repertoire diversity. Thus, whereas a diverse repertoire may be important for recognition of epitope variants, its effect on the response to cognate pMHC recognition appears minimal.

Altered CD8+ T Cell Immunodominance after Vaccinia Virus Infection and the Naive Repertoire in Inbred and F1 Mice

Previous studies of CD8+ T cell immunodominance after primary virus infection of F1 mice compared with their inbred parents have generally concluded that no dramatic changes occur. In this study, we revisit this issue using vaccinia virus (VACV), which has a large genome, a recently defined immunodominance hierarchy in mice, and is a candidate vector for vaccines. We found that immunogenicity of VACV peptides defined using inbred mice was highly variable in F1 progeny: some peptides were equally immunogenic in F1 and inbred, whereas others elicited responses that were reduced by >90% in F1 mice. Furthermore, the dominance of a peptide in the relevant inbred parent did not predict whether it would be poorly immunogenic in F1 mice. This result held using F1 hybrids of MHC-congenic mice, suggesting that MHC differences alone were responsible. It was also extended to foreign epitopes expressed by an rVACV vaccine. F1 mice were less able to mount responses to the poorly immunogenic peptides when used as a sole immunogen, ruling out immunodomination. In addition, conserved TCR Vβ usage between inbred and F1 mice did not always correlate with strong responses in F1 mice. However, direct estimation of naive precursor numbers showed that these were reduced in F1 compared with inbred mice for specificities that were poorly immunogenic in the hybrids. These data have implications for our understanding of the extent to which MHC diversity alters the range of epitopes that are immunogenic in outbred populations.

Reproducible selection of high avidity CD8+ T-cell clones following secondary acute virus infection

Significance Exactly how memory cells are selected into a recall response to acute viral infection (stochastic or deterministic) remains unresolved. This paper demonstrates definitively that selection of virus-specific CD8+ T cells from memory occurs via active selection of particular T-cell clones after secondary virus infection and that this selection appears to be based on the avidity of the T-cell receptor (TCR) for the virus-derived peptide (p) + major histocompatibility complex class I molecule. We also show that despite clear clonal preferences, there is no global narrowing of epitope-specific TCR diversity in the recall response. We propose that the immune system has evolved such a strategy to optimize cytotoxic T-lymphocyte responses while safeguarding TCR repertoire diversity. The recall of memory CD8+ cytotoxic T lymphocytes (CTLs), elicited by prior virus infection or vaccination, is critical for immune protection. The extent to which this arises as a consequence of stochastic clonal expansion vs. active selection of particular clones remains unclear. Using a parallel adoptive transfer protocol in combination with single cell analysis to define the complementarity determining region (CDR) 3α and CDR3β regions of individual T-cell receptor (TCR) heterodimers, we characterized the antigen-driven recall of the same memory CTL population in three individual recipients. This high-resolution analysis showed reproducible enrichment (or diminution) of particular TCR clonotypes across all challenged animals. These changes in clonal composition were TCRα− and β chain–dependent and were directly related to the avidity of the TCR for the virus-derived peptide (p) + major histocompatibility complex class I molecule. Despite this shift in clonotype representation indicative of differential selection, there was no evidence of overall repertoire narrowing, suggesting a strategy to optimize CTL responses while safeguarding TCR diversity.

The Influenza Virus–Specific CTL Immunodominance Hierarchy in Mice Is Determined by the Relative Frequency of High-Avidity T Cells

Virus-specific CTL responses typically fall into reproducible hierarchies with particular epitopes eliciting either immunodominant or subdominant responses after viral challenge. The recently acquired capacity to directly enumerate naive CTL precursors (CTLps) in both mice and humans has implicated CTLp frequency as a key predictor of immune response magnitude after Ag challenge. However, recent studies have indicated that naive CTLp frequencies do not necessarily predict the size of the Ag-driven response, indicating an important role for differential CTLp recruitment and/or expansion. This study characterizes the early emergence of various influenza epitope-specific CTL responses at multiple sites in C57BL/6 mice, and probes the role of Ag dose and TCR avidity in dictating immune response hierarchies. Despite large naive CTLp numbers, subdominance was found to arise largely as a consequence of the abrupt and premature cessation of CTL proliferation, at least for one epitope specificity. Investigation into the possible drivers of the poor proliferation observed for subdominant specificities showed that the immunodominance hierarchy endured irrespective of epitope abundance, and correlated with the prevalence of high-avidity T cells in both the naive and immune compartments. Our study strongly indicates that the quality, and not simply the quantity, of antiviral CTLs dictate response magnitude.

Paired TCRαβ analysis of virus-specific CD8(+) T cells exposes diversity in a previously defined 'narrow' repertoire.

T‐cell receptor (TCR) usage has an important role in determining the outcome of CD8+ cytotoxic T‐lymphocyte responses to viruses and other pathogens. However, the characterization of TCR usage from which such conclusions are drawn is based on exclusive analysis of either the TCRα chain or, more commonly, the TCRβ chain. Here, we have used a multiplexed reverse transcription‐PCR protocol to analyse the CDR3 regions of both TCRα and β chains from single naive or immune epitope‐specific cells to provide a comprehensive picture of epitope‐specific TCR usage and selection into the immune response. Analysis of TCR repertoires specific for three influenza‐derived epitopes (DbNP366, DbPA224 and DbPB1‐F262) showed preferential usage of particular TCRαβ proteins in the immune repertoire relative to the naive repertoire, in some cases, resulting in a complete shift in TRBV preference or CDR3 length, and restricted repertoire diversity. The NP366‐specific TCRαβ repertoire, previously defined as clonally restricted based on TCRβ analysis, was similarly diverse as the PA224‐ and PB1‐F262‐specific repertoires. Intriguingly, preferred TCR characteristics (variable gene usage, CDR3 length and junctional gene usage) appeared to be able to confer specificity either independently or in concert with one another, depending on the epitope specificity. These data have implications for established correlations between the nature of the TCR repertoire and response outcomes after infection, and suggest that analysis of a subset of cells or a single TCR chain does not accurately depict the nature of the antigen‐specific TCRαβ repertoire.

Heightened self-reactivity associated with selective survival, but not expansion, of naïve virus-specific CD8+ T cells in aged mice.

Significance Compromised CD8+ T-cell immunity is associated with significant morbidity and mortality in the elderly. Whereas the number of naïve CD8+ T cells declines with age, the drivers of loss and consequences for clonal composition are unclear. We show that aging disproportionately impacts small naïve CD8+ T-cell populations. For one CD8+ T-cell population, loss of diversity was minimally attributable to expansion but rather was associated with diminished cell number and selective retention of cells exhibiting markers of heightened self, but not foreign, recognition. Thus, vaccine formulations for the elderly may benefit from targeting naïve antigen-specific populations with relatively high precursor frequency and self-reactivity, and retention of high-quality T cells may be achieved through repeated low-level T-cell receptor stimulation. In advanced age, decreased CD8+ cytotoxic T-lymphocyte (CTL) responses to novel pathogens and cancer is paralleled by a decline in the number and function of naïve CTL precursors (CTLp). Although the age-related fall in CD8+ T-cell numbers is well established, neither the underlying mechanisms nor the extent of variation for different epitope specificities have been defined. Furthermore, naïve CD8+ T cells expressing high levels of CD44 accumulate with age, but it is unknown whether this accumulation reflects their preferential survival or an age-dependent driver of CD8+ T-cell proliferation. Here, we track the number and phenotype of four influenza A virus (IAV)-specific CTLp populations in naïve C57BL/6 (B6) mice during aging, and compare T-cell receptor (TCR) clonal diversity for the CD44hi and CD44lo subsets of one such population. We show differential onset of decline for several IAV-specific CD8+ T-cell populations with advanced age that parallel age-associated changes in the B6 immunodominance hierarchy, suggestive of distinct impacts of aging on different epitope-specific populations. Despite finding no evidence of clonal expansions in an aged, epitope-specific TCR repertoire, nonrandom alterations in TCR usage were observed, along with elevated CD5 and CD8 coreceptor expression. Collectively, these data demonstrate that naïve CD8+ T cells expressing markers of heightened self-recognition are selectively retained, but not clonally expanded, during aging.

Narrowed TCR diversity for immunised mice challenged with recombinant influenza A-HIV Env311-320 virus

Understanding CD8+ T cell responses generated by live virus vectors is critical for the rational design of next generation HIV CTL-based vaccines. We used recombinant influenza viruses expressing the HIV Env(311-320) peptide in the neuraminidase stalk to study response magnitude, cytokine production and repertoire diversity for the elicited CD8+ D(d)Env(311) CTL set. The insertion of the CD8+ D(d)Env(311) epitope into the NA stalk resulted in a decrease in viral fitness that was reflected in lower lung viral titres. While not affecting the magnitude of endogenous primary influenza-specific responses, the introduction of the D(d)Env(311) CD8+ T cell epitope altered the hierarchy of responses following secondary challenge. The CD8+ K(d)NP(147) response increased 9-fold in the spleen following secondary infection whereas the CD8+ D(d)Env(311) response increased 15-fold in the spleen. Moreover, this study is the first to describe narrowing of CD8+ TCR repertoire diversity in the context of an evolving secondary immune response against influenza A virus. Analysis of Vbeta bias for CD8+ D(d)Env(311) T cell responses showed a narrowing of CD8+ Vbeta8.1/8.2 D(d)Env(311) TCR repertoire diversity. This work further emphasizes the importance of understanding vaccine-induced CD8+ T cell responses.

Multiplexed combinatorial tetramer staining in a mouse model of virus infection

Use of fluorescently labelled multimers, particularly tetramers of peptide and MHC class I glycoprotein (pMHC-I) complexes, is essential for the analysis of CD8+ T cell immunity in basic research and clinical settings. A recently described combinatorial approach using pMHC-I multimers coupled to a unique combination of distinct fluorochromes has facilitated the simultaneous screening of multiple T cell specificities within a single human blood sample. The present analysis establishes that this multiplexed tetramer staining protocol can also be applied in mouse models of a disease to detect multiple subdominant CD8+ T cell specificities in the presence of prominent immunodominant T cell sets at different stages of infection. We have established a modified protocol that concurrently identified influenza-specific CD8+ T cells at the acute and long-term memory phases of influenza virus infection in B6 mice. Highly dominant (D(b)NP(366)+CD8+ and D(b)PA(224)+CD8+) and subdominant (K(b)PB1(703)+CD8+, D(b)PB1-F2(62)+CD8+ and K(b)NS2(114)+CD8+) T cell responses can be detected simultaneously at levels comparable to the conventional tetramer staining with this combinatorial approach. The technique proved particularly useful with aged mice, where we used 5-fold fewer animals, making the detection of multiple T cell specificities more cost-effective and less time-consuming. Overall, our study establishes that this comprehensive concurrent analysis of multiple T cell specificities is of value for analysing mouse models of disease, especially in situations where sample size and/or response magnitude is limiting.

Diversity and clonotypic composition of influenza-specific CD8+ TCR repertoires remain unaltered in the absence of Aire.

TCR repertoire diversity is important for the protective efficacy of CD8+ T cells, limiting viral escape and cross‐reactivity between unrelated epitopes. The exact mechanism for selection of restricted versus diverse TCR repertoires is far from clear, although one thought is that the epitopes resembling self‐peptides might select a limited array of TCR due to the deletion of autoreactive TCR. The molecule Aire promotes the expression of tissue‐specific Ag on thymic medullary epithelial cells and the deletion of autoreactive cells, and in the absence of Aire autoreactive cells persist. However, the contribution of Aire‐dependent peptides to the selection of the Ag‐specific TCR repertoire remains unknown. In this study, we dissect restricted (DbNP366%+CD8+) and diverse (DbPA224%+CD8+, KdNP147%+CD8+) TCR repertoires responding to three influenza‐derived peptides in Aire‐deficient mice on both B6 and BALB/c backgrounds. Our study shows that the number, qualitative characteristics and TCR repertoires of all influenza‐specific, DbNP366%+CD8+, DbPA224%+CD8+ and KdNP147%+CD8+ T cells are not significantly altered in the absence of Aire. This provides the first demonstration that the selection of an Ag‐specific T‐cell repertoire is not significantly perturbed in the absence of Aire.