Coimmunization with an optimized IL-15 plasmid results in enhanced function and longevity of CD8 T cells that are partially independent of CD4 T cell help

DNA vaccines are a promising technology for the induction of Ag-specific immune responses, and much recent attention has gone into improving their immune potency. In this study we test the feasibility of delivering a plasmid encoding IL-15 as a DNA vaccine adjuvant for the induction of improved Ag-specific CD8(+) T cellular immune responses. Because native IL-15 is poorly expressed, we used PCR-based strategies to develop an optimized construct that expresses 80-fold higher than the native IL-15 construct. Using a DNA vaccination model, we determined that immunization with optimized IL-15 in combination with HIV-1gag DNA constructs resulted in a significant enhancement of Ag-specific CD8(+) T cell proliferation and IFN-gamma secretion, and strong induction of long-lived CD8(+) T cell responses. In an influenza DNA vaccine model, coimmunization with plasmid expressing influenza A PR8/34 hemagglutinin with the optimized IL-15 plasmid generated improved long term CD8(+) T cellular immunity and protected the mice against a lethal mucosal challenge with influenza virus. Because we observed that IL-15 appeared to mostly adjuvant CD8(+) T cell function, we show that in the partial, but not total, absence of CD4(+) T cell help, plasmid-delivered IL-15 could restore CD8 secondary immune responses to an antigenic DNA plasmid, supporting the idea that the effects of IL-15 on CD8(+) T cell expansion require the presence of low levels of CD4 T cells. These data suggest a role for enhanced plasmid IL-15 as a candidate adjuvant for vaccine or immunotherapeutic studies.     

Immunological and antitumor effects of IL-23 as a cancer vaccine adjuvant

The promising, but modest, clinical results of many human cancer vaccines indicate a need for vaccine adjuvants that can increase both the quantity and the quality of vaccine-induced, tumor-specific T cells. In this study we tested the immunological and antitumor effects of the proinflammatory cytokine, IL-23, in gp100 peptide vaccine therapy of established murine melanoma. Neither systemic nor local IL-23 alone had any impact on tumor growth or tumor-specific T cell numbers. Upon specific vaccination, however, systemic IL-23 greatly increased the relative and absolute numbers of vaccine-induced CD8(+) T cells and enhanced their effector function at the tumor site. Although IL-23 specifically increased IFN-gamma production by tumor-specific T cells, IFN-gamma itself was not a primary mediator of the vaccine adjuvant effect. The IL-23-induced antitumor effect and accompanying reversible weight loss were both partially mediated by TNF-alpha. In contrast, local expression of IL-23 at the tumor site maintained antitumor activity in the absence of weight loss. Under these conditions, it was also clear that enhanced effector function of vaccine-induced CD8(+) T cells, rather than increased T cell number, is a primary mechanism underlying the antitumor effect of IL-23. Collectively, these results suggest that IL-23 is a potent vaccine adjuvant for the induction of therapeutic, tumor-specific CD8(+) T cell responses.     

Regulation of CD28 expression on umbilical cord blood and adult peripheral blood CD8+ T cells by interleukin(IL)-15/IL-21

Interleukin (IL)-15 and IL-21, both belonging to common γ-chain-signaling cytokine family, have an important role to maintain homeostatic proliferation of CD8(+) T cells. CD28, an essential co-stimulatory molecule on T cells, may be a marker of replicative senescence. We investigated the effect of IL-15 and IL-21, alone or in combination, on activation, apoptosis, cytokine production and cytotoxic function of magnetic bead purified umbilical cord blood (UCB) and adult peripheral blood (APB) CD8(+) T cells with regards to their CD28 expression. We established that (1) IL-15-induced CD8(+) T cell proliferation was associated with a preferential expansion of CD28(-) population in UCB, which could be partially counteracted by IL-21; (2) UCB CD8(+) T cells were more readily responsive to IL-15 compared to their adult counterparts in terms of CD69 expression, with the majority of CD69-bearing CD8(+) T cells were CD28(-); (3) IL-21 further promoted interferon-gamma, but not tumor necrosis factor-alpha production from IL-15 treated CD8(+) T cells; (4) IL-21 also synergized with IL-15 to enhance perforin and granzyme B expression of CD8(+) T cells, especially in APB CD8(+)CD28(-) subsets; (5) IL-21 resulted in CD8(+) T cells apoptosis both in APB and UCB cells, mainly in CD8(+)CD28(-) subsets. Taken together, we demonstrate differential IL-15/IL-21 response in UCB CD8(+) T cells with regards to CD28 expression. Our results suggest that combining IL-21 and IL-15 immunotherapy may be better than IL-15 alone to ameliorate graft-versus-host disease while preserving antitumor effect in the post-UCB transplantation period.     

IL-2/anti-IL-2 antibody complex enhances vaccine-mediated antigen-specific CD8+ T cell responses and increases the ratio of effector/memory CD8+ T cells to regulatory T cells

IL-2 is well described as a cytokine with two markedly distinct functionalities: as a necessary signal during CD4(+) and CD8(+) T cell activation/expansion and as an essential cytokine for the maintenance of CD4(+)CD25(+)FoxP3(+) T cells (regulatory T (T(REG)) cells) during homeostasis. In this study we demonstrate for the first time that, compared with the use of IL-2 alone, a complex of IL-2 and anti-IL-2 Ab (IL-2 complex) enhances the effectiveness of a viral vaccine in a mouse model with known Ag specificity. IL-2 complex led to an increase in the number of Ag-specific effector/memory CD8(+) T cells, cytokine production, and CTL lysis following Ag-specific restimulation in a vaccination setting. Our results further demonstrate that this effect is temporary and declines over the course of a few days after the IL-2 complex treatment cycle. Moreover, in contrast to the use of IL-2 alone, IL-2 complex greatly increased the ratio of effector/memory CD8(+) T cells to T(REG) cells. This phenomenon can thus potentially be used in the enhancement of immune responses to vaccination.     

Listeriolysin O expressed in a bacterial vaccine suppresses CD4+CD25high regulatory T cell function in vivo

CD4(+)CD25(high) regulatory T cells (Treg) protect the host from autoimmune diseases but are also obstacles against cancer therapies. An ideal cancer vaccine would stimulate specific cytotoxic responses and reduce/suppress Treg function. In this study, we showed that Escherichia coli expressing listeriolysin O and OVA (E. coli LLO/OVA) demonstrated remarkable levels of protection against OVA-expressing tumor cells. By contrast, E. coli expressing OVA only (E. coli OVA) showed poor protection. High-avidity OVA-specific CTL were induced in E. coli LLO/OVA-vaccinated mice, and CD8(+) depletion--but not NK cell depletion, abolished the antitumor activity of the E. coli LLO/OVA vaccine. Phenotypic analysis of T cells following vaccination with either vaccine revealed preferential generation of CD44(high)CD62L(low) CD8(+) effector memory T cells over CD44(high)CD62L(high) central memory T cells. Unexpectedly, CD4(+) depletion turned E. coli OVA into a vaccine as effective as E. coli LLO/OVA suggesting that a subset of CD4(+) cells suppressed the CD8(+) T cell-mediated antitumor response. Further depletion experiments demonstrated that these suppressive cells consisted of CD4(+)CD25(high) regulatory T cells. We therefore assessed these vaccines for Treg function and found that although CD4(+)CD25(high) expansion and Foxp3 expression within this population was similar in all groups of mice, Treg cells from E. coli LLO/OVA-vaccinated animals were unable to suppress conventional T cells proliferation. These findings provide the first evidence that LLO expression affects Treg cell function and may have important implications for enhancing antitumor vaccination strategies in humans.     

Adjuvant IL-15 does not enhance the efficacy of tumor cell lysate-pulsed dendritic cell vaccines for active immunotherapy of T cell lymphoma

There has been a recent interest in using IL-15 to enhance antitumor activity in several models because of its ability to stimulate CD8⁺ T cell expansion, inhibit apoptosis and promote memory T cell survival and maintenance. Previously, we reported that C6VL tumor lysate-pulsed dendritic cell vaccines significantly enhanced the survival of tumor-bearing mice by stimulating a potent tumor-specific CD8⁺ T cell response. In this study, we determined whether IL-15 used as immunologic adjuvant would augment vaccine-primed CD8⁺ T cell immunity against C6VL and further improve the survival of tumor-bearing mice. We report that IL-15 given after C6VL lysate-pulsed dendritic cell vaccines stimulated local and systemic expansion of NK, NKT and CD8⁺ CD44^hi T cells. IL-15 did not, however, augment innate or cellular responses against the tumor. T cells from mice infused with IL-15 following vaccination did not secrete increased levels of tumor-specific TNF-α or IFN-γ or have enhanced C6VL-specific CTL activity compared to T cells from recipients of the vaccine alone. Lastly, IL-15 did not enhance the survival of tumor-bearing vaccinated mice. Thus, while activated- and memory-phenotype CD8⁺ T cells were dramatically expanded by IL-15 infusion, vaccine-primed CD8⁺ T cell specific for C6VL were not significantly expanded. This is the first account of using IL-15 as an adjuvant in a therapeutic model of active immunotherapy where there was not a preexisting pool of tumor-specific CD8⁺ T cells. Our results contrast the recent studies where IL-15 was successfully used to augment tumor-reactivity of adoptively transferred transgenic CD8⁺ T cells. This suggests that the adjuvant potential of IL-15 may be greatest in settings where it can augment the number and activity of preexisting tumor-specific CD8⁺ T cells.     

IL-15 transpresentation augments CD8+ T cell activation and is required for optimal recall responses by central memory CD8+ T cells

Although the adaptive immune system has a remarkable ability to mount rapid recall responses to previously encountered pathogens, the cellular and molecular signals necessary for memory CD8(+) T cell reactivation are poorly defined. IL-15 plays a critical role in memory CD8(+) T cell survival; however, whether IL-15 is also involved in memory CD8(+) T cell reactivation is presently unclear. Using artificial Ag-presenting surfaces prepared on cell-sized microspheres, we specifically addressed the role of IL-15 transpresentation on mouse CD8(+) T cell activation in the complete absence of additional stimulatory signals. In this study we demonstrate that transpresented IL-15 is significantly more effective than soluble IL-15 in augmenting anti-CD3epsilon-induced proliferation and effector molecule expression by CD8(+) T cells. Importantly, IL-15 transpresentation and TCR ligation by anti-CD3epsilon or peptide MHC complexes exhibited synergism in stimulating CD8(+) T cell responses. In agreement with previous studies, we found that transpresented IL-15 preferentially stimulated memory phenotype CD8(+) T cells; however, in pursuing this further, we found that central memory (T(CM)) and effector memory (T(EM)) CD8(+) T cells responded differentially to transpresented IL-15. T(CM) CD8(+) T cells undergo Ag-independent proliferation in response to transpresented IL-15 alone, whereas T(EM) CD8(+) T cells are relatively unresponsive to transpresented IL-15. Furthermore, upon Ag-specific stimulation, T(CM) CD8(+) T cell responses are enhanced by IL-15 transpresentation, whereas T(EM) CD8(+) T cell responses are only slightly affected, both in vitro and in vivo. Thus, our findings distinguish the role of IL-15 transpresentation in the stimulation of distinct memory CD8(+) T cell subsets, and they also have implications for ex vivo reactivation and expansion of Ag-experienced CD8(+) T cells for immunotherapeutic approaches.     

Cell-associated double-stranded RNA enhances antitumor activity through the production of type I IFN

The efficacy of tumor cell vaccination largely depends on the maturation and activation status of the dendritic cell. Here we investigated the ability of soluble and tumor cell-associated dsRNA to serve as an adjuvant in the induction of protective adaptive antitumor responses. Our data showed that cell-associated dsRNA, but not soluble dsRNA, enhanced both tumor-specific CD8(+) and CD4(+) T cell responses. The cell-associated dsRNA increased the clonal burst of tumor-specific CD8(+) T cells and endowed them with an enhanced capacity for expansion upon a secondary encounter with tumor Ags, even when the CD8(+) T cells were primed in the absence of CD4(+) T cell help. The adjuvant effect of cell-associated dsRNA was fully dependent on the expression of TLR3 by the APCs and their subsequent production of type I IFNs, as the adjuvant effect of cell-associated dsRNA was completely abrogated in mice deficient in TLR3 or type I IFN signaling. Importantly, treatment with dsRNA-associated tumor cells increased the number of tumor-infiltrating lymphocytes and enhanced the survival of tumor-bearing mice. The data from our studies suggest that using cell-associated dsRNA as a tumor vaccine adjuvant may be a suitable strategy for enhancing vaccine efficacy for tumor cell therapy in cancer patients.     

Increased CD8+ T cell memory to concurrent infection at the expense of increased erosion of pre-existing memory: the paradoxical role of IL-15

The use of cytokines during vaccination, particularly IL-15, is being considered due to the unique ability of IL-15 to enhance the proliferation of memory CD8(+) T cells. However, as homeostatic mechanisms limit excessive lymphocyte expansion, we addressed the consequences of this enhancement of T cell memory by IL-15. Infection of mice with either recombinant Mycobacterium bovis (BCG) expressing IL-15 (BCG-IL-15) or BCG and purified IL-15 resulted in an increased CD44, IL-2Rbeta expression and increased frequency of IFN-gamma-secreting CD8(+) T cells. Surprisingly, the enhancement of memory to concurrent infection by IL-15 exacerbated the attrition of pre-existing memory. Infection of mice with Listeria monocytogenes expressing OVA resulted in potent OVA(257-264)-specific CD8(+) T cell memory, and a challenge of these mice with either BCG-IL-15 or BCG and purified IL-15 resulted in an increased erosion of OVA(257-264)-specific CD8(+) T cell memory, relative to BCG. Enhancement in the erosion of OVA-specific CD8(+) T cell memory by BCG-IL-15 resulted in a consequently greater impairment in protection against a challenge with OVA-expressing tumor cells. We thus raise important questions regarding vaccinations that are aimed at maximizing T cell memory without considering the impact on pre-existing T cell memory.     

Distinct effects of IL-18 on the engraftment and function of human effector CD8 T cells and regulatory T cells

IL-18 has pleotropic effects on the activation of T cells during antigen presentation. We investigated the effects of human IL-18 on the engraftment and function of human T cell subsets in xenograft mouse models. IL-18 enhanced the engraftment of human CD8(+) effector T cells and promoted the development of xenogeneic graft versus host disease (GVHD). In marked contrast, IL-18 had reciprocal effects on the engraftment of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) in the xenografted mice. Adoptive transfer experiments indicated that IL-18 prevented the suppressive effects of Tregs on the development of xenogeneic GVHD. The IL-18 results were robust as they were observed in two different mouse strains. In addition, the effects of IL-18 were systemic as IL-18 promoted engraftment and persistence of human effector T cells and decreased Tregs in peripheral blood, peritoneal cavity, spleen and liver. In vitro experiments indicated that the expression of the IL-18Ralpha was induced on both CD4 and CD8 effector T cells and Tregs, and that the duration of expression was less sustained on Tregs. These preclinical data suggest that human IL-18 may have use as an adjuvant for immune reconstitution after cytotoxic therapies, and to augment adoptive immunotherapy, donor leukocyte infusions, and vaccine strategies.     

A novel functional CTL avidity/activity compartmentalization to the site of mucosal immunization contributes to protection of macaques against simian/human immunodeficiency viral depletion of mucosal CD4+ T cells

The presence of high-avidity CTLs in the right compartment can greatly affect clearance of a virus infection (for example, AIDS viral infection of and dissemination from mucosa). Comparing mucosal vs systemic immunization, we observed a novel compartmentalization of CTL avidity and proportion of functionally active Ag-specific CD8(+) T cells to tissues proximal to sites of immunization. Whereas both s.c. and intrarectal routes of immunization induced tetramer(+) cells in the spleen and gut, the mucosal vaccine induced a higher percentage of functioning IFN-gamma(+) Ag-specific CD8(+) T cells in the gut mucosa in mice. Translating to the CD8(+) CTL avidity distribution in rhesus macaques, intrarectal vaccination induced more high-avidity mucosal CTL than s.c. vaccination and protection of mucosal CD4(+) T cells from AIDS viral depletion, whereas systemic immunization induced higher avidity IFN-gamma-secreting cells in the draining lymph nodes but no protection of mucosal CD4(+) T cells, after mucosal challenge with pathogenic simian/human immunodeficiency virus. Mucosal CD4(+) T cell loss is an early critical step in AIDS pathogenesis. The preservation of CD4(+) T cells in colonic lamina propria and the reduction of virus in the intestine correlated better with high-avidity mucosal CTL induced by the mucosal AIDS vaccine. This preferential localization of high-avidity CTL may explain previous differences in vaccination results and may guide future vaccination strategy.     

IL-12 gene as a DNA vaccine adjuvant in a herpes mouse model: IL-12 enhances Th1-type CD4+ T cell-mediated protective immunity against herpes simplex virus-2 challenge

IL-12 has been shown to enhance cellular immunity in vitro and in vivo. Recent reports have suggested that combining DNA vaccine approach with immune stimulatory molecules delivered as genes may significantly enhance Ag-specific immune responses in vivo. In particular, IL-12 molecules could constitute an important addition to a herpes vaccine by amplifying specific immune responses. Here we investigate the utility of IL-12 cDNA as an adjuvant for a herpes simplex virus-2 (HSV-2) DNA vaccine in a mouse challenge model. Direct i.m. injection of IL-12 cDNA induced activation of resting immune cells in vivo. Furthermore, coinjection with IL-12 cDNA and gD DNA vaccine inhibited both systemic gD-specific Ab and local Ab levels compared with gD plasmid vaccination alone. In contrast, Th cell proliferative responses and secretion of cytokines (IL-2 and IFN-gamma) and chemokines (RANTES and macrophage inflammatory protein-1alpha) were significantly increased by IL-12 coinjection. However, the production of cytokines (IL-4 and IL-10) and chemokine (MCP-1) was inhibited by IL-12 coinjection. IL-12 coinjection with a gD DNA vaccine showed significantly better protection from lethal HSV-2 challenge compared with gD DNA vaccination alone in both inbred and outbred mice. This enhanced protection appears to be mediated by CD4+ T cells, as determined by in vivo CD4+ T cell deletion. Thus, IL-12 cDNA as a DNA vaccine adjuvant drives Ag-specific Th1 type CD4+ T cell responses that result in reduced HSV-2-derived morbidity as well as mortality.     

Interleukin-15 but not interleukin-7 abrogates vaccine-induced decrease in virus level in simian immunodeficiency virus mac251-infected macaques

The loss of CD4(+) T cells and the impairment of CD8(+) T cell function in HIV infection suggest that pharmacological treatment with IL-7 and IL-15, cytokines that increase the homeostatic proliferation of T cells and improve effector function, may be beneficial. However, these cytokines could also have a detrimental effect in HIV-1-infected individuals, because both cytokines increase HIV replication in vitro. We assessed the impact of IL-7 and IL-15 treatment on viral replication and the immunogenicity of live poxvirus vaccines in SIV(mac251)-infected macaques (Macaca mulatta). Neither cytokine augmented the frequency of vaccine-expanded CD4(+) or CD8(+) memory T cells, clonal recruitment to the SIV-specific CD8(+) T cell pool, or CD8(+) T cell function. Vaccination alone transiently decreased the viral set point following antiretroviral therapy suspension. IL-15 induced massive proliferation of CD4(+) effector T cells and abrogated the ability of vaccination to decrease set point viremia. In contrast, IL-7 neither augmented nor decreased the vaccine effect and was associated with a decrease in TGF-beta expression. These results underscore the importance of testing immunomodulatory approaches in vivo to assess potential risks and benefits for HIV-1-infected individuals.     

CD8+ CTL priming by exact peptide epitopes in incomplete Freund's adjuvant induces a vanishing CTL response, whereas long peptides induce sustained CTL reactivity

Therapeutic vaccination trials, in which patients with cancer were vaccinated with minimal CTL peptide in oil-in-water formulations, have met with limited success. Many of these studies were based on the promising data of mice studies, showing that vaccination with a short synthetic peptide in IFA results in protective CD8(+) T cell immunity. By use of the highly immunogenic OVA CTL peptide in IFA as a model peptide-based vaccine, we investigated why minimal CTL peptide vaccines in IFA performed so inadequately to allow full optimization of peptide vaccination. Injection of the minimal MHC class I-binding OVA(257-264) peptide in IFA transiently activated CD8(+) effector T cells, which eventually failed to undergo secondary expansion or to kill target cells, as a result of a sustained and systemic presentation of the CTL peptides gradually leaking out of the IFA depot without systemic danger signals. Complementation of this vaccine with the MHC class II-binding Th peptide (OVA(323-339)) restored both secondary expansion and in vivo effector functions of CD8(+) T cells. Simply extending the CTL peptide to a length of 30 aa also preserved these CD8(+) T cell functions, independent of T cell help, because the longer CTL peptide was predominantly presented in the locally inflamed draining lymph node. Importantly, these functional differences were reproduced in two additional model Ag systems. Our data clearly show why priming of CTL with minimal peptide epitopes in IFA is suboptimal, and demonstrate that the use of longer versions of these CTL peptide epitopes ensures the induction of sustained effector CD8(+) T cell reactivity in vivo.     

Preferential migration of T regulatory cells induced by IL-16

As a natural ligand for CD4, IL-16 has been shown to preferentially induce migration in Th1 cells, and, in long-term cultures with IL-2, IL-16 facilitates the expansion of CD4(+)CD25(+) cells. In addition, IL-16 has an immunomodulatory role in asthmatic inflammation, as exogenous administration significantly reduces inflammation and airway hyperreactivity. The mechanism for this, however, is not clear. Based on its functional characteristics and potential immunomodulatory role, we investigated the ability of IL-16 to recruit and influence the development of T regulatory (Treg) cells. We now demonstrate that IL-16 preferentially induces migration in a CD25(+)CTLA-4(+) human T cell subset and that responding cells produce IFNgamma and TGFbeta but not IL-10. These cells are relatively unresponsive to antigenic stimulation and can suppress proliferation and IL-5, but not IFNgamma, production by autologous T cells. We further demonstrate that IL-16-recruited cells are enriched for Forkhead box P3 (Foxp3). In addition, we find that IL-16 stimulation may facilitate de novo induction of Foxp3(+) Treg cells, because the stimulation of FoxP3-negative T cells for 48 h results in the expression of FoxP3 mRNA and protein. These data indicate that at sites of inflammation IL-16 may contribute to selective Treg cell expansion through the preferential induction of a migratory response from existing Treg cells, as well as by the induction of de novo generation of FoxP3(+) cells. These findings offer a potential mechanism for the immunosuppressive effects of IL-16 seen in Th2-mediated inflammation.     

CD4(+) T cells are required for the development of cytotoxic CD8(+) T cells during Mycobacterium tuberculosis infection.

The control of acute and chronic Mycobacterium tuberculosis infection is dependent on CD4(+) T cells. In a variety of systems CD8(+) T cell effector responses are dependent on CD4(+) T cell help. The development of CD8(+) T cell-mediated immune responses in the absence of CD4(+) T cells was investigated in a murine model of acute tuberculosis. In vitro and in vivo, priming of mycobacteria-specific CD8(+) T cells was unaffected by the absence of CD4(+) T cells. Infiltration of CD8(+) T cells into infected lungs of CD4(-/-) or wild-type mice was similar. IFN-gamma production by lung CD8(+) T cells in CD4(-/-) and wild-type mice was also comparable, suggesting that emergence of IFN-gamma-producing mycobacteria-specific CD8(+) T cells in the lungs was independent of CD4(+) T cell help. In contrast, cytotoxic activity of CD8(+) T cells from lungs of M. tuberculosis-infected mice was impaired in CD4(-/-) mice. Expression of mRNA for IL-2 and IL-15, cytokines critical for the development of cytotoxic effector cells, was diminished in the lungs of M. tuberculosis-infected CD4(-/-) mice. As tuberculosis is frequently associated with HIV infection and a subsequent loss of CD4(+) T cells, understanding the interaction between CD4(+) and CD8(+) T cell subsets during the immune response to M. tuberculosis is imperative for the design of successful vaccination strategies.     

Vaccination without autoantigen protects against collagen II-induced arthritis via immune deviation and regulatory T cells

Anti-inflammation immunotherapy has been successfully applied for the treatment of autoimmune diseases. Mucosal vaccines against autoimmune disorders are beneficial by influencing the regulatory compartment of gut and systemic adaptive immune systems. A Salmonella vector expressing colonization factor Ag I (CFA/I), shown to behave as an anti-inflammatory vaccine, stimulates the production of CD4(+)CD25(+) T cells and regulatory cytokines. In this work, we queried whether Salmonella-CFA/I can protect DBA/1 mice from collagen-induced arthritis. The incidence of arthritis and cartilage loss in vaccinated DBA/1 mice was remarkably lower when compared with unprotected mice. Clinical findings were accompanied by the suppression of inflammatory cytokines TNF-alpha, IL-1beta, IL-6, and IL-27. Vaccination evoked a multi-tier response consisting of IL-4 producing Th2 cells, an increased production of TGF-beta by CD4(+) T cells, and suppression of collagen II-specific CD4(+) T cell proliferation. To assess the contribution of Salmonella-CFA/I-primed CD4(+) T cells, adoptive transfer studies with total CD4(+), CD4(+)CD25(-), or CD4(+)CD25(+) T cells were performed 15 days postchallenge. Mice receiving either subset showed reduced disease incidence and low clinical scores; however, mice receiving total CD4(+) T cells showed delayed disease onset by 10 days with reduced clinical scores, reduced IL-17 and IL-27, but enhanced IL-4, IL-10, IL-13, and TGF-beta. Inhibition of TGF-beta or IL-4 compromised protective immunity. These data show that Salmonella-CFA/I vaccination of DBA/1 mice protects against collagen-induced arthritis by stimulating TGF-beta- and IL-4-producing regulatory CD4(+) T cells.     

Rapid clonal expansion and prolonged maintenance of memory CD8+ T cells of the effector (CD44highCD62Llow) and central (CD44highCD62Lhigh) phenotype by an archaeosome adjuvant independent of TLR2

Vaccines capable of eliciting long-term T cell immunity are required for combating many diseases. Live vectors can be unsafe whereas subunit vaccines often lack potency. We previously reported induction of CD8(+) T cells to Ag entrapped in archaeal glycerolipid vesicles (archaeosomes). In this study, we evaluated the priming, phenotype, and functionality of the CD8(+) T cells induced after immunization of mice with OVA-Methanobrevibacter smithii archaeosomes (MS-OVA). A single injection of MS-OVA evoked a profound primary response but the numbers of H-2K(b)OVA(257-264)-specific CD8(+) T cells declined by 14-21 days, and <1% of primarily central phenotype (CD44(high)CD62L(high)) cells persisted. A booster injection of MS-OVA at 3-11 wk promoted massive clonal expansion and a peak effector response of approximately 20% splenic/blood OVA(257-264)-specific CD8(+) T cells. Furthermore, contraction was protracted and the memory pool (IL-7Ralpha(high)) of approximately 5% included effector (CD44(high)CD62L(low)) and central (CD44(high)CD62L(high)) phenotype cells. Recall response was observed even at >300 days. CFSE-labeled naive OT-1 (OVA(257-264) TCR transgenic) cells transferred into MS-OVA-immunized recipients cycled profoundly (>90%) within the first week of immunization indicating potent Ag presentation. Moreover, approximately 25% cycling of Ag-specific cells was seen for >50 days, suggesting an Ag depot. In vivo, CD8(+) T cells evoked by MS-OVA killed >80% of specific targets, even at day 180. MS-OVA induced responses similar in magnitude to Listeria monocytogenes-OVA, a potent live vector. Furthermore, protective CD8(+) T cells were induced in TLR2-deficient mice, suggesting nonengagement of TLR2 by archaeal lipids. Thus, an archaeosome adjuvant vaccine represents an alternative to live vectors for inducing CD8(+) T cell memory.     

A regulatory T cell-dependent novel function of CD25 (IL-2Ralpha) controlling memory CD8(+) T cell homeostasis

A massive systemic expansion of CD8(+) memory T (T(M)) cells and a remarkable increase in circulating IL-2 were observed only in IL-2Ralpha (CD25) knockout (KO) mice but not in IL-2 KO and scurfy mice, although all three mutants lack regulatory T (Treg) cells. However, both phenotypes were suppressed by the transfer of Treg cells. The data presented indicate that Treg cell deficiency drives naive T cells to T(M) cells. The lack of high-affinity IL-2R in IL-2Ralpha KO mice increases circulating IL-2 that is then preferentially used by CD8(+) T(M) cells through its abundant low-affinity IL-2R, resulting in systemic CD8(+) T(M) cell dominance. Our study demonstrates the critical control of CD8(+) T(M) cell homeostasis by a Treg cell-dependent novel function of CD25 and resolves its mechanism of action.