Differential CD40/CD40L expression results in counteracting antitumor immune responses

Establishment of host-protective memory T cells against tumors is the objective of an antitumor immunoprophylactic strategy such as reinforcing T cell costimulation via CD40-CD40L interaction. Previous CD40-targeted strategies assumed that T cell costimulation is an all-or-none phenomenon. It was unknown whether different levels of CD40L expression induce quantitatively and qualitatively different effector T cell responses. Using mice expressing different levels of CD40L, we demonstrated that the greater the T cell CD40L expression the less tumor growth occurred; the antitumor T cell response was host-protective. Lower levels of CD40L expression on T cells induced IL-10-mediated suppression of tumor-regressing effector CD8(+) T cells and higher productions of IL-4 and IL-10. Using mice expressing different levels of CD40 or by administering different doses of anti-CD40 Ab, similar observations were recorded implying that the induction of protumor or antitumor T cell responses was a function of the extent of CD40 cross-linking. IL-10 neutralization during priming with tumor Ags resulted in a stronger tumor-regressing effector T cell response. Using IL-10(-/-) DC for priming of mice expressing different levels of CD40L and subsequent transfer of the T cells from the primed mice to nu/nu mice, we demonstrated the protumor role of IL-10 in the induction of tumor-promoting T cells. Our results demonstrate that a dose-dependent cross-linking of a costimulatory molecule dictates the functional phenotype of the elicited effector T cell response. The T cell costimulation is a continuum of a function that induces not only graded T cell responses but also two counteracting responses at two extremes.     

IL-12 augments CD8+ T cell development for contact hypersensitivity responses and circumvents anti-CD154 antibody-mediated inhibition.

During sensitization with dinitrofluorobenzene for contact hypersensitivity (CHS) responses, hapten-specific CD8(+) T cells develop into IFN-gamma-producing cells, and CD4(+) T cells develop into IL-4/IL-5-producing cells. Administration of IL-12 during sensitization skews CD4(+) T cell development to IFN-gamma-producing cells, resulting in exaggerated CHS responses. In the current report we tested the role of IL-12 on CD8(+) T cell development during sensitization and elicitation of CHS to dinitrofluorobenzene. Administration of IL-12 during hapten sensitization induced the expression of IL-12Rbeta2 on both CD4(+) and CD8(+) T cells, augmented IFN-gamma production by these T cell populations, and increased the magnitude and duration of the CHS response to hapten challenge. CHS responses were virtually identical in wild-type and IL-12 p40(-/-) mice. Since engagement of CD40 on APC may stimulate IL-12 production, we also tested the role of CD40-CD154 interactions on the development of IFN-gamma-producing CD4(+) and CD8(+) T cells following hapten sensitization. Development of IFN-gamma-producing CD4(+) T cells during hapten sensitization was absent in wild-type mice treated with anti-CD154 mAb or in CD154(-/-) mice. In contrast, the absence of CD40-CD154 signaling had little or no impact on the development of IFN-gamma-producing CD8(+) T cells. These results demonstrate that the development of hapten-specific Th1 effector CD4(+) T cells in CHS requires both CD40-CD154 interactions and IL-12, whereas the development of IFN-gamma-producing effector CD8(+) T cells can occur independently of these pathways.     

CD4+ and CD8+ T cell priming for contact hypersensitivity occurs independently of CD40-CD154 interactions

The primary effector cells of contact hypersensitivity (CHS) responses to dintrofluorobenzene (DNFB) are IFN-gamma-producing CD8(+) T cells, whereas CD4(+) T cells regulate the magnitude and duration of the response. The requirement for CD40-CD154 engagement during CD8(+) and CD4(+) T cell priming by hapten-presenting Langerhans cells (hpLC) is undefined and was tested in the current study. Similar CHS responses to DNFB were elicited in wild-type and CD154(-/-) animals. DNFB sensitization of CD154(-/-) mice primed IFN-gamma-producing CD8(+) T cells and IL-4-producing CD4(+) T cells. However, anti-CD154 mAb MR1 given during hapten sensitization inhibited hapten-specific CD8(+), but not CD4(+), T cell development and the CHS response to challenge. F(ab')(2) of MR1 failed to inhibit CD8(+) T cell development and the CHS response suggesting that the mechanism of inhibition is distinct from that of CD40-CD154 blockade. Furthermore, anti-CD154 mAb did not inhibit CD8(+) T cell development and CHS responses in mice depleted of CD4(+) T cells or in CD4(-/-) mice. During in vitro proliferation assays, hpLC from mice treated with anti-CD154 mAb during DNFB sensitization were less stimulatory for hapten-primed T cells than hpLC from either control mice or mice depleted of CD4(+) T cells before anti-CD154 mAb administration. These results demonstrate that development of IFN-gamma-producing CD8(+) T cells and the CHS response are not dependent on CD40-CD154 interactions. This study proposes a novel mechanism of anti-CD154 mAb-mediated inhibition of CD8(+) T cell development where anti-CD154 mAb acts indirectly through CD4(+) T cells to impair the ability of hpLC to prime CD8(+) T cells.     

Potent tumor-specific protection ignited by adoptively transferred CD4+ T cells

Administration of anti-CD25 mAb before an aggressive murine breast tumor inoculation provoked effective antitumor immunity. Compared with CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that did not reject tumor, CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that rejected tumor stimulated by dendritic cells (DCs) produced more IFN-gamma and IL-2, and less IL-17 in vitro, and ignited protective antitumor immunity in vivo in an adoptive transfer model. Tumor Ag-loaded DCs activated naive CD8(+) T cells in the presence of these CD4(+) T cells in vitro. Tumor Ag and adoptively transferred CD4(+) T cells were both required for inducing a long-term tumor-specific IFN-gamma-producing cellular response and potent protective antitumor activity. Although adoptively transferred CD4(+) T cells ignited effective tumor-specific antitumor immunity in wild-type mice, they failed to do so in endogenous NK cell-depleted, Gr-1(+) cell-depleted, CD40(-/-), CD11c(+) DC-depleted, B cell(-/-), CD8(+) T cell-depleted, or IFN-gamma(-/-) mice. Collectively, the data suggest that adoptively transferred CD4(+) T cells orchestrate both endogenous innate and adaptive immunity to generate effective tumor-specific long-term protective antitumor immunity. The data also demonstrate the pivotal role of endogenous DCs in the tumor-specific protection ignited by adoptively transferred CD4(+) T cells. Thus, these findings highlight the importance of adoptively transferred CD4(+) T cells, as well as host immune components, in generating effective tumor-specific long-term antitumor activity.     

CD40 ligand promotes priming of fully potent antitumor CD4(+) T cells in draining lymph nodes in the presence of apoptotic tumor cells.

The presence or absence of CD4(+) T cell help can determine the direction of adaptive immune responses toward either cross-priming or cross-tolerance. It has been demonstrated that interactions of CD40-CD40 ligand can replace CD4(+) T cell help and enable dendritic cells to prime cytotoxic T cells. Here, we demonstrate that antitumor reactivity induced in regional lymph nodes (LNs) by s.c. injection of CD40 ligand (CD40L)-transduced tumor (MCA205 CD40L) showed far superior therapeutic efficacy against established brain tumors of a weakly immunogenic fibrosarcoma, MCA205, when adoptively transferred. Coinjection of apoptotic, but not necrotic parental tumor cells with CD40L-expressing tumor cells caused a strong synergistic induction of antitumor reactivity in tumor-draining LNs. Freshly isolated T cells from LNs immunized with apoptotic parental tumor cells and MCA205 CD40L were capable of mediating regression of the parental tumor in vivo. In contrast, T cells derived from LNs immunized without MCA205 CD40L required ex vivo anti-CD3/IL-2 activation to elicit therapeutic activity. On anti-CD3/IL-2 activation, cells from LNs immunized with MCA205 CD40L exhibited superior per cell antitumor reactivity. An in vitro depletion study revealed that either CD4(+) or CD8(+) T cells could mediate therapeutic efficacy but that the antitumor efficacy mediated by CD4(+) T cells was far superior. Cytosolic flow cytometric analyses indicated that priming of CD4(+) cells in LNs draining CD40L-expressing tumors was polarized to the Th1 type. This is the first report that fully potent antitumor CD4(+) T cell priming was promoted by s.c. injection of CD40L-transduced tumor in the presence of apoptotic tumor cells.     

CD40 engagement enhances antigen-presenting langerhans cell priming of IFN-gamma-producing CD4+ and CD8+ T cells independently of IL-12

The delivery of CD40 signaling to APCs during T cell priming enhances many T cell-mediated immune responses. Although CD40 signaling up-regulates APC production of IL-12, the impact of this increased production on T cell priming is unclear. In this study an IL-12-independent T cell-mediated immune response, contact hypersensitivity (CHS), was used to further investigate the effect of CD40 ligation on the phenotypic development of Ag-specific CD4(+) and CD8(+) T cells. Normally, sensitization for CHS responses induces hapten-specific CD4(+) T cells producing type 2 cytokines and CD8(+) T cells producing IFN-gamma. Treatment of mice with agonist anti-CD40 mAb during sensitization with the hapten 2,4-dinitrofluorobenzene resulted in CHS responses of increased magnitude and duration. These augmented responses in anti-CD40 Ab-treated mice correlated with increased numbers of hapten-specific CD4(+) and CD8(+) T cells producing IFN-gamma in the skin draining lymph nodes. Identical results were observed using IL-12(-/-) mice, indicating that CD40 ligation promotes CHS responses and development of IFN-gamma-producing CD4(+) and CD8(+) T cells in the absence of IL-12. Engagement of CD40 on hapten-presenting Langerhans cells (hpLC) up-regulated the expression of both class I and class II MHC and promoted hpLC migration into the T cell priming site. These results indicate that hpLC stimulated by CD40 ligation use a mechanism distinct from increased IL-12 production to promote Ag-specific T cell development to IFN-gamma-producing cells.     

Selective requirement for CD40-CD154 in drug-induced type 1 versus type 2 responses to trinitrophenyl-ovalbumin

CD154 is transiently expressed by activated T cells and interacts with CD40 on B cells, dendritic cells, macrophages, and monocytes. This costimulatory receptor-ligand couple seems decisive in Ag-driven immune responses but may be differentially involved in type 1 vs type 2 responses. We studied the importance of CD40-CD154 in both responses using the reporter Ag popliteal lymph node assay in which selectively acting drugs generate clearly polarized type 1 (streptozotocin) or type 2 (D-penicillamine, diphenylhydantoin) responses to a constant coinjected Ag in the same mouse strain. Treatment of mice with anti-CD154 reduced characteristic immunological parameters in type 2 responses (B and CD4(+) T cell proliferation, IgG1 and IgE Abs, and IL-4 secretion) and only slightly affected the type 1 response (small decrease in IFN-gamma production, influx of CD11c(+) and F4/80(+) cells, and prevention of architectural disruption of the lymph node, but no effect on IgG2a Ab and TNF-alpha secretion or B and CD4(+) T cell proliferation). The findings indicate that the CD40-CD154 costimulatory interaction is a prerequisite in drug-induced type 2 responses and is only marginally involved in type 1 responses. The observed expression patterns of CD80 and CD86 on different APC (B cells in type 2 and dendritic cells in type 1) may be responsible for this discrepancy.     

IFN-gamma mediates CD4+ T-cell loss and impairs secondary antitumor responses after successful initial immunotherapy

Protective cell-mediated immune responses in cancer are critically dependent on T-helper type 1 (T(H)1) cytokines such as interferon-gamma (IFN-gamma). We have previously shown that the combination of CD40 stimulation and interleukin-2 (IL-2) leads to synergistic antitumor responses in several models of advanced metastatic disease. We now report that after this treatment and other immunotherapy regimens, the CD4+ T-cell population, in contrast to CD8+ T cells, did not significantly increase but rather exhibited a substantial level of apoptosis that was dependent on IFN-gamma. Mice immunized with tumor cells and treated with an immunotherapy regimen that was initially protective were later unable to mount effective memory responses compared with immunized mice not receiving immunotherapy. Immunotherapy given to tumor-bearing Ifngr-/- mice resulted in restoration of secondary responses. Thus, although immunotherapeutic regimens inducing strong IFN-gamma responses can lead to successful early antitumor efficacy, they may also impair the development of durable antitumor responses.     

The role of CD4+ T cell help and CD40 ligand in the in vitro expansion of HIV-1-specific memory cytotoxic CD8+ T cell responses

CD4(+) T cells have been shown to play a critical role in the maintenance of an effective anti-viral CD8(+) CTL response in murine models. Recent studies have demonstrated that CD4(+) T cells provide help to CTLs through ligation of the CD40 receptor on dendritic cells. The role of CD4(+) T cell help in the expansion of virus-specific CD8(+) memory T cell responses was examined in normal volunteers recently vaccinated to influenza and in HIV-1 infected individuals. In recently vaccinated normal volunteers, CD4(+) T cell help was required for optimal in vitro expansion of influenza-specific CTL responses. Also, CD40 ligand trimer (CD40LT) enhanced CTL responses and was able to completely substitute for CD4(+) T cell help in PBMCs from normal volunteers. In HIV-1 infection, CD4(+) T cell help was required for optimal expansion of HIV-1-specific memory CTL in vitro in 9 of 10 patients. CD40LT could enhance CTL in the absence of CD4(+) T cell help in the majority of patients; however, the degree of enhancement of CTL responses was variable such that, in some patients, CD40LT could not completely substitute for CD4(+) T cell help. In those HIV-1-infected patients who demonstrated poor responses to CD40LT, a dysfunction in circulating CD8(+) memory T cells was demonstrated, which was reversed by the addition of cytokines including IL-2. Finally, it was demonstrated that IL-15 produced by CD40LT-stimulated dendritic cells may be an additional mechanism by which CD40LT induces the expansion of memory CTL in CD4(+) T cell-depleted conditions, where IL-2 is lacking.     

Invariant NKT cells promote CD8+ cytotoxic T cell responses by inducing CD70 expression on dendritic cells

Activation of invariant NK T (iNKT) cells with the glycolipid alpha-galactosylceramide promotes CD8(+) cytotoxic T cell responses, a property that has been used to enhance the efficacy of antitumor vaccines. Using chimeric mice, we now show that the adjuvant properties of iNKT cells require that CD40 triggering and Ag presentation to CD8(+) T cells occur on the same APCs. We demonstrate that injection of alpha-galactosylceramide triggers CD70 expression on splenic T cell zone dendritic cells and that this is dependent on CD40 signaling. Importantly, we show that blocking the interaction between CD70 and CD27, its costimulatory receptor on T cells, abrogates the ability of iNKT cells to promote a CD8(+) T cell response and abolishes the efficacy of alpha-GalCer as an adjuvant for antitumor vaccines. These results define a key role for CD70 in linking the innate response of iNKT cells to the activation of CD8(+) T cells.     

Th cells act via two synergistic pathways to promote antiviral CD8+ T cell responses

The mechanisms of how Th cells promote CD8(+) T cell responses during viral infections are largely unknown. In this study, we unraveled the mechanisms of T cell help for CD8(+) T cell responses during vaccinia virus infection. Our results demonstrate that Th cells promote vaccinia virus-specific CD8(+) T cell responses via two interconnected synergistic pathways: First, CD40L expressed by activated CD4(+) T cells instructs dendritic cells to produce bioactive IL-12p70, which is directly sensed by Ag-specific CD8(+) T cells, resulting in increased IL-2Rα expression. Second, Th cells provide CD8(+) T cells with IL-2, thereby enhancing their survival. Thus, Th cells are at the center of an important communication loop with a central role for IL-2/IL-2R and bioactive IL-12.     

Differential regulation of antigen-specific CD8+ T cell responses by IL-12p40 in a dose-dependent manner

IL-12p40 is a natural antagonist which inhibits IL-12- and IL-23-mediated biological activity by blocking the binding of IL-12/23 to their receptors. Recently, IL-12p40 was also shown to have immune-enhancing activity through the activation of macrophages or dendritic cells. In this study, we investigated the effects of IL-12p40 as a genetic adjuvant on immune modulation using recombinant adenoviruses expressing IL-12p40 (rAd/IL-12p40) and OVA (rAd/OVA). Coimmunization of rAd/IL-12p40 at a low dose (1 x 10(4) PFU) with rAd/OVA resulted in OVA-specific immune enhancement, while a high dose of rAd/IL-12p40 (1 x 10(8) PFU) caused significant suppression of CD8(+) T cell responses. In addition, the enhancement and suppression of OVA-specific CD8(+) T cell responses correlated with antitumor activity against E.G7-OVA tumor challenge, which subsequently affected the survival rate. Moreover, the differential CD8(+) T cell response by IL-12p40 was still observed in IL-12Rbeta2 knockout (IL-12Rbeta2KO), but not in IL-12Rbeta1 knockout (IL-12Rbeta1KO) mice, indicating that IL-12p40 is a cytokine which can modulate Ag-specific T cell responses depending on IL-12Rbeta1. Our findings provide a novel insight on the physiological role of IL-12p40, which can be informative in the design of vaccine strategies and therapeutic regimens.     

Prolonged antitumor NK cell reactivity elicited by CXCL10-expressing dendritic cells licensed by CD40L+ CD4+ memory T cells

Immunotherapy using dendritic cells (DCs) has the potential to activate both T cells and NK cells. We previously demonstrated the long-lasting antitumor responses by NK cells following immunization with bone marrow-derived DCs. In the current study, we demonstrate that long-term antitumor NK responses require endogenous DCs and a subset of effector memory CD4(+) T (CD4(+) T(EM)) cells. One month after DC immunization, injection of a tumor into DC-immunized mice leads to an increase in the expression of CXCL10 by endogenous DCs, thus directing NK cells into the white pulp where the endogenous DCs bridged CD4(+) T(EM) cells and NK cells. In this interaction, CD4(+) T(EM) cells express CD40L, which matures the endogenous DCs, and produce cytokines, such as IL-2, which activates NK cells. These findings suggest that DC vaccination can sustain long-term innate NK cell immunity but requires the participation of the adaptive immune system.     

Induction of CD70 on dendritic cells through CD40 or TLR stimulation contributes to the development of CD8+ T cell responses in the absence of CD4+ T cells

The expansion of CD8(+) T cells in response to Ag can be characterized as either dependent or independent of CD4(+) T cells. The factors that influence this dichotomy are poorly understood but may be dependent upon the degree of inflammation associated with the Ag. Using dendritic cells derived from MHC class II-deficient mice to avoid interaction with CD4(+) T cells in vivo, we have compared the immunogenicity of peptide-pulsed dendritic cells stimulated with molecules associated with infection to those stimulated via CD40. In the absence of CD4(+) T cell help, the expansion of primary CD8(+) T cells after immunization with TNF-alpha- or poly(I:C)-stimulated dendritic cells was minimal. In comparison, LPS- or CpG-stimulated dendritic cells elicited substantial primary CD8(+) T cell responses, though not to the same magnitude generated by immunization with CD40L-stimulated dendritic cells. Remarkably, mice immunized with any stimulated dendritic cell population generated fully functional recall CD8(+) T cells without the aid of CD4(+) T cell help. The observed hierarchy of immunogenicity was closely correlated with the expression of CD70 (CD27L) on the stimulated dendritic cells, and Ab-mediated blockade of CD70 substantially prevented the CD4(+) T cell-independent expansion of primary CD8(+) T cells. These results indicate that the expression of CD70 on dendritic cells is an important determinant for helper-dependence of primary CD8(+) T cell expansion and provide an explanation for the ability of a variety of pathogens to stimulate primary CD8(+) T cell responses in the absence of CD4(+) T cells.     

Augmented IL-15Rα expression by CD40 activation is critical in synergistic CD8 T cell-mediated antitumor activity of anti-CD40 antibody with IL-15 in TRAMP-C2 tumors in mice

IL-15 has potential as an immunotherapeutic agent for cancer treatment because it is a critical factor for the proliferation and activation of NK and CD8(+) T cells. However, monotherapy of patients with malignancy with IL-15 that has been initiated may not be optimal, because of the limited expression of the private receptor, IL-15Rα. We demonstrated greater CD8 T cell-mediated therapeutic efficacy using a combination regimen of murine IL-15 administered with an agonistic anti-CD40 Ab (FGK4.5) that led to increased IL-15Rα expression on dendritic cells (DCs), as well as other cell types, in a syngeneic established TRAMP-C2 tumor model. Seventy to one hundred percent of TRAMP-C2 tumor-bearing wild-type C57BL/6 mice in the combination group manifested sustained remissions, whereas only 0-30% in the anti-CD40-alone group and none in the murine IL-15-alone group became tumor free (p < 0.001). However, the combination regimen showed less efficacy in TRAMP-C2 tumor-bearing IL-15Rα(-/-) mice than in wild-type mice. The combination regimen significantly increased the numbers of TRAMP-C2 tumor-specific SPAS-1/SNC9-H(8) tetramer(+)CD8(+) T cells, which were associated with the protection from tumor development on rechallenge with TRAMP-C2 tumor cells. Using an in vitro cytolytic assay that involved NK cells primed by wild-type or IL-15Rα(-/-) bone marrow-derived DCs, we demonstrated that the expression of IL-15Rα by DCs appeared to be required for optimal IL-15-induced NK priming and killing. These findings support the view that anti-CD40-mediated augmented IL-15Rα expression was critical in IL-15-associated sustained remissions observed in TRAMP-C2 tumor-bearing mice receiving combination therapy.     

IL-27 mediates complete regression of orthotopic primary and metastatic murine neuroblastoma tumors: role for CD8+ T cells

We have shown previously that IFN-gamma-inducing cytokines such as IL-12 can mediate potent antitumor effects against murine solid tumors. IL-27 is a newly described IL-12-related cytokine that potentiates various aspects of T and/or NK cell function. We hypothesized that IL-27 might also mediate potent antitumor activity in vivo. TBJ neuroblastoma cells engineered to overexpress IL-27 demonstrated markedly delayed growth compared with control mice, and complete durable tumor regression was observed in >90% of mice bearing either s.c. or orthotopic intra-adrenal tumors, and 40% of mice bearing induced metastatic disease. The majority of mice cured of their original TBJ-IL-27 tumors were resistant to tumor rechallenge. Furthermore, TBJ-IL-27 tumors were heavily infiltrated by CD8(+) T cells, and draining lymph node-derived lymphocytes from mice bearing s.c. TBJ-IL-27 tumors are primed to proliferate more readily when cultured ex vivo with anti-CD3/anti-CD28 compared with lymphocytes from mice bearing control tumors, and to secrete higher levels of IFN-gamma. In addition, marked enhancement of local IFN-gamma gene expression and potent up-regulation of cell surface MHC class I expression are noted within TBJ-IL-27 tumors compared with control tumors. Functionally, these alterations occur in conjunction with the generation of tumor-specific CTL reactivity in mice bearing TBJ-IL-27 tumors, and the induction of tumor regression via mechanisms that are critically dependent on CD8(+), but not CD4(+) T cells or NK cells. Collectively, these studies suggest that IL-27 could be used therapeutically to potentiate the host antitumor immune response in patients with malignancy.     

No intrinsic deficiencies in CD8+ T cell-mediated antitumor immunity with aging

Aging is associated with a decline in immune function, particularly within the T cell compartment. Because CD8(+) T cells are critical mediators of protective immunity against cancer, which arises more frequently with advancing age, it is important to understand how aging affects T cell-based antitumor responses. We used our DUC18 T cell/CMS5 tumor model system to examine the ability of both aged APCs and aged, tumor-specific CD8(+) T cells to mount protective responses to tumors in vivo. An assessment of aged DUC18 T cells in vitro showed a naive phenotype, but impaired proliferation in response to anti-CD3 and anti-CD28 stimulation. We found that DCs from young and old recipient mice are comparable phenotypically, and endogenous APCs in these mice are equally able to prime adoptively transferred young DUC18 T cells. Even when aged DUC18 T cells are transferred into aged CMS5-challenged mice, Ag-specific proliferation and CD25 expression are similar to those found when young DUC18 T cells are transferred into young mice. Although trafficking to tumor sites appears unequal, old and young DUC18 T cells reject primary CMS5 challenges to the same degree and with similar kinetics. Overall, we found no loss of endogenous APC function or intrinsic defects in CD8(+) DUC18 T cells with advanced age. Therefore, when young and old tumor-specific T cell populations are equivalently sized, CD8(+) T cell-mediated antitumor immunity in our system is not impaired by age, a finding that has positive implications for T cell-based immunotherapies.     

OX40 ligation of CD4+ T cells enhances virus-specific CD8+ T cell memory responses independently of IL-2 and CD4+ T regulatory cell inhibition

We have previously shown that CD4(+) T cells are required to optimally expand viral-specific memory CD8(+) CTL responses using a human dendritic cell-T cell-based coculture system. OX40 (CD134), a 50-kDa transmembrane protein of the TNFR family, is expressed primarily on activated CD4(+) T cells. In murine models, the OX40/OX40L pathway has been shown to play a critical costimulatory role in dendritic cell/T cell interactions that may be important in promoting long-lived CD4(+) T cells, which subsequently can help CD8(+) T cell responses. The current study examined whether OX40 ligation on ex vivo CD4(+) T cells can enhance their ability to "help" virus-specific CTL responses in HIV-1-infected and -uninfected individuals. OX40 ligation of CD4(+) T cells by human OX40L-IgG1 enhanced the ex vivo expansion of HIV-1-specific and EBV-specific CTL from HIV-1-infected and -uninfected individuals, respectively. The mechanism whereby OX40 ligation enhanced help of CTL was independent of the induction of cytokines such as IL-2 or any inhibitory effect on CD4(+) T regulatory cells, but was associated with a direct effect on proliferation of CD4(+) T cells. Thus, OX40 ligation on CD4(+) T cells represents a potentially novel immunotherapeutic strategy that should be investigated to treat and prevent persistent virus infections, such as HIV-1 infection.     

Priming in the presence of IL-10 results in direct enhancement of CD8+ T cell primary responses and inhibition of secondary responses

Although IL-10 acts as an inhibitory cytokine for APC and CD4(+) T cell function, its effects on CD8(+) T cells are unclear. Additionally, little is known about whether initial priming in the presence of IL-10 can have long-lasting effects and influence subsequent CD8(+) T cell responses that occur in the absence of the cytokine. In the present study, we clarified the role of IL-10 during primary responses and examined whether exposure to IL-10 during initial priming of CD8(+) T cells impacted secondary responses. To determine the effect of IL-10 on Ag-specific T cell responses, peptide-pulsed IL-10R2(-/-) splenic dendritic cells were used to prime T cells from OT-I CD8(+) TCR transgenic mice. During the primary response, the presence of IL-10 resulted in enhancement of CD8(+) T cell numbers without detectable alterations in the kinetics or percentage of cells that underwent proliferation. A modest increase in survival, not attributable to Bcl-2 or Bcl-x(L), was also observed with IL-10 treatment. Other parameters of CD8(+) T cell function, including IL-2, IFN-gamma, TNF-alpha, and granzyme production, were unaltered. In contrast, initial exposure to IL-10 during the primary response resulted in decreased OT-I expansion during secondary stimulation. This was accompanied by lowered IL-2 levels and reduced percentages of proliferating BrdU(+) cells and OT-I cells that were CD25(high). IFN-gamma, TNF-alpha, and granzyme production were unaltered. These data suggest that initial exposure of CD8(+) T cells to IL-10 may be temporarily stimulatory; however, programming of the cells may be altered, resulting in diminished overall responses.     

A new dynamic model of CD8+ T effector cell responses via CD4+ T helper-antigen-presenting cells

A long-standing paradox in cellular immunology has been the conditional requirement for CD4(+) Th cells in priming of CD8(+) CTL responses. We propose a new dynamic model of CD4(+) Th cells in priming of Th-dependent CD8(+) CTL responses. We demonstrate that OT II CD4(+) T cells activated by OVA-pulsed dendritic cells (DC(OVA)) are Th1 phenotype. They acquire the immune synapse-composed MHC II/OVAII peptide complexes and costimulatory molecules (CD54 and CD80) as well as the bystander MHC class I/OVAI peptide complexes from the DC(OVA) by DC(OVA) stimulation and thus also the potential to act themselves as APCs. These CD4(+) Th-APCs stimulate naive OT I CD8(+) T cell proliferation through signal 1 (MHC I/OVAI/TCR) and signal 2 (e.g., CD54/LFA-1 and CD80/CD28) interactions and IL-2 help. In vivo, they stimulate CD8(+) T cell proliferation and differentiation into CTLs and induce effective OVA-specific antitumor immunity. Taken together, this study demonstrates that CD4(+) Th cells carrying acquired DC Ag-presenting machinery can, by themselves, efficiently stimulate CTL responses. These results have substantial implications for research in antitumor and other aspects of immunity.