HA-specific CD4 Research Articles

Proteolytic processing of TNF-α by influenza-specific CD8+ T cells is required for alveolar epithelial cell MIP-2 production and severe lung injury in a transgenic mouse model of influenza infection (67.3)

Abstract Adoptive transfer of influenza hemagglutinin (HA)-specific CD8+ T cells into transgenic mice expressing influenza HA on the alveolar epithelium results in epithelial cell production of monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-2, the subsequent mononuclear cell infiltration and diffuse alveolar damage. The lung injury observed in this model is mediated by CD8+ T cell production of tumor necrosis factor (TNF)-α. TNF-α is expressed as a transmembrane (tm) protein that is proteolytically processed to produce soluble (s) TNF-α. To determine the roles of tmTNF-α and sTNF-α in CD8+ T cell mediated lung injury, we generated HA-specific CD8+ T cells that only express a noncleavable tmTNF-α (TM). We found that proteolytic processing of tmTNF-α to sTNF-α was required for alveolar epithelial cell production of MIP-2 but not MCP-1 in vitro. Furthermore, this epithelial cell production of MIP-2 required ERK activation that was dependent on TNF-α signaling through TNF-α receptor II. Finally, we adoptively transferred HA-specific TM CD8+ T cells into HA transgenic mice and found that TM CD8+ T cells failed to induce alveolar epithelial cell MIP-2 production and neutrophil infiltration, and produced milder lung injury than transferred wild-type CD8+ T cells. Taken together, these findings suggest that proteolytic processing of tmTNF-α to sTNF-α is a critical event regulating CD8+ T cell mediated injury in a transgenic mouse model of influenza infection.

Monocytes promote the formation of autoreactive Th17 cells in inflammatory arthritis (101.23)

Abstract Th17 cells have been implicated in a number of autoimmune diseases and their animal models including inflammatory arthritis. How these cells arise in the context of autoimmunity is not well understood. We recently described a mouse model of autoimmune arthritis in which mice expressing influenza hemagglutinin (HA) as a self-antigen under the control of a MHC Class II promoter (HACII), and co-expressing an HA-specific CD4+ TCR (TS1), spontaneously develop inflammatory arthritis by an IL-17-dependent mechanism. Here we have used these TS1xHACII mice to investigate the role of specific cell types in the activation of autoreactive CD4+ Th17 cells. We have found that monocytes are selectively enriched in the spleens and LNs of arthritic TS1xHACII mice relative to mice expressing the HACII transgene alone. Moreover, monocytes purified from the spleens of arthritic TS1xHACII and HACII mice had profound differences in their abilities to activate HA-specific T cells in vitro: monocytes from HACII mice were potent inducers of Th1 cells, whereas those from TS1xHACII mice were significantly more efficient at inducing Th17 cells. Monocytes from TS1xHACII mice also had an enhanced capacity to induce Th17 cells relative to B cells and cDCs isolated in parallel from the same organs. Collectively, these data suggest that monocytes can play an important role in the development of Th17 cells in vivo, and can participate in an autoimmune response that results in inflammatory arthritis.

Vectors Based on Modified Vaccinia Ankara Expressing Influenza H5N1 Hemagglutinin Induce Substantial Cross-Clade Protective Immunity

BackgroundNew highly pathogenic H5N1 influenza viruses are continuing to evolve with a potential threat for an influenza pandemic. So far, the H5N1 influenza viruses have not widely circulated in humans and therefore constitute a high risk for the non immune population. The aim of this study was to evaluate the cross-protective potential of the hemagglutinins of five H5N1 strains of divergent clades using a live attenuated modified vaccinia Ankara (MVA) vector vaccine.Methodology/Principal FindingsThe replication-deficient MVA virus was used to express influenza hemagglutinin (HA) proteins. Specifically, recombinant MVA viruses expressing the HA genes of the clade 1 virus A/Vietnam/1203/2004 (VN/1203), the clade 2.1.3 virus A/Indonesia/5/2005 (IN5/05), the clade 2.2 viruses A/turkey/Turkey/1/2005 (TT01/05) and A/chicken/Egypt/3/2006 (CE/06), and the clade 2.3.4 virus A/Anhui/1/2005 (AH1/05) were constructed. These experimental live vaccines were assessed in a lethal mouse model. Mice vaccinated with the VN/1203 hemagglutinin-expressing MVA induced excellent protection against all the above mentioned clades. Also mice vaccinated with the IN5/05 HA expressing MVA induced substantial protection against homologous and heterologous AH1/05 challenge. After vaccination with the CE/06 HA expressing MVA, mice were fully protected against clade 2.2 challenge and partially protected against challenge of other clades. Mice vaccinated with AH1/05 HA expressing MVA vectors were only partially protected against homologous and heterologous challenge. The live vaccines induced substantial amounts of neutralizing antibodies, mainly directed against the homologous challenge virus, and high levels of HA-specific IFN-γ secreting CD4 and CD8 T-cells against epitopes conserved among the H5 clades and subclades.Conclusions/SignificanceThe highest level of cross-protection was induced by the HA derived from the VN/1203 strain, suggesting that pandemic H5 vaccines utilizing MVA vector technology, should be based on the VN/1203 hemagglutinin. Furthermore, the recombinant MVA-HA-VN, as characterized in the present study, would be a promising candidate for such a vaccine.

Local Induction of Immunosuppressive CD8+ T Cells in the Gut-Associated Lymphoid Tissues

BackgroundIn contrast to intestinal CD4+ regulatory T cells (Tregs), the generation and function of immunomodulatory intestinal CD8+ T cells is less well defined. To dissect the immunologic mechanisms of CD8+ T cell function in the mucosa, reactivity against hemagglutinin (HA) expressed in intestinal epithelial cells of mice bearing a MHC class-I-restricted T-cell-receptor specific for HA was studied.Methodology and Principal FindingsHA-specific CD8+ T cells were isolated from gut-associated tissues and phenotypically and functionally characterized for the expression of Foxp3+ and their suppressive capacity. We demonstrate that intestinal HA expression led to peripheral induction of HA-specific CD8+Foxp3+ T cells. Antigen-experienced CD8+ T cells in this transgenic mouse model suppressed the proliferation of CD8+ and CD4+ T cells in vitro. Gene expression analysis of suppressive HA-specific CD8+ T cells revealed a specific up-regulation of CD103, Nrp1, Tnfrsf9 and Pdcd1, molecules also expressed on CD4+ Treg subsets. Finally, gut-associated dendritic cells were able to induce HA-specific CD8+Foxp3+ T cells.Conclusion and SignificanceWe demonstrate that gut specific antigen presentation is sufficient to induce CD8+ Tregs in vivo which may maintain intestinal homeostasis by down-modulating effector functions of T cells.

Hemagglutinin Displayed Baculovirus Protects Against Highly Pathogenic Influenza

Baculovirus (BV) replicating in insect cells can express a foreign gene product as part of its genome. The influenza hemagglutinin (HA) can be expressed from BV and displayed on the surface of baculovirus (HA-DBV). In this study we first generated six recombinant baculoviruses that expressed chimeric HAs with segments of the BV glycoprotein (gp64). The signal peptide (SP) and cytoplasmic tail (CT) domains of gp64 can enhance the display of HA from A/PR8/34 on BV surface, while the transmembrane (TM) domain of gp64 impairs HA display. Different doses of either live or β-propiolactone (BPL)-inactivated HA-DBV were administered to BALB/c mice. Live HA-DBV elicited higher hemagglutination-inhibition (HAI) titers than BPL-inactivated HA-DBV, and provided sterilizing protection. A second generation recombinant BV simultaneously displaying four HAs derived from four subclades of H5N1 influenza viruses was constructed. This tetravalent H5N1 HA-DBV vaccine elicited HAI titers against all four homologous H5N1 viruses, significantly decreasing viral lung titers of challenged mice and providing 100% protection against lethal doses of homologous H5N1 viruses. Moreover, mice vaccinated with HA-DBV had high levels of IFNγ-secreting and HA-specific CD8+ T cells. Taken together, this study demonstrates that HA-DBV can stimulate strong humoral, as well as cellular immune responses, and is an effective vaccine candidate for influenza.

A Pandemic Influenza H1N1 Live Vaccine Based on Modified Vaccinia Ankara Is Highly Immunogenic and Protects Mice in Active and Passive Immunizations

BackgroundThe development of novel influenza vaccines inducing a broad immune response is an important objective. The aim of this study was to evaluate live vaccines which induce both strong humoral and cell-mediated immune responses against the novel human pandemic H1N1 influenza virus, and to show protection in a lethal animal challenge model.Methodology/Principal FindingsFor this purpose, the hemagglutinin (HA) and neuraminidase (NA) genes of the influenza A/California/07/2009 (H1N1) strain (CA/07) were inserted into the replication-deficient modified vaccinia Ankara (MVA) virus - a safe poxviral live vector – resulting in MVA-H1-Ca and MVA-N1-Ca vectors. These live vaccines, together with an inactivated whole virus vaccine, were assessed in a lung infection model using immune competent Balb/c mice, and in a lethal challenge model using severe combined immunodeficient (SCID) mice after passive serum transfer from immunized mice. Balb/c mice vaccinated with the MVA-H1-Ca virus or the inactivated vaccine were fully protected from lung infection after challenge with the influenza H1N1 wild-type strain, while the neuraminidase virus MVA-N1-Ca induced only partial protection. The live vaccines were already protective after a single dose and induced substantial amounts of neutralizing antibodies and of interferon-γ-secreting (IFN-γ) CD4- and CD8 T-cells in lungs and spleens. In the lungs, a rapid increase of HA-specific CD4- and CD8 T cells was observed in vaccinated mice shortly after challenge with influenza swine flu virus, which probably contributes to the strong inhibition of pulmonary viral replication observed. In addition, passive transfer of antisera raised in MVA-H1-Ca vaccinated immune-competent mice protected SCID mice from lethal challenge with the CA/07 wild-type virus.Conclusions/SignificanceThe non-replicating MVA-based H1N1 live vaccines induce a broad protective immune response and are promising vaccine candidates for pandemic influenza.

Effects and Regulation of Autoreactive CD8 + T Cells in a Transgenic Mouse Model of Autoimmune Hepatitis

Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease of unknown etiology. Autoreactive T cells are thought to mediate liver injury, but the pathogenesis of AIH is poorly understood because of the lack of suitable animal models. We established a mouse model to investigate liver-specific T-cell responses and assess the effects and regulation of autoreactive CD8(+) T cells in the pathogenesis of AIH. We generated transgenic mice expressing the influenza virus hemagglutinin (HA) autoantigen under control of mouse albumin regulatory elements and alpha-fetoprotein enhancers (Alb) specifically in the liver (Alb-HA mice); they were crossed with mice that express a specific T-cell receptor (TCR) (CL4-TCR). CL4-TCR transgenic CD8(+) T cells were also adoptively transferred into Alb-HA mice. We investigated immunologic mechanisms of CD8(+) T cell-induced liver damage and of counteracting peripheral tolerance. The double-transgenic mice (Alb-HA/CL4-TCR) spontaneously developed chronic, autoimmune-mediated hepatitis, characterized by necroinflammatory lesions, hepatic fibrosis, and increased levels of aminotransferase; these features resembled those of AIH. Interestingly, most liver-infiltrating, HA-specific CD8(+) T cells had an anergic phenotype; regulatory CD4(+)CD25(+)Foxp3(+) T cells accumulated in the inflamed liver. The continuous development of a few, HA-specific CD8(+) effector T cells is sufficient to induce chronic hepatitis. Peripheral tolerance mechanisms such as induction of T-cell anergy and accumulation of regulatory CD4(+)CD25(+)Foxp3(+) T cells protect the liver from severe damage. Our mouse model that spontaneously develops chronic autoimmune-mediated hepatitis provides a new tool to investigate autoantigen-specific T-cell responses and regulatory mechanisms involved in the prevention and pathogenesis of AIH.

Impact of bortezomib-induced proteasome inhibition in vivo on anti-tumor T cell responses (101.1)

Abstract The perturbation of cellular homeostasis offers a new approach for cancer therapy. Our recent reports showed that proteasome inhibitor bortezomib (Velcade/PS-341) not only sensitizes tumors to death receptor-mediated apoptosis but also increases their susceptibility to NK cell lysis. However, the effects of bortezomib on T cell anti-tumor responses are not known. Thus, in mice established with Renca-HA tumor expressing influenza virus hemagglutinin (HA) as a surrogate tumor antigen, we evaluated the anti-tumor functions of adoptively transferred monoclonal HA-specific CD4 and CD8 T cells following bortezomib administration. Interestingly, no inhibition of T cell proliferation or cytolytic activity in vivo was observed following bortezomib treatments. Nor were there any defects in dendritic cell activation or antigen presentation in bortezomib-treated mice. However, a decrease in total T cell yield and a blockade of cellular homeostatic proliferation was noted in these mice. Thus, bortezomib-inhibitory effects on T cell responses appeared quantitative rather than qualitative. Moreover, combined treatment of mice established with Renca-HA tumor with adoptive T cell transfer and bortezomib resulted in superior anti-tumor responses than either therapy alone. These data demonstrate that, with an appropriate scheduling, it may be possible to combine bortezomib-induced sensitization of tumors to apoptosis with tumor-specific T cell adoptive transfers to improve cancer immunotherapy.

Antigen‐specific Treg impair CD8+ T‐cell priming by blocking early T‐cell expansion

Foxp3(+) Treg are crucial for the maintenance of self-tolerance and have been shown to control CD8(+) T-cell effector functions. In addition, Treg are thought to control the priming of CD8(+) T cells, which recognize the same antigens as Treg. Taking advantage of our model of peripheral tolerance induction to influenza hemagglutinin (HA) after HA gene transfer, we found that HA-specific Treg suppress antigen-linked CTL responses through early blockade of CD8(+) T-cell expansion. Confronted with their cognate antigen, Treg expand more rapidly than CD8(+) T cells and are highly suppressive only during the initial stages of immune priming. They nullify HA-specific CD8(+) T-cell responses, local inflammatory responses and rejection of HA transduced cells. When HA gene transfer is performed with extensive tissue inflammation, HA-specific Treg are less effective but still reduce the frequency of newly primed HA-specific CD8(+) T cells and the ensuing frequency of memory CD8(+) T cells. Our results demonstrate that Treg control CTL priming in an antigen-specific manner at the level of T-cell expansion, highlighting how self-reactive Treg could prevent the induction of autoimmune responses through selective blockade of autoreactive T-cell proliferation.

TLR2 Engagement on Dendritic Cells Promotes High Frequency Effector and Memory CD4 T Cell Responses

Ligation of TLR by distinct pathogen components provides essential signals for T cell priming, although how individual TLR engagement affects primary and memory T cell responses is not well defined. In this study, we demonstrate distinct effects of TLR2 vs TLR4 engagement on primary and memory CD4 T cell responses due to differential effects on APC. Priming of influenza hemagglutinin (HA)-specific naive CD4 T cells with HA peptide and the TLR2 agonist Pam3CysK in vivo resulted in a high frequency of activated HA-specific CD4 T cells that predominantly produced IL-2 and IL-17, whereas priming with HA peptide and the TLR4 agonist LPS yielded a lower frequency of HA-specific CD4 T cells and predominant IFN-gamma producers. TLR2 agonist priming depended on TLR2 expression by APC, as wild-type CD4 T cells did not expand in response to peptide and Pam3CysK in TLR2-deficient hosts. TLR2-mediated priming also led to an increased frequency of Ag-specific memory CD4 T cells compared with TLR4 priming and mediated enhanced secondary responses to influenza challenge. Our results show that TLR engagement on APC influences both primary and secondary CD4 T cell responses, and suggest that long-term functional capacities of T cells are set by innate signals during early phases of an infection.

Towards Identification of the Mechanisms of Action of Parasite-Derived Peptide GK1 on the Immunogenicity of an Influenza Vaccine

Previous studies have shown that the synthetic peptide GK1, derived from Taenia crassiceps cysticerci, enhances the immunogenicity of the commercial inactivated influenza vaccine Fluzone in both young and aged mice. In particular, antibody responses were much improved. Since GK1 is a peptide and is rapidly cleared from the body, it offers the possibility to improve vaccine performance without undesirable effects. This study was therefore designed to understand the mechanisms of action involved in the adjuvant properties of GK1. For this, transgenic mice expressing a T-cell receptor specific for an epitope from the influenza virus hemagglutinin (HA) protein were employed. The GK1 peptide significantly increased the in vivo proliferative response of HA-specific CD4+ T cells when it was coimmunized with the HA epitope. Dendritic cells treated in vitro with GK1 were capable of enhancing T-cell activation. Furthermore, in synergy with lipopolysaccharide, GK1 enhanced the expression of major histocompatibility complex class II and costimulatory molecules of dendritic cells and promoted the secretion of proinflammatory cytokines and chemokines upon antigen-driven T-cell interaction. These data provide important insights into the mechanism that underlies the GK1 adjuvant capacity observed previously and underline the feasibility of using the transgenic mouse model described herein as a tool for investigation of the modes of action of different influenza vaccine adjuvants.

Immunogenicity and Protective Efficacy of a Live Attenuated H5N1 Vaccine in Nonhuman Primates

The continued spread of highly pathogenic H5N1 influenza viruses among poultry and wild birds, together with the emergence of drug-resistant variants and the possibility of human-to-human transmission, has spurred attempts to develop an effective vaccine. Inactivated subvirion or whole-virion H5N1 vaccines have shown promising immunogenicity in clinical trials, but their ability to elicit protective immunity in unprimed human populations remains unknown. A cold-adapted, live attenuated vaccine with the hemagglutinin (HA) and neuraminidase (NA) genes of an H5N1 virus A/VN/1203/2004 (clade 1) was protective against the pulmonary replication of homologous and heterologous wild-type H5N1 viruses in mice and ferrets. In this study, we used reverse genetics to produce a cold-adapted, live attenuated H5N1 vaccine (AH/AAca) that contains HA and NA genes from a recent H5N1 isolate, A/Anhui/2/05 virus (AH/05) (clade 2.3), and the backbone of the cold-adapted influenza H2N2 A/AnnArbor/6/60 virus (AAca). AH/AAca was attenuated in chickens, mice, and monkeys, and it induced robust neutralizing antibody responses as well as HA-specific CD4+ T cell immune responses in rhesus macaques immunized twice intranasally. Importantly, the vaccinated macaques were fully protected from challenge with either the homologous AH/05 virus or a heterologous H5N1 virus, A/bar-headed goose/Qinghai/3/05 (BHG/05; clade 2.2). These results demonstrate for the first time that a cold-adapted H5N1 vaccine can elicit protective immunity against highly pathogenic H5N1 virus infection in a nonhuman primate model and provide a compelling argument for further testing of double immunization with live attenuated H5N1 vaccines in human trials.

Post-exposure vaccination against influenza (132.4)

Abstract Vaccines currently licensed for the prevention of influenza induce antibodies against the influenza hemagglutinin (HA) and neuraminidase (NA) contained in the vaccine preparation but require at least two weeks after immunization for the development of protective immunity. We have developed a novel vaccine based on recombinant vesicular stomatitis virus which expresses the influenza hemagglutinin (rVSV HA) and protects mice from lethal influenza challenge when the vaccine is administered intramuscularly shortly after delivery of the influenza challenge virus. To our knowledge ours is the first vaccine which effectively protects animals from lethal influenza challenge when delivered by a systemic route after influenza exposure has occurred. The mechanism(s) by which rVSV HA protects remain to be determined but induction of HA-specific immune responses is essential, because animals immunized with an empty rVSV vector were not protected equally. Our data are consistent with a model in which post-exposure rVSV HA vaccination induces an immediate antiviral cytokine response, followed by expansion of HA-specific cytotoxic CD8 T cells, which circulate to the respiratory tract and clear infected cells. Vaccinated animals do not have detectable serum neutralizing Ab to influenza during the acute phase of the infection, but antibody may be required at later timepoints for complete clearance of influenza.

Myeloid-Derived Suppressor Cells in Inflammatory Bowel Disease: A New Immunoregulatory Pathway

CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs) have been shown to cause T-cell tolerance in tumor-bearing mice; however, little is known about the role of MDSCs in chronic inflammation. Here, for the first time, we have identified and analyzed their role in inflammatory bowel disease (IBD). Repetitive adoptive transfer of clone 4/T-cell receptor (CL4-TCR) transgenic CD8(+) T cells into VILLIN-hemagglutinin (HA) transgenic mice was performed on days 1, 12, and 27. Recipient mice were analyzed for immunopathology, HA-specific CD8(+) T-cell responses, and CD11b(+)Gr-1(+) MDSCs (frequency, phenotype, expression analysis, and in vitro as well as in vivo function). In addition, peripheral blood from patients with active Crohn's disease and ulcerative colitis was examined for the presence and function of human MDSCs denoted as CD14(+)HLA-DR(-/low) cells. Repetitive transfer of HA-specific CD8(+) T cells prevented VILLIN-HA recipient mice from development of severe enterocolitis, which is seen after a single transfer of T cells. Repeated transfer of antigen-specific T cells led to an increase in the frequency of nitric oxide synthase 2 and arginase-expressing CD11b(+)Gr-1(+) MDSCs in spleen and intestine of VILLIN-HA mice with immunosuppressive function. Cotransfer of MDSCs with HA-specific CD8(+) T cells into naive VILLIN-HA mice ameliorated enterocolitis, indicating a direct immune regulatory effect of MDSCs on induction of IBD by antigen-specific T cells. Finally, an increase in the frequency of human MDSCs with suppressor function was observed in peripheral blood from patients with IBD. These results identify MDSCs as a new immune regulatory pathway in IBD.

Enumeration of Haemagglutinin‐specific CD8+ T Cells after Influenza Vaccination Using MHC Class I Peptide Tetramers

With emergence of MHC class I tetramers loaded with CD8+ T-cell viral epitopes, it is possible to study virus-specific CD8 cells in humans during infection and after vaccination. MHC class I tetramers was used to detect the frequency of haemagglutinin (HA)-specific T cells in 26 healthy influenza-vaccinated humans. Peripheral blood was collected before, and 7, 14 and 28 days after vaccination. Four-colour flow cytometry was used for monitoring of vaccine induced T-cell response. In 15 donors, two- to fivefold increase in frequency of HA-specific T cells was observed 7 days after vaccination. In addition, in 12 of these donors, this increase was accompanied with fourfold increase of H1N1 antibody titre. The increase in frequency of HA-specific CD8+/IFN-gamma+ cells was low and peaked 28 days after vaccination in three of the six donors tested. Frequencies of HA-specific CD8+ T cells and antibody titre returned to prevaccination values 1 year after vaccination. Subunit influenza vaccines have the ability to induce HA-specific CD8+ cells. As the immune response to this vaccine decreased significantly after 1 year, our results confirm the importance of annual immunization for adequate protection.

Antigen-specific immune responses to influenza vaccine in utero

Initial immune responses to allergens may occur before birth, thereby modulating the subsequent development of atopy. This paradigm remains controversial, however, due to the inability to identify antigen-specific T cells in cord blood. The advent of MHC tetramers has revolutionized the detection of antigen-specific T cells. Tetramer staining of cord blood after CMV infection has demonstrated that effective CD8(+) antigen-specific immune responses can follow intrauterine viral infections. We hypothesized that sensitization to antigens occurs in utero in humans. We studied cord blood B and T cell immune responses following vaccination against influenza during pregnancy. Anti-Fluzone and anti-matrix protein IgM antibodies were detected in 38.5% (27 of 70) and 40.0% (28 of 70), respectively, of cord blood specimens. Using MHC tetramers, HA-specific CD4(+) T cells were detected among 25.0% (3 of 12) and 42.9% (6 of 14) of cord blood specimens possessing DRB1*0101 and DRB1*0401 HLA types, respectively, and were detected even when the DRB1 HLA type was inherited from the father. Matrix protein-specific CD8(+) T cells were detected among 10.0% (2 of 20) of HLA-A*0201(+) newborns. These results suggest that B and T cell immune responses occur in the fetus following vaccination against influenza and have important implications for determining when immune responses to environmental exposures begin.

Spontaneous Autoreactive Memory B Cell Formation Driven by a High Frequency of Autoreactive CD4+ T Cells

Although somatically mutated autoantibodies are characteristic of many autoimmune diseases, the processes that can lead to their development remain poorly understood. We have examined the formation of autoreactive memory B cells in PevHA mice, which express the influenza virus PR8 hemagglutinin (HA) as a transgenic membrane bound neo-self-Ag. Using a virus immunization strategy, we show that PR8 HA-specific memory B cell formation can occur in PevHA mice, even though a major subset of PR8 HA-specific B cells is negatively selected from the primary repertoire. Moreover, PR8 HA-specific memory B cells develop spontaneously in TS1 x PevHA mice, which coexpress a transgenic PR8 HA-specific TCR and contain a high frequency of HA-specific CD4(+) T cells. Notably, autoreactive memory B cell formation occurred in TS1 x PevHA mice even though approximately half of the HA-specific CD4(+) T cells were CD25(+)Foxp3(+) cells that could significantly attenuate, but did not completely abolish HA-specific autoantibody production in an adoptive transfer setting. The findings provide evidence that a high frequency of autoreactive CD4(+) T cells can be sufficient to promote autoreactive memory B cell formation in the absence of signals provided by overt immunization or infection and despite the presence of abundant autoantigen-specific CD4(+)CD25(+)Foxp3(+) regulatory T cells.

Lung resident memory CD4 T cells provide optimal protective immunity to influenza challenge (43.4)

Abstract Diverse subsets of CD4 memory T cells in lymphoid and non-lymphoid tissue are known to persist after infection; however, the ability of these subsets to mediate protective immunity against viral challenge is not known. We previously showed that a population of influenza hemagglutinin (HA)-specific memory CD4 T cells exhibits diverse migration properties with spleen and lung-derived memory CD4 T cells exhibiting tissue-specific homing. Here, we examined the ability of purified lung and spleen-resident HA-specific memory CD4 T cells to mediate protection upon influenza challenge. We found that lung-derived memory CD4 T cells facilitated more rapid clearance of virus from infected lung compared to equal numbers of spleen-derived memory T cells. Moreover, viral clearance in mice receiving lung memory occurred with reduced weight loss pathology, whereas naive mice or mice receiving splenic HA-specific memory T cells displayed precipitous weight loss and ruffled appearance. We found intrinsic functional differences in proliferation and effector capacity of lung compared to spleen-derived memory cells, including reduced overall expansion and IL-2 production from lung versus spleen subsets. Our results demonstrate an enhanced ability of lung-derived memory CD4 T cells to mediate protective responses with minimal pathology compared to lymphoid memory T cells, with important implications for vaccine design. Supported by NIH-AI-50632

Dendritic cells program non‐immunogenic prostate‐specific T cell responses beginning at early stages of prostate tumorigenesis

Prostate cancer promotes the development of T cell tolerance towards prostatic antigens, potentially limiting the efficacy of prostate cancer vaccines targeting these antigens. Here, we sought to determine the stage of disease progression when T cell tolerance develops, as well as the role of steady state dendritic cells (DC) and CD4(+)CD25(+) T regulatory cells (Tregs) in programming tolerance. The response of naïve HA-specific CD4(+) T cells were analyzed following adoptive transfer into Pro-HA x TRAMP transgenic mice harboring variably-staged HA-expressing prostate tumors on two genetic backgrounds that display different patterns and kinetics of tumorigenesis. The role of DC and Tregs in programming HA-specific CD4 cell responses were assessed via depletion. HA-specific CD4 cells underwent non-immunogenic responses at all stages of tumorigenesis in both genetic backgrounds. These responses were completely dependent on DC, but not appreciably influenced by Tregs. These results suggest that tolerogenicity is an early and general property of prostate tumors.

Coimmunization with Antigens Shared between Neoplastic and Normal B Cells Suppresses Induction of Strictly Tumor-Specific Cytotoxic T Cells through In Situ Activation of Regulatory T Cells.

CD4+CD25+ regulatory T cells (Treg) recognize autoantigens and inhibit autoreactive immune responses in a cell contact-dependent manner. In cancer-bearing patients, expansion and functional aberrations of Treg may inhibit immune responses against the tumor. The available evidence suggests that such Treg recognize self antigens expressed by the tumor and argues that induction of anti-tumor T cell responses might be more successful if true tumor-specific rather than lineage-restricted or shared antigens are used for active immunotherapy. Indeed, we have observed a preferential recognition of tumor-individual over shared epitopes by vaccination-induced T cells after immunization of B-NHL patients with recombinant lymphoma idiotype (Bertinetti et al., Cancer Res. 2006). To study this phenomenon in an exemplary fashion, we immunized BALB/c mice with dendritic cells loaded with H-2K-restricted peptides of the immunoglobulin of the A20 lymphoma. A J region-derived peptide served as a model for a shared antigen; a heteroclitic peptide from the CDR3 region represented a tumor-specific antigen. Both peptides bind H-2Kd with similar affinity. Compared to a highly immunogenic influenza HA peptide, the CDR3 peptide was similarly efficient in inducing specific cytotoxic T cells as analyzed by tetramer staining, IFNγ release to peptide stimulation, and in vitro and in vivo cytotoxicity assays with CFSE-labelled, peptide-loaded splenocytes. In contrast, no effector cells were detected with any assay after J immunization. After in vitro restimulation with peptide, however, antigen-specific IFNγ-secreting effector populations were demonstrated for each vaccination, suggesting in vivo inhibition, possibly mediated by Treg, rather than total absence of J-specific T cells. No difference in numbers and the TCR repertoire of CD4+CD25+FoxP3+ cells in the draining lymph node could be detected. However, activation of Treg by J immunization was indicated by potent suppression of antigen-specific splenic effectors compared to CDR3-immunized animals, and by a 4fold higher spontaneous proliferation of FoxP3+ cells from the draining lymph node in vitro. In contrast to CDR3-derived Treg, the addition of J-induced Treg to effector cells resulted in a dose-dependent production of IL-10 in mixed cultures, independently of the antigen specificity of the effectors. Finally, coimmunization with HA and J peptides led to inhibition of the proliferation of HA-specific CD8+ effectors in vivo as demonstrated by adoptive transfer and subsequent flow cytometry analysis of CFSE-labelled TCR-transgenic T cells. This inhibition was absent after coimmunization with HA and CDR3 peptides and could be largely abolished by prior in vivo depletion of Treg with an αCD25 antibody. These data demonstrate in a non-transgenic model that coimmunization with shared and individual, strictly MHC I-restricted tumor antigens leads to a potent inhibition of tumor-specific CD8+ T cells through rapid in situ activation of CD4+FoxP3+ Treg elicited by the shared tumor antigen. It is postulated that these Treg recognize MHC II-restricted self antigens presumably derived from non-neoplastic cells as a consequence of an aborted immune response to the shared antigen. These experiments provide direct evidence that active immunotherapy of malignant tumors exclusively with true tumor-specific antigens has a greater chance of success since the presence of shared antigens will prevent tumor-specific immune responses through Treg activation.