Peroxiredoxin II Regulates Effector and Secondary Memory CD8+ T Cell Responses

Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in cellular responses. However, the effect of increased H₂O₂ on an antigen-specific CD8⁺ T cell response was unknown. Following T cell receptor (TCR) stimulation, the expression and oxidation of peroxiredoxin II (PrdxII), a critical antioxidant enzyme, increased in CD8⁺ T cells. Deletion of PrdxII increased ROI, S phase entry, division, and death during in vitro division. During primary acute viral and bacterial infection, the number of effector CD8⁺ T cells in PrdxII-deficient mice was increased, while the number of memory cells were similar to those of the wild-type cells. Adoptive transfer of P14 TCR transgenic cells demonstrated that the increased expansion of effector cells was T cell autonomous. After rechallenge, effector CD8⁺ T cells in mutant animals were more skewed to memory phenotype than cells from wild-type mice, resulting in a larger secondary memory CD8⁺ T cell pool. During chronic viral infection, increased antigen-specific CD8⁺ T cells accumulated in the spleens of PrdxII mutant mice, causing mortality. These results demonstrate that PrdxII controls effector CD8⁺ T cell expansion, secondary memory generation, and immunopathology.     

Superior In Vitro Stimulation of Human CD8+ T-Cells by Whole Virus versus Split Virus Influenza Vaccines

Pandemic and seasonal influenza viruses cause considerable morbidity and mortality in the general human population. Protection from severe disease may result from vaccines that activate antigen-presenting DC for effective stimulation of influenza-specific memory T cells. Special attention is paid to vaccine-induced CD8⁺ T-cell responses, because they are mainly directed against conserved internal influenza proteins thereby presumably mediating cross-protection against circulating seasonal as well as emerging pandemic virus strains. Our study showed that influenza whole virus vaccines of major seasonal A and B strains activated DC more efficiently than those of pandemic swine-origin H1N1 and pandemic-like avian H5N1 strains. In contrast, influenza split virus vaccines had a low ability to activate DC, regardless which strain was investigated. We also observed that whole virus vaccines stimulated virus-specific CD8⁺ memory T cells much stronger compared to split virus counterparts, whereas both vaccine formats activated CD4⁺ Th cell responses similarly. Moreover, our data showed that whole virus vaccine material is delivered into the cytosolic pathway of DC for effective activation of virus-specific CD8⁺ T cells. We conclude that vaccines against seasonal and pandemic (-like) influenza strains that aim to stimulate cross-reacting CD8⁺ T cells should include whole virus rather than split virus formulations.     

PC61 (Anti-CD25) Treatment Inhibits Influenza A Virus-Expanded Regulatory T Cells and Severe Lung Pathology during a Subsequent Heterologous Lymphocytic Choriomeningitis Virus Infection

Prior immunity to influenza A virus (IAV) in mice changes the outcome to a subsequent lymphocytic choriomeningitis virus (LCMV) infection and can result in severe lung pathology, similar to that observed in patients that died of the 1918 H1N1 pandemic. This pathology is induced by IAV-specific memory CD8⁺ T cells cross-reactive with LCMV. Here, we discovered that IAV-immune mice have enhanced CD4⁺ Foxp3⁺ T-regulatory (Treg) cells in their lungs, leading us to question whether a modulation in the normal balance of Treg and effector T-cell responses also contributes to enhancing lung pathology upon LCMV infection of IAV-immune mice. Treg cell and interleukin-10 (IL-10) levels remained elevated in the lungs and mediastinal lymph nodes (mLNs) throughout the acute LCMV response of IAV-immune mice. PC61 treatment, used to decrease Treg cell levels, did not change LCMV titers but resulted in a surprising decrease in lung pathology upon LCMV infection in IAV-immune but not in naive mice. Associated with this decrease in pathology was a retention of Treg in the mLN and an unexpected partial clonal exhaustion of LCMV-specific CD8⁺ T-cell responses only in IAV-immune mice. PC61 treatment did not affect cross-reactive memory CD8⁺ T-cell proliferation. These results suggest that in the absence of IAV-expanded Treg cells and in the presence of cross-reactive memory, the LCMV-specific response was overstimulated and became partially exhausted, resulting in a decreased effector response. These studies suggest that Treg cells generated during past infections can influence the characteristics of effector T-cell responses and immunopathology during subsequent heterologous infections. Thus, in humans with complex infection histories, PC61 treatment may lead to unexpected results.     

Memory CD4 T Cells Direct Protective Responses to Influenza Virus in the Lungs through Helper-Independent Mechanisms

Memory CD4 T cells specific for influenza virus are generated from natural infection and vaccination, persist long-term, and recognize determinants in seasonal and pandemic influenza virus strains. However, the protective potential of these long-lived influenza virus-specific memory CD4 T cells is not clear, including whether CD4 T-cell helper or effector functions are important in secondary antiviral responses. Here we demonstrate that memory CD4 T cells specific for H1N1 influenza virus directed protective responses to influenza virus challenge through intrinsic effector mechanisms, resulting in enhanced viral clearance, recovery from sublethal infection, and full protection from lethal challenge. Mice with influenza virus hemagglutinin (HA)-specific memory CD4 T cells or polyclonal influenza virus-specific memory CD4 T cells exhibited protection from influenza virus challenge that occurred in the presence of CD8-depleting antibodies in B-cell-deficient mice and when CD4 T cells were transferred into lymphocyte-deficient RAG2^−/− mice. Moreover, the presence of memory CD4 T cells mobilized enhanced T-cell recruitment and immune responses in the lung. Neutralization of gamma interferon (IFN-γ) production in vivo abrogated memory CD4 T-cell-mediated protection from influenza virus challenge by HA-specific memory T cells and heterosubtypic protection by polyclonal memory CD4 T cells. Our results indicate that memory CD4 T cells can direct enhanced protection from influenza virus infection through mobilization of immune effectors in the lung, independent of their helper functions. These findings have important implications for the generation of universal influenza vaccines by promoting long-lived protective CD4 T-cell responses.Influenza virus poses substantial threats to world health due to the emergence of new pandemic strains through viral mutation and reassortment, including the 2009 H1N1 pandemic strain. Developing effective vaccines that can provide immune-mediated protection to multiple influenza virus strains remains a major challenge, as current vaccines generate neutralizing antibodies directed against the highly variable hemagglutinin (HA) and neuraminidase (NA) surface viral glycoproteins (18). These vaccines are only partially effective at protecting individuals from succumbing to seasonal strains and are largely ineffective at protecting individuals from new pandemics. In contrast, T lymphocytes have the potential to provide long-term cross-strain protection, through their recognition of invariant viral determinants (3, 9), generation of effector responses to coordinate both cellular and humoral immunity, and development of memory populations that persist for decades (34). In humans, influenza virus-specific CD4 and CD8 T cells recognize internal polymerase, matrix, and nucleoprotein components of influenza virus which are conserved in multiple strains (3). Influenza virus-specific memory T cells generated from virus exposure and vaccines can be detected readily in the peripheral blood of healthy older children and adults (16, 30). Elucidating the protective capacities of memory T cells in antiviral immunity and their underlying mechanisms is therefore crucial to understanding clinical responses to influenza and to developing strategies to boost T-cell-mediated immunity for the next emerging pandemic.The potent cytolytic responses of virus-specific CD8 T cells and their roles in antiviral primary and secondary responses have been well established (58); however, considerably less is known about the function of memory CD4 T cells in antiviral immunity. Memory CD4 T cells have the potential to play more diverse roles in coordinating secondary responses than those of memory CD8 T cells via their ability to “help” or promote cellular and humoral immunity, and also through direct effector functions. Compared to CD8 T-cell responses, memory CD4 T-cell responses in humans were found to recognize a more diverse array of influenza virus-specific epitopes (46-48) and to exhibit cross-reactivities with new pandemic strains, including avian H5N1 and 2009 H1N1 “swine flu” strains (23, 28, 36, 48). In addition, antiviral memory CD4 T cells generated as a result of influenza vaccination (22) were found to persist longer than CD8 T cells in vivo following smallpox vaccination (29). These findings suggest that memory CD4 T-cell responses could be potential targets for boosting long-term cellular immunity following vaccination, although their protective capacity remains undefined.The role of CD4 T cells in anti-influenza virus immunity has been elucidated mainly for primary responses, and less is known about the protective potential and mechanisms underlying memory CD4 T-cell-directed secondary responses. In primary influenza virus infection, CD4 T cells promote antibody production by B cells necessary for complete viral clearance (2, 17, 19, 39, 40, 57) and also promote the generation of memory CD8 T cells (4). Whether memory CD4 T cells have a similar helper-intensive role in promoting B cells and CD8 T cells in secondary influenza responses or whether effector responses predominate is not known. In this study, we investigated the mechanisms by which memory CD4 T cells mediate secondary responses and promote recovery from influenza virus infection in the clinically relevant scenario of a persisting CD4 T-cell response but no preexisting antibody response to a new influenza virus strain. We demonstrate that both influenza virus HA-specific and polyclonal influenza virus-specific memory CD4 T cells direct rapid lung viral clearance and protect from lethality via secondary antiviral responses in the absence of CD8 T cells, B cells, or any lymphocytes. Unlike primary responses to influenza virus, which can mediate protection independent of gamma interferon (IFN-γ), memory CD4 T-cell-mediated protection in the lung is dependent on secreted IFN-γ and is associated with localized interactions with lung airways and foci of T-cell-directed responses. Our findings reveal that memory CD4 T cells drive antiviral protection in the lung through a qualitatively distinct mechanism and have important implications for exploiting the protective role of persisting memory CD4 T cells in vaccines and immunotherapies.     

Gamma Interferon Regulates Contraction of the Influenza Virus-Specific CD8 T Cell Response and Limits the Size of the Memory Population

The factors that regulate the contraction of the CD8 T cell response and the magnitude of the memory cell population against localized mucosal infections such as influenza are important for generation of efficient vaccines but are currently undefined. In this study, we used a mouse model of influenza to demonstrate that the absence of gamma interferon (IFN-γ) or IFN-γ receptor 1 (IFN-γR1) leads to aberrant contraction of antigen-specific CD8 T cell responses. The increased accumulation of the effector CD8 T cell population was independent of viral load. Reduced contraction was associated with an increased fraction of CD8 T cells expressing the interleukin-7 receptor (IL-7R) at the peak of the response, resulting in enhanced numbers of memory/memory precursor cells in IFN-γ^−/− and IFN-γR^−/− compared to wild-type (WT) mice. Blockade of IL-7 within the lungs of IFN-γ^−/− mice restored the contraction of influenza virus-specific CD8 T cells, indicating that IL-7R is important for survival and is not simply a consequence of the lack of IFN-γ signaling. Finally, enhanced CD8 T cell recall responses and accelerated viral clearance were observed in the IFN-γ^−/− and IFN-γR^−/− mice after rechallenge with a heterologous strain of influenza virus, confirming that higher frequencies of memory precursors are formed in the absence of IFN-γ signaling. In summary, we have identified IFN-γ as an important regulator of localized viral immunity that promotes the contraction of antigen-specific CD8 T cells and inhibits memory precursor formation, thereby limiting the size of the memory cell population after an influenza virus infection.     

Vaccination with Embryonic Stem Cells Protects against Lung Cancer: Is a Broad-Spectrum Prophylactic Vaccine against Cancer Possible?

The antigenic similarity between tumors and embryos has been appreciated for many years and reflects the expression of embryonic gene products by cancer cells and/or cancer-initiating stem cells. Taking advantage of this similarity, we have tested a prophylactic lung cancer vaccine composed of allogeneic murine embryonic stem cells (ESC). Naïve C57BL/6 mice were vaccinated with ESC along with a source of granulocyte macrophage-colony stimulating factor (GM-CSF) in order to provide immunostimulatory adjuvant activity. Vaccinated mice were protected against subsequent challenge with implantable Lewis lung carcinoma (LLC). ESC-induced anti-tumor immunity was not due to a non-specific “allo-response” as vaccination with allogeneic murine embryonic fibroblasts did not protect against tumor outgrowth. Vaccine efficacy was associated with robust tumor-reactive primary and memory CD8⁺ T effector responses, Th1 cytokine response, higher intratumoral CD8⁺ T effector/CD4⁺CD25⁺Foxp3⁺ T regulatory cell ratio, and reduced myeloid derived suppressor cells in the spleen. Prevention of tumorigenesis was found to require a CD8-mediated cytotoxic T lymphocyte (CTL) response because in vivo depletion of CD8⁺ T lymphocytes completely abrogated the protective effect of vaccination. Importantly, this vaccination strategy also suppressed the development of lung cancer induced by the combination of carcinogen administration and chronic pulmonary inflammation. Further refinement of this novel vaccine strategy and identification of shared ESC/tumor antigens may lead to immunotherapeutic options for lung cancer patients and, perhaps more importantly, could represent a first step toward the development of prophylactic cancer vaccines.     

In vitro generation of influenza-specific polyfunctional CD4+ T cells suitable for adoptive immunotherapy

Background aimsInfluenza viruses cause potentially fatal respiratory infections in stem cell transplant patients. Specific T cells provide long-lived host adaptive immunity to influenza viruses, and the potential for generating such cells for clinical use was investigatedMethodsThe inactivated influenza vaccine (Fluvax) approved for human use was used as the antigen source. Monocyte-derived dendritic cells pulsed with Fluvax were used to stimulate autologous peripheral blood mononuclear cells (PBMC) on days 0 and 7. Cells were expanded with interleukin (IL)-2 from day 7 onwards. Cell numbers and phenotype were assessed on day 21. The presence of influenza virus-specific cells was assessed by cytokine production and proliferative responses following restimulation with influenza antigensResultsOver 21 days of culture, a mean fold increase of 26.3 in cell number was observed (n = 7). Cultures were predominantly effector and central memory CD4⁺ cells, and expressed a phenotype characteristic of activated antigen-specific cells capable of B-cell helper function. Cytotoxic CD4⁺ and CD8⁺ cells specific for influenza and a high percentage of CD4⁺ cells specific for each of three influenza viruses targeted by Fluvax (H1N1, H3N2 and Brisbane viruses) were generated. In addition, T cells expanded when restimulated with antigens derived from influenza viruses.ConclusionsWe have demonstrated a clinically usable method for producing influenza virus-specific T cells that yield high numbers of highly reactive CD4⁺ cells suitable for adoptive immunotherapy. We propose that reconstructing host immunity through adoptive transfer of influenza virus-specific T cells will reduce the frequency of influenza-related deaths in the period of severe immune suppression that follows stem cell transplantation.     

Agonistic Anti-CD40 Enhances the CD8+ T Cell Response during Vesicular Stomatitis Virus Infection

Intracellular pathogens are capable of inducing vigorous CD8⁺ T cell responses. However, we do not entirely understand the factors driving the generation of large pools of highly protective memory CD8⁺ T cells. Here, we studied the generation of endogenous ovalbumin-specific memory CD8⁺ T cells following infection with recombinant vesicular stomatitis virus (VSV) and Listeria monocytogenes (LM). VSV infection resulted in the generation of a large ovalbumin-specific memory CD8⁺ T cell population, which provided minimal protective immunity that waned with time. In contrast, the CD8⁺ T cell population of LM-ova provided protective immunity and remained stable with time. Agonistic CD40 stimulation during CD8⁺ T cell priming in response to VSV infection enabled the resultant memory CD8⁺ T cell population to provide strong protective immunity against secondary infection. Enhanced protective immunity by agonistic anti-CD40 was dependent on CD70. Agonistic anti-CD40 not only enhanced the size of the resultant memory CD8⁺ T cell population, but enhanced their polyfunctionality and sensitivity to antigen. Our data suggest that immunomodulation of CD40 signaling may be a key adjuvant to enhance CD8⁺ T cell response during development of VSV vaccine strategies.     

Increase in IFNγ?IL-2+ Cells in Recent Human CD4 T Cell Responses to 2009 Pandemic H1N1 Influenza

Human CD4 T cell recall responses to influenza virus are strongly biased towards Type 1 cytokines, producing IFNγ, IL-2 and TNFα. We have now examined the effector phenotypes of CD4 T cells in more detail, particularly focusing on differences between recent versus long-term, multiply-boosted responses. Peptides spanning the proteome of temporally distinct influenza viruses were distributed into pools enriched for cross-reactivity to different influenza strains, and used to stimulate antigen-specific CD4 T cells representing recent or long-term memory. In the general population, peptides unique to the long-circulating influenza A/New Caledonia/20/99 (H1N1) induced Th1-like responses biased toward the expression of IFNγ⁺TNFα⁺ CD4 T cells. In contrast, peptide pools enriched for non-cross-reactive peptides of the pandemic influenza A/California/04/09 (H1N1) induced more IFNγ⁻IL-2⁺TNFα⁺ T cells, similar to the IFNγ⁻IL-2⁺ non-polarized, primed precursor T cells (Thpp) that are a predominant response to protein vaccination. These results were confirmed in a second study that compared samples taken before the 2009 pandemic to samples taken one month after PCR-confirmed A/California/04/09 infection. There were striking increases in influenza-specific TNFα⁺, IFNγ⁺, and IL-2⁺ cells in the post-infection samples. Importantly, peptides enriched for non-cross-reactive A/California/04/09 specificities induced a higher proportion of Thpp-like IFNγ⁻IL-2⁺TNFα⁺ CD4 T cells than peptide pools cross-reactive with previous influenza strains, which induced more Th1 (IFNγ⁺TNFα⁺) responses. These IFNγ⁻IL-2⁺TNFα⁺ CD4 T cells may be an important target population for vaccination regimens, as these cells are induced upon infection, may have high proliferative potential, and may play a role in providing future effector cells during subsequent infections.     

High,Broad, Polyfunctional,and Durable T Cell Immune Responses Induced in Mice by a Novel Hepatitis C Virus (HCV) Vaccine Candidate (MVA-HCV) Based on Modified Vaccinia Virus Ankara Expressing the Nearly Full-Length HCV Genome

A major goal in the control of hepatitis C infection is the development of a vaccine. Here, we have developed a novel HCV vaccine candidate based on the highly attenuated poxvirus vector MVA (referred to as MVA-HCV) expressing the nearly full-length (7.9-kbp) HCV sequence, with the aim to target almost all of the T and B cell determinants described for HCV. In infected cells, MVA-HCV produces a polyprotein that is subsequently processed into the structural and nonstructural HCV proteins, triggering the cytoplasmic accumulation of dense membrane aggregates. In both C57BL/6 and transgenic HLA-A2-vaccinated mice, MVA-HCV induced high, broad, polyfunctional, and long-lasting HCV-specific T cell immune responses. The vaccine-induced T cell response was mainly mediated by CD8 T cells; however, although lower in magnitude, the CD4⁺ T cells were highly polyfunctional. In homologous protocol (MVA-HCV/MVA-HCV) the main CD8⁺ T cell target was p7+NS2, whereas in heterologous combination (DNA-HCV/MVA-HCV) the main target was NS3. Antigenic responses were also detected against other HCV proteins (Core, E1-E2, and NS4), but the magnitude of the responses was dependent on the protocol used. The majority of the HCV-induced CD8⁺ T cells were triple or quadruple cytokine producers. The MVA-HCV vaccine induced memory CD8⁺ T cell responses with an effector memory phenotype. Overall, our data showed that MVA-HCV induced broad, highly polyfunctional, and durable T cell responses of a magnitude and quality that might be associated with protective immunity and open the path for future considerations of MVA-HCV as a prophylactic and/or therapeutic vaccine candidate against HCV.     

Acetyl CoA Carboxylase 2 Is Dispensable for CD8+ T Cell Responses

Differentiation of T cells is closely associated with dynamic changes in nutrient and energy metabolism. However, the extent to which specific metabolic pathways and molecular components are determinative of CD8⁺ T cell fate remains unclear. It has been previously established in various tissues that acetyl CoA carboxylase 2 (ACC2) regulates fatty acid oxidation (FAO) by inhibiting carnitine palmitoyltransferase 1 (CPT1), a rate-limiting enzyme of FAO in mitochondria. Here, we explore the cell-intrinsic role of ACC2 in T cell immunity in response to infections. We report here that ACC2 deficiency results in a marginal increase of cellular FAO in CD8⁺ T cells, but does not appear to influence antigen-specific effector and memory CD8⁺ T cell responses during infection with listeria or lymphocytic choriomeningitis virus. These results suggest that ACC2 is dispensable for CD8⁺ T cell responses.     

Different Arms of the Adaptive Immune System Induced by a Combination Vaccine Work in Concert to Provide Enhanced Clearance of Influenza

Current split influenza virus vaccines that induce strain-specific neutralising antibodies provide some degree of protection against influenza infection but there is a clear need to improve their effectiveness. The constant antigenic drift of influenza viruses means that vaccines are often not an exact match to the circulating strain and so levels of relevant antibodies may not be sufficiently high to afford protection. In the situation where the emergent influenza virus is completely novel, as is the case with pandemic strains, existing vaccines may provide no benefit. In this study we tested the concept of a combination vaccine consisting of sub-optimal doses of split influenza virus vaccine mixed with a cross-protective T-cell inducing lipopeptide containing the TLR2 ligand Pam2Cys. Mice immunised with combination vaccines showed superior levels of lung viral clearance after challenge compared to either split virus or lipopeptide alone, mediated through activation of enhanced humoral and/or additional cellular responses. The mechanism of action of these vaccines was dependent on the route of administration, with intranasal administration being superior to subcutaneous and intramuscular routes, potentially through the induction of memory CD8⁺ T cells in the lungs. This immunisation strategy not only provides a mechanism for minimising the dose of split virus antigen but also, through the induction of cross-protective CD8⁺ T cells, proves a breadth of immunity to provide potential benefit upon encounter with serologically diverse influenza isolates.     

Chronic Parasitic Infection Maintains High Frequencies of Short-Lived Ly6C+CD4+ Effector T Cells That Are Required for Protection against Re-infection

In contrast to the ability of long-lived CD8⁺ memory T cells to mediate protection against systemic viral infections, the relationship between CD4⁺ T cell memory and acquired resistance against infectious pathogens remains poorly defined. This is especially true for T helper 1 (Th1) concomitant immunity, in which protection against reinfection coincides with a persisting primary infection. In these situations, pre-existing effector CD4 T cells generated by ongoing chronic infection, not memory cells, may be essential for protection against reinfection. We present a systematic study of the tissue homing properties, functionality, and life span of subsets of memory and effector CD4 T cells activated in the setting of chronic Leishmania major infection in resistant C57Bl/6 mice. We found that pre-existing, CD44⁺CD62L⁻T-bet⁺Ly6C⁺ effector (T_EFF) cells that are short-lived in the absence of infection and are not derived from memory cells reactivated by secondary challenge, mediate concomitant immunity. Upon adoptive transfer and challenge, non-dividing Ly6C⁺ T_EFF cells preferentially homed to the skin, released IFN-γ, and conferred protection as compared to CD44⁺CD62L⁻Ly6C⁻ effector memory or CD44⁺CD62L⁺Ly6C⁻ central memory cells. During chronic infection, Ly6C⁺ T_EFF cells were maintained at high frequencies via reactivation of T_CM and the T_EFF themselves. The lack of effective vaccines for many chronic diseases may be because protection against infectious challenge requires the maintenance of pre-existing T_EFF cells, and is therefore not amenable to conventional, memory inducing, vaccination strategies.     

Intratumoral interleukin-2/agonist CD40 antibody drives CD4<Superscript>+</Superscript>-independent resolution of treated-tumors and CD4<Superscript>+</Superscript>-dependent systemic and memory responses

Targeting interleukin-2 (IL-2) and/or agonist anti-CD40 antibody (Ab) into tumors represents an effective vaccination strategy that avoids systemic toxicity and resolves treated-site tumors. Here, we examined IL-2 and/or anti-CD40 Ab-driven local versus systemic T cell function and the installation of T cell memory. Single tumor studies showed that IL-2 induced a potent CD4⁺ and CD8⁺ T cell response that was limited to the draining lymph node and treated-site tumor, and lymph node tumor-specific CD8⁺ T cells did not upregulate CD44. A two-tumor model showed that while IL-2-treated-site tumors resolved, distal tumors continued to grow, implying limited systemic immunity. In contrast, anti-CD40 Ab treatment with or without IL-2 expanded the systemic T cell response to non-draining lymph nodes, and distal tumors resolved. Tumor-specific T cells in lymph nodes of anti-CD40 Ab ± IL-2-treated mice upregulated CD44, demonstrating activation and transition to effector/memory migratory cells. While CD40-activated CD4⁺ T cells were not required for eradicating treated-site tumors, they, plus CD8⁺ T cells, were crucial for removing distal tumors. Rechallenge/depletion experiments showed that the effector/memory phase required the presence of previously CD40/IL-2-activated CD4⁺ and CD8⁺ T cells to prevent recurrence. These novel findings show that different T cell effector mechanisms can operate for the eradication of local treated-site tumors versus untreated distal tumors and that signaling through CD40 generates a whole of body, effector/memory CD4⁺ and CD8⁺ T cell response that is amplified and prolonged via IL-2. Thus, successful immunotherapy needs to generate collaborating CD4⁺ and CD8⁺ T cells for a complete long-term protective cure.     

Increased Cell Surface Free Thiols Identify Effector CD8+ T Cells Undergoing T Cell Receptor Stimulation

Recognition of peptide Major Histocompatibility Complexes (MHC) by the T cell receptor causes rapid production of reactive oxygen intermediates (ROI) in naïve CD8⁺ T cells. Because ROI such as H₂O₂ are membrane permeable, mechanisms must exist to prevent overoxidation of surface proteins. In this study we used fluorescently labeled conjugates of maleimide to measure the level of cell surface free thiols (CSFT) during the development, activation and differentiation of CD8⁺ T cells. We found that during development CSFT were higher on CD8 SP compared to CD4 SP or CD4CD8 DP T cells. After activation CSFT became elevated prior to division but once proliferation started levels continued to rise. During acute viral infection CSFT levels were elevated on antigen-specific effector cells compared to memory cells. Additionally, the CSFT level was always higher on antigen-specific CD8⁺ T cells in lymphoid compared to nonlymphoid organs. During chronic viral infection, CSFT levels were elevated for extended periods on antigen-specific effector CD8⁺ T cells. Finally, CSFT levels on effector CD8⁺ T cells, regardless of infection, identified cells undergoing TCR stimulation. Taken together these data suggest that CD8⁺ T cells upregulate CSFT following receptor ligation and ROI production during infection to prevent overoxidation of surface proteins.     

A role for the chemokine RANTES in regulating CD8 T cell responses during chronic viral infection

RANTES (CCL5) is a chemokine expressed by many hematopoietic and non-hematopoietic cell types that plays an important role in homing and migration of effector and memory T cells during acute infections. The RANTES receptor, CCR5, is a major target of anti-HIV drugs based on blocking viral entry. However, defects in RANTES or RANTES receptors including CCR5 can compromise immunity to acute infections in animal models and lead to more severe disease in humans infected with west Nile virus (WNV). In contrast, the role of the RANTES pathway in regulating T cell responses and immunity during chronic infection remains unclear. In this study, we demonstrate a crucial role for RANTES in the control of systemic chronic LCMV infection. In RANTES^−/− mice, virus-specific CD8 T cells had poor cytokine production. These RANTES^−/− CD8 T cells also expressed higher amounts of inhibitory receptors consistent with more severe exhaustion. Moreover, the cytotoxic ability of CD8 T cells from RANTES^−/− mice was reduced. Consequently, viral load was higher in the absence of RANTES. The dysfunction of T cells in the absence of RANTES was as severe as CD8 T cell responses generated in the absence of CD4 T cell help. Our results demonstrate an important role for RANTES in sustaining CD8 T cell responses during a systemic chronic viral infection.     

Vaccine-Elicited CD8+ T Cells Protect against Respiratory Syncytial Virus Strain A2-Line19F-Induced Pathogenesis in BALB/c Mice

CD8⁺ T cells may contribute to vaccines for respiratory syncytial virus (RSV). Compared to CD8⁺ T cells responding to RSV infection, vaccine-elicited anti-RSV CD8⁺ T cells are less well defined. We used a peptide vaccine to test the hypothesis that vaccine-elicited RSV-specific CD8⁺ T cells are protective against RSV pathogenesis. BALB/c mice were treated with a mixture (previously termed TriVax) of an M2_82-90 peptide representing an immunodominant CD8 epitope, the Toll-like receptor (TLR) agonist poly(I·C), and a costimulatory anti-CD40 antibody. TriVax vaccination induced potent effector anti-RSV CD8⁺ cytotoxic T lymphocytes (CTL). Mice were challenged with RSV strain A2-line19F, a model of RSV pathogenesis leading to airway mucin expression. Mice were protected against RSV infection and against RSV-induced airway mucin expression and cellular lung inflammation when challenged 6 days after vaccination. Compared to A2-line19F infection alone, TriVax vaccination followed by challenge resulted in effector CD8⁺ T cells with greater cytokine expression and the more rapid appearance of RSV-specific CD8⁺ T cells in the lung. When challenged 42 days after TriVax vaccination, memory CD8⁺ T cells were elicited with RSV-specific tetramer responses equivalent to TriVax-induced effector CD8⁺ T cells. These memory CD8⁺ T cells had lower cytokine expression than effector CD8⁺ T cells, and protection against A2-line19F was partial during the memory phase. We found that vaccine-elicited effector anti-RSV CD8⁺ T cells protected mice against RSV infection and pathogenesis, and waning protection correlated with reduced CD8⁺ T cell cytokine expression.     

CD154 and IL-2 Signaling of CD4+ T Cells Play a Critical Role in Multiple Phases of CD8+ CTL Responses Following Adenovirus Vaccination

Adenoviral (AdV) vectors represent most commonly utilized viral vaccines in clinical studies. While the role of CD8⁺ cytotoxic T lymphocyte (CTL) responses in mediating AdV-induced protection is well understood, the involvement of CD4⁺ T cell-provided signals in the development of functional CD8⁺ CTL responses remain unclear. To explore CD4⁺ T helper signals required for AdVova-stimulated CTL responses, we established an adoptive transfer system by transferring CD4⁺ T cells derived from various knock out and transgenic mice into wild-type and/or CD4-deficient animals, followed by immunizing with recombinant ovalbumin (OVA)-expressing AdVova vector. Without CD4⁺ T help, both primary and memory CTL responses were greatly reduced in this model, and were associated with increased PD-1 expression. The provision of OVA-specific CD4⁺ T help in CD4⁺ T cell-deficient mice restored AdVova-induced primary CTL responses, and supported survival and recall responses of AdVova-stimulated memory CTLs. These effects were specifically mediated by CD4⁺ T cell-produced IL-2 and CD154 signals. Adoptive transfer of “helped” or “unhelped” effector and memory CTLs into naïve CD4⁺ T cell-deficient or -sufficient mice also revealed an additional role for polyclonal CD4⁺ T cell environment in the survival of AdVova-stimulated CTLs, partially explaining the extension of CTL contraction phase. Finally, during recall responses, CD4⁺ T cell environment, particularly involving memory CD4⁺ T cells, greatly enhanced expansion of memory CTLs. Collectively, our data strongly suggest a critical role for CD4⁺ T help in multiple phases of AdV-stimulated CTL responses, and could partially explain certain failures in AdV-based immunization trials targeting malignant tumors and chronic diseases that are often associated with compromised CD4⁺ T cell population and function.