TCR-independent activation of extrathymically developed, self antigen-specific T cells by IL-2/IL-15

Naive intrathymically developed T cells, which express foreign Ag-specific TCR, do not express IL-2R. After antigenic stimulation, they express high affinity IL-2R, which enables IL-2 to be used as an autocrine growth factor. On the contrary, extrathymically developed T cells, which express self Ag-specific TCR but are unresponsive to antigenic stimulation, spontaneously express low affinity IL-2R. In this study, we compared the responses of these two subsets of T cells to IL-2R stimulation and examined the influences of TCR-mediated signaling on the responses. IL-2 or IL-15 augmented the proliferative response of Ag-stimulated, intrathymically developed T cells. On the other hand, extrathymically developed T cells proliferated in response to IL-2 or IL-15, independently of Ag stimulation. Furthermore, both IL-2 and IL-15 induced IFN-gamma production of these T cells, which is strikingly augmented by the presence of IL-12. These results revealed functional differences between intrathymically developed, foreign Ag-specific T cells and extrathymically developed, self Ag-specific T cells. The latter can be activated by some inflammatory cytokines, in an Ag-independent manner, similar to NK cells.     

Bacillus Calmette-Guérin vaccination of human newborns induces T cells with complex cytokine and phenotypic profiles

The immune response to vaccination with bacillus Calmette-Guérin (BCG), the only tuberculosis vaccine available, has not been fully characterized. We used multiparameter flow cytometry to examine specific T cell cytokine production and phenotypic profiles in blood from 10-wk-old infants routinely vaccinated with BCG at birth. Ex vivo stimulation of whole blood with BCG for 12 h induced expression of predominantly IFN-gamma, IL-2, and TNF-alpha in CD4+ T cells in seven distinct cytokine combinations. IL-4 and IL-10 expression was detected in CD4+ T cells at low frequencies and only in cells that did not coexpress type 1 cytokines. Specific CD8+ T cells were less frequent than CD4+ T cells and produced mainly IFN-gamma and/or IL-2 and less TNF-alpha, IL-4, and IL-10. Importantly, many mycobacteria-specific CD4+ and CD8+ T cells did not produce IFN-gamma. The predominant phenotype of BCG-specific type 1 T cells was that of effector cells, i.e., CD45RA-CCR7-CD27+, which may reflect persistence of Mycobacterium bovis BCG in infants until 10 wk of age. Among five phenotypic patterns of CD4+ T cells, central memory cells were more likely to be IL-2+ and effector cells were more likely to be IFN-gamma+. We concluded that neonatal vaccination with BCG induces T cells with a complex pattern of cytokine expression and phenotypes. Measuring IFN-gamma production alone underestimates the magnitude and complexity of the host cytokine response to BCG vaccination and may not be an optimal readout in studies of BCG and novel tuberculosis vaccination.     

Simultaneous Th1-type cytokine expression is a signature of peritoneal CD4+ lymphocytes responding to infection with Listeria monocytogenes

The robust murine response to infection with Listeria monocytogenes makes an excellent model to study the functional development of immune cells. We investigated the cellular immune response to i.p. infection using intracellular cytokine staining to identify Ag-specific lymphocytes. CD4(+) peritoneal exudate cells obtained 10 days postinfection predominantly coexpressed TNF-alpha, IFN-gamma, and IL-2 after polyclonal or Ag stimulation. A population of cells simultaneously making TNF-alpha and IFN-gamma was also detected but at a lower frequency. By following the kinetics of the response to Listeria, we found that CD4(+) lymphocytes coexpressing TNF-alpha and IFN-gamma dominated on day 6 postinfection and then declined. From days 10-27, TNF-alpha(+)IFN-gamma(+)IL-2(+) (triple-positive) was the most prevalent cytokine phenotype, and the frequency steadily declined. These characteristic cytokine expression patterns were observed in both primary and secondary responses to Listeria infection and developed even when infection was terminated with antibiotic treatment. A cytokine-assisted immunization procedure resulted in both double- and triple-positive cells, but the clear predominance of triple-positive cells required Listeria infection. Triple-positive cells were preferentially noted in the peritoneal cavity tissue site; spleen cells displayed a predominant population of double-positive T cells (TNF-alpha(+)IFN-gamma(+)). We speculate that the appearance of triple-positive cells represents a functionally significant subset important in host defense at nonlymphoid tissue sites.     

IL-12 and type-I IFN synergize for IFN-gamma production by CD4 T cells, whereas neither are required for IFN-gamma production by CD8 T cells after Listeria monocytogenes infection

Differentiation of Ag-specific T cells into IFN-gamma producers is essential for protective immunity to intracellular pathogens. In addition to stimulation through the TCR and costimulatory molecules, IFN-gamma production is thought to require other inflammatory cytokines. Two such inflammatory cytokines are IL-12 and type I IFN (IFN-I); both can play a role in priming naive T cells to produce IFN-gamma in vitro. However, their role in priming Ag-specific T cells for IFN-gamma production during experimental infection in vivo is less clear. In this study, we examine the requirements for IL-12 and IFN-I, either individually or in combination, for priming Ag-specific T cell IFN-gamma production after Listeria monocytogenes (Lm) infection. Surprisingly, neither individual nor combined defects in IL-12 or IFN-I signaling altered IFN-gamma production by Ag-specific CD8 T cells after Lm infection. In contrast, individual defects in either IL-12 or IFN-I signaling conferred partial ( approximately 50%) reductions, whereas combined deficiency in both IL-12 and IFN-I signaling conferred more dramatic (75-95%) reductions in IFN-gamma production by Ag-specific CD4 T cells. The additive effects of IL-12 and IFN-I signaling on IFN-gamma production by CD4 T cells were further demonstrated by adoptive transfer of transgenic IFN-IR(+/+) and IFN-IR(-/-) CD4 T cells into normal and IL-12-deficient mice, and infection with rLm. These results demonstrate an important dichotomy between the signals required for priming IFN-gamma production by CD4 and CD8 T cells in response to bacterial infection.     

Lipopolysaccharides from distinct pathogens induce different classes of immune responses in vivo.

The adaptive immune system has evolved distinct responses against different pathogens, but the mechanism(s) by which a particular response is initiated is poorly understood. In this study, we investigated the type of Ag-specific CD4(+) Th and CD8(+) T cell responses elicited in vivo, in response to soluble OVA, coinjected with LPS from two different pathogens. We used Escherichia coli LPS, which signals through Toll-like receptor 4 (TLR4) and LPS from the oral pathogen Porphyromonas gingivalis, which does not appear to require TLR4 for signaling. Coinjections of E. coli LPS + OVA or P. gingivalis LPS + OVA induced similar clonal expansions of OVA-specific CD4(+) and CD8(+) T cells, but strikingly different cytokine profiles. E. coli LPS induced a Th1-like response with abundant IFN-gamma, but little or no IL-4, IL-13, and IL-5. In contrast, P. gingivalis LPS induced Th and T cell responses characterized by significant levels of IL-13, IL-5, and IL-10, but lower levels of IFN-gamma. Consistent with these results, E. coli LPS induced IL-12(p70) in the CD8alpha(+) dendritic cell (DC) subset, while P. gingivalis LPS did not. Both LPS, however, activated the two DC subsets to up-regulate costimulatory molecules and produce IL-6 and TNF-alpha. Interestingly, these LPS appeared to have differences in their ability to signal through TLR4; proliferation of splenocytes and cytokine secretion by splenocytes or DCs from TLR4-deficient C3H/HeJ mice were greatly impaired in response to E. coli LPS, but not P. gingivalis LPS. Therefore, LPS from different bacteria activate DC subsets to produce different cytokines, and induce distinct types of adaptive immunity in vivo.     

Dynamic relationship between IFN-gamma and IL-2 profile of Mycobacterium tuberculosis-specific T cells and antigen load

Distinct IFN-gamma and IL-2 profiles of Ag-specific CD4(+) T cells have recently been associated with different clinical disease states and Ag loads in viral infections. We assessed the kinetics and functional profile of Mycobacterium tuberculosis Ag-specific T cells secreting IFN-gamma and IL-2 in 23 patients with untreated active tuberculosis when bacterial and Ag loads are high and after curative treatment, when Ag load is reduced. The frequencies of M. tuberculosis Ag-specific IFN-gamma-secreting T cells declined during 28 mo of follow-up with an average percentage decline of 5.8% per year (p = 0.005), while the frequencies of Ag-specific IL-2-secreting T cells increased during treatment (p = 0.02). These contrasting dynamics for the two cytokines led to a progressive convergence of the frequencies of IFN-gamma- and IL-2-secreting cells over 28 mo. Simultaneous measurement of IFN-gamma and IL-2 secretion at the single-cell level revealed a codominance of IFN-gamma-only secreting and IFN-gamma/IL-2 dual secreting CD4(+) T cells in active disease that shifted to dominance of IFN-gamma/IL-2-secreting CD4(+) T cells and newly detectable IL-2-only secreting CD4(+) T cells during and after treatment. These distinct T cell functional signatures before and after treatment suggest a novel immunological marker of mycobacterial load and clinical status in tuberculosis that now requires validation in larger prospective studies.     

Enhancement of HIV-specific CD8 T cell responses by dual costimulation with CD80 and CD137L

HIV-specific CD8 T cell responses are defective in chronic HIV infection. In this study, we report that costimulation with either CD137L (4-1BBL) or CD80 (B7.1) enhanced the Ag-specific expansion and acquisition of effector function by HIV-specific memory CD8 T cells. Ag-specific T cells from recently infected donors showed maximal expansion with single costimulatory molecules. Dual costimulation of T cells from recently infected donors or from healthy donors responding to influenza epitopes led to enhanced responses when the accumulation of cytokines was measured. However, accumulation of regulatory cytokines, particularly IFN-gamma, led to inhibition of further Ag-specific CD8 T cell expansion in the cultures. This inhibition was relieved by neutralization of IFN-gamma or of IFN-gamma, TNF, and IL-10. Thus, strong costimulation of T cells in vitro can lead to induction of regulatory cytokines at levels that limit further T cell expansion. In marked contrast, T cells from long-term (>4 years) infected HIV+ donors exhibited reduced Ag-specific CD8 T cell expansion, reduced CD4 T cell responses, and minimal cytokine accumulation. Dual costimulation with both 4-1BBL and B7.1 enhanced responses of T cells from long-term infected subjects to a level similar to that obtained with T cells from early in HIV infection. Experiments with purified CD8 T cells showed that B7.1 and 4-1BBL could act directly and synergistically on CD8 T cells. Taken together, these data suggest that 4-1BBL and B7.1 have additive or synergistic effects on HIV-specific CD8 T cell responses and represent a promising combination for therapeutic vaccination for HIV.     

IL-21 influences the frequency, phenotype, and affinity of the antigen-specific CD8 T cell response

IL-21, a newly described cytokine belonging to the IL-2 gamma-chain receptor cytokine family (that includes IL-2, IL-7, and IL-15), has been described as an important regulator of the cellular immune response. In this study, the role of IL-21 in the generation of a human Ag-specific CD8+ T cell response is characterized by tracking a rare, but measurable population of self-Ag-specific T cells in vitro. Autologous dendritic cells pulsed with the melanoma antigen recognized T cells 1 self-peptide were used to stimulate CD8+ T cells from HLA-A2+ healthy donors and melanoma patients. We demonstrate that exposure to IL-21 increased the total number of MART-1-specific CD8+ T cells that could be elicited by >20-fold and, at the clonal level, enriched for a population of high-affinity CD8+ T cells with a peptide dose requirement more than 1 log(10)-fold less than their untreated counterparts. Phenotypic analysis of T cells from IL-21-treated cultures revealed a unique population of CD45RO+ CD28(high) CD8+ T cells, a phenotype that was stable for at least 4 wk after IL-21 exposure. These CD28(high) CD8+ T cells produced IL-2 upon Ag stimulation and represent potential helper-independent CTLs. Our studies demonstrate a significant role for IL-21 in the primary Ag-specific human CTL response and support the use of IL-21 in the ex vivo generation of potent Ag-specific CTLs for adoptive therapy or as an adjuvant cytokine during in vivo immunization against tumor Ags.     

A Whole Recombinant Yeast-Based Therapeutic Vaccine Elicits HBV X,S and Core Specific T Cells in Mice and Activates Human T Cells Recognizing Epitopes Linked to Viral Clearance

Chronic hepatitis B infection (CHB) is characterized by sub-optimal T cell responses to viral antigens. A therapeutic vaccine capable of restoring these immune responses could potentially improve HBsAg seroconversion rates in the setting of direct acting antiviral therapies. A yeast-based immunotherapy (Tarmogen) platform was used to make a vaccine candidate expressing hepatitis B virus (HBV) X, surface (S), and Core antigens (X-S-Core). Murine and human immunogenicity models were used to evaluate the type and magnitude of HBV-Ag specific T cell responses elicited by the vaccine. C57BL/6J, BALB/c, and HLA-A*0201 transgenic mice immunized with yeast expressing X-S-Core showed T cell responses to X, S and Core when evaluated by lymphocyte proliferation assay, ELISpot, intracellular cytokine staining (ICS), or tumor challenge assays. Both CD4⁺ and CD8⁺ T cell responses were observed. Human T cells transduced with HBc18–27 and HBs183–91 specific T cell receptors (TCRs) produced interferon gamma (IFNγ following incubation with X-S-Core-pulsed dendritic cells (DCs). Furthermore, stimulation of peripheral blood mononuclear cells (PBMCs) isolated from CHB patients or from HBV vaccine recipients with autologous DCs pulsed with X-S-Core or a related product (S-Core) resulted in pronounced expansions of HBV Ag-specific T cells possessing a cytolytic phenotype. These data indicate that X-S-Core-expressing yeast elicit functional adaptive immune responses and supports the ongoing evaluation of this therapeutic vaccine in patients with CHB to enhance the induction of HBV-specific T cell responses.     

Activation of virus-specific CD8+ T cells by lipopolysaccharide-induced IL-12 and IL-18

Virus-specific T cells represent a hallmark of Ag-specific, adaptive immunity. However, some T cells also demonstrate innate functions, including non-Ag-specific IFN-gamma production in response to microbial products such as LPS or exposure to IL-12 and/or IL-18. In these studies we examined LPS-induced cytokine responses of CD8(+) T cells directly ex vivo. Following acute viral infection, 70-80% of virus-specific T cells will produce IFN-gamma after exposure to LPS-induced cytokines, and neutralization experiments indicate that this is mediated almost entirely through production of IL-12 and IL-18. Different combinations of these cytokines revealed that IL-12 decreases the threshold of T cell activation by IL-18, presenting a new perspective on IL-12/IL-18 synergy. Moreover, memory T cells demonstrate high IL-18R expression and respond effectively to the combination of IL-12 and IL-18, but cannot respond to IL-18 alone, even at high cytokine concentrations. This demonstrates that the synergy between IL-12 and IL-18 in triggering IFN-gamma production by memory T cells is not simply due to up-regulation of the surface receptor for IL-18, as shown previously with naive T cells. Together, these studies indicate how virus-specific T cells are able to bridge the gap between innate and adaptive immunity during unrelated microbial infections, while attempting to protect the host from cytokine-induced immunopathology and endotoxic shock.     

A unique mechanism for innate cytokine promotion of T cell responses to viral infections

The kinetics of CD8 T cell IFN-gamma responses as they occur in situ are defined here during lymphocytic choriomeningitis virus (LCMV) infections, and a unique mechanism for the innate cytokines IFN-alphabeta and IL-18 in promoting these responses is defined. Infections of mice with Armstrong or WE strains of LCMV induced an unexpectedly early day 4 IFN-gamma response detectable in serum samples and spleen and liver homogenates. Production of IFN-gamma was MHC class I/CD8 dependent, but did not require IL-12, NK cells, TCR-gammadelta T cells, MHC class II, or CD4 T cells. Peak response required specific Ag recognition, as administration of antagonist peptide partially impaired day 4 IFN-gamma induction, and viral peptide stimulation enhanced CD8 T cell IFN-gamma expression in culture. The IFN-gamma response was associated with IL-18 and IFN-alphabeta expression. Furthermore, both factors augmented peptide-driven IFN-gamma production in culture, and mice lacking IL-18 or IFN-alphabeta functions had reduced day 4 IFN-gamma. Collectively, these results demonstrate that during viral infections, there is a dramatic in vivo CD8 T cell response preceding maximal expansion of these cells, and that the mechanism supporting this response is dependent on endogenous innate cytokines. Because stimulation by microbial products is linked to innate cytokine expression, the studies also suggest a pathway for precisely limiting T cell functions to times of need.     

Activated and memory CD8+ T cells can be distinguished by their cytokine profiles and phenotypic markers

Dissecting the mechanisms of T cell-mediated immunity requires the identification of functional characteristics and surface markers that distinguish between activated and memory T lymphocytes. In this study, we compared the rates of cytokine production by virus-specific primary and memory CD8+ T cells directly ex vivo. Ag-specific IFN-gamma and TNF-alpha production by both primary and long-term memory T cells was observed in 相似文献   

Gliadin-specific type 1 regulatory T cells from the intestinal mucosa of treated celiac patients inhibit pathogenic T cells

Celiac disease (CD) results from a permanent intolerance to dietary gluten and is due to a massive T cell-mediated immune response to gliadin, the main component of gluten. In this disease, the regulation of immune responses to dietary gliadin is altered. Herein, we investigated whether IL-10 could modulate anti-gliadin immune responses and whether gliadin-specific type 1 regulatory T (Tr1) cells could be isolated from the intestinal mucosa of CD patients in remission. Short-term T cell lines were generated from jejunal biopsies, either freshly processed or cultured ex vivo with gliadin in the presence or absence of IL-10. Ex vivo stimulation of CD biopsies with gliadin in the presence of IL-10 resulted in suppression of Ag-specific proliferation and cytokine production, indicating that pathogenic T cells are susceptible to IL-10-mediated immune regulation. T cell clones generated from intestinal T cell lines were tested for gliadin specificity by cytokine production and proliferative responses. The majority of gliadin-specific T cell clones had a Th0 cytokine production profile with secretion of IL-2, IL-4, IFN-gamma, and IL-10 and proliferated in response to gliadin. Tr1 cell clones were also isolated. These Tr1 cells were anergic, restricted by DQ2 (a CD-associated HLA), and produced IL-10 and IFN-gamma, but little or no IL-2 or IL-4 upon activation with gliadin or polyclonal stimuli. Importantly, gliadin-specific Tr1 cell clones suppressed proliferation of pathogenic Th0 cells. In conclusion, dietary Ag-specific Tr1 cells are present in the human intestinal mucosa, and strategies to boost their numbers and/or function may offer new therapeutic opportunities to restore gut homeostasis.     

CC chemokine receptor 9 expression defines a subset of peripheral blood lymphocytes with mucosal T cell phenotype and Th1 or T-regulatory 1 cytokine profile

The chemokine receptor CCR9 is expressed on most small intestinal lamina propria and intraepithelial lymphocytes and on a small subset of peripheral blood lymphocytes. CCR9-expressing lymphocytes may play an important role in small bowel immunity and inflammation. We studied the phenotype and functional characteristics of CCR9(+) lymphocytes in blood from normal donors. A subset of CCR9(+) T cells have a phenotype of activated cells and constitutively express the costimulatory molecules CD40L and OX-40. In contrast to CCR9(-), CCR9(+)CD4(+) peripheral blood T cells proliferate to anti-CD3 or anti-CD2 stimulation and produce high levels of IFN-gamma and IL-10. IL-10-producing cells were exclusively detected within the CCR9(+) subset of CD4(+) T cells by intracellular staining and were distinct from IL-2- and IFN-gamma-producing cells. Moreover, memory CCR9(+)CD4(+) lymphocytes respond to CD2 stimulation with proliferation and IFN-gamma/IL-10 production, whereas memory CCR9(-)CD4(+) cells were unresponsive. In addition, memory CCR9(+)CD4(+) T cells support Ig production by cocultured CD19(+) B cells in the absence of prior T cell activation or addition of exogenous cytokines. Our data show that the memory subset of circulating CCR9(+)CD4(+) T cells has characteristics of mucosal T lymphocytes and contains cells with either Th1 or T-regulatory 1 cytokine profiles. Studies on the cytokine profile and Ag specificity of this cell subset could provide important insight into small intestinal immune-mediated diseases and oral tolerance in humans.     

Soluble form of T cell Ig mucin 3 is an inhibitory molecule in T cell-mediated immune response

T cell Ig mucin 3 (Tim-3) has been found to play an important role in Th1-mediated auto- and alloimmune responses, but the function of soluble form of Tim-3 (sTim-3) remains to be elucidated. In this study, we report the inhibitory effect of sTim-3 on T cell-mediated immune response. In this study, sTim-3 mRNA was found, among different tissues and organs, only in splenic cells, and the activation of splenocytes resulted in up-regulated production of both sTim-3 mRNA and protein. We constructed a eukaryotic expression plasmid, psTim-3, which expresses functional murine sTim-3. In C57BL/6 mice inoculated with B16F1 melanoma cells, the growth of tumor was facilitated by the expression of this plasmid in vivo. Furthermore, sTim-3 inhibited the responses of T cells to Ag-specific stimulation or anti-CD3 mAb plus anti-CD28 mAb costimulation and the production of cytokines IL-2 and IFN-gamma in vitro. In tumor rejection model, sTim-3 significantly impaired T cell antitumor immunity, evidenced by decreased antitumor CTL activity and reduced amount of tumor-infiltrating lymphocytes in tumor. Real-time PCR analysis of gene expression in tumor microenvironment revealed the decreased expression of Th1 cytokine genes and the unchanged profile of the genes related to T regulatory cell function, suggesting that the inhibitory effect of sTim-3 on the generation of Ag-specific T cells in vivo is dominated by T effector cells rather than T regulatory cells. Our studies thus define sTim-3 as an immunoregulatory molecule that may be involved in the negative regulation of T cell-mediated immune response.     

Prevention of allergen-specific, Th2-biased immune responses in vivo: role of increased IL-12 and IL-18 responsiveness

The factors that control development of adaptive responses to exogenous Ag remain incompletely understood. An ability to selectively direct immunity toward a specific phenotype would be of clinical benefit in numerous immunological disorders. Administration of chemically modified allergen glutaraldehyde-polymerized OVA (OA-POL) leads to >90% reductions in murine IgE and >500-fold increases in IgG2c responses that develop upon subsequent immunization with native Ag. In the present study, we examine the mechanisms underlying this reorientation of the type 2 dominant response that would normally develop. Lack of endogenous IL-12 or IFN-gamma results in markedly reduced induction of IgG2c responses following OA-POL treatment, but only IFN-gamma(-/-) mice demonstrate reduced capacity to prevent IgE induction. This indicates that while both IL-12 and IFN-gamma are critical promoters of type 1 immunity, only IFN-gamma is required to maximally inhibit development of type 2 immune responses. Compared with OVA-immunized mice, CD69(+) T cells from OA-POL-immunized mice demonstrate elevated IL-12Rbeta(2), IL-18Ralpha, and IL-18Rbeta mRNA levels, as well as increased IFN-gamma production in response to rIL-12 or rIL-18 stimulation. Collectively, these data indicate that preventing induction of type 2 immune responses is critically dependent on altered T cell responsiveness to these cytokines. The finding that targeted, Ag-specific manipulation of IL-12 and IL-18 responsiveness can be used to shape the phenotype of the dominant immune response that develops suggests that specifically targeting IL-12 and IL-18 receptor expression may offer clinical options for clinical prophylaxis or intervention.     

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.