NKT cells inhibit antigen-specific effector CD8 T cell induction to skin viral proteins

We recently demonstrated that CD1d-restricted NKT cells resident in skin can inhibit CD8 T cell-mediated graft rejection of human papillomavirus E7-expressing skin through an IFN-γ-dependent mechanism. In this study, we examined the role of systemically derived NKT cells in regulating the rejection of skin grafts expressing viral proteins. In lymph nodes draining transplanted skin, Ag-specific CD8 T cell proliferation, cytokine production, and cytotoxic activity were impaired by NKT cells. NKT cell suppression was mediated via CD11c(+) dendritic cells. Inhibition of CD8 T cell function did not require Foxp3(+) regulatory T cells or NKT cell-secreted IFN-γ, IL-10, or IL-17. Thus, following skin grafting or immunization with human papillomavirus-E7 oncoprotein, NKT cells reduce the capacity of draining lymph node-resident APCs to cross-present Ag to CD8 T cell precursors, as evidenced by impaired expansion and differentiation to Ag-specific CD8 T effector cells. Therefore, in the context of viral Ag challenge in the skin, systemic NKT cells limit the capacity for effective priming of adaptive immunity.     

Cutting edge: regulation of CD8+ T cell effector population size

Naive CD8(+) T cells are activated on encounter with Ag presented on dendritic cells and proliferate rapidly. To investigate the regulation of naive CD8(+) T cells proliferation, we adoptively transferred TCR-transgenic CD8(+) T cells into intact mice together with Ag-pulsed dendritic cells. Regardless of the number of cells initially transferred, the expansion of activated Ag-specific CD8(+) T cells was limited to a ceiling of effector cells. This limit was reached from a wide range of T cell doses, including a physiological number of precursor cells, and was not altered by changing the amount of Ag or APCs. The total Ag-specific response was composed of similar numbers of host and donor transgenic cells regardless of donor cell input, suggesting that these populations were independently regulated. Regulation of the transgenic donor cell population was TCR specific. We hypothesize that a clone-specific regulatory mechanism controls the extent of CD8(+) T cell responses to Ag.     

Innate immunity together with duration of antigen persistence regulate effector T cell induction

Proliferation of T cells is important for the expansion of specific T cell clones during immune responses. In addition, for the establishment of protective immunity against viruses, bacteria, and tumors, the expanded T cells must differentiate into effector T cells. Here we show that effector T cell generation is driven by activation of APCs and duration of antigenic stimulation. Adoptively transferred TCR-transgenic T cells extensively proliferated upon immunization. However, these T cells failed to differentiate into effector cells and died within 1 wk after immunization unless antigenic peptides persisted for >1 day or were presented by activated APCs. The induction of protective immunity in a nontransgenic system was more stringent, since activation of APCs or prolonged Ag persistence alone was not sufficient to drive immunity. In contrast, Ag had to be presented for several days by activated APCs to trigger protective T cell responses. Thus, activation of APCs and duration of Ag presentation together regulate the induction of protective T cell responses.     

Disruption of innate-mediated proinflammatory cytokine and reactive oxygen species third signal leads to antigen-specific hyporesponsiveness

Successful Ag activation of naive T helper cells requires at least two signals consisting of TCR and CD28 on the T cell interacting with MHC II and CD80/CD86, respectively, on APCs. Recent evidence demonstrates that a third signal consisting of proinflammatory cytokines and reactive oxygen species (ROS) produced by the innate immune response is important in arming the adaptive immune response. In an effort to curtail the generation of an Ag-specific T cell response, we targeted the synthesis of innate immune response signals to generate Ag-specific hyporesponsiveness. We have reported that modulation of redox balance with a catalytic antioxidant effectively inhibited the generation of third signal components from the innate immune response (TNF-alpha, IL-1beta, ROS). In this study, we demonstrate that innate immune-derived signals are necessary for adaptive immune effector function and disruption of these signals with in vivo CA treatment conferred Ag-specific hyporesponsiveness in BALB/c, NOD, DO11.10, and BDC-2.5 mice after immunization. Modulating redox balance led to decreased Ag-specific T cell proliferation and IFN-gamma synthesis by diminishing ROS production in the APC, which affected TNF-alpha levels produced by CD4(+) T cells and impairing effector function. These results demonstrate that altering redox status can be effective in T cell-mediated diseases such as autoimmune diabetes to generate Ag-specific immunosuppression because it inhibits the third signal necessary for CD4(+) T cells to transition from expansion to effector function.     

Endogenous dendritic cells are required for amplification of T cell responses induced by dendritic cell vaccines in vivo

Dendritic cells (DCs) loaded in vitro with Ag are used as cellular vaccines to induce Ag-specific immunity. These cells are thought to be responsible for direct stimulation of Ag-specific T cells, which may subsequently mediate immunity. In this study, in transgenic mouse models with targeted MHC class II expression specifically on DCs, we show that the DC vaccine is responsible only for partial CD4(+) T cell activation, but to obtain optimal expansion of T cells in vivo, participation of endogenous (resident) DCs, but not endogenous B cells, is crucial. Transfer of Ag to endogenous DCs seems not to be mediated by simple peptide diffusion, but rather by DC-DC interaction in lymph nodes as demonstrated by histological analysis. In contrast, injection of apoptotic or necrotic DC vaccines does not induce T cell responses, but rather represents an immunological null event, which argues that viability of DC vaccines can be crucial for initial triggering of T cells. We propose that viable DCs from the DC vaccine must migrate to the draining lymph nodes and initiate a T cell response, which thereafter requires endogenous DCs that present transferred Ag in order induce optimal T cell expansion. These results are of specific importance with regard to the applicability of DC vaccinations in tumor patients, where the function of endogenous DCs is suppressed by either tumors or chemotherapy.     

The immunogenicity of dendritic cell-based vaccines is not hampered by doxorubicin and melphalan administration

Immunization of cancer patients is most effective in tumor-free conditions or in the presence of minimal residual disease. In the attempt to develop new strategies able to control tumor recurrence while allowing the development of protective immunity, we have investigated the immunogenic potential of two distinct vaccine formulations when provided alone or upon single and repeated treatment with chemotherapeutics drugs. Vaccine-induced T cell responses were first investigated by tracing Ag-specific T cell responses in mice bearing detectable frequencies of Ag-specific TCR transgenic CD4 and CD8 T cells. These studies indicated that immunization with peptide-pulsed dendritic cells and soluble Ag plus adjuvant elicited a comparable expansion and differentiation of CD4 and CD8 effector cells in the peripheral lymphoid tissues when provided alone or shortly after Doxorubicin or Melphalan administration. We also analyzed the potency of the combined vaccination in transgenic adenocarcinoma mouse prostate mice, which develop spontaneous prostate cancer. Dendritic cell-based vaccination elicited potent tumor-specific cytotoxic responses in mice bearing prostate intraepithelial neoplasia both in the absence and in the presence of Doxorubicin. Together our results indicate that Doxorubicin- or Melphalan-based chemotherapy and Ag-specific vaccination can be combined for adjuvant treatments of cancer patients.     

Antigen-experienced CD4 T cells display a reduced capacity for clonal expansion in vivo that is imposed by factors present in the immune host

It is thought that protective immunity is mediated in part by Ag-experienced T cells that respond more quickly and vigorously than naive T cells. Using adoptive transfer of OVA-specific CD4 T cells from TCR transgenic mice as a model system, we show that Ag-experienced CD4 T cells accumulate in lymph nodes more rapidly than naive T cells after in vivo challenge with Ag. However, the magnitude of clonal expansion by Ag-experienced T cells was much less than that of naive T cells, particularly at early times after primary immunization. Ag-experienced CD4 T cells quickly reverted to the slower but more robust clonal expansion behavior of naive T cells after transfer into a naive environment. Conversely, the capacity for rapid clonal expansion was acquired by naive CD4 T cells after transfer into passively immunized recipients. These results indicate that rapid in vivo response by Ag-experienced T cells is facilitated by Ag-specific Abs, whereas the limited capacity for clonal expansion is imposed by some other factor in the immune environment, perhaps residual Ag.     

Antigen targeting to plasmacytoid dendritic cells via Siglec-H inhibits Th cell-dependent autoimmunity

Plasmacytoid dendritic cells (PDCs) have been shown to present Ags and to contribute to peripheral immune tolerance and to Ag-specific adaptive immunity. However, modulation of adaptive immune responses by selective Ag targeting to PDCs with the aim of preventing autoimmunity has not been investigated. In the current study, we demonstrate that in vivo Ag delivery to murine PDCs via the specifically expressed surface molecule sialic acid binding Ig-like lectin H (Siglec-H) inhibits Th cell and Ab responses in the presence of strong immune stimulation in an Ag-specific manner. Correlating with sustained low-level MHC class II-restricted Ag presentation on PDCs, Siglec-H-mediated Ag delivery induced a hyporesponsive state in CD4(+) T cells leading to reduced expansion and Th1/Th17 cell polarization without conversion to Foxp3(+) regulatory T cells or deviation to Th2 or Tr1 cells. Siglec-H-mediated delivery of a T cell epitope derived from the autoantigen myelin oligodendrocyte glycoprotein to PDCs effectively delayed onset and reduced disease severity in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis by interfering with the priming phase without promoting the generation or expansion of myelin oligodendrocyte glycoprotein-specific Foxp3(+) regulatory T cells. We conclude that Ag delivery to PDCs can be harnessed to inhibit Ag-specific immune responses and prevent Th cell-dependent autoimmunity.     

Duration of infection and antigen display have minimal influence on the kinetics of the CD4+ T cell response to Listeria monocytogenes infection

The T cell response to infection consists of clonal expansion of effector cells, followed by contraction to memory levels. It was previously thought that the duration of infection determines the magnitude and kinetics of the T cell response. However, recent analysis revealed that transition between the expansion and contraction phases of the Ag-specific CD8+ T cell response is not affected by experimental manipulation in the duration of infection or Ag display. We studied whether the duration of infection and Ag display influenced the kinetics of the Ag-specific CD4+ T cell response to Listeria monocytogenes (LM) infection. We found that truncating infection and Ag display with antibiotic treatment as early as 24 h postinfection had minimal impact on the expansion or contraction of CD4+ T cells; however, the magnitudes of the Ag-specific CD4+ and CD8+ T cell responses were differentially affected by the timing of antibiotic treatment. Treatment of LM-infected mice with antibiotics at 24 h postinfection did not prevent generation of detectable CD4+ and CD8+ memory T cells at 28 days after infection, vigorous secondary expansion of these memory T cells, or protection against a subsequent LM challenge. These results demonstrate that events within the first few days of infection stimulate CD4+ and CD8+ T cell responses that are capable of carrying out the full program of expansion and contraction to functional memory, independently of prolonged infection or Ag display.     

Low-dose antigen promotes induction of FOXP3 in human CD4+ T cells

Low Ag dose promotes induction and persistence of regulatory T cells (Tregs) in mice, yet few studies have addressed the role of Ag dose in the induction of adaptive CD4(+)FOXP3(+) Tregs in humans. To this end, we examined the level of FOXP3 expression in human CD4(+)CD25(-) T cells upon activation with autologous APCs and varying doses of peptide. Ag-specific T cells expressing FOXP3 were identified by flow cytometry using MHC class II tetramer (Tmr). We found an inverse relationship between Ag dose and the frequency of FOXP3(+) cells for both foreign Ag-specific and self Ag-specific T cells. Through studies of FOXP3 locus demethylation and helios expression, we determined that variation in the frequency of Tmr(+)FOXP3(+) T cells was not due to expansion of natural Tregs, but instead, we found that induction, proliferation, and persistence of FOXP3(+) cells was similar in high- and low-dose cultures, whereas proliferation of FOXP3(-) T cells was favored in high Ag dose cultures. The frequency of FOXP3(+) cells positively correlated with suppressive function, indicative of adaptive Treg generation. The frequency of FOXP3(+) cells was maintained with IL-2, but not upon restimulation with Ag. Together, these data suggest that low Ag dose favors the transient generation of human Ag-specific adaptive Tregs over the proliferation of Ag-specific FOXP3(-) effector T cells. These adaptive Tregs could function to reduce ongoing inflammatory responses and promote low-dose tolerance in humans, especially when Ag exposure and tolerance is transient.     

Induction of rapid T cell activation, division, and recirculation by intratracheal injection of dendritic cells in a TCR transgenic model

Dendritic cells (DCs) are thought to be responsible for sensitization to inhaled Ag and induction of adaptive immunity in the lung. The characteristics of T cell activation in the lung were studied after transfer of Ag-pulsed bone marrow-derived DCs into the airways of naive mice. Cell division of Ag-specific T cells in vivo was followed in a carboxyfluorescein diacetate succinimidyl ester-labeled cohort of naive moth cytochrome c-reactive TCR transgenic T cells. Our adoptive transfer system was such that transferred DCs were the only cells expressing the MHC molecule required for presentation of cytochrome c to transgenic T cells. Ag-specific T cell activation and proliferation occurred rapidly in the draining lymph nodes of the lung, but not in nondraining lymph nodes or spleen. No bystander activation of non-Ag-specific T cells was induced. Division of Ag-specific T cells was accompanied by transient expression of CD69, while up-regulation of CD44 increased with each cell division. Divided cells had recirculated to nondraining lymph nodes and spleen by day 4 of the response. In vitro restimulation with specific Ag revealed that T cells were primed to proliferate more strongly and to produce higher amounts of cytokines per cell. These data are consistent with the notion that DCs in the lung are extremely efficient in selecting Ag-reactive T cells from a diverse repertoire. The response is initially localized in the mediastinal lymph nodes, but subsequently spreads systemically. This system should allow us to study the early events leading to sensitization to inhaled Ag.     

Efficient priming of CD4+ and CD8+ T cells by DNA vaccination depends on appropriate targeting of sufficient levels of immunologically relevant antigen to appropriate processing pathways

The initial cellular events and interactions that occur following DNA immunization are likely to be key to determining the character and magnitude of the resulting immune response, and as such, a better understanding of these events could ultimately lead to the design of more effective pathogen-appropriate DNA vaccines. Therefore, we have used a variety of sensitive cell-based techniques to study the induction of adaptive immunity in vivo. We examined the efficacy of induction of Ag-specific CD4(+) and CD8(+) T cell responses in vivo by the adoptive transfer of fluorescently labeled Ag-specific TCR transgenic T cells and have demonstrated how such approaches can be used to study the effect of simple DNA construct manipulations on immunological priming. OVA-specific CD8(+) and CD4(+) T cells were activated and divided in vivo following immunization with DNA constructs that targeted OVA expression to different subcellular locations; however, the kinetics and degree of cell proliferation were dependent on the cellular location of the expressed protein. DNA vectors encoding cell-associated OVA resulted in greater CD8(+) T cell division compared with other forms of OVA. In contrast, soluble secreted OVA targeted to the classical secretory pathway enhanced division of CD4(+) T cells. Furthermore, the inclusion of mammalian introns to enhance protein expression increased the ability of poorly immunogenic forms of Ag to activate naive T cells, indicating that not only the location, but also the amount of Ag expression, is important for efficient T cell priming following DNA injection.     

Engagement of OX40 enhances antigen-specific CD4(+) T cell mobilization/memory development and humoral immunity: comparison of alphaOX-40 with alphaCTLA-4.

Increasing the long-term survival of memory T cells after immunization is key to a successful vaccine. In the past, the generation of large numbers of memory T cells in vivo has been difficult because Ag-stimulated T cells are susceptible to activation-induced cell death. Previously, we reported that OX40 engagement resulted in a 60-fold increase in the number of Ag-specific CD4(+) memory T cells that persisted 60 days postimmunization. In this report, we used the D011.10 adoptive transfer model to examine the kinetics of Ag-specific T cell entry into the peripheral blood, the optimal route of administration of Ag and alphaOX40, and the Ag-specific Ab response after immunization with soluble OVA and alphaOX40. Finally, we compared the adjuvant properties of alphaOX40 to those of alphaCTLA-4. Engagement of OX-40 in vivo was most effective when the Ag was administered s.c. Time course studies revealed that it was crucial for alphaOX40 to be delivered within 24-48 h after Ag exposure. Examination of anti-OVA Ab titers revealed a 10-fold increase in mice that received alphaOX40 compared with mice that received OVA alone. Both alphaOX40 and alphaCTLA-4 increased the percentage of OVA-specific CD4(+) T cells early after immunization (day 4), but alphaOX40-treated mice had much higher percentages of OVA-specific memory CD4(+) T cells from days 11 to 29. These studies demonstrate that OX40 engagement early after immunization with soluble Ag enhances long-term T cell and humoral immunity in a manner distinct from that provided by blocking CTLA-4.     

A specific role for B cells in the generation of CD8 T cell memory by recombinant Listeria monocytogenes

In this study, we investigated whether B cells play a role in the induction and maintenance of CD8 T cell memory after immunization with an intracellular bacterium, Listeria monocytogenes. Our results show that B cells play a minimal role in the initial activation and Ag-driven expansion of CD8 T lymphocytes. However, absence of B cells results in increased death of activated CD8 T cells during the contraction phase, leading to a lower level of Ag-specific CD8 T cell memory. Once memory is established, B cells are no longer required for the long-term maintenance and rapid recall response of memory CD8 T cells. Increased contraction of Ag-specific CD8 T cells in B cell-deficient mice is not due to impaired CD4 T cell responses since priming of epitope-specific CD4 T cell responses is normal in B cell-deficient mice following L. monocytogenes infection. Furthermore, no exaggerated contraction of Ag-specific CD8 T cells is evident in CD4 knockout mice. Thus, B cells play a specific role in modulating the contraction of CD8 T cell responses following immunization. Elucidation of factors that regulate the death phase may allow us to manipulate this process to increase the level of immunological memory and thus, vaccine efficacy.     

In vivo depletion of CD11c+ cells delays the CD4+ T cell response to Mycobacterium tuberculosis and exacerbates the outcome of infection

Although dendritic cells (DC) are potent APC that prime T cells against many pathogens, there is no direct evidence that DC are required for immunity to Mycobacterium tuberculosis (Mtb) infection. The requirement for DC to prime the CD4+ T cell response following Mtb infection was investigated using pCD11c-diptheria toxin receptor/GFP transgenic mice, in which DC can be transiently ablated in vivo. We show a critical role for DC in initiation of the CD4+ T cell response to the mycobacterial Ag early secretory Ag of tuberculosis 6. The delay in initiating the Ag-specific T cell response led to impaired control of Mtb replication. Interestingly, DC were not required for the secondary CD4+ T cell response following Mtb infection in peptide-vaccinated mice. Thus, this study shows that DC are essential for the initiation of the adaptive T cell response to the human pathogen Mtb.     

Distinct effects of saracatinib on memory CD8+ T cell differentiation

Immunologic memory involving CD8(+) T cells is a hallmark of an adaptive Ag-specific immune response and constitutes a critical component of protective immunity. Designing approaches that enhance long-term T cell memory would, for the most part, fortify vaccines and enhance host protection against infectious diseases and, perhaps, cancer immunotherapy. A better understanding of the cellular programs involved in the Ag-specific T cell response has led to new approaches that target the magnitude and quality of the memory T cell response. In this article, we show that T cells from TCR transgenic mice for the nucleoprotein of influenza virus NP68 exhibit the distinct phases--priming, expansion, contraction, and memory--of an Ag-specific T cell response when exposed in vitro to the cognate peptide. Saracatinib, a specific inhibitor of Src family kinases, administered at low doses during the expansion or contraction phases, increased CD62L(high)/CD44(high) central memory CD8(+) T cells and IFN-γ production but suppressed immunity when added during the priming phase. These effects by saracatinib were not accompanied by the expected decline of Src family kinases but were accompanied by Akt-mammalian target of rapamycin suppression and/or mediated via another pathway. Increased central memory cells by saracatinib were recapitulated in mice using a poxvirus-based influenza vaccine, thus underscoring the importance of dose and timing of the inhibitor in the context of memory T cell differentiation. Finally, vaccine plus saracatinib treatment showed better protection against tumor challenge. The immune-potentiating effects on CD8(+) T cells by a low dose of saracatinib might afford better protection from pathogens or cancer when combined with vaccine.     

Rapid development of T cell memory

Prime-boost immunization is a promising strategy for inducing and amplifying pathogen- or tumor-specific memory CD8 T cell responses. Although expansion of CD8 T cell populations following the second Ag dose is integral to the prime-boost strategy, it remains unclear when, after priming, memory T cells become competent to proliferate. In this study, we show that Ag-specific CD8 T cells with the capacity to undergo extensive expansion are already present at the peak of the primary immune response in mice. These early memory T cells represent a small fraction of the primary immune response and, at early time points, their potential to proliferate is obscured by large effector T cell populations that rapidly clear Ag upon reimmunization. With sufficient Ag boosting, however, secondary expansion of these memory cells can be induced as early as 5-7 days following primary immunization. Importantly, both early and delayed boosting result in similar levels of protective immunity to subsequent pathogen challenge. Early commitment and differentiation of memory T cells during primary immunization suggest that a short duration between priming and boosting is feasible, providing potential logistic advantages for large-scale prime-boost vaccination of human populations.     

The CD8+ T cell population elicited by recombinant adenovirus displays a novel partially exhausted phenotype associated with prolonged antigen presentation that nonetheless provides long-term immunity

We have previously reported that the CD8+ T cell response elicited by recombinant adenovirus vaccination displayed a delayed contraction in the spleen. In our current study, we demonstrate that this unusual kinetic is a general phenomenon observed in multiple tissues. Phenotypic analysis of transgene-specific CD8+ T cells present 30 days postimmunization with recombinant adenovirus revealed a population with evidence of partial exhaustion, suggesting that the cells had been chronically exposed to Ag. Although Ag expression could no longer be detected 3 wk after immunization, examination of Ag presentation within the draining lymph nodes demonstrated that APCs were loaded with Ag peptide for at least 40 days postimmunization, suggesting that Ag remains available to the system for a prolonged period, although the exact source of this Ag remains to be determined. At 60 days postimmunization, the CD8+ T cell population continued to exhibit a phenotype consistent with partially exhausted effector memory cells. Nonetheless, these CD8+ T cells conferred sterilizing immunity against virus challenge 7-12 wk postimmunization, suggesting that robust protective immunity can be provided by CD8+ T cells with an exhausted phenotype. These data demonstrate that prolonged exposure to Ag may not necessarily impair protective immunity and prompt a re-evaluation of the impact of persistent exposure to Ag on T cell function.     

Conventional dendritic cells are required for the activation of helper-dependent CD8 T cell responses to a model antigen after cutaneous vaccination with lentiviral vectors

Cutaneous vaccination with lentiviral vectors generates systemic CD8 T cell responses that have the potential to eradicate tumors for cancer immunotherapy. However, although s.c. immunization with <1 million lentiviral particles clearly primes cytotoxic T cells, vaccination with much higher doses has routinely been used to define the mechanisms of T cell activation by lentiviral vectors. In particular, experiments to test presentation of lentiviral Ags by dendritic cells (DC) require injection of high viral titers, which may result in aberrant transduction of different DC populations. We exploited inducible murine models of DC depletion to investigate which DC prime the lentiviral response after s.c. immunization with low doses of lentiviral particles. In this article, we demonstrate that conventional DC are required to present Ag to CD8 T cells in draining lymph nodes. Langerhans cells are not required to activate the effector response, and neither Langerhans cells nor plasmacytoid DC are sufficient to prime Ag-specific T cells. Immunization drives the generation of endogenous long-lived memory T cells that can be reactivated to kill Ag-specific targets in the absence of inflammatory challenge. Furthermore, lentiviral vaccination activates expansion of endogenous CD4 Th cells, which are required for the generation of effector CD8 T cells that produce IFN-γ and kill Ag-specific targets. Collectively, we demonstrate that after cutaneous immunization with lentiviral particles, CD4-licensed lymph node conventional DC present Ag to CD8 T cells, resulting in the generation of protective endogenous antitumor immunity that may be effective for cancer immunotherapy.     

A mechanistic study of immune system activation by fusion of antigens with the ligand-binding domain of CTLA4

Fusion proteins consisting of the ligand-binding domain of CTLA4 covalently attached to an antigen (Ag) are potent immunogens. This fusion strategy effectively induces Ag-specific immunity both when introduced as a DNA-based vaccine and as a recombinant protein. CTLA4 is a ligand for B7 molecules expressed on the surface of antigen-presenting cells (APCs), and this interaction is critical for the fusion protein to stimulate Ag-specific immunity. We show that interaction of the fusion protein with either B7-1 or B7-2 is sufficient to stimulate immune activity, and that T cells are essential for the development of IgG responses. In addition, we demonstrate that human dendritic cells (DCs) pulsed with CTLA4–Ag fusion proteins can efficiently present Ag to T cells and induce an Ag-specific immune response in vitro. These studies provide further mechanistic understanding of the process by which CTLA4–Ag fusion proteins stimulate the immune system, and represent an efficient means of generating Ag-specific T cells for immunotherapy.Dhanalakshmi Chinnasamy and Matt Tector contributed equally to this work