4-1BB Signaling in Conventional T Cells Drives IL-2 Production That Overcomes CD4+CD25+FoxP3+ T Regulatory Cell Suppression

Costimulation with the recombinant SA-4-1BBL agonist of 4-1BB receptor on conventional CD4⁺ T cells (Tconvs) overcomes the suppression mediated by naturally occurring CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs). The mechanistic basis of this observation has remained largely unknown. Herein we show that Tconvs, but not Tregs, are the direct target of SA-4-1BBL-mediated evasion of Treg suppression. IL-2 produced by Tconvs in response to 4-1BB signaling is both necessary and sufficient for overcoming Treg suppression. Supernatant from Tconvs stimulated with SA-4-1BBL contains high levels of IL-2 and overcomes Treg suppression in ex vivo Tconv:Treg cocultures. Removal of IL-2 from such supernatant restores Treg suppression and repletion of Tconv:Treg cocultures with exogenous recombinant IL-2 overcomes suppression. This study establishes 4-1BB signaling as a key circuit that regulates physical and functional equilibrium between Tregs and Tconvs with important implications for immunotherapy for indications where a fine balance between Tregs and Teffs plays a decisive role.     

The Late Domain of Human Immunodeficiency Virus Type 1 p6 Promotes Virus Release in a Cell Type-Dependent Manner

The p6 domain of human immunodeficiency virus type 1 (HIV-1) is located at the C terminus of the Gag precursor protein Pr55(Gag). Previous studies indicated that p6 plays a critical role in HIV-1 particle budding from virus-expressing HeLa cells. In this study, we performed a detailed mutational analysis of the N terminus of p6 to map the sequences required for efficient virus release. We observed that the highly conserved P-T/S-A-P motif located near the N terminus of p6 is remarkably sensitive to change; even conservative mutations in this sequence imposed profound virus release defects in HeLa cells. In contrast, single and double amino acid substitutions outside the P-T/S-A-P motif had no significant effect on particle release. The introduction of stop codons one or two residues beyond the P-T/S-A-P motif markedly impaired virion release, whereas truncation four residues beyond P-T/S-A-P had no effect on particle production in HeLa cells. By examining the effects of p6 mutation in biological and biochemical analyses and by electron microscopy, we defined the role of p6 in particle release and virus replication in a panel of T-cell and adherent cell lines and in primary lymphocytes and monocyte-derived macrophages. We demonstrated that the effects of p6 mutation on virus replication are markedly cell type dependent. Intriguingly, even in T-cell lines and primary lymphocytes in which p6 mutations block virus replication, these changes had little or no effect on particle release. However, p6-mutant particles produced in T-cell lines and primary lymphocytes exhibited a defect in virion-virion detachment, resulting in the production of tethered chains of virions. Virus release in monocyte-derived macrophages was markedly inhibited by p6 mutation. To examine further the cell type-specific virus release defect in HeLa versus T cells, transient heterokaryons were produced between HeLa cells and the Jurkat T-cell line. These heterokaryons display a T-cell-like phenotype with respect to the requirement for p6 in particle release. The results described here define the role of p6 in virus replication in a wide range of cell types and reveal a strong cell type-dependent requirement for p6 in virus particle budding.     

Prognostic Impact of FoxP3+ Regulatory T Cells in Relation to CD8+ T Lymphocyte Density in Human Colon Carcinomas

Background

T-lymphocyte infiltration into colon carcinomas can influence clinical outcome, and interactions among T cell subsets may be more informative than either subset alone. Our objective was to examine the prognostic impact of tumor-infiltrating FoxP3⁺ regulatory T cells (Tregs) in relation to cytotoxic CD8⁺ T lymphocytes in patients with colon carcinomas characterized by DNA mismatch repair (MMR) status who participated in adjuvant chemotherapy trials.

Methods

FoxP3⁺ and CD8⁺ densities in tumor epithelial and stromal compartments were analyzed by immunohistochemistry and quantified in resected, stage II and III colonic carcinomas (N = 216). Immune marker density was dichotomized at the median and categorized as high vs low. MMR status was classified as MMR deficient (dMMR) or proficient (pMMR). Cox models were adjusted for age, stage, and tumor grade.

Results

The density of FoxP3+ infiltration was similar in tumor stroma and epithelia, whereas CD8+ was higher in stroma. The prognostic impact of FoxP3+ and CD8+ T cell infiltration was stronger in stroma vs epithelia, and the density of each marker in stroma was independently associated with improved overall survival (OS). However, the impact of FoxP3+ on survival was dependent upon CD8+ density (P interaction  = .040). Among CD8+_low tumors, FoxP3+_high cases had significantly improved OS compared to FoxP3+_low cases after adjustment for covariates (hazard ratio 0.43; 95% confidence interval 0.19 to 0.95; P = .030). In contrast, FoxP3+ was not prognostic among CD8+_high tumors. FoxP3+ remained prognostic in CD8+_low tumors after further adjustment for MMR or BRAF ^V600E mutation status. Additionally, these immune markers identified a pMMR subgroup with a similarly favorable OS as for dMMR tumors.

Conclusions

The prognostic impact of FoxP3+ and CD8+ T cell density are inter-dependent, whereby FoxP3+ exerts a favorable influence on survival only in colon cancers with low CD8+ infiltration.     

Circulating FoxP3+ Regulatory T and Interleukin17-Producing Th17 Cells Actively Influence HBV Clearance in De Novo Hepatitis B Virus Infected Patients after Orthotopic Liver Transplantation

Objective

To longitudinally investigate the role of FoxP3+ Regulatory T cells (Treg) and interleukin17-producing T helper 17 cells (Th17) in De Novo Hepatitis B Virus infection after orthotopic Liver Transplantation (DNHB-OLT), and analyze the possible correlation between these cells and HBV clearance of the disease.

Methods

We enrolled 12 control cases after orthotopic Liver Transplantation (OLT) and 24 patients, including 12 diagnosed with DNHB-OLT and 12 diagnosed with Acute Hepatitis B Virus infection (AHB), into the study from the liver transplantation and research center at Beijing 302 Hospital. Flow cytometry was used to detect the frequencies of Treg and Th17, and ELISA was applied to detect the concentration of IL6, IL22, TGF-β and IL2 in peripheral blood. We also measured the gene expression level by real time-quantitative PCR and protein expression using immunohistochemistry and western-blot. Furthermore, we divided DNHB-OLT patients into the clearance and non-clearance groups and examined longitudinally Th17, Treg cells at different times.

Results

The percentage of Treg cells, expression of FoxP3 mRNA and related anti-inflammatory cytokines such as IL2 and TGF-β1 in the DNHB-OLT group were significantly higher than that in the AHB and OLT groups. The percentage of Th17 cells, expression of RORγt mRNA and related pro-inflammatory cytokines such as IL17 and IL22 in the DNHB-OLT group were significantly lower than that in the AHB group, but the levels of these cytokines are very similar to the OLT group. The ratios of Treg to Th17 in the DNHB-OLT group were significantly higher than that in the OLT and AHB groups. Treg frequencies significantly correlated with HBV DNA, whereas IL17 frequencies didn’t significantly correlate with ALT. In DNHB-OLT patients, the clearance group was accompanied by a rapid increase in the Th17 cells during the first 4^th week and afterwards continuously decrease to the control group, together with a continuously decrease in Treg cells from the onset time point, which lead to a significant reduction in the ratios of Treg to Th17. The non-clearance group was accompanied by an increase in the Treg cells during the first 4^th week and afterwards sharply decrease, together with a relatively stable and unchanged Th17 cells, which lead to a significant change in the ratios. In addition, compared to clearance group, the ratios of Treg to Th17 in non-clearance group were significantly higher at the onset point, 4^th and 12^th week, but no difference at 24^th week.

Conclusion

DNHB-OLT patients possessed a favorable Treg differentiation environment, accompanied by a sustained higher preferentially Treg frequencies and up-regulation of related anti-inflammatory cytokines. The immune imbalance of the ratios between Treg and Th17 existed in DNHB-OLT patients. The changes of the ratios during the DNHB-OLT events were associated with HBV clearance, which suppressed immune inflammation reaction as well as inhibited ability of specific HBV clearance and led to immune escape and chronicity.     

A Human CD4+ T Cell Epitope in the Influenza Hemagglutinin Is Cross-Reactive to Influenza A Virus Subtypes and to Influenza B Virus

The hemagglutinin protein (HA) of the influenza virus family is a major antigen for protective immunity. Thus, it is a relevant target for developing vaccines. Here, we describe a human CD4(+) T cell epitope in the influenza virus HA that lies in the fusion peptide of the HA. This epitope is well conserved in all 16 subtypes of the HA protein of influenza A virus and the HA protein of influenza B virus. By stimulating peripheral blood mononuclear cells (PBMCs) from a healthy adult donor with peptides covering the entire HA protein based on the sequence of A/Japan/305/1957 (H2N2), we generated a T cell line specific to this epitope. This CD4(+) T cell line recognizes target cells infected with influenza A virus seasonal H1N1 and H3N2 strains, a reassortant H2N1 strain, the 2009 pandemic H1N1 strain, and influenza B virus in cytotoxicity assays and intracellular-cytokine-staining assays. It also lysed target cells infected with avian H5N1 virus. We screened healthy adult PBMCs for T cell responses specific to this epitope and found individuals who had ex vivo gamma interferon (IFN-γ) responses to the peptide epitope in enzyme-linked immunospot (ELISPOT) assays. Almost all donors who responded to the epitope had the HLA-DRB1*09 allele, a relatively common HLA allele. Although natural infection or standard vaccination may not induce strong T and B cell responses to this highly conserved epitope in the fusion peptide, it may be possible to develop a vaccination strategy to induce these CD4(+) T cells, which are cross-reactive to both influenza A and B viruses.     

Decline of FoxP3+ Regulatory CD4 T Cells in Peripheral Blood of Children Heavily Exposed to Malaria

FoxP3+ regulatory CD4 T cells (T_regs) help to maintain the delicate balance between pathogen-specific immunity and immune-mediated pathology. Prior studies suggest that T_regs are induced by P. falciparum both in vivo and in vitro; however, the factors influencing T_reg homeostasis during acute and chronic infections, and their role in malaria immunopathogenesis, remain unclear. We assessed the frequency and phenotype of T_regs in well-characterized cohorts of children residing in a region of high malaria endemicity in Uganda. We found that both the frequency and absolute numbers of FoxP3+ T_regs in peripheral blood declined markedly with increasing prior malaria incidence. Longitudinal measurements confirmed that this decline occurred only among highly malaria-exposed children. The decline of T_regs from peripheral blood was accompanied by reduced in vitro induction of T_regs by parasite antigen and decreased expression of TNFR2 on T_regs among children who had intense prior exposure to malaria. While T_reg frequencies were not associated with protection from malaria, there was a trend toward reduced risk of symptomatic malaria once infected with P. falciparum among children with lower T_reg frequencies. These data demonstrate that chronic malaria exposure results in altered T_reg homeostasis, which may impact the development of antimalarial immunity in naturally exposed populations.     

CD4+CD25+FoxP3+ 调节性T 细胞与黑龙江省HIV/AIDS 患者病情 相关性的研究

目的:比较黑龙江省HIV/AIDS患者与健康对照者(healthy controls,HCs)外周血CD4+CD25+FoxP3+调节性T细胞数量、免疫抑制功能的变化,探讨CD4+CD25+FoxP3+调节性T细胞在HIV/AIDS感染过程中的作用。方法:采用流式细胞仪检测21例HIV/AIDS患者及20例健康对照组的外周血CD4+CD25+FoxP3+调节性T细胞数量的百分比及绝对数量;采用共同培养方法检测HIV/AIDS患者外周血CD4+CD25+FoxP3+调节性T细胞免疫抑制功能的变化;实时荧光定量聚合酶链反应(RT-FQ-PCR)检测HIV/AIDS患者外周血CD4+CD25+FoxP3+调节性T细胞中FoxP3mRNA的表达。结果:黑龙江省HIV/AIDS患者外周血CD4+CD25+FoxP3+调节性T细胞比率明显高于HCs(P<0.01),而CD4+CD25+FoxP3+调节性T细胞的绝对计数显著下降,且与CD4+T细胞绝对计数成反比;混合淋巴细胞共同培养结果显示,HIV/AIDS患者外周血CD4+CD25+FoxP3+调节性T细胞的抑制功能无明显变化;HIV/AIDS患者外周血CD4+CD25+FoxP3+调节性T细胞的FoxP3 mRNA相对表达量无显著变化。结论:黑龙江省HIV/AIDS患者CD4+CD25+FoxP3+调节性T细胞的数量变化与病情相关。     

Retention of CD4+ CD25+ FoxP3+ regulatory T cells in the liver after therapy-induced hepatitis C virus eradication in humans

Following infection with the hepatitis C virus (HCV), in most cases immunity fails to eradicate the virus, resulting in slowly progressing immunopathology in the HCV-infected liver. We are the first to examine intrahepatic T cells and CD4(+) CD25(+) FoxP3(+) regulatory T cells (Treg) in patients chronically infected with HCV (chronic HCV patients) during and after antiviral therapy by collecting multiple aspiration biopsy samples from the liver at different time points. We found that intrahepatic Treg frequencies were increased upon alpha interferon and ribavirin administration in about 50% of chronic HCV patients, suggesting stronger regulation of intrahepatic immunity by Treg during antiviral therapy. After cessation of antiviral therapy, the frequency of intrahepatic Treg remained above baseline in the large majority of livers of individuals who successfully cleared the virus. The phenotype of those Treg that were retained in the liver months after therapy-induced clearance of HCV RNA indicated a reduced contribution of effector memory cells. Our findings, gathered by multiple samplings of the liver, indicate that successful antiviral therapy of chronic HCV patients does not lead to normalization of the local immune response to a resting state comparable to that for healthy livers. The continuous presence of high numbers of Treg, with a phenotype reflecting a relatively weak suppressive activity, suggests ongoing residual regulation of immunopathology. These findings provide important insight into the dynamics of the immune response to HCV, as well as the effect of therapy on intrahepatic immunity.     

FoxP3+ Regulatory T Cells Determine Disease Severity in Rodent Models of Inflammatory Neuropathies

Inflammatory neuropathies represent disabling human autoimmune disorders with considerable disease variability. Animal models provide insights into defined aspects of their disease pathogenesis. Forkhead box P3 (FoxP3)+ regulatory T lymphocytes (Treg) are anti-inflammatory cells that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. Dysfunction or a reduced frequency of Tregs have been associated with different human autoimmune disorders. We here analyzed the functional relevance of Tregs in determining disease manifestation and severity in murine models of autoimmune neuropathies. We took advantage of the DEREG mouse system allowing depletion of Treg with high specificity as well as anti-CD25 directed antibodies to deplete Tregs in mice in actively induced experimental autoimmune neuritis (EAN). Furthermore antibody-depletion was performed in an adoptive transfer model of chronic neuritis. Early Treg depletion increased clinical EAN severity both in active and adoptive transfer chronic neuritis. This was accompanied by increased proliferation of myelin specific T cells and histological signs of peripheral nerve inflammation. Late stage Treg depletion after initial disease manifestation however did not exacerbate inflammatory neuropathy symptoms further. We conclude that Tregs determine disease severity in experimental autoimmune neuropathies during the initial priming phase, but have no major disease modifying function after disease manifestation. Potential future therapeutic approaches targeting Tregs should thus be performed early in inflammatory neuropathies.     

Induction of FoxP3+CD4+CD25+ regulatory T cells by a bone marrow population distinct from plasmacytoid-DC

Facilitating cells (FC) are bone marrow-derived cells that facilitate allogeneic hematopoietic stem cell (SC) engraftment and induce transplantation tolerance without causing graft vs. host disease. Although there is evidence for FC directing the development of FoxP3⁺CD4⁺CD25⁺ regulatory T cells, the specific FC subsets that control regulatory T cell development have not been defined. The current study investigates the role of FC-CD3ε⁺ and FC-CD3ε⁻ subpopulations in the development of FoxP3⁺CD4⁺CD25⁺ regulatory T cells. Here, we demonstrate that the induction of FoxP3⁺CD4⁺CD25⁺ regulatory T cells in coculture is mediated by not only the FC-CD3ε⁻ subset but also the FC-CD3ε⁺ subset, which is distinct from plasmacytoid precursor dendritic cells (p-preDC). The identification of cell populations distinct from p-preDC that efficiently induce the generation of FoxP3⁺CD4⁺CD25⁺ regulatory T cells may prove useful for future therapeutic applications for the induction of tolerance following allogeneic SC transplantation.     

Nicotine Promotes Late Endothelial Progenitor Cells Functional Activity in a PI 3-Kinase-Dependent Manner

Recent studies have shown that endothelial progenitor cells (EPCs) participated in angiogenic effects of nicotine and nicotine dose dependently increased the functional activity of early EPCs. The effects of nicotine on late EPCs remain to be determined. Therefore, we investigated whether nicotine had influences on the functional activity of late EPCs. Late EPCs were isolated from human umbilical cord blood and characterized. Late EPCs of 3–5 passages were treated for 32 h with either vehicle or nicotine. The proliferative, migratory, and in vitro vasculogenesis activities of late EPCs were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, modified Boyden chamber assay, and in matrigel, respectively. Late EPCs adhesion assay was performed by replating cells on fibronectin-coated dishes, and then adherent cells were counted. Nicotine enhanced proliferative, migratory, adhesive, and in vitro vasculogenesis capacities of late EPCs. These effects were significantly reduced in the presence of phosphatidylinositol (PI) 3-kinase inhibitor.     

IL-21 Promotes Late Activator APC-Mediated T Follicular Helper Cell Differentiation in Experimental Pulmonary Virus Infection

IL-21 is a type-I cytokine that has pleiotropic immuno-modulatory effects. Primarily produced by activated T cells including NKT and T_FH cells, IL-21 plays a pivotal role in promoting T_FH differentiation through poorly understood cellular and molecular mechanisms. Here, employing a mouse model of influenza A virus (IAV) infection, we demonstrate that IL-21, initially produced by NKT cells, promotes T_FH differentiation by promoting the migration of late activator antigen presenting cell (LAPC), a recently identified T_FH inducer, from the infected lungs into the draining lymph nodes (dLN). LAPC migration from IAV-infected lung into the dLN is CXCR3-CXCL9 dependent. IL-21-induced TNF-α production by conventional T cells is critical to stimulate CXCL9 expression by DCs in the dLN, which supports LAPC migration into the dLN and ultimately facilitates T_FH differentiation. Our results reveal a previously unappreciated mechanism for IL-21 modulation of T_FH responses during respiratory virus infection.     

Plasmodium vivax: Induction of CD4+CD25+FoxP3+ Regulatory T Cells during Infection Are Directly Associated with Level of Circulating Parasites

Circulation CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Tregs) have been associated with the delicate balancing between control of overwhelming acute malaria infection and prevention of immune pathology due to disproportionate inflammatory responses to erythrocytic stage of the parasite. While the role of Tregs has been well-documented in murine models and P. falciparum infection, the phenotype and function of Tregs in P. vivax infection is still poorly characterized. In the current study, we demonstrated that patients with acute P. vivax infection presented a significant augmentation of circulating Tregs producing anti-inflammatory (IL-10 and TGF-β) as well as pro-inflammatory (IFN-γ, IL-17) cytokines, which was further positively correlated with parasite burden. Surface expression of GITR molecule and intracellular expression of CTLA-4 were significantly upregulated in Tregs from infected donors, presenting also a positive association between either absolute numbers of CD4⁺CD25⁺FoxP3⁺GITR⁺ or CD4⁺CD25⁺FoxP3⁺CTLA-4⁺ and parasite load. Finally, we demonstrate a suppressive effect of Treg cells in specific T cell proliferative responses of P. vivax infected subjects after antigen stimulation with Pv-AMA-1. Our findings indicate that malaria vivax infection lead to an increased number of activated Treg cells that are highly associated with parasite load, which probably exert an important contribution to the modulation of immune responses during P. vivax infection.     

An Increase in CD3+CD4+CD25+ Regulatory T Cells after Administration of Umbilical Cord-Derived Mesenchymal Stem Cells during Sepsis

Sepsis remains an important cause of death worldwide, and vigorous immune responses during sepsis could be beneficial for bacterial clearance but at the price of collateral damage to self tissues. Mesenchymal stem cells (MSCs) have been found to modulate the immune system and attenuate sepsis. In the present study, MSCs derived from bone marrow and umbilical cord were used and compared. With a cecal ligation and puncture (CLP) model, the mechanisms of MSC-mediated immunoregulation during sepsis were studied by determining the changes of circulating inflammation-associated cytokine profiles and peripheral blood mononuclear cells 18 hours after CLP-induced sepsis. In vitro, bone marrow-derived MSCs (BMMSCs) and umbilical cord-derived MSCs (UCMSCs) showed a similar morphology and surface marker expression. UCMSCs had stronger potential for osteogenesis but lower for adipogenesis than BMMSCs. Compared with rats receiving PBS only after CLP, the percentage of circulating CD3+CD4+CD25+ regulatory T (Treg) cells and the ratio of Treg cells/T cells were elevated significantly in rats receiving MSCs. Further experiment regarding Treg cell function demonstrated that the immunosuppressive capacity of Treg cells from rats with CLP-induced sepsis was decreased, but could be restored by administration of MSCs. Compared with rats receiving PBS only after CLP, serum levels of interleukin-6 and tumor necrosis factor-α were significantly lower in rats receiving MSCs after CLP. There were no differences between BMMSCs and UCMSCs. In summary, this work provides the first in vivo evidence that administering BMMSCs or UCMSCs to rats with CLP-induced sepsis could increase circulating CD3+CD4+CD25+ Treg cells and Treg cells/T cells ratio, enhance Treg cell suppressive function, and decrease serum levels of interleukin-6 and tumor necrosis factor-α, suggesting the immunomodulatory association of Treg cells and MSCs during sepsis.     

Isolation of Highly Suppressive CD25+FoxP3+ T Regulatory Cells from G-CSF-Mobilized Donors with Retention of Cytotoxic Anti-Viral CTLs: Application for Multi-Functional Immunotherapy Post Stem Cell Transplantation

Previous studies have demonstrated the effective control of cytomegalovirus (CMV) infections post haematopoietic stem cell transplant through the adoptive transfer of donor derived CMV-specific T cells (CMV-T). Strategies for manufacturing CMV immunotherapies has involved a second leukapheresis or blood draw from the donor, which in the unrelated donor setting is not always possible. We have investigated the feasibility of using an aliquot of the original G-CSF-mobilized graft as a starting material for manufacture of CMV-T and examined the activation marker CD25 as a targeted approach for identification and isolation following CMVpp65 peptide stimulation. CD25+ cells isolated from G-CSF-mobilized apheresis revealed a significant increase in the proportion of FoxP3 expression when compared with conventional non-mobilized CD25+ cells and showed a superior suppressive capacity in a T cell proliferation assay, demonstrating the emergence of a population of Tregs not present in non-mobilized apheresis collections. The expansion of CD25+ CMV-T in short-term culture resulted in a mixed population of CD4+ and CD8+ T cells with CMV-specificity that secreted cytotoxic effector molecules and lysed CMVpp65 peptide-loaded phytohaemagglutinin-stimulated blasts. Furthermore CD25 expanded cells retained their suppressive capacity but did not maintain FoxP3 expression or secrete IL-10. In summary our data indicates that CD25 enrichment post CMV stimulation in G-CSF-mobilized PBMCs results in the simultaneous generation of both a functional population of anti-viral T cells and Tregs thus illustrating a potential single therapeutic strategy for the treatment of both GvHD and CMV reactivation following allogeneic haematopoietic stem cell transplantation. The use of G-CSF-mobilized cells as a starting material for cell therapy manufacture represents a feasible approach to alleviating the many problems incurred with successive donations and procurement of cells from unrelated donors. This approach may therefore simplify the clinical application of adoptive immunotherapy and broaden the approach for manufacturing multi-functional T cells.     

Multiple Distinct Forms of CD8+ T Cell Cross-Reactivity and Specificities Revealed after 2009 H1N1 Influenza A Virus Infection in Mice

Influenza primed mice are protected against lethal infection with H1N1 A/CA/04/E3/09 virus, and T depletion and serum transfer studies suggest a T-dependent mechanism. We therefore set out to investigate the quality of the cross-reactive T cell response to CA/E3/09 in mice primed with H3N2 influenza A/Hong Kong/X31 virus. Sequences of the immunodominant nucleoprotein (NP) NP366–374 and acid polymerase (PA) PA224–233 CD8 epitopes from X31 each differ from the CA/E3/09 virus by one amino acid: an M371V substitution at position 6 of the NP peptide, and an S224P substitution at position 1 of the PA peptide, raising questions about the role of these epitopes in protection. PA224–233 peptides from either virus could elicit IFN-γ spot forming cells from mice infected with X31, indicating cross-reactivity of these two peptides. However, no T cell responses to either PA224–233 peptide were detectable after primary CA/E3/09 infection, suggesting it is cryptic in this virus. In contrast, primary responses to the NP366 peptides were detectable after infection with either virus, but did not cross-react in vitro. Similarly, H2-D^b tetramers of each NP epitope stained CD8+ T cells from each respective virus infection, but did not obviously cross-react. Early after lethal CA/E3/09 challenge, X31 primed mice had enhanced IFN-γ responses toward both NP366 peptides, as well as recall responses to a set of subdominant NP and PA peptides not detectable after primary X31 infection alone. Furthermore, dual-tetramer staining revealed an expanded population of CD8 T cells reactive to both NP366 variant peptides also not seen after the priming infection alone. These observations demonstrate unusual CD8+ T cell cross-reactivity and specificity are elicited after primary and secondary CA/E3/09 influenza virus infections.