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.     

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.     

IL-1 receptor signaling is required at multiple stages of sensitization and elicitation of the contact hypersensitivity response

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to hapten skin sensitization and challenge. The points at which IL-1R signaling is required during this complex, multistep immune response have not been clearly delineated. The role of IL-1R signaling during 2, 4 dinitro-1-fluorobenezene (DNFB) sensitization to induce hapten-specific CD8 effector T cells and in the trafficking of the effector T cells to the DNFB challenge site to elicit the response were investigated using IL-1R deficient mice. DNFB-sensitized IL-1R(-/-) mice had low CHS responses to hapten challenge that were caused in part by marked decreases in hapten-specific CD8 T cell development to IL-17- and IFN-γ-producing cells during sensitization. Hapten-primed wild type CD8 T cell transfer to naive IL-1R(-/-) mice did not result in T cell activation in response to hapten challenge, indicating a need for IL-1R signaling for the localization or activation, or both, of the CD8 T cells at the challenge site. Decreased CD8 T cell priming in sensitized IL-1R(-/-) mice was associated with marked decreases in hapten-presenting dendritic cell migration from the sensitized skin to draining lymph nodes. Transfer of hapten-presenting dendritic cells from wild type donors to naive IL-1R(-/-) mice resulted in decreased numbers of the dendritic cells in the draining lymph nodes and decreased priming of hapten-specific CD8 T cells compared with dendritic cell transfer to naive wild type recipients. These results indicate that IL-1R signaling is required at multiple steps during the course of sensitization and challenge to elicit CHS.     

CD4+ T cells regulate CD8+ T cell-mediated cutaneous immune responses by restricting effector T cell development through a Fas ligand-dependent mechanism

The magnitude and duration of CD8(+) T cell-mediated responses in the skin to hapten sensitization and challenge, contact hypersensitivity (CHS), is negatively regulated by CD4(+) T cells through an unknown mechanism. In this study we show that CD4(+) T cells restrict the development and expansion of hapten-specific CD8(+) T cells mediating CHS responses to 2,4-dinitrofluorobenzene. In the absence of CD4(+) T cells, high numbers of hapten-specific CD8(+) T cells producing IFN-gamma were detected in the skin-draining lymph nodes on day 5 postsensitization, and these numbers decreased slightly, but were maintained through day 9, correlating with the increased magnitude and duration of CHS responses observed in these mice. In the presence of CD4(+) T cells, the number of hapten-specific CD8(+) T cells producing IFN-gamma detected on day 5 postsensitization was lower and quickly fell to background levels by day 7. The limited development of effector CD8(+) T cells was associated with decreased numbers of hapten-presenting dendritic cells in the lymphoid priming site. This form of immunoregulation was absent after sensitization of Fas ligand-defective gld mice. Transfer of wild-type CD4(+) T cells to gld mice restored the negative regulation of CD8(+) T cell priming and the immune response to hapten challenge in gld-recipient mice. These results indicate that CD4(+) T cells restrict hapten-specific CD8(+) T cell priming for CHS responses through a Fas ligand-dependent mechanism.     

The CD154-CD40 T cell costimulation pathway is required for host sensitization of CD8(+) T cells by skin grafts via direct antigen presentation

Although the CD154-CD40 T cell costimulation pathway has been shown to mediate alloimmune responses in normal recipients, little is known about its role in sensitized hosts. In this work, by using novel models of cardiac allograft rejection in skin-sensitized CD154- and CD40-deficient mice, we reaffirm the key role of CD154-CD40 signaling in host sensitization to alloantigen in vivo. First, we identified CD8(+) T cells as principal effectors in executing accelerated rejection in our model. Disruption of CD154-CD40 signaling in recipients at the T cell side (CD154-deficient) but not at the APC side (CD40-deficient) abrogated accelerated (<2 days) rejection and resulted in long-term (>100 days) graft survival. This suggests that the CD154-dependent mechanism in host CD8(+) T cell sensitization operates via the direct Ag presentation. Then, in comparative studies of alloimmune responses in CD154-deficient and wild-type recipients, we showed that, although alloreactive B cell responses were inhibited, alloreactive T cell responses were down-regulated selectively in the CD8(+) T cell compartment, leaving CD4(+) T cells largely unaffected. This unique alteration in host alloreactivity, seen not only in peripheral lymphocytes but also in allograft infiltrate, may represent the key mechanism by which disruption of CD154-CD40 signaling prevents sensitization to alloantigen in vivo and leads to long-term allograft survival.     

Neutrophil expression of fas ligand and perforin directs effector CD8 T cell infiltration into antigen-challenged skin

Contact hypersensitivity (CHS) is a T cell response to hapten skin challenge of sensitized individuals proposed to be mediated by hapten-primed CD8 cytolytic T cells. Effector CD8 T cell recruitment into hapten challenge sites to elicit CHS requires prior CXCL1- and CXCL2-mediated neutrophil infiltration into the site. We investigated whether neutrophil activities directing hapten-primed CD8 T cell skin infiltration in response to 2,4-dinitro-1-fluorobenzene (DNFB) required Fas ligand (FasL) and perforin expression. Although DNFB sensitization of gld/perforin(-/-) mice induced hapten-specific CD8 T cells producing IFN-γ and IL-17, these T cells did not infiltrate the DNFB challenge site to elicit CHS but did infiltrate the challenge site and elicit CHS when transferred to hapten-challenged naive wild-type recipients. Hapten-primed wild-type CD8 T cells, however, did not elicit CHS when transferred to naive gld/perforin(-/-) recipients. Wild-type bone marrow neutrophils expressed FasL and perforin, and when transferred to sensitized gld/perforin(-/-) mice, they restored hapten-primed CD8 T cell infiltration into the challenge site and CHS. The FasL/perforin-mediated activity of wild-type neutrophils induced the expression of T cell chemoattractants, CCL1, CCL2, and CCL5, within the hapten-challenged skin. These results indicate FasL/perforin-independent functions of hapten-primed CD8 T cells in CHS and identify new functions for neutrophils in regulating effector CD8 T cell recruitment and immune responses in the skin.     

Critical role for IL-4 in the development of transplant arteriosclerosis in the absence of CD40-CD154 costimulation.

Blockade of the CD40-CD154 pathway can inhibit CD4(+) T cell activation but is unable to prevent immune responses mediated by CD8(+) T cells. However, even in the absence of CD8(+) T cells, inhibition of the CD40-CD154 pathway is insufficient to prevent the development of transplant arteriosclerosis. This study investigated the mechanisms of transplant arteriosclerosis in the absence of the CD40 pathway. C57BL/6 CD40(-/-) (H2(b)) recipients were transplanted with MHC-mismatched BALB/c (H2(d)) aortas. Transplant arteriosclerosis was evident in both CD40(-/-) and CD40(+/-) mice (intimal proliferation was 59 +/- 5% for CD40(-/-) mice vs 58 +/- 4% for CD40(+/-) mice) in the presence or absence of CD8(+) T cells (intimal proliferation was 46 +/- 7% for CD40(-/-) anti-CD8-treated mice vs 50 +/- 10% for CD40(+/-) anti-CD8-treated mice), confirming that CD8(+) T cells are not essential effector cells for the development of this disease. In CD40(-/-) recipients depleted of CD8(+) T cells, the number of eosinophils infiltrating the graft was markedly increased (109 +/- 24 eosinophils/grid for CD40(-/-) anti-CD8-treated mice vs 28 +/- 7 for CD40(+/-) anti-CD8-treated mice). The increased presence of eosinophils correlated with augmented intragraft production of IL-4. To test the hypothesis that IL-4 was responsible for the intimal proliferation, CD8 T cell-depleted CD40(-/-) recipients were treated with anti-IL-4 mAb. This resulted in significantly reduced eosinophil infiltration into the graft (12 +/- 5 eosinophils/grid for CD40(-/-) anti-CD8(+), anti-IL-4-treated mice vs 109 +/- 24 for CD40(-/-) anti-CD8-treated mice), intragraft eotaxin, CCR3 mRNA production, and the level of intimal proliferation (18 +/- 5% for CD40(-/-) anti-CD8(+)-, anti-IL-4-treated mice vs 46 +/- 7% for CD40(-/-) anti-CD8-treated mice). In conclusion, elevated intragraft IL-4 production results in an eosinophil infiltrate and is an important mechanism for CD8(+) T cell-independent transplant arteriosclerosis in the absence of CD40-CD154 costimulation.     

Oral administration of hapten inhibits in vivo induction of specific cytotoxic CD8+ T cells mediating tissue inflammation: a role for regulatory CD4+ T cells

We investigated whether oral tolerance could block the development of an inflammatory response mediated by CD8+ T cells, using a mouse model of oral tolerance of contact sensitivity (CS) to the hapten 2, 4-dinitrofluorobenzene (DNFB). In this system, the skin inflammatory response is initiated by hapten-specific class I-restricted cytotoxic CD8+ T (CTL) cells, independently of CD4 help. Oral delivery of DNFB before skin sensitization blocked the CS response by impairing the development of DNFB-specific CD8+ effector T cells in secondary lymphoid organs. This was shown by complete inhibition of DNFB-specific CTL and proliferative responses of CD8+ T cells, lack of specific IFN-gamma-producing CD8+ T cells, and inability of CD8+ T cells to transfer CS in RAG20/0 mice. RT-PCR and immunohistochemical analysis confirmed that recruitment of CD8+ effectors of CS in the skin at the site of hapten challenge was impaired in orally tolerized mice. Sequential anti-CD4 Ab treatment showed that only depletion of CD4+ T cells during the afferent phase of CS abrogated oral tolerance induction by restoring high numbers of specific CD8+ effectors in lymphoid organs, whereas CD4 depletion during the efferent phase of CS did not affect oral tolerance. These data demonstrate that a single intragastric administration of hapten can block in vivo induction of DNFB-specific CD8+ CTL responsible for tissue inflammation and that a subset of regulatory CD4+ T cells mediate oral tolerance by inhibiting expansion of specific CD8+ effectors in lymph nodes.     

CD4+ Th1 and CD8+ type 1 cytotoxic T cells both play a crucial role in the full development of contact hypersensitivity

The role of CD4(+) vs CD8(+) T cells in contact hypersensitivity (CHS) remains controversial. In this study, we used gene knockout (KO) mice deficient in CD4(+) or CD8(+) T cells to directly address this issue. Mice lacking either CD4(+) or CD8(+) T cells demonstrated depressed CHS responses to dinitrofluorobenzene and oxazolone compared with wild-type C57BL/6 mice. The depression of CHS was more significant in CD8 KO mice than in CD4 KO mice. Furthermore, in vivo depletion of either CD8(+) T cells from CD4 KO mice or CD4(+) T cells from CD8 KO mice virtually abolished CHS responses. Lymph node cells (LNCs) from hapten-sensitized CD4 and CD8 KO mice showed a decreased capacity for transferring CHS. In vitro depletion of either CD4(+) T cells from CD8 KO LNCs or CD8(+) T cells from CD4 KO LNCs resulted in a complete loss of CHS transfer. LNCs from CD4 and CD8 KO mice produced significant amounts of IFN-gamma, indicating that both CD4(+) and CD8(+) T cells are able to secrete IFN-gamma. LNCs from CD8, but not CD4, KO mice were able to produce IL-4 and IL-10, suggesting that IL-4 and IL-10 are mainly derived from CD4(+) T cells. Intracellular cytokine staining of LNCs confirmed that IFN-gamma-positive cells consisted of CD4(+) (Th1) and CD8(+) (type 1 cytotoxic T) T cells, whereas IL-10-positive cells were exclusively CD4(+) (Th2) T cells. Collectively, these results suggest that both CD4(+) Th1 and CD8(+) type 1 cytotoxic T cells are crucial effector cells in CHS responses to dinitrofluorobenzene and oxazolone in C57BL/6 mice.     

Differential requirement for the CD40-CD154 costimulatory pathway during Th cell priming by CD8 alpha+ and CD8 alpha- murine dendritic cell subsets

Dendritic cells (DCs) regulate the development of distinct Th populations and thereby provoke appropriate immune responses to various kinds of Ags. In the present work, we investigated the role CD40-CD154 interactions play during the process of Th cell priming by CD8 alpha(+) and CD8 alpha(-) murine DC subsets, which have been reported to differently regulate the Th response. Adoptive transfer of Ag-pulsed CD8 alpha(+) DCs induced a Th1 response and the production of IgG2a Abs, whereas transfer of CD8 alpha(-) DCs induced Th2 cells and IgE Abs in vivo. Induction of distinct Th populations by each DC subset was also confirmed in vitro. Although interruption of CD80/CD86-CD28 interactions inhibited Th cell priming by both DC subsets, disruption of CD40-CD154 interactions only inhibited the induction of the Th1 response by CD8 alpha(+) DCs in vivo. CD40-CD154 interactions were not required for the proliferation of Ag-specific naive Th cells stimulated by either DC subset, but were indispensable in the production of IL-12 from CD8 alpha(+) DCs and their induction of Th1 cells in vitro. Taken together, in our immunization model of Ag-pulsed DC transfer, CD40-CD154 interactions play an important role in the development of CD8 alpha(+) DC-driven Th1 responses but not CD8 alpha(-) DC-driven Th2 responses to protein Ags.     

CD8+ IL-17-producing T cells are important in effector functions for the elicitation of contact hypersensitivity responses

Allergen-induced contact hypersensitivity (CHS) is a T cell-mediated delayed-type immune response which has been considered to be primarily mediated by CD8+ T cytotoxic type I (Tc1) cells. IFN-gamma, the prototype Tc1 (Th1) cytokine, has been implicated as the primary inflammatory cytokine for CHS. In this study, we demonstrate that neutralization of IL-17 rather than IFN-gamma suppresses the elicitation of CHS. The suppression does not result from inhibition of the proliferation of allergen-activated T cells. Allergen sensitization induces the development of distinct CD8+ T cell subpopulations that produce IFN-gamma or IL-17. Although CD8+ IL-17-producing cells are stimulated by IL-23, they are inhibited by IL-12, a prototypical stimulator of IFN-gamma-producing Tc1 cells. This indicates that CD8+ IL-17-producing cells are distinct from Tc1 cells and are important in effector functions at the elicitation of CHS. These studies provide insights into a novel mechanism for CHS.     

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.     

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

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

DNA vaccines,combining form of antigen and method of delivery to raise a spectrum of IFN-gamma and IL-4-producing CD4+ and CD8+ T cells

DNA-based immunizations have been used to determine the patterns of type 1 and type 2 cytokines that can be induced in vivo for Ag-specific CD4(+) and CD8(+) T cells. IL-4 was used as a signature cytokine for a type 2 T cell response and IFN-gamma as the signature cytokine for a type 1 response. Gene gun deliveries of secreted Ags were used to bias responses toward type 2 and saline injections of cell-associated Ags to bias responses toward type 1. The studies revealed that gene gun bombardments of DNAs expressing secreted Ags strongly biased responses toward type 2, inducing IL-4-producing CD8(+) as well as CD4(+) T cells. Saline injections of DNAs expressing cell-associated Ags strongly biased responses toward type 1, inducing IFN-gamma-producing CD8(+) and CD4(+) cells. A mixed type 1/type 2 response of IFN-gamma-producing CD8(+) T cells and IL-4-producing CD4(+) T cells was found for gene gun deliveries of cell-associated Ags. Saline injections of secreted Ags raised a weakly type 1-biased response characterized by only slightly higher frequencies of IFN-gamma- than IL-4-producing CD4(+) and CD8(+) T cells. Studies in B cell knockout and hen egg lysozyme Ig transgenic mice revealed that B cells were required for the generation of IL-4-producing CD8(+) T cells.     

IL-12 contributes to allergen-induced airway inflammation in experimental asthma

Lack of sufficient IL-12 production has been suggested to be one of the basic underlying mechanisms in atopy, but a potential role of IL-12 in established allergic airway disease remains unclear. We took advantage of a mouse model of experimental asthma to study the role of IL-12 during the development of bronchial inflammation. Administration of anti-IL-12p35 or anti-IL-12p40 mAb to previously OVA-sensitized BALB/c mice concomitantly with exposure to nebulized OVA, abolished both the development of bronchial hyperresponsiveness to metacholine as well as the eosinophilia in bronchoalveolar lavage fluid and peripheral blood. Anti-IL-12 treatment reduced CD4(+) T cell numbers and IL-4, IL-5, and IL-13 levels in the bronchoalveolar lavage fluid and the mRNA expression of IL-10, eotaxin, RANTES, MCP-1, and VCAM-1 in the lung. Anti-IL-12p35 treatment failed to show these effects in IFN-gamma knockout mice pointing to the essential role of IFN-gamma in IL-12-induced effects. Neutralization of IL-12 during the sensitization process aggravated the subsequent development of allergic airway inflammation. These data together with recent information on the role of dendritic cells in both the sensitization and effector phase of allergic respiratory diseases demonstrate a dual role of IL-12. Whereas IL-12 counteracts Th2 sensitization, it contributes to full-blown allergic airway disease upon airway allergen exposure in the postsensitization phase, with enhanced recruitment of CD4(+) T cells and eosinophils and with up-regulation of Th2 cytokines, chemokines, and VCAM-1. IFN-gamma-producing cells or cells dependent on IFN-gamma activity, play a major role in this unexpected proinflammatory effect of IL-12 in allergic airway disease.     

Cutting edge: IL-12 induces CD4+CD25- T cell activation in the presence of T regulatory cells

IL-12p40 cytokines have been implicated in the development of organ-specific autoimmune diseases as well as pathogen-specific adaptive immunity. In addition to inducing IFN-gamma, IL-12 stimulates effector CD4(+) T cells to express adhesion molecules and homing receptors that facilitate their migration to sites of inflammation. In this study, we expand upon those observations by demonstrating an alternative pathway by which IL-12 could promote Th1 inflammatory responses in mice, namely, by restoring proliferation and cytokine expression by effector T cells in the presence of CD4(+)CD25(+) regulatory T cells (Treg). This effect of IL-12 was not replicated by IL-23 or IFN-gamma and was dependent on signaling through the IL-12R expressed on CD25(-) responder cells, but not on Treg. Our studies suggest that IL-12 could act in concert with other proinflammatory factors to stimulate CD4(+)CD25(-) T cell activation in the presence of Treg.     

Ultraviolet light exposure suppresses contact hypersensitivity by abrogating endothelial intercellular adhesion molecule-1 up-regulation at the elicitation site

Hapten sensitization through UV-exposed skin induces systemic immune suppression, which is experimentally demonstrated by inhibition of contact hypersensitivity (CHS). Although this UV-induced effect has been shown to be mediated by inhibition of the afferent phase of the CHS, the UV effects on the efferent (elicitation) phase remain unknown. In this study, UV effects on endothelial ICAM-1 expression at elicitation sites were first examined. Mice were sensitized by hapten application onto UV-exposed back skin, and ears were challenged 5 days later. ICAM-1 up-regulation at nonirradiated elicitation sites following hapten challenge was eliminated by UV exposure on sensitization sites distant from elicitation sites. To assess whether loss of the ICAM-1 up-regulation at elicitation sites contributed to UV-induced immunosuppression, we examined CHS responses in UV-exposed ICAM-1-deficient (ICAM-1(-/-)) mice that genetically lacked the ICAM-1 up-regulation. ICAM-1(-/-) mice exhibited reduced CHS responses without UV exposure, but UV exposure did not further reduce CHS responses in ICAM-1(-/-) mice. Furthermore, ICAM-1 deficiency did not affect the afferent limb, because ICAM-1(-/-) mice had normal generation of hapten-specific suppressor and effector T cells. This UV-induced immunosuppression was associated with a lack of TNF-alpha production after Ag challenge at elicitation sites. Local TNF-alpha injection before elicitation abrogated the UV-induced CHS inhibition with increased endothelial ICAM-1 expression. TNF-alpha production at elicitation sites was down-regulated by IL-10, a possible mediator produced by hapten-specific suppressor T cells that are generated by UV exposure. These results indicate that UV exposure inhibits CHS by abrogating up-regulation of endothelial ICAM-1 expression after Ag challenge at elicitation sites.     

Full development of Th2 immunity requires both innate and adaptive sources of CD154

The CD40-CD154 interaction is critical for Th2 response generation during helminth infection and following immunization with helminth-conditioned dendritic cells, yet the key cellular sources of these molecules have still to be defined in vivo. In this study, we demonstrate that the requirement for CD40 expression during murine Th2 response induction is restricted exclusively to the Ag-bearing dendritic cells. In contrast, development of full Th2 immunity required CD154 expression on multiple populations. In this respect, optimal production of IL-5, IL-10, and IL-13 was dependent upon CD154 expression by both CD4(+) T cells and non-lymphoid cells. IL-4 production had less stringent costimulatory requirements, with expression of CD154 on either non-lymphoid cells or T cells alone being sufficient to enable production of this archetypal Th2 cytokine. Disparities in CD154 requirements for T cell and B cell responses were revealed during experimental schistosomiasis where, even in the face of robust Th2 generation, B cell class-switching was entirely dependent upon expression of CD154 by the lymphoid compartment. These data help define the costimulatory interactions that occur during the generation of Th2 immunity, and challenge the widely held view that CD154 expressing T cells are the sole contributors in this process.     

Dependency of direct pathway CD4+ T cells on CD40-CD154 costimulation is determined by nature and microenvironment of primary contact with alloantigen

Blockade of the CD40-CD154 costimulatory pathway can inhibit CD4(+) T cell-mediated alloimmune responses. The aim of this study was to define the in vivo requirement for CD40-CD154 costimulation by CD4(+) T cells that respond to alloantigen following direct recognition. We used TCR-transgenic CD4(+) T cells that are reactive to the MHC class II alloantigen, H2A(s). An experimental in vivo model was established that allowed direct comparison of the fate of a trace population of H2A(s)-reactive CD4(+) T cells when challenged with different forms of H2A(s+) alloantigen under conditions of CD40-CD154 costimulation blockade. In this study, we demonstrate that an i.v. infusion of H2A(s+) leukocytes in combination with anti-CD154 therapy rapidly deletes H2A(s)-reactive CD4(+) T cells. In contrast, following transplantation of an H2A(s+) cardiac allograft, H2A(s)-reactive CD4(+) T cell responses were unaffected by blocking CD40-CD154 interactions. Consistent with these findings, combined treatment with donor leukocytes and anti-CD154 therapy was found to be more effective in prolonging the survival of cardiac allografts compared with CD154 mAb treatment alone. The dominant mechanism by which donor leukocyte infusion and anti-CD154 therapy facilitate allograft acceptance is deletion of donor-reactive direct pathway T cells. No evidence for the generation of regulatory cells by this combined therapy was found. Taken together, these results clearly demonstrate that naive alloreactive CD4(+) T cells have distinct requirements for CD40-CD154 costimulation depending on the form and microenvironment of primary alloantigen contact.