The roles of IL-12 and IL-23 in CD8 T cell-mediated immunity against Listeria monocytogenes: Insights from a DC vaccination model

Listeria monocytogenes infection induces a strong inflammatory response characterized by the production of IL-12 and IFN-γ and protective immunity against this pathogen is dependent on CD8⁺ T cells (CTL). Recent studies have suggested that these inflammatory cytokines affect the rate of memory CD8⁺ T cell generation as well as the number of short-lived effector cells generated. The role of the closely related cytokine, IL-23, in this response has not been examined. We hypothesized that IL-12 and IL-23 produced by dendritic cells collectively enhance the generation and function of memory cells. To test this hypothesis, we employed a DC vaccination approach. Mice lacking IL-12 and IL-23 were vaccinated with wild-type (WT), IL-12^−/−, or IL-12/23^−/− DC and protection to Lm was monitored. Mice vaccinated with WT and IL-12^−/− DC were resistant to lethal challenge with Lm. Surprisingly, mice vaccinated with IL-12/23^−/− DC exhibited significantly reduced protection when challenged. Protection correlated with the relative size of the memory pools generated. In summary, these data indicate that IL-23 can partially compensate for the lack of IL-12 in the generation protective immunity against Lm.     

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.     

Purified PTP1 protein induces antigen-specific protective immunity against Encephalitozoon cuniculi

Microsporidiosis poses a problem for immunocompromised individuals including patients with HIV infection as well as those with organ transplantation. Recent reports from Africa have suggested that microsporidiosis with diarrhea is an independent risk factor for malnutrition in children. Previous studies from our laboratory have demonstrated that CD8⁺ T cells are an essential component of protective immunity against the microsporidium Encephalitozoon cuniculi. Mutant mice lacking this T cell subset or cytotoxic function are unable to clear the infection and ultimately succumb to the disease. However, information regarding the antigens involved in the elicitation of CD8⁺ T cell response is not available. In this study, we report that immunization of animals with Encephalitozoon hellem polar tube protein 1 (rEhPTP1) induces a strong T cell response in vaccinated animals. Splenic dendritic cells pulsed with rEhPTP1 are able to induce E. cuniculi specific CD8⁺ T cell response with no effect on the CD4⁺ T cell subset. This is the first report identifying a protein capable of inducing CD8⁺ T cell immunity, which is conserved in other microsporidial species of human importance.     

A pathway to cure chronic infection with Toxoplasma gondii through immunological intervention

Toxoplasma gondii, an obligate intracellular protozoan parasite, can establish a chronic infection in the brain by forming tissue cysts. This chronic infection is widespread in humans worldwide including developed countries, with up to one third of the population being estimated to be infected with this parasite. Diagnosis of this chronic infection is usually conducted by serological detection of IgG antibodies against this parasite. Since infected individuals remain positive for these antibodies for years, it has generally been considered that this infection is a lifelong infection. It is also often considered that this chronic infection is “latent” or “quiescent”. However, recent discovery of the capability of perforin-dependent, CD8⁺ T cell-mediated immune responses to eliminate T. gondii cysts in collaboration with phagocytes illustrated dynamic interplays between T. gondii cysts and host immune system during this chronic infection. Importantly, the cytotoxic T cell-mediated protective immunity is able to remove mature cysts of the parasite. It is now clear that chronic T. gondii infection is not “latent” or “quiescent”. Elucidating the mechanisms of the dynamic host-pathogen interactions between the anti-cyst protective immunity and T. gondii cysts and identifying the pathway to appropriately activate anti-cyst CD8⁺ cytotoxic T cells would be able to open a door for eradicating T. gondii cysts and curing chronic infection with this parasite.     

Induction of IL-12p40 and type 1 immunity by Toxoplasma gondii in the absence of the TLR-MyD88 signaling cascade

Toxoplasma gondii is an orally acquired pathogen that induces strong IFN-γ based immunity conferring protection but that can also be the cause of immunopathology. The response in mice is driven in part by well-characterized MyD88-dependent signaling pathways. Here we focus on induction of less well understood immune responses that do not involve this Toll-like receptor (TLR)/IL-1 family receptor adaptor molecule, in particular as they occur in the intestinal mucosa. Using eYFP-IL-12p40 reporter mice on an MyD88^-/- background, we identified dendritic cells, macrophages, and neutrophils as cellular sources of MyD88-independent IL-12 after peroral T. gondii infection. Infection-induced IL-12 was lower in the absence of MyD88, but was still clearly above noninfected levels. Overall, this carried through to the IFN-γ response, which while generally decreased was still remarkably robust in the absence of MyD88. In the latter mice, IL-12 was strictly required to induce type I immunity. Type 1 and type 3 innate lymphoid cells (ILC), CD4⁺ T cells, and CD8⁺ T cells each contributed to the IFN-γ pool. We report that ILC3 were expanded in infected MyD88^-/- mice relative to their MyD88^+/+ counterparts, suggesting a compensatory response triggered by loss of MyD88. Furthermore, bacterial flagellin and Toxoplasma specific CD4⁺ T cell populations in the lamina propria expanded in response to infection in both WT and KO mice. Finally, we show that My88-independent IL-12 and T cell mediated IFN-γ production require the presence of the intestinal microbiota. Our results identify MyD88-independent intestinal immune pathways induced by T. gondii including myeloid cell derived IL-12 production, downstream type I immunity and IFN-γ production by ILC1, ILC3, and T lymphocytes. Collectively, our data reveal an underlying network of immune responses that do not involve signaling through MyD88.     

IL-4 and IL-13 receptors: Roles in immunity and powerful vaccine adjuvants

The roles of interleukin (IL)-4 and IL-13 during both innate and adaptive Th2 mediated immunity have received considerable scrutiny, however, mechanisms by which these cytokines influence the cellular interactions involved in negatively modulating the development of effective Th1 immunity are poorly characterized. In this article we discuss the recent advances in IL-4/IL-13 biology, mainly (i) role of these cytokines in allergic inflammation, atopic dermatitis, cancer, transplant rejection, bacterial/viral infections, and specifically the therapeutic potential of IL-13Rα2, (ii) insights into how “alarmin” stimulation activate IL-4/IL-13 at the lung mucosae, (iii) how these two cytokines modulate antigen-specific CD8⁺ T cell quality/avidity in a vaccine route dependent manner and (iv) finally discuss the potential of using transient inhibition of IL-4 and/or IL-13 at the vaccination site as a platform vaccine technology to induce strong sustained high quality CD8⁺ T cell immunity for protection against many chronic mucosal pathogens such as HIV-1.     

Induction of IL-10-producing regulatory B cells following Toxoplasma gondii infection is important to the cyst formation

Toxoplasma gondii is a protozoan parasite that infects humans and animals via congenital or postnatal routes. During parasite infection, IL-10-producing Bregs are stimulated as part of the parasite-induced host immune responses that favor infection. In this study, we investigated whether T. gondii infection induces immune regulatory cells including IL-10-producing CD1d^highCD5⁺ regulatory B cells (Bregs) and whether Breg induction is critical for the development of chronic infection of T. gondii. Furthermore, B cell-deficient (μMT) mice revealed that the IL-10-producing B cells might be associated with the development of chronic T. gondii infection. To better understand the mechanism underlying the accumulation of IL-10-producing B cells upon T. gondii infection, we determined the effect of products released by T. gondii on the induction and differentiation of IL-10-producing B cells during the acute stage of infection using transgenic green fluorescent protein (GFP)-expressing T. gondii strain. We demonstrated that products secreted at the stage of cell lysis by fully replicated tachyzoites induced the differentiation of naive B cells to IL-10-producing Bregs. Our results indicated that the downregulation of the immune response via Bregs during T. gondii infection is related to cyst formation in the host brain and to the establishment of chronic infection.     

Helminth Infections Coincident with Active Pulmonary Tuberculosis Inhibit Mono- and Multifunctional CD4+ and CD8+ T Cell Responses in a Process Dependent on IL-10

Tissue invasive helminth infections and tuberculosis (TB) are co-endemic in many parts of the world and can trigger immune responses that might antagonize each other. We have previously shown that helminth infections modulate the Th1 and Th17 responses to mycobacterial-antigens in latent TB. To determine whether helminth infections modulate antigen-specific and non-specific immune responses in active pulmonary TB, we examined CD4⁺ and CD8⁺ T cell responses as well as the systemic (plasma) cytokine levels in individuals with pulmonary TB with or without two distinct helminth infections—Wuchereria bancrofti and Strongyloides stercoralis infection. By analyzing the frequencies of Th1 and Th17 CD4⁺ and CD8⁺ T cells and their component subsets (including multifunctional cells), we report a significant diminution in the mycobacterial–specific frequencies of mono- and multi–functional CD4⁺ Th1 and (to a lesser extent) Th17 cells when concomitant filarial or Strongyloides infection occurs. The impairment in CD4⁺ and CD8⁺ T cell cytokine responses was antigen-specific as polyclonal activated T cell frequencies were equivalent irrespective of helminth infection status. This diminution in T cell responses was also reflected in diminished circulating levels of Th1 (IFN-γ, TNF-α and IL-2)- and Th17 (IL-17A and IL-17F)-associated cytokines. Finally, we demonstrate that for the filarial co-infections at least, this diminished frequency of multifunctional CD4⁺ T cell responses was partially dependent on IL-10 as IL-10 blockade significantly increased the frequencies of CD4⁺ Th1 cells. Thus, co-existent helminth infection is associated with an IL-10 mediated (for filarial infection) profound inhibition of antigen-specific CD4⁺ T cell responses as well as protective systemic cytokine responses in active pulmonary TB.     

Atg5 but not Atg7 in dendritic cells enhances IL-2 and IFN-γ production by Toxoplasma gondii-reactive CD4+ T cells

The autophagy proteins (Atg) modulate not only innate but also adaptive immunity against pathogens. We examined the role of dendritic cell Atg5 and Atg7 in the production of IL-2 and IFN-γ by Toxoplasma gondii-reactive CD4⁺ T cells. T. gondii-reactive mouse CD4⁺ T cells exhibited unimpaired production of IL-2 and IFN-γ when stimulated with Atg7-deficient mouse dendritic cells that were infected with T. gondii or pulsed with T. gondii lysate antigens. In marked contrast, dendritic cells deficient in Atg5 induced diminished CD4⁺ T cell production of IL-2 and IFN-γ. This defect was not accompanied by changes in costimulatory ligand expression on dendritic cells or impaired production of IL-12 p70, IL-1β or TNF-α. Knockdown of Irg6a in dendritic cells did not affect CD4⁺ T cell cytokine production. These results indicate that Atg5 and Atg7 in dendritic cells play differential roles in the modulation of IL-2 and IFN-γ production by T. gondii-reactive CD4⁺ T cells.     

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-17 Induction by ArtinM is Due to Stimulation of IL-23 and IL-1 Release and/or Interaction with CD3 in CD4+ T Cells

ArtinM is a D-mannose-binding lectin extracted from the seeds of Artocarpus heterophyllus that interacts with TLR2 N-glycans and activates antigen-presenting cells (APCs), as manifested by IL-12 production. In vivo ArtinM administration induces Th1 immunity and confers protection against infection with several intracellular pathogens. In the murine model of Candida albicans infection, it was verified that, in addition to Th1, ArtinM induces Th17 immunity manifested by high IL-17 levels in the treated animals. Herein, we investigated the mechanisms accounting for the ArtinM-induced IL-17 production. We found that ArtinM stimulates the IL-17 production by spleen cells in BALB/c or C57BL/6 mice, a response that was significantly reduced in the absence of IL-23, MyD88, or IL-1R. Furthermore, we showed that ArtinM directly induced the IL-23 mRNA expression and the IL-1 production by macrophages. Consistently, in cell suspensions depleted of macrophages, the IL-17 production stimulated by ArtinM was reduced by 53% and the exogenous IL-23 acted synergistically with ArtinM in promoting IL-17 production by spleen cell suspensions. We verified that the absence of IL-23, IL-1R, or MyD88 inhibited, but did not block, the IL-17 production by ArtinM-stimulated spleen cells. Therefore, we investigated whether ArtinM exerts a direct effect on CD4⁺ T cells in promoting IL-17 production. Indeed, spleen cell suspensions depleted of CD4⁺ T cells responded to ArtinM with very low levels of IL-17 release. Likewise, isolated CD4⁺ T cells under ArtinM stimulus augmented the expression of TGF-β mRNA and released high levels of IL-17. Considering the observed synergism between IL-23 and ArtinM, we used cells from IL-23 KO mice to assess the direct effect of lectin on CD4⁺ T cells. We verified that ArtinM increased the IL-17 production significantly, a response that was inhibited when the CD4⁺ T cells were pre-incubated with anti-CD3 antibody. In conclusion, ArtinM stimulates the production of IL-17 by CD4⁺ T cells in two major ways: (I) through the induction of IL-23 and IL-1 by APCs and (II) through the direct interaction with CD3 on the CD4⁺ T cells. This study contributes to elucidation of mechanisms accounting for the property of ArtinM in inducing Th17 immunity and opens new perspectives in designing strategies for modulating immunity by using carbohydrate recognition agents.     

IL-15 Superagonist Expands mCD8+ T,NK and NKT Cells after Burn Injury but Fails to Improve Outcome during Burn Wound Infection

Background

Severely burned patients are highly susceptible to opportunistic infections and sepsis, owing to the loss of the protective skin barrier and immunological dysfunction. Interleukin-15 (IL-15) belongs to the IL-2 family of common gamma chain cytokines and stimulates the proliferation and activation of T (specifically memory CD8), NK and NKT cells. It has been shown to preserve T cell function and improve survival during cecal ligation and puncture (CLP)-induced sepsis in mice. However, the therapeutic efficacy of IL-15 or IL-15 superagonist (SA) during infection after burn injury has not been evaluated. Moreover, very few, if any, studies have examined, in detail, the effect of burn injury and infection on the adaptive immune system. Thus, we examined the effect of burn and sepsis on adaptive immune cell populations and the effect of IL-15 SA treatment on the host response to infection.

Methods

Mice were subjected to a 35% total body surface area burn, followed by wound infection with Pseudomonas aeruginosa. In some experiments, IL-15 SA was administered after burn injury, but before infection. Leukocytes in spleen, liver and peritoneal cavity were characterized using flow cytometry. Bacterial clearance, organ injury and survival were also assessed.

Results

Burn wound infection led to a significant decline in total white blood cell and lymphocyte counts and induced organ injury and sepsis. Burn injury caused decline in CD4⁺ and CD8⁺ T cells in the spleen, which was worsened by infection. IL-15 treatment inhibited this decline and significantly increased cell numbers and activation, as determined by CD69 expression, of CD4⁺, CD8⁺, B, NK and NKT cells in the spleen and liver after burn injury. However, IL-15 SA treatment failed to prevent burn wound sepsis-induced loss of CD4⁺, CD8⁺, B, NK and NKT cells and failed to improve bacterial clearance and survival.

Conclusion

Cutaneous burn injury and infection cause significant adaptive immune dysfunction. IL-15 SA does not augment host resistance to burn wound sepsis in mice despite inducing proliferation and activation of lymphocyte subsets.     

Activation and IL-10 production of specific CD4+ T cells are regulated by IL-27 during chronic infection with Plasmodium chabaudi

IL-27, a regulatory cytokine, plays critical roles in the prevention of immunopathology during Plasmodium infection. We examined these roles in the immune responses against Plasmodium chabaudi infection using the Il-27ra^−/− mice. While IL-27 was expressed at high levels during the early phase of the infection, enhanced CD4⁺ T cell function and reduction in parasitemia were observed mainly during the chronic phase in the mutant mice. In mice infected with P. chabaudi and cured with drug, CD4⁺ T cells in the Il-27ra^−/− mice exhibited enhanced CD4⁺ T-cell responses, indicating the inhibitory role of IL-27 on the protective immune responses. To determine the role of IL-27 in detail, we performed CD4⁺ T-cell transfer experiments. The Il-27ra^−/− and Il27p28^−/− mice were first infected with P. chabaudi and then cured using drug treatment. Plasmodium-antigen primed CD4⁺ T cells were prepared from these mice and transferred into the recipient mice, followed by infection with the heterologous parasite P. berghei ANKA. Il-27ra^−/− CD4⁺ T cells in the infected recipient mice did not produce IL-10, indicating that IL-10 production by primed CD4⁺ T cells is IL-27 dependent. Il27p28^−/− CD4⁺ T cells that were primed in the absence of IL-27 exhibited enhanced recall responses during the challenge infection with P. berghei ANKA, implying that IL-27 receptor signaling during the primary infection affects recall responses in the long-term via the regulation of the memory CD4⁺ T cell generation. These features highlighted direct and time-transcending roles of IL-27 in the regulation of immune responses against chronic infection with Plasmodium parasites.     

Regulation of CD8 T cell responses to infection with parasitic protozoa

There are over 10,000 species of parasitic protozoa, a subset of which can cause considerable disease in humans. Here we examine in detail the complex immune response generated during infection with a subset of these parasites: Trypanosoma cruzi, Leishmania sp., Toxoplasma gondii, and Plasmodium sp. While these particular species perhaps represent the most studied parasites in terms of understanding how T cells function during infection, it is clear that the lessons learned from this body of work are also relevant to the other protozoa known to induce a CD8⁺ T cell response. This review will highlight some of the key studies that established that CD8⁺ T cells play a major role in protective immunity to protozoa, the factors that promote the generation as well as maintenance of the CD8⁺ T cell response during these infections, and draw attention to some of the gaps in our knowledge. Moreover, the development of new tools, including MHC-Class I tetramer reagents and the use of TCR transgenic mice or genetically modified parasites, has provided a better appreciation of how parasite specific CD8⁺ T cell responses are initiated and new insights into their phenotypic plasticity     

IL-4Rα-Associated Antigen Processing by B Cells Promotes Immunity in Nippostrongylus brasiliensis Infection

In this study, B cell function in protective T_H2 immunity against N. brasiliensis infection was investigated. Protection against secondary infection depended on IL-4Rα and IL-13; but not IL-4. Protection did not associate with parasite specific antibody responses. Re-infection of B cell-specific IL-4Rα^−/− mice resulted in increased worm burdens compared to control mice, despite their equivalent capacity to control primary infection. Impaired protection correlated with reduced lymphocyte IL-13 production and B cell MHC class II and CD86 surface expression. Adoptive transfer of in vivo N. brasiliensis primed IL-4Rα expressing B cells into naïve BALB/c mice, but not IL-4Rα or IL-13 deficient B cells, conferred protection against primary N. brasiliensis infection. This protection required MHC class II compatibility on B cells suggesting cognate interactions by B cells with CD4⁺ T cells were important to co-ordinate immunity. Furthermore, the rapid nature of these protective effects by B cells suggested non-BCR mediated mechanisms, such as via Toll Like Receptors, was involved, and this was supported by transfer experiments using antigen pulsed Myd88^−/− B cells. These data suggest TLR dependent antigen processing by IL-4Rα-responsive B cells producing IL-13 contribute significantly to CD4⁺ T cell-mediated protective immunity against N. brasiliensis infection.     

IL-15 Augments TCR-Induced CD4+ T Cell Expansion In Vitro by Inhibiting the Suppressive Function of CD25High CD4+ T Cells

Due to its critical role in NK cell differentiation and CD8⁺ T cell homeostasis, the importance of IL-15 is more firmly established for cytolytic effectors of the immune system than for CD4⁺ T cells. The increased levels of IL-15 found in several CD4⁺ T cell-driven (auto-) immune diseases prompted us to examine how IL-15 influences murine CD4⁺ T cell responses to low dose TCR-stimulation in vitro. We show that IL-15 exerts growth factor activity on both CD4⁺ and CD8⁺ T cells in a TCR-dependent and Cyclosporin A-sensitive manner. In CD4⁺ T cells, IL-15 augmented initial IL-2-dependent expansion and once IL-15Rα was upregulated, IL-15 sustained the TCR-induced expression of IL-2/15Rβ, supporting proliferation independently of secreted IL-2. Moreover, IL-15 counteracts CD4⁺ T cell suppression by a gradually expanding CD25^HighCD4⁺ T cell subset that expresses Foxp3 and originates from CD4⁺CD25⁺ Tregs. These in vitro data suggest that IL-15 may dramatically strengthen the T cell response to suboptimal TCR-triggering by overcoming an activation threshold set by Treg that might create a risk for autoimmune pathology.     

T cell-derived interferon-γ is required for host defense to Toxoplasma gondii

Interferon-γ (IFN-γ) is important for host defense against various intracellular organisms including a protozoan pathogen Toxoplasma gondii. Various immune cells are recently shown to produce IFN-γ in T. gondii infection, however, it remains elusive which cell types are important for anti-T. gondii host defense so far. Here we generate a new IFN-γ reporter "GREVEN" mouse line in which a fusion protein of Venus and NanoLuc to analyze IFN-γ producing cells during T. gondii infection and find that CD4⁺, CD8⁺, γδ T cells and natural killer cells express Venus in a time dependent manner. Furthermore, Lck-Cre/Ifng^fl/fl mice are highly susceptible to T. gondii infection. Taken together, our results demonstrate that T cell-derived IFN-γ plays an important role in anti-T. gondii host defense.     

A Two-Component DNA-Prime/Protein-Boost Vaccination Strategy for Eliciting Long-Term,Protective T Cell Immunity against Trypanosoma cruzi

In this study, we evaluated the long-term efficacy of a two-component subunit vaccine against Trypanosoma cruzi infection. C57BL/6 mice were immunized with TcG2/TcG4 vaccine delivered by a DNA-prime/Protein-boost (D/P) approach and challenged with T. cruzi at 120 or 180 days post-vaccination (dpv). We examined whether vaccine-primed T cell immunity was capable of rapid expansion and intercepting the infecting T. cruzi. Our data showed that D/P vaccine elicited CD4⁺ (30-38%) and CD8⁺ (22-42%) T cells maintained an effector phenotype up to 180 dpv, and were capable of responding to antigenic stimulus or challenge infection by a rapid expansion (CD8>CD4) with type 1 cytokine (IFNγ⁺ and TFNα⁺) production and cytolytic T lymphocyte (CTL) activity. Subsequently, challenge infection at 120 or 180 dpv, resulted in 2-3-fold lower parasite burden in vaccinated mice than was noted in unvaccinated/infected mice. Co-delivery of IL-12- and GMCSF-encoding expression plasmids provided no significant benefits in enhancing the anti-parasite efficacy of the vaccine-induced T cell immunity. Booster immunization (bi) with recombinant TcG2/TcG4 proteins 3-months after primary vaccine enhanced the protective efficacy, evidenced by an enhanced expansion (1.2-2.8-fold increase) of parasite-specific, type 1 CD4⁺ and CD8⁺ T cells and a potent CTL response capable of providing significantly improved (3-4.5-fold) control of infecting T. cruzi. Further, CD8⁺T cells in vaccinated/bi mice were predominantly of central memory phenotype, and capable of responding to challenge infection 4-6-months post bi by a rapid expansion to a poly-functional effector phenotype, and providing a 1.5-2.3-fold reduction in tissue parasite replication. We conclude that the TcG2/TcG4 D/P vaccine provided long-term anti-T. cruzi T cell immunity, and bi would be an effective strategy to maintain or enhance the vaccine-induced protective immunity against T. cruzi infection and Chagas disease.