Immune profiling of influenza-specific B- and T-cell responses in macaques using flow cytometry-based assays.

Influenza remains a significant global public health burden, despite substantial annual vaccination efforts against circulating virus strains. As a result, novel vaccine approaches are needed to generate long-lasting and universal broadly cross-reactive immunity against distinct influenza virus strains and subtypes. Several new vaccine candidates are currently under development and/or in clinical trials. The successful development of new vaccines requires testing in animal models, other than mice, which capture the complexity of the human immune system. Importantly, following vaccination or challenge, the assessment of adaptive immunity at the antigen-specific level is particularly informative. In this study, using peripheral blood mononuclear cells (PBMCs) from cynomolgus macaques, we describe detection methods and in-depth analyses of influenza virus-specific B cells by recombinant hemagglutinin probes and flow cytometry, as well as the detection of influenza virus-specific CD8+ and CD4+ T cells by stimulation with live influenza A virus and intracellular cytokine staining. We highlight the potential of these assays to be used with PBMCs from other macaque species, including rhesus macaques, pigtail macaques and African green monkeys. We also demonstrate the use of a human cytometric bead array kit in detecting inflammatory cytokines and chemokines from cynomolgus macaques to assess cytokine/chemokine milieu. Overall, the detection of influenza virus-specific B and T cells, together with inflammatory responses, as described in our study, provides useful insights for evaluating novel influenza vaccines. Our data deciphering immune responses toward influenza viruses can be also adapted to understanding immunity to other infections or vaccination approaches in macaque models.

Quantitative and functional diversity of cross-reactive EBV-specific CD8+ T cells in a longitudinal study cohort of lung transplant recipients.

BACKGROUND: Cross-reactive antiviral memory T cells constitute a significant proportion of the alloresponse, potentially playing a pivotal role in adverse posttransplant outcomes in human leukocyte antigen (HLA)-mismatched allografts. We explored the longitudinal dynamics of cross-reactive HLA-B8-restricted Epstein-Barr virus-specific CD8+ T cells directed toward the EBNA3A epitope FLRGRAYGL (FLR) in lung transplant recipients (LTRs) to determine whether their corecognition of HLA-B*4402 expressed on the allograft contributed to poorer posttransplant outcomes. METHODS: Cross-reactive FLR-specific CD8+ T cells were measured in the peripheral blood mononuclear cells and bronchoalveolar lavage fluid in 11 HLA-B8+ LTR, who had received HLA-B44+ lung allograft, after in vitro autologous (FLR pulsed) or allogeneic stimulation by multiparameter flow cytometry. RESULTS: FLR-specific CD8+ T cells were detectable ex vivo and after 13 days following in vitro peptide stimulation of peripheral blood mononuclear cells. Individual LTR and demonstrated diverse functional profiles of either cytokine production and/or cytotoxic potential (interferon-g+, interferon-g+CD107a+ and CD107a+ subsets). However, cells isolated from bronchoalveolar lavage exhibited a skewed functional phenotype toward CD107a expression alone, indicating cytotoxic-producing but not cytokine-producing capabilities. In addition, our findings suggested that the presence of cross-reactive FLR-specific CD8+ T cells may influence the alloreactive hierarchy directed against the allograft, although they were not associated with poorer short- or long-term clinical outcomes in the absence of Epstein-Barr virus reactivation and in the setting of current immunosuppression and antiviral prophylaxis protocols. CONCLUSION: We report, for the first time, the longitudinal measurement of cross-reactive FLR-specific CD8 T cells within a clinical transplantation framework.