Anton V. Gorbachev

Alloreactive T Cell Responses and Acute Rejection of Single Class II MHC-Disparate Heart Allografts Are under Strict Regulation by CD4+CD25+ T Cells

Skin but not vascularized cardiac allografts from B6.H-2bm12 mice are acutely rejected by C57BL/6 recipients in response to the single class II MHC disparity. The underlying mechanisms preventing acute rejection of B6.H-2bm12 heart allografts by C57BL/6 recipients were investigated. B6.H-2bm12 heart allografts induced low levels of alloreactive effector T cell priming in C57BL/6 recipients, and this priming was accompanied by low-level cellular infiltration into the allograft that quickly resolved. Recipients with long-term-surviving heart allografts were unable to reject B6.H-2bm12 skin allografts, suggesting potential down-regulatory mechanisms induced by the cardiac allografts. Depletion of CD25+ cells from C57BL/6 recipients resulted in 15-fold increases in alloreactive T cell priming and in acute rejection of B6.H-2bm12 heart grafts. Similarly, reconstitution of B6.Rag−/− recipients with wild-type C57BL/6 splenocytes resulted in acute rejection of B6.H-2bm12 heart grafts only if CD25+ cells were depleted. These results indicate that acute rejection of single class II MHC-disparate B6.H-2bm12 heart allografts by C57BL/6 recipients is inhibited by the emergence of CD25+ regulatory cells that restrict the clonal expansion of alloreactive T cells.

The intensity of neutrophil infiltration controls the number of antigen-primed CD8 T cells recruited into cutaneous antigen challenge sites.

Recruitment of antigen‐specific T cells into the skin is a critical initiating event during immune responses to many parasites and tumors as well as T cell‐mediated, cutaneous, allergic responses and autoimmune diseases. Mechanisms directing T cell trafficking into skin remain largely undefined. Here, we show that cutaneous contact with reactive antigen induces KC/CXC chemokine ligand 1 production and neutrophil infiltration in an antigen, dose‐dependent manner. The intensity of neutrophil infiltration into cutaneous antigen challenge sites, in turn, controls the number of antigen‐primed T cells recruited into the site and the magnitude of the immune response elicited. The absence of responses in immune animals challenged with suboptimal doses of antigen is overcome by manipulating neutrophil infiltration that then directs antigen‐primed T cell infiltration into the challenge site. This inflammation also directs T cells primed to one antigen (dinitrofluorobenzene) into the site when challenged with a completely different antigen (oxazolone). These results identify the intensity of neutrophil infiltration into cutaneous, antigen‐deposition sites as a critical parameter for the level of antigen‐primed T cell recruitment to mediate the adaptive immune response. This interplay between the innate and adaptive responses suggests a strategy to modulate, in a positive or negative manner, antigen‐primed T cell infiltration into cutaneous inflammation sites.

Inhibition of Functional T Cell Priming and Contact Hypersensitivity Responses by Treatment with Anti-Secondary Lymphoid Chemokine Antibody During Hapten Sensitization

Recent studies have suggested a pivotal role for secondary lymphoid chemokine (SLC) in directing dendritic cell trafficking from peripheral to lymphoid tissues. As an extension of these studies, we examined the consequences of anti-SLC Ab treatment during Ag priming on T cell function in an inflammatory response. We used a model of T cell-mediated inflammation, contact hypersensitivity (CHS), where priming of the effector T cells is dependent upon epidermal dendritic cell, Langerhans cells, and migration from the hapten sensitization site in the skin to draining lymph nodes. A single injection of anti-SLC Ab given at the time of sensitization with FITC inhibited Langerhans cell migration into draining lymph nodes for at least 3 days. The CHS response to hapten challenge was inhibited by anti-SLC Ab treatment in a dose-dependent manner. Despite the inhibition of CHS, T cells producing IFN-γ following in vitro stimulation with anti-CD3 mAb or with hapten-labeled cells were present in the skin-draining lymph nodes of mice treated with anti-SLC Ab during hapten sensitization. These T cells were unable, however, to passively transfer CHS to naive recipients. Animals treated with anti-SLC Ab during hapten sensitization were not tolerant to subsequent sensitization and challenge with the hapten. In addition, anti-SLC Ab did not inhibit CHS responses when given at the time of hapten challenge. These results indicate an important role for SLC during sensitization for CHS and suggest a strategy to circumvent functional T cell priming for inflammatory responses through administration of an Ab inhibiting dendritic cell trafficking.

CD8+ T cells produce IL‐2, which is required for CD4+CD25+ T cell regulation of effector CD8+ T cell development for contact hypersensitivity responses

Interleukin (IL)‐2 functions to promote, as well as down‐regulate, expansion of antigen‐reactive CD4+ and CD8+ T cells, but the role of IL‐2 in hapten‐specific CD8+ T cell priming for contact hypersensitivity (CHS) responses remains untested. Using enzyme‐linked immunospot to enumerate numbers of hapten‐specific CD4+ and CD8+ T cells producing IL‐2 in hapten‐sensitized mice, the number of IL‐2‐producing CD8+ T cells was tenfold that of CD4+ T cells. Hapten‐primed D4+ T cells produced low amounts of IL‐2 during culture with hapten‐presenting Langerhans cells, whereas production by hapten‐primed CD8+ T cells was fivefold greater. CD8+ T cells did not express CD25 during hapten priming, but treatment with anti‐IL‐2 or anti‐CD25 monoclonal antibodies during hapten sensitization increased hapten‐specific effector CD8+ T cells as well as the magnitude and duration of the CHS response. These results indicate that CD8+ T cells are the primary source of IL‐2 and that this IL‐2 is required for the function of a population of CD4+CD25+ T cells to restrict the development of the hapten‐reactive effector CD8+ T cells that mediate CHS responses.

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-γ 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-γ 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.

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-γ-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-γ-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-γ-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.

CXC Chemokine Ligand 9/Monokine Induced by IFN-γ Production by Tumor Cells Is Critical for T Cell-Mediated Suppression of Cutaneous Tumors

The role of tumor-produced chemokines in the growth of malignancies remains poorly understood. We retrieved an in vivo growing MCA205 fibrosarcoma and isolated tumor cell clones that produce both CXCL9/monokine induced by IFN-γ (Mig) and CXCL10/IFN-γ-inducible protein 10 following stimulation with IFN-γ and clones that produce IFN-γ-inducible protein 10 but not Mig. The Mig-deficient variants grew more aggressively as cutaneous tumors in wild-type mice than the Mig-producing tumor cells. The growth of Mig-expressing, but not Mig-deficient, tumor cells was suppressed by NK and T cell activity. Transduction of Mig-negative variants to generate constitutive tumor cell production of Mig resulted in T cell-dependent rejection of the tumors and in induction of protective tumor-specific CD8+ T cell responses to Mig-deficient tumors. The results indicate a critical role for tumor-derived Mig in T cell-mediated responses to cutaneous fibrosarcomas and suggest the loss of Mig expression as a mechanism used by tumor cells to evade these responses.

Regulatory role of CD4+ T cells during the development of contact hypersensitivity responses

Contac thypersensitivity (CHS) is a T cell-mediated immune response to cutaneous sensitization and subsequent challenge with haptens such as dinitrofluorobenzene and oxazolone. Many aspects concerning the development and regulation of CHS remain unknown. Using CHS as a model of T cell-mediated immune responses to antigens deposited in the skin we have studied the development and function of effector and regulatory T cell componenets of this response. These studies have revealed the effector role of hapten-specific CD8+ T cells in this response. In contrast, hapten-specific CD4+ T cells negatively regulate the magnitude and duration of the response. In this article we propose a model in which the CD4+ T cells during sensitization for CHS and discuss potential mechanisms that CD4+ T cells might utilize to mediate this regulation.

Quinacrine inhibits the epidermal dendritic cell migration initiating T cell-mediated skin inflammation

Quinacrine (QC) is an anti‐inflammatory drug that has been used for the treatment of malaria and rheumatoid diseases. The mechanism(s) underlying the anti‐inflammatory activity of QC remains poorly understood. We recently reported the QC‐mediated inhibition of the NF‐κB pathway using an in vitro model. To test this potential mechanism in vivo, we used the contact hypersensitivity response (CHS) to chemical allergen sensitization and challenge in mice as a model of skin inflammation. The results indicated that QC treatment inhibited NF‐κB activation in the skin during allergen sensitization. This inhibition was reflected by decreased mRNA expression and protein production of the NF‐κB‐dependent cytokines TNF‐α and IL‐1β and the chemokine CCL21 in the skin. The decreases in these cytokines resulted in reduced migration of allergen‐presenting dendritic cells from the skin into skin‐draining lymph nodes and markedly decreased activation of effector CD8+ T cells for the CHS response to allergen challenge (inhibitory concentration 50% or IC50 was 55 mg/kg). These findings reveal a previously unrecognized mechanism of QC‐mediated inhibition of inflammation.

IL‐15 Induces Alloreactive CD28− Memory CD8 T Cell Proliferation and CTLA4‐Ig Resistant Memory CD8 T Cell Activation

The presence of CD28− memory CD8 T cells in the peripheral blood of renal transplant patients is a risk factor for graft rejection and resistance to CTLA‐4Ig induction therapy. In vitro analyses have indicated poor alloantigen‐induced CD28− memory CD8 T cell proliferation, raising questions about mechanisms mediating their clonal expansion in kidney grafts to mediate injury. Candidate proliferative cytokines were tested for synergy with alloantigen in stimulating CD28− memory CD8 T cell proliferation. Addition of IL‐15, but not IL‐2 or IL‐7, to co‐cultures of CD28− or CD28+ memory CD8 T cells and allogeneic B cells rescued proliferation of the CD28− and enhanced CD28+ memory T cell proliferation. Proliferating CD28− memory CD8 T cells produced high amounts of interferon gamma and tumor necrosis factor alpha and expressed higher levels of the cytolytic marker CD107a than CD28+ memory CD8 T cells. CTLA‐4Ig inhibited alloantigen‐induced proliferation of CD28+ memory CD8 T cell proliferation but had no effect on alloantigen plus IL‐15‐induced proliferation of either CD28− or CD28+ memory CD8 T cells. These results indicate the ability of IL‐15, a cytokine produced by renal epithelial during inflammation, to provoke CD28− memory CD8 T cell proliferation and to confer memory CD8 T cell resistance to CTLA‐4Ig‐mediated costimulation blockade.