Flu-specific CD8 Research Articles

Cellular senescence is a double-edged sword in regulating aged immune responses to influenza.

Clearance of senescent cells has demonstrated therapeutic potential in the context of chronic age-related diseases. Little is known, however, how clearing senescent cells affects the ability to respond to an acute infection and form quality immunological memory. We aimed to probe the effects of clearing senescent cells in aged mice on the immune response to influenza (flu) infection. We utilized a p16 trimodality reporter mouse model (p16-3MR) to allow for identification and selective clearance of p16-expressing cells upon administration of ganciclovir (GCV). While p16-expressing cells may exacerbate dysfunctional responses to a primary infection, our data suggest they may play a role in fostering memory cell generation. We demonstrate that although clearance of p16-expressing cells enhanced viral clearance, this also severely limited antibody production in the lungs of flu-infected aged mice. 30 days later, there were fewer flu-specific CD8 memory T cells and lower levels of flu-specific antibodies in the lungs of GCV-treated mice. Furthermore, GCV-treated mice were unable to mount an optimal memory response and demonstrated increased viral load following heterosubtypic challenge. These results suggest that targeting senescent cells may potentiate primary responses while limiting the ability to form durable and protective immune memory with age.

P16-EXPRESSING SENESCENT CELLS ARE A DOUBLE-EDGED SWORD IN SHAPING IMMUNE RESPONSES WITH AGE

Abstract Aging results in the accumulation of senescent cells which can cause dysfunction in many contexts but the effects on immune responses remain unclear. Here, we aimed to probe the effects of clearing senescent cells in aged mice on the immune response to influenza infection. We utilized a powerful p16 trimodality reporter mouse model (p16-3MR): under the control of the p16 promoter, these mice express cassettes encoding luciferase, RFP, and herpesvirus thymidine kinase (HSV-TK). p16 is commonly upregulated in senescent cells so this model allows us to selectively delete those cells by treating with ganciclovir (GCV), which will induce apoptosis in cells expressing HSV-TK. We hypothesized that while p16-expressing senescent cells may exacerbate dysfunctional responses to a primary infection, they may play a protective role in resolving inflammation and fostering memory cell generation. We found that deletion of p16-expressing cells enhanced viral clearance and decreased infiltration of pro-inflammatory flu-specific CD8 T cells during the primary response to infection. Conversely, at 30 days post infection, there were fewer flu-specific CD8 memory T cells and lower amounts of anti-viral antibodies in the lungs of GCV treated mice. We also observed perturbations in memory T cell trafficking in GCV treated mice. Furthermore, GCV treated mice were unable to mount an effective memory response and were unable to control viral load following a heterosubtypic challenge. This suggests that targeting senescent cells may potentiate primary responses while limiting the ability to form durable and protective immune memory with age.

Chronic genotype 1 hepatitis C along with cirrhosis drives a persistent imprint in virus-specific CD8+ T cells after direct-acting antiviral therapies.

Chronic hepatitis C virus (HCV) infection impairs HCV CD8+ T-cell responses, while it could influence immune responses towards unrelated viruses/vaccines (e.g. cytomegalovirus, CMV, and influenza, Flu). The aim of our study was to delineate whether restoration of these virus-specific CD8+ T cells occurs after direct-acting antiviral (DAA) therapies and particularly in patients with cirrhosis. We performed longitudinal analysis (baseline, week 4, follow-up [FU] 12 and FU48) of virus-specific CD8+ T cells by multicolour flow cytometry in HCV-cirrhotic patients undergoing DAA therapy (n=26) after in vitro expansion with immunodominant HCV, CMV and Flu epitopes restricted by HLA-A*02. HCV noncirrhotic patients (n=9) and healthy individuals (n=10) served as controls. We found that the proliferative capacity of HCV-specific CD8+ T cells increased from baseline up to FU48 in a significant proportion of cirrhotic and noncirrhotic patients. Nevertheless, these cells remained poor cytokine producers in both patient groups, regardless of the down-regulation of inhibitory co-regulatory receptors in HCV-cirrhotic patients at FU48. Likewise, high expression levels of these exhaustion markers were detected in CMV-/Flu-specific CD8+ T cells in HCV-cirrhotic patients at all time points, albeit without affecting their proliferative capacity or cytokine production. We conclude that DAA therapies induce restoration of the proliferative capacity of HCV-specific CD8+ T cells. However, these cells remain phenotypically and functionally impaired. Contrarily, the 'exhausted' phenotype in CMV-/Flu-specific CD8+ T cells in HCV-cirrhotic patients did not associate with their functions. Larger studier with longer follow-up may elucidate whether this complex interplay influences the outcome of cirrhotic patients.

Protection from lethal Flu infections: lessons from a systemic lupus mouse model

Abstract Patients with systemic lupus erythematosus (SLE) are more susceptible to infections than healthy subjects but it is unknown whether this is due to disease or treatment. Using mice expressing three SLE susceptibility loci (triple congenics hereafter called TC mice) we unexpectedly found that these mice are resistant to an otherwise lethal respiratory Flu PR8 (H1N1) infection. At the naïve state, TC mice had not only more activated plasmacytoid dendritic cells, inflammatory monocytes and natural killer cells but also more activated CD8+ and CD4+ T cells with higher TNF and IFNg-producing capacity than C57BL/6J controls. At the primary effector phase following intranasal Flu PR8 infection, TC mice mounted poor antibody responses but their Flu-specific CD8+ T cell responses were greater effectors at the membrane level (higher frequencies of KLRG1+IL7R− and KLRG1+IL7R+) and at the transcriptional level (higher T-bet expression) than CD8+ T cells from C57BL/6J counterparts. PR8-immune TC mice also developed more functional Flu-specific CD8+ T cell responses than C57BL/6J mice following to a heterosubtypic Flu X-31 (H3N2) challenge. While immunosuppressive treatments of SLE in patients might be responsible of mounting poor anti-microbial immune responses, lessons from this SLE mouse model could be taken to prevent and protect against severe Flu infections.

Pulmonary monocytes promote the establishment of lung tissue-resident CD8 T cell memory following influenza infection

Abstract Lung tissue resident memory CD8 T cells (TRM) are critical for protection against heterologous influenza virus challenge by promoting rapid viral clearance. Unlike other mucosal tissues, we have found that local environmental signals are insufficient for the establishment of lung TRM, and that pulmonary antigen encounter is necessary for TRM to differentiate in the lung. Therefore, we investigated the role of lung antigen presenting cell (APC) subsets in the establishment of lung CD8 TRM following influenza infection. The initial appearance of flu-specific CD8 T cells in the lung with a TRM phenotype begins around day 10 post-infection, which coincides with viral clearance. Correspondingly, while most lung APC subsets contain influenza antigens over the course of infection, monocytes are the most numerous lung APC subset containing influenza antigens at the time of TRM appearance. Furthermore, imaging shows direct interaction between monocytes and T cells in the lung, supporting the idea that monocytes can act as drivers of lung TRM differentiation. In a murine influenza model, CCR2−/− mice, which have a defect in monocyte recruitment to the lung, had significantly fewer flu-specific lung CD8 TRM than WT controls. Notably, this defect was not observed in the flu-specific CD8 memory T cells in the lung vasculature or mediastinal lymph node, indicating this effect is specific for tissue-resident memory cells. In addition, this phenotype is not driven by a lack of CCR2 on T cells, as adoptive transfer of wild-type OT-I T cells into CCR2−/− mice yields the same deficiency of lung TRM. Thus, these data show that antigen presentation by lung CCR2+ monocytes has an important role in the establishment of lung CD8 TRM.

Tissue-resident monocytes promote the establishment of lung-resident CD8 T cell memory following influenza infection

Abstract Lung tissue resident memory CD8 T cells (TRM) have been shown to be critical in protecting against heterologous influenza virus challenge by limiting early viral replication and promoting more rapid viral clearance. Unlike other mucosal tissues, we have found that environmental signals are insufficient for the establishment of lung TRM, and that pulmonary antigen encounter is necessary for TRM to differentiate in the lung. This indicates a likely role for pulmonary antigen presenting cells (APC’s) in the process of lung TRM development, and thus we investigated the role of lung APC subsets on the establishment of lung CD8 TRM following influenza infection. Although many lung APC subsets contain flu nucleoprotein (FluNP) over the course of infection, most lung APC’s do not contain FluNP once virus has been cleared by day 10 post-infection. In contrast, lung monocytes continue to have high numbers of FluNP-bearing cells through day 15 post infection, the same time we observe the appearance of flu-specific CD8 T cells with a TRM phenotype. As these data suggest a potential role for monocytes as drivers of TRM differentiation, we tested mice deficient for the chemokine receptor CCR2 (CCR2−/−), which have a significant defect in the recruitment of monocytes to the lung. In our murine influenza model these CCR2−/− mice had significantly less flu-specific lung CD8 TRM than WT controls. Notably, this defect was not observed in the flu-specific CD8 memory T cells in the lung vasculature or mediastinal lymph node (MedLN), indicating this effect is specific for tissue-resident memory cells. Thus, these data show that antigen presentation by CCR2+ monocytes have a likely role in the lung in the establishment of lung CD8 TRM.

Heterosubtypic cross‐protection induced by whole inactivated influenza virus vaccine in mice: influence of the route of vaccine administration

Development of influenza vaccines capable of inducing broad protection against different virus subtypes is necessary given the ever-changing viral genetic landscape. Previously, we showed that vaccination with whole inactivated virus (WIV) induces heterosubtypic protection against lethal virus infection in mice. Whole inactivated virus-induced cross-protection was found to be mediated primarily by flu-specific CD8+ T cells. As it has been demonstrated that the route of vaccine administration strongly influences both the quantity and quality of vaccine-induced immunity, in this study, we determined which route of WIV administration induces optimal heterosubtypic cross-protection. We compared the magnitude of the immune response and heterosubtypic protection against lethal A/PR/8/34 (H1N1) infection after subcutaneous (SC), intramuscular (IM), and intranasal (IN) vaccination with A/NIBRG-14 (H5N1) WIV. Subcutaneous and IM administration was superior to IN administration of influenza WIV in terms of flu-specific CD8+ T-cell induction and protection of mice against lethal heterosubtypic challenge. Surprisingly, despite the very low flu-specific CD8+ T-cell responses detected in IN-vaccinated mice, these animals were partially protected, most likely due to cross-reactive IgA antibodies. The results of this study show that the magnitude of WIV-induced flu-specific CD8+ T-cell activity depends on the applied vaccination route. We conclude that parenteral administration of WIV vaccine, in particular IM injection, is superior to IN vaccine delivery for the induction of heterosubtypic cross-protection and generally appears to elicit stronger immune responses than mucosal vaccination with WIV.

Co-infection with Streptococcus pneumoniae and influenza A virus leads to increased disease and a decrease in the size and functional capacity of the influenza specific CD8+ T cell response (P6363)

Abstract Most of our understanding of influenza A virus and its interaction with the adaptive immune response has been generated in the context of infection with a single pathogen. It is clear however that infection with influenza virus can lead to increased susceptibility to subsequent infection, often Streptococcus pneumoniae. The potential for a second infection to regulate the ongoing anti-influenza response is not clear. To address this important question we have developed an in vivo co-infection model to test the effects of pneumococcus on the ongoing CD8+ influenza specific T cell response. We show that co-infection with A/PR 8/34 (H1N1) and the non-invasive EF3030 strain of S. pneumoniae leads to increased disease. Within the lungs of the co-infected animals we observe a significant decrease in the size of the flu specific CD8+ T cell response. This decrease is not present in the lung draining lymph nodes, indicating selective regulation at the site of bacterial/viral co-infection. In addition to the decrease in size, the effector function of these CD8+ T cells is qualitatively diminished as measured by their ability to co-produce interferon γ and tissue necrosis factor α. These results demonstrate a dynamic interplay between the two concurrent pathogens and the immune system and emphasize the importance of increasing our understanding of how polymicrobial infections modulate the immune response to individual pathogens.

Lack of full CD8 functional restoration after antiviral treatment for acute and chronic hepatitis C virus infection

BackgroundHepatitis C virus (HCV) persistence is associated with impaired CD8 functions. Whether functional restoration of CD8 T cells chronically exposed to antigen can be obtained once the antigen is removed...

The proapoptotic protein Bim is not differentially regulated in CD8+ T cells from resolved or chronic HCV infection

Background: CD8 T cells play an important role in the antiviral immune response against HCV. Nevertheless, these cells fail to control the viral replication in patients with chronic infection either due to mutational escape of the virus in targeted epitopes or a dysfunctional phenotype of virus-specific CD8 T cells. Moreover, deletion of virus-specific CD8 T cells due to premature apoptosis upon their activation in the liver has been suggested. The pro-apoptotic BH3-only protein Bim has been found to be upregulated in CD8+ T cells activated by hepatocytes in mouse models as well as in patients chronically infected with HBV. Here, we addressed whether this molecule also plays a role in in HCV infection. Methods: PBMC were collected from anti-HCV positive injection drug users with chronic or spontaneously resolved HCV infection. From this cohort 13 “resolvers” and 13 “chronics” with detectable CD8 T cells by staining with HLA/peptide-multimers ex vivo were used for this study. The expression of Bim and the activation marker CD38 was analysed on HCV-, CMV- and Flu-specific CD8 T cells ex vivo and after 7 days of antigen-specific expansion via flow cytometry. Results: No significant difference in the expression level of Bim or CD38 could be observed in HCV-specific CD8+ T cells from patients with resolved infection compared to those with chronic infection ex vivo. After 7 days of antigen-specific expansion, both Bim and CD38 were upregulated on antigen-specific CD8 T cells, irrespective of the disease status. Both molecules were also upregulated in bulk CD8 T cells after unspecific activation with anti-CD3. There was also no difference between the Bim expression in HCV-specific CD8 T cells and Flu- or CMV-specific CD8 T cells. Conclusions: The pro-apoptotic molecule Bim is upregulated in CD8 T cells upon their activation, however, we find no evidence for differential regulation in HCV-specific cells from patients with chronic or spontaneously resolved HCV-infection.

Investigating the role of 4-1BB and 4-1BBL in dendritic cell immunobiology (113.6)

Abstract The tumour necrosis factor (TNF) receptor family member 4-1BB, and its TNF family ligand, 4-1BBL, have been thoroughly investigated as potent modulators of the CD8 T cell response. During a viral infection, 4-1BBL on APC, binding to 4-1BB on activated T cells mediates survival signaling in activated and memory CD8 T cells. Similarly to T cells, 4-1BB is also induced on dendritic cells (DC) following activation; however, how 4-1BB and 4-1BBL modulate DC biology is poorly understood. We provide evidence that DC express both 4-1BB and 4-1BBL upon activation with a microbial stimulus in vitro, and that these molecules interact on maturing DC. Using an in vivo DC vaccination model, we demonstrate that in the absence of 4-1BBL on the DC, there is a 2-3 fold defect in the ability of these DC to generate influenza-specific CD8 T cells in a WT host. This defect is not observed when 4-1BB-/- hosts are vaccinated with WT DC, suggesting that 4-1BBL on the DC is not acting through 4-1BB in the host, but intrinsically on the DC. Even more striking is that 4-1BB-/- DC do not show this defect in the generation of flu-specific CD8 T cells, raising the possibility of a 4-1BB-independent 4-1BBL mechanism. In that regard, we further show that anti-4-1BBL antibodies can induce Erk activation in WT DC, but not in 4-1BBL-/- DC. These results identify 4-1BBL as a DC modulator and have overall implications for vaccine design.

Ex vivo analysis of HCV-specific CD8+ T cell antiviral efficacy using a novel immunological model

Virus-specific CD8+ T cells play an important role in the outcome (elimination versus persistence) and pathogenesis of hepatitis C virus (HCV) infection. Due to the lack of a suitable model, it is currently unclear whether and to what extent HCV-specific CD8+ T cells are able to exert direct antiviral efficacy. To address this important issue we utilized a novel immunological model based on a subgenomic HCV-replicating cell line stably transduced with the HLA-A2 gene (Huh7A2HCV) (Gastroenterology 2009). In this study, we compared the antiviral efficacy of HCV-specific CD8+ T cells to CMV-, EBV- and Flu-specific CD8+ T cells of the same patient. PBMCs from 15 chronically HCV infected patients were analyzed for virus-specific CD8+ T cells by tetramer staining and co-cultured with Huh7A2HCV cells pulsed with the respective peptides at a (E: T) ratio of 2: 1 for 72h. Inhibition of HCV RNA replication was measured by a decrease in luciferase activity.

The immunoregulatory role of CD244 in chronic hepatitis B infection and its inhibitory potential on virus-specific CD8+ T-cell function

Multiple inhibitory receptors may play a role in the weak or absent CD8+ T-cell response in chronic hepatitis B virus (HBV) infection. Yet few receptors have been characterized in detail and little is known about their complex regulation. In the present study, we investigated the role of the signaling lymphocyte activation molecule (SLAM)-related receptor CD244 and of programmed death 1 (PD-1) in HBV infection in 15 acutely and 66 chronically infected patients as well as 9 resolvers and 21 healthy controls. The expression of CD244, PD-1, and T-cell immunoglobulin domain and mucin domain 3 (TIM-3) was analyzed in virus-specific CD8+ T-cells derived from peripheral blood or liver using major histocompatibility complex class I pentamers targeting immunodominant epitopes of HBV, Epstein-Barr-virus (EBV), or influenza virus (Flu). In chronic HBV infection, virus-specific CD8+ T-cells expressed higher levels of CD244 both in the peripheral blood and liver in comparison to the acute phase of infection or following resolution. CD244 was expressed at similarly high levels in EBV infection, but was low on Flu-specific CD8+ T-cells. In chronic HBV infection, high-level CD244 expression coincided with an increased expression of PD-1. The inhibition of the CD244 signaling pathway by antibodies directed against either CD244 or its ligand CD48 resulted in an increased virus-specific proliferation and cytotoxicity as measured by the expression of CD107a, interferon-γ, and tumor necrosis factor-α in CD8+ T-cells. CD244 and PD-1 are highly coexpressed on virus-specific CD8+ T-cells in chronic HBV infection and blocking CD244 or its ligand CD48 may restore T-cell function independent of the PD-1 pathway. CD244 may thus be another potential target for immunotherapy in chronic viral infections.

Perforin and IL-2 Upregulation Define Qualitative Differences among Highly Functional Virus-Specific Human CD8+ T Cells

The prevailing paradigm of T lymphocyte control of viral replication is that the protective capacity of virus-specific CD8+ T cells is directly proportional to the number of functions they can perform, with IL-2 production capacity considered critical. Having recently defined rapid perforin upregulation as a novel effector function of antigen-specific CD8+ T cells, here we sought to determine whether new perforin production is a component of polyfunctional CD8+ T cell responses that contributes to the control of several human viral infections: cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza (flu), and adenovirus (Ad). We stimulated normal human donor PBMC with synthetic peptides whose amino acid sequences correspond to defined CTL epitopes in the aforementioned viruses, and then used polychromatic flow cytometry to measure the functional capacity and the phenotype of the responding CD8+ T cells. While EBV and flu-specific CD8+ T cells rarely upregulate perforin, CMV-specific cells often do and Ad stimulates an exceptionally strong perforin response. The differential propensity of CD8+ T cells to produce either IL-2 or perforin is in part related to levels of CD28 and the transcription factor T-bet, as CD8+ T cells that rapidly upregulate perforin harbor high levels of T-bet and those producing IL-2 express high amounts of CD28. Thus, “polyfunctional” profiling of antigen-specific CD8+ T cells must not be limited to simply the number of functions the cell can perform, or one particular memory phenotype, but should actually define which combinations of memory markers and functions are relevant in each pathogenic context.

Universal CD137 Expression upon Activation Allows Efficient Isolation of a Broad Repertoire of Virus-Specific CD8+ and CD4+ T Cells for Adoptive Immunotherapy.

Reactivation of adenovirus (ADV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) can cause serious morbidity and mortality during the prolonged period of immune deficiency following allogeneic stem cell transplantation. It has been shown that adoptive transfer of donor-derived virus-specific T cells can be a successful strategy to control viral reactivation. To provide safe and effective anti-viral immunotherapy, we aimed to generate combined CD8+ and CD4+ T cell lines with high specificity for a broad range of viral epitopes. Isolation by the IFNg capture assay of virus-specific T cells that produce IFNg upon activation allows the generation of highly specific T cell lines without the need for extensive culture. However, it has been recently shown that specific upregulation of the co-stimulatory molecule CD137 upon antigen-specific activation of CD8+ and CD4+ T cells can also be used for isolation. We therefore analyzed IFNg production and CD137 expression by CD8+ and CD4+ T cells upon incubation of peripheral blood mononuclear cells (PBMC) from seropositive donors with peptides corresponding to 17 defined MHC class I restricted minimal epitopes from 10 different ADV, CMV, EBV and influenza (FLU) proteins, and 15-mer or 30-mer peptides containing MHC class II restricted epitopes from CMV pp65 or ADV hexon. Using tetramer and intracellular IFNg staining we first determined the fraction of CD8+ T cells that produced IFNg upon activation with the minimal epitopes. Specific IFNg production was observed for 58–100% of tetramer+ CD8+ T cells specific for CMV pp65 (n=6), and 83% for FLU (n=1), but only 18–58% for CMV pp50 (n=3) or IE-1 (n=3), 4–91% for EBV latent (n=3) and lytic (n=3) epitopes, and 41–63% for ADV hexon (n=2). In contrast to the variation in the fraction of IFNg-producing cells, we observed homogeneous upregulation of CD137 by the virus-specific tetramer+ T cell populations upon activation. In 2 cases where no CD137 expression by tetramer+ T cells could be detected, no IFNg production was observed either. These data suggest that the majority of CD8+ T cells specific for CMV pp65 or FLU can be isolated on basis of IFNg production, but only part of CD8+ T cell populations specific for other viral proteins, while complete virus-specific CD8+ T cell populations may be isolated on basis of CD137 expression. Activation of CD4+ T cells specific for CMV pp65 or ADV hexon with 15-mer or 30-mer peptides induced both specific IFNg production and CD137 expression. To investigate whether multiple virus-specific T cell populations could be isolated simultaneously, we next determined the kinetics of IFNg production after activation with defined MHC class I epitopes or peptides containing MHC class II epitopes. CMV- and EBV-specific CD8+ T cells and CMV-specific CD4+ T cells showed a rapid induction of IFNg production, which peaked after 4 hours and decreased thereafter. In contrast, ADV- and FLU-specific CD8+ T cells and ADV-specific CD4+ T cells, predominantly having a more early differentiation phenotype (CD27+CD28+) compared to CMV- and EBV-specific T cells, showed peak IFNg production after 8 hours that continued for more than 48 hours. This difference in phenotype and IFNg kinetics may suggest that the persistent and frequent presentation of CMV and EBV epitopes in vivo, in contrast to an intermittent exposure to ADV and FLU epitopes, drives differentiation and shapes the kinetics of the IFNg response of specific T cells. Kinetic analysis of CD137 expression showed uniform upregulation by virus-specific CD8+ T cell populations from day 1 to day 4 after activation, which peaked at day 2, suggesting that this may be the optimal time point for CD137-based isolation. In a limited number of experiments, virus-specific CD8+ and CD4+ T cells could be isolated based on CD137 expression within the same timeframe. These data indicate that virus-specific T cell populations can be more efficiently isolated at one time point on basis of CD137 expression than on basis of IFNg production, due to differences in IFNg kinetics. In conclusion, this study shows that T cell lines generated by CD137 isolation may comprise a significant number of virus-specific T cells which do not produce IFNg, but may have other effector functions. Furthermore, CD137-based enrichment may be more robust and allows the efficient simultaneous isolation of multiple virus-specific T cell populations due to uniform kinetics of CD137 expression.

Engineered ovarian cancer artificial antigen presenting cells (aAPCs) support CD8+T cells growth and function

Ovarian cancer is one of the leading causes of cancer death in women. Immunotherapy may play a potential role as an ovarian cancer treatment. In this study, we demonstrated that the ovarian cancer cell line OV79m can be engineered to function as an aAPC, by using lentiviral vectors to introduce the costimulatory molecules CD64, CD86 and 41BBL (OV79m-aAPCs). The OV79m-aAPCs support long-term polyclonal expansion of human CD8+ T cells from normal donors in vitro, and the cell expansion does not require exogenous cytokines. Flow cytometric analysis has shown that (1) polyclonal expanded CD8+ T cells from normal donors have an early effectors’ CD8+ T cell phenotype, (2) the expanded CD8+ T cells produce the cytokines IL-2 and IFNγ, (3) CD107a expression can be detected on the cell surface of CD8+ T cells following activation. Further studies have shown that OV79m-aAPCs transduced to express influenza matrix protein support long term expansion of Flu-specific CD8+ T cells from normal donors. The expanded Flu-specific CD8+T cells produce IFNγ and are CD107a positive. In conclusion, our data suggest that ovarian cancer cell lines can be engineered as effective APCs; therefore they have therapeutic potential for ovarian cancer patients. Future studies need to examine if the OV79m-aAPCs can expand tumor-specific T cells from patient tumor infiltrating T cells (TILs), and if the expanded TILs are functional in vivo.

CD8 + T cells specific for both persistent and non-persistent viruses display distinct differentiation phenotypes but have similar level of PD-1 expression in healthy Chinese individuals

Anti-viral CD8 + T cell responses involve an initial expansion and effector phase, followed by contraction phase and formation of CD8 + memory T cells. During this contraction phase, increased surface expression of the negative regulator PD-1 is associated with functional exhaustion of CD8 + T cells. Although its role in T cell suppression has been established, the importance of PD-1 in the differentiation of CD8 + T cells remains unclear. In this study, we examine PD-1 expression in relation to viral specificity of CD8 + T cells against persistent or non-persistent viruses, and further define differentiation phenotypes of CD8 + T cells by CD27 and CD28 expression. Surprisingly, the inhibitory receptor PD-1 was expressed by Flu-specific CD8 + T cells in a level comparable to HCMV-and EBV-specific cells. Moreover, in virus-specific CD8 + T cells, CD127 +/CD127 − and CD62L +/CD62L − cells expressed similar levels of PD-1 molecules. These results suggest that the PD-1/PD-L1 pathway may play a regulatory role in memory T cell subsets in addition to its association with T-cell exhaustion.

Persistent Depots of Influenza Antigen Fail To Induce a Cytotoxic CD8 T Cell Response

Encounter with Ag during chronic infections results in the generation of phenotypically and functionally heterogeneous subsets of Ag-specific CD8 T cells. Influenza, an acute infection, results in the generation of similar CD8 T cell heterogeneity, which may be attributed to long-lived depots of flu Ags that stimulate T cell proliferation well after virus clearance. We hypothesized that the heterogeneity of flu-specific CD8 T cells and maintenance of T cell memory required the recruitment of new CD8 T cells to persistent depots of flu Ag, as was the case for flu-specific CD4 T cell responses. However, robust expansion and generation of highly differentiated cytolytic effectors and memory T cells only occurred when naive CD8 T cells were primed during the first week of flu infection. Priming of new naive CD8 T cells after the first week of infection resulted in low numbers of poorly functional effectors, with little to no cytolytic activity, and a negligible contribution to the memory pool. Therefore, although the presentation of flu Ag during the late stages of infection may provide a mechanism for maintaining an activated population of CD8 T cells in the lung, few latecomer CD8 T cells are recruited into the functional memory T cell pool.

Inducing mammaglobin-A (mamA) specific HLA class I tetramer positive CD8+cytotoxic T-lymphocytes (CTL) against breast cancer by DNA vaccination

2522 Background: We recently reported that DNA vaccination using MamA, a highly expressed human (hu) breast cancer specific protein, leads to immunity against breast cancer. Here we demonstrate that the induction of breast cancer immunity following MamA DNA vaccination occurs through the expansion of CD8+ CTL by using HLA class I MamA peptide tetramers. Methods: HLA-A2+huCD8+ double transgenic mice received four (100μg) two-weekly IM injections of PCI-neo vector cloned with hu MamA gene. HLA-A2 tetramers (tet) carrying an immunodominant Mam2.1 peptide (LIYDSSLCDL) were used to monitor development of mam-A specific CD8+ T cells. Specificity of tetramers was tested against CD8+ CTL lines developed in vitro by stimulation of normal hu HLA-A2+ peripheral blood lymphocytes with TAP-2 deficient T2 cells pulsed with pooled MamA HLA-A2 binding peptides. Results: There was a sequential expansion of Mam2.1 tet+CD8+ T cells during stimulation of hu PBLs with MamA peptides in vitro (<1% pre-stimulation to >15% after six stimulations). These Mam2.1 tet+CD8+ T cells were HLA-A2 restricted and MamA specific since they revealed cytotoxicity only towards UACC-822 (HLA-A2+MamA+, 75% lysis) but not MCF-7 (A2+MamA−) or DU-4475 (A2−MamA+) breast cancer cell lines. In contrast, there was no binding of MamA peptide tetramers to control Flu-specific CD8+ CTL lines (<1%). MamA DNA vaccination, but not PCI-neo vector alone, of HLA-A2+huCD8+ mice lead to the expansion of Mam2.1 tet+CD8+ T cells (<0.7% pre-vaccination to >2.5% post-vaccination) in the peripheral blood that revealed in vitro cytotoxicity against UACC-822 (70% lysis) but not against DU-4475 or MCF-7 breast cancer cell lines. Adoptive transfer of both whole splenocytes or fractionated CD8+ T cells from vaccinated mice into immunodeficient SCID-beige mice with previously established subcutaneous hu breast cancer colonies lead to tumor infiltration of tet+CD8+ CTLs and, at 4-weeks, at least 80% tumor regression of UACC-822 but not DU4475 or MCF-7 colonies. Conclusions: MamA DNA vaccination resulted in the expansion of CD8+ CTL. The CD8+ CTL were HLA class I MamA peptide tetramer positive, HLA class I restricted and specifically lysed mamA+ human breast cancer cells both in vitro and in vivo. No significant financial relationships to disclose.

Selective accumulation of differentiated CD8+ T cells specific for respiratory viruses in the human lung

The lungs are frequently challenged by viruses, and resident CD8+ T cells likely contribute to the surveillance of these pathogens. To obtain insight into local T cell immunity to respiratory viruses in humans, we determined the specificity, phenotype, and function of lung-residing CD8+ T cells and peripheral blood CD8+ T cells in a paired analysis. The lung contained markedly higher frequencies of influenza (FLU)-specific and respiratory syncytial virus (RSV)-specific CD8+ T cells when compared with the circulation. This contrasted with an equal distribution of cytomegalovirus- and Epstein-Bar virus–specific CD8+ T cells. Noticeably, a substantial fraction of the lung-residing FLU- and RSV-specific CD8+ T cells had progressed to a relatively late differentiation phenotype, reflected by low expression of CD28 and CD27. Lung-derived FLU-specific CD8+ T cells had low activation requirements, as expansion of these cells could be initiated by cognate peptide in the absence of helper cell–derived signals. Thus, the human lung contains high numbers of differentiated FLU- and RSV-specific memory CD8+ T cells that can readily expand upon reexposure to virus. Resident lung T cells may provide immediate immunological protection against pulmonary virus infections.