Potential killers exposed: tracking endogenous influenza-specific CD8+ T cells.

Current influenza A virus (IAV) vaccines stimulate antibody responses that are directed against variable regions of the virus, and are therefore ineffective against divergent strains. As CD8+ T cells target the highly conserved, internal IAV proteins, they have the potential to increase heterosubtypic immunity. Early T-cell priming events influence lasting memory, which is required for long-term protection. However, the early responding, IAV-specific cells are difficult to monitor because of their low frequencies. Here, we tracked the dissemination of endogenous IAV-specific CD8+ T cells during the initial phases of the immune response following IAV infection. We exposed a significant population of recently activated, CD25+ CD43+ IAV-specific T cells that were not detected by tetramer staining. By tracking this population, we found that initial T-cell priming occurred in the mediastinal lymph nodes, which gave rise to the most expansive IAV-specific CD8+ T-cell population. Subsequently, IAV-specific CD8+ T cells dispersed to the bronchoalveolar lavage and blood, followed by spleen and liver, and finally to the lung. These data provide important insight into the priming and tissue dispersion of an endogenous CD8+ T-cell response. Importantly, the CD25+ CD43+ phenotype identifies an inclusive population of early responding CD8+ T cells, which may provide insight into TCR repertoire selection and expansion. A better understanding of this response is critical for designing improved vaccines that target CD8+ T cells.

Highly pathological influenza A virus infection is associated with augmented expression of PD-1 by functionally compromised virus-specific CD8+ T cells.

One question that continues to challenge influenza A research is why some strains of virus are so devastating compared to their more mild counterparts. We approached this question from an immunological perspective, investigating the CD8(+) T cell response in a mouse model system comparing high- and low-pathological influenza virus infections. Our findings reveal that the early (day 0 to 5) viral titer was not the determining factor in the outcome of disease. Instead, increased numbers of antigen-specific CD8(+) T cells and elevated effector function on a per-cell basis were found in the low-pathological infection and correlated with reduced illness and later-time-point (day 6 to 10) viral titer. High-pathological infection was associated with increased PD-1 expression on influenza virus-specific CD8(+) T cells, and blockade of PD-L1 in vivo led to reduced virus titers and increased CD8(+) T cell numbers in high- but not low-pathological infection, though T cell functionality was not restored. These data show that high-pathological acute influenza virus infection is associated with a dysregulated CD8(+) T cell response, which is likely caused by the more highly inflamed airway microenvironment during the early days of infection. Therapeutic approaches specifically aimed at modulating innate airway inflammation may therefore promote efficient CD8(+) T cell activity. We show that during a severe influenza virus infection, one type of immune cell, the CD8 T cell, is less abundant and less functional than in a more mild infection. This dysregulated T cell phenotype correlates with a lower rate of virus clearance in the severe infection and is partially regulated by the expression of a suppressive coreceptor called PD-1. Treatment with an antibody that blocks PD-1 improves T cell functionality and increases virus clearance.

Paired analysis of TCRα and TCRß chains at the single-cell level in mice.

Characterizing the TCRα and TCRß chains expressed by T cells responding to a given pathogen or underlying autoimmunity helps in the development of vaccines and immunotherapies, respectively. However, our understanding of complementary TCRα and TCRß chain utilization is very limited for pathogen- and autoantigen-induced immunity. To address this problem, we have developed a multiplex nested RT-PCR method for the simultaneous amplification of transcripts encoding the TCRα and TCRß chains from single cells. This multiplex method circumvented the lack of antibodies specific for variable regions of mouse TCRα chains and the need for prior knowledge of variable region usage in the TCRß chain, resulting in a comprehensive, unbiased TCR repertoire analysis with paired coexpression of TCRα and TCRß chains with single-cell resolution. Using CD8+ CTLs specific for an influenza epitope recovered directly from the pneumonic lungs of mice, this technique determined that 25% of such effectors expressed a dominant, nonproductively rearranged Tcra transcript. T cells with these out-of-frame Tcra mRNAs also expressed an alternate, in-frame Tcra, whereas approximately 10% of T cells had 2 productive Tcra transcripts. The proportion of cells with biallelic transcription increased over the course of a response, a finding that has implications for immune memory and autoimmunity. This technique may have broad applications in mouse models of human disease.

Physiological numbers of CD4+ T cells generate weak recall responses following influenza virus challenge.

Naive and recall CD4(+) T cell responses were probed with recombinant influenza A viruses incorporating the OVA OT-II peptide. The extent of OT-II-specific CD4(+) T cell expansion was greater following primary exposure, with secondary challenge achieving no significant increase in numbers, despite higher precursor frequencies. Adoptive transfer experiments with OT-II TCR-transgenic T cells established that the predominant memory set is CD62L(hi), whereas the CD62L(lo) precursors make little contribution to the recall response. Unlike the situation described by other investigators, in which the transfer of very large numbers of in vitro-activated CD4 effectors can modify the disease process, providing CD62L(hi) or CD62L(lo) OT-II-specific T cells at physiological levels neither enhanced virus clearance nor altered clinical progression. Some confounding effects of the transgenic model were observed, with decreasing primary expansion efficiency correlating with greater numbers of transferred cells. This was associated with increased levels of mRNA for the proapoptotic molecule Bim in cells recovered following high-dose transfer. However, even with very low numbers of transferred cells, memory T cells did not expand significantly following secondary challenge. A similar result was recorded in mice primed and boosted to respond to an endogenous IA(b)-restricted epitope derived from the influenza virus hemagglutinin glycoprotein. Depletion of CD8(+) T cells during secondary challenge generated an increased accumulation of OT-II-specific T cells but only at the site of infection. Taken together, significant expansion was not a feature of these secondary influenza-specific CD4 T cell responses and the recall of memory did not enhance recovery.

Protective memory responses are modulated by priming events prior to challenge.

Human infections with highly pathogenic H5N1 avian influenza A viruses in the last decade have legitimized fears of a long-predicted pandemic. We thus investigated the response to secondary infections with an engineered, but still highly virulent, H5N1 influenza A virus in the C57BL/6 mouse model. Mice primed with the H1N1 A/Puerto Rico/8/34 (PR8) virus were partially protected from lethality following respiratory infection with the modified H5N1 virus A/Vietnam/1203/04 (DeltaVn1203). In contrast, those that had been comparably exposed to the HKx31 (H3N2) virus succumbed to the DeltaVn1203 challenge, despite similarities in viral replication, weight loss, and secondary CD8(+)-T-cell response characteristics. All three viruses share the internal genes of PR8 that are known to stimulate protective CD8(+)-T-cell-mediated immunity. This differential survival of PR8- and HKx31-primed mice was also apparent for antibody-deficient mice challenged with the DeltaVn1203 virus. The relative protection afforded by PR8 priming was abrogated in tumor necrosis factor-deficient (TNF(-/-)) mice, although lung fluids from the B6 HKx31-primed mice contained more TNF early after challenge. These data demonstrate that the nature of the primary infection can influence pathological outcomes following virulent influenza virus challenge, although the effect is not clearly correlated with classical measures of CD8(+)-T-cell-mediated immunity.

Screening monoclonal antibodies for cross-reactivity in the ferret model of influenza infection.

Influenza virus infections carry a high public health cost, and pandemics are potentially catastrophic. Though the ferret is generally regarded as the best model for human influenza, few reagents are available for the analysis of cellular immunity. We thus screened monoclonal antibodies (mAbs) made for identifying immune cells in other species to see if any were cross-reactive. Flow cytometric analysis of lymphocytes isolated from blood, spleen, and lung of normal and virus-infected ferrets indicated that several mouse mAbs bound to the corresponding antigens in ferrets. Typing bronchoalveolar lavage populations from pneumonic ferrets with mAb to human CD8 showed the massive CD8+ T cell enrichment characteristic of this infection in mice. The availability of this, and several other mAbs that showed cross-reactivity, should allow us to begin the dissection of cell-mediated immunity in the ferret, which, at least from these early results, looks similar to the situation in mice.

Hidden epitopes emerge in secondary influenza virus-specific CD8+ T cell responses.

Influenza A virus-specific CD8+ T cell responses in H2(b) mice are characterized by reproducible hierarchies. Compensation by the D(b)PB1-F2(62) epitope is apparent following infection with a variant H3N2 virus engineered to disrupt the prominent D(b)NP(366) and D(b)PA(224) epitopes (a double knockout or DKO). Analysis with a "triple" knockout (TKO) virus, which also compromises D(b)PB1-F2(62), did not reveal further compensation to the known residual, minor, and predicted epitopes. However, infection with this deletion mutant apparently switched protective immunity to an alternative Ab-mediated pathway. As expected, TKO virus clearance was significantly delayed in Ab-deficient MHC class II(-/-) and Ig(-/-) mice, which were much more susceptible following primary, intranasal infection with the TKO, but not DKO, virus. CD8+ T cell compensation was detected in DKO, but not TKO, infection of Ig-deficient mice, suggestive of cooperation among CD8+ T cell responses. However, after priming with a TKO H1N1 mutant, MHC II(-/-) mice survived secondary intranasal exposure to the comparable H3N2 TKO virus. Such prime/challenge experiments with the DKO and TKO viruses allowed the emergence of two previously unknown epitopes. The contrast between the absence of compensatory effect following primary exposure and the substantial clonal expansion after secondary challenge suggests that the key factor limiting the visibility of these "hidden" epitopes may be very low naive T cell precursor frequencies. Overall, these findings suggest that vaccine approaches using virus vectors to deliver an Ag may be optimized by disrupting key peptides in the normal CD8+ T cell response associated with common HLA types.