D. Keith Bishop

CD8+ Th17 Mediate Costimulation Blockade-Resistant Allograft Rejection in T-bet-Deficient Mice

While studying Th responses induced by cardiac transplantation, we observed that mice deficient in the Th1 transcription factor T-bet (T-bet−/−) mount both Th1 and Th17 responses, whereas wild-type recipients mount only Th1 responses. Cells producing both IFN-γ and IL-17 were readily detectable within the rejecting graft of T-bet−/− recipients, but were absent from the spleen, indicating that the in vivo microenvironment influences Th function. In addition, disrupting CD40-CD40L costimulatory interactions was highly effective at prolonging allograft survival in WT mice, but ineffective in T-bet−/− recipients. In this study, we report that CD8+ Th17 mediate costimulation blockade-resistant rejection in T-bet−/− allograft recipients. Depleting CD8+ cells or neutralizing IL-17 or the Th17-inducing cytokine IL-6 ablated the Th17 response and reversed costimulation blockade-resistant graft rejection. Neutralizing IL-4 in IFN-γ−/− allograft recipients did not induce Th17, suggesting that T-bet, rather than IL-4 and IFN-γ (known inhibitors of Th17), plays a critical role in negatively regulating Th17 in the transplant setting.

Immunobiology of Allograft Rejection in the Absence of IFN-γ: CD8+ Effector Cells Develop Independently of CD4+ Cells and CD40-CD40 Ligand Interactions

Both wild-type (WT) and IFN-γ-deficient (IFN-γ−/−) C57BL/6 mice can rapidly reject BALB/c cardiac allografts. When depleted of CD8+ cells, both WT and IFN-γ−/− mice rejected their allografts, indicating that these mice share a common CD4-mediated, CD8-independent mechanism of rejection. However, when depleted of CD4+ cells, WT mice accepted their allografts, while IFN-γ−/− recipients rapidly rejected them. Hence, IFN-γ−/−, but not WT mice developed an unusual CD8-mediated, CD4-independent, mechanism of allograft rejection. Allograft rejection in IFN-γ−/− mice was associated with intragraft accumulation of IL-4-producing cells, polymorphonuclear leukocytes, and eosinophils. Furthermore, this form of rejection was resistant to treatment with anti-CD40 ligand (CD40L) mAb, which markedly prolonged graft survival in WT mice. T cell depletion studies verified that anti-CD40L treatment failed to prevent CD8-mediated allograft rejection in IFN-γ−/− mice. However, anti-CD40L treatment did prevent CD4-mediated rejection in IFN-γ−/− mice, although grafts were eventually rejected when CD8+ T cells repopulated the periphery. The IL-4 production and eosinophil influx into the graft that occurred during CD8-mediated rejection were apparently epiphenomenal, since treatment with anti-IL-4 mAb blocked intragraft accumulation of eosinophils, but did not interfere with allograft rejection. These studies demonstrate that a novel, CD8-mediated mechanism of allograft rejection, which is resistant to experimental immunosuppression, can develop when IFN-γ is limiting. An understanding of this mechanism is confounded by its association with Th2-like immune events, which contribute unique histopathologic features to the graft but are apparently unnecessary for the process of allograft rejection.

Immunogenetic Therapy of Human Melanoma Utilizing Autologous Tumor Cells Transduced to Secrete Granulocyte-Macrophage Colony-Stimulating Factor

We performed a clinical study of five patients with melanoma to evaluate the immunobiological effects of retrovirally transduced autologous tumor cells given as a vaccine to prime draining lymph nodes. Patients were inoculated with both wild-type (WT) and GM-CSF gene-transduced tumor cells in different extremities. Approximately 7 days later, vaccine-primed lymph nodes (VPLNs) were removed. There was an increased infiltration of dendritic cells (DCs) in the GM-CSF-secreting vaccine sites compared with the WT vaccine sites. This resulted in a greater number of cells harvested from the GM-CSF-VPLNs compared with the WT-VPLNs at a time when serum levels of GM-CSF were not detectable. Four of five patients proceeded to have the adoptive transfer of GM-CSF-VPLN cells secondarily activated and expanded ex vivo with anti-CD3 MAb and IL-2. One patient had a durable complete remission of metastatic tumor. Utilizing cytokine (IFN-gamma, GM-CSF, IL-10) release assays, GM-CSF-VPLN T cells manifested diverse responses when exposed to tumor antigen in vitro. In two of two patients, GM-CSF-VPLN T cell responses were different from those of matched WT-VPLN cells. This study documents measurable immunobiologic differences of GM-CSF-transduced tumor cells given as a vaccine compared with WT tumor cells. The complete tumor remission in one patient provides a rationale to pursue this approach further.

Interferon-gamma expression by intraepithelial lymphocytes results in a loss of epithelial barrier function in a mouse model of total parenteral nutrition

ObjectiveTo determine the etiology of the loss of epithelial barrier function observed with the administration of total parenteral nutrition (TPN) in a mouse model. Summary Background DataRemoval of enteral nutrition with the administration of TPN is associated with a loss of intestinal epithelial barrier function. The etiology of this barrier loss is not clear. Because intraepithelial lymphocytes (IELs) produce a number of cytokines that may alter epithelial permeability, the authors investigated IEL cytokine expression in a mouse model of TPN. MethodsAdult C57BL/6 mice received TPN or enteral diet for 7 days. IELs were subsequently harvested and the mRNA expression of cytokines was measured. Epithelial barrier function was assessed in vitro with 51Cr-EDTA in Ussing chambers and was expressed as the permeability coefficient (Papp). ResultsIEL mRNA expression of interferon-gamma (IFN-&ggr;) rose from 0.14 ± 0.07 in the control (enterally fed) group to 0.44 ± 0.11 attomoles/&mgr;L in the TPN group (P < .05). Transforming growth factor-&bgr;1 declined slightly but not significantly, from 0.75 ± 0.35 to 0.55 ± 0.18 attomoles/&mgr;L in the control and TPN groups, respectively. Epithelial barrier function declined significantly with TPN compared to controls. To assess the relevance of IFN-&ggr; changes, permeability in IFN-&ggr; knockout mice was studied. Barrier function was significantly higher in IFN-&ggr; knockout mice on TPN compared to C57BL/6 mice that received TPN. ConclusionsIEL cytokine expression changes significantly with TPN administration. The partial correction with IFN-&ggr; knockout mice suggests that an upregulation of IFN-&ggr; expression is one mechanism responsible for the loss of the epithelial barrier associated with TPN.

New Insights on OX40 in the Control of T Cell Immunity and Immune Tolerance In Vivo

OX40 is a T cell costimulatory molecule that belongs to the TNFR superfamily. In the absence of immune activation, OX40 is selectively expressed by Foxp3+ regulatory T cells (Tregs), but not by resting conventional T cells. The exact role of OX40 in Treg homeostasis and function remains incompletely defined. In this study, we demonstrate that OX40 engagement in vivo in naive mice induces initial expansion of Foxp3+ Tregs, but the expanded Tregs have poor suppressive function and exhibit features of exhaustion. We also show that OX40 enables the activation of the Akt and Stat5 pathways in Tregs, resulting in transient proliferation of Tregs and reduced levels of Foxp3 expression. This creates a state of relative IL-2 deficiency in naive mice that further impacts Tregs. This exhausted Treg phenotype can be prevented by exogenous IL-2, as both OX40 and IL-2 agonists drive further expansion of Tregs in vivo. Importantly, Tregs expanded by both OX40 and IL-2 agonists are potent suppressor cells, and in a heart transplant model, they promote long-term allograft survival. Our data reveal a novel role for OX40 in promoting immune tolerance and may have important clinical implications.

IL-12 p40 Homodimer-Dependent Macrophage Chemotaxis and Respiratory Viral Inflammation Are Mediated through IL-12 Receptor β1

Leukocyte recruitment to the airway lumen is a central feature of inflammatory conditions such as asthma and respiratory viral infection. Characterization of mediators that regulate leukocyte recruitment in these conditions revealed increased IL-12 p40 homodimer (p80) levels were associated with enhanced airway macrophage accumulation. To examine this association, we used in vivo and in vitro assays to demonstrate p80, but not IL-12 or p40, provided a macrophage chemoattractant signal. Macrophages from genetically deficient mice indicated p80-dependent chemotaxis was independent of IL-12 and required IL-12Rβ1 (Rβ1) expression. Furthermore, analysis of murine cell lines and primary culture macrophages revealed Rβ1 expression, with an intact cytoplasmic tail, was necessary and sufficient to mediate p80-dependent chemotaxis. To examine the role for Rβ1 in mediating macrophage accumulation in vivo, we contrasted Sendai virus-driven airway inflammation in wild-type and Rβ1-deficient mice. Despite similar viral burden and production of the macrophage chemoattractant p80, the Rβ1-deficient mice displayed a selective decrease in airway macrophage accumulation and resistance to viral-dependent mortality. Thus, Rβ1 mediates p80-dependent macrophage chemotaxis and inhibition of the p80-Rβ1 interaction may provide a novel anti-inflammatory strategy to manipulate the inflammation associated with asthma and respiratory viral infection.

TISSUE-SPECIFIC CONSEQUENCES OF THE ANTI-ADENOVIRAL IMMUNE RESPONSE: IMPLICATIONS FOR CARDIAC TRANSPLANTS

The immune response to adenoviral vectors can induce inflammation and loss of transgene expression in transfected tissues. This would limit the use of adenovirus-mediated gene transfer in disease states in which long-term gene expression is required. While studying the effect of the anti-adenoviral immune response in transplantation, we found that transgene expression persisted in cardiac isografts transfected with an adenovirus encoding β-galactosidase. Transfected grafts remained free of inflammation, despite the presence of an immune response to the vector. Thus, adenovirus-mediated gene transfer may have therapeutic value in cardiac transplantation and heart diseases. Furthermore, immunological limitations of adenoviral vectors for gene therapy are not universal for all tissue types.

Role of T Cell TGFβ Signaling and IL-17 in Allograft Acceptance and Fibrosis Associated with Chronic Rejection

Chronic allograft rejection (CR) is the main barrier to long-term transplant survival. CR is a progressive disease defined by interstitial fibrosis, vascular neointimal development, and graft dysfunction. The underlying mechanisms responsible for CR remain poorly defined. TGFβ has been implicated in promoting fibrotic diseases including CR, but is beneficial in the transplant setting due to its immunosuppressive activity. To assess the requirement for T cell TGFβ signaling in allograft acceptance and the progression of CR, we used mice with abrogated T cell TGFβ signaling as allograft recipients. We compared responses from recipients that were transiently depleted of CD4+ cells (that develop CR and express intragraft TGFβ) with responses from mice that received anti-CD40L mAb therapy (that do not develop CR and do not express intragraft TGFβ). Allograft acceptance and suppression of graft-reactive T and B cells were independent of T cell TGFβ signaling in mice treated with anti-CD40L mAb. In recipients transiently depleted of CD4+ T cells, T cell TGFβ signaling was required for the development of fibrosis associated with CR, long-term graft acceptance, and suppression of graft-reactive T and B cell responses. Furthermore, IL-17 was identified as a critical element in TGFβ-driven allograft fibrosis. Thus, IL-17 may provide a therapeutic target for preventing graft fibrosis, a measure of CR, while sparing the immunosuppressive activity of TGFβ.

The Immunobiology of Inductive Anti-CD40L Therapy in Transplantation: Allograft Acceptance is Not Dependent Upon the Deletion of Graft-Reactive T Cells

CD40–CD40L costimulatory interactions are crucial for allograft rejection, in that treatment with anti‐CD40L mAb markedly prolongs allograft survival in several systems. Recent reports indicate that costimulatory blockade results in deletion of graft‐reactive cells, which leads to allograft tolerance. To assess immunologic parameters that were influenced by inductive CD40–CD40L blockade, cardiac allograft recipients were treated with multiple doses of the anti‐CD40L mAb MR1, which was remarkably effective at prolonging allograft survival. Acute allograft rejection responses such as IL‐2 producing helper cell priming, Th1 priming, and alloantibody production were abrogated by anti‐CD40L treatment. Interestingly, the spleens of mice bearing long‐term cardiac allografts following inductive anti‐CD40L treatment retained precursor donor alloantigen‐reactive CTL, IL‐2 producing helper cells, and Th1 in numbers comparable to those observed in naïve mice. These mice retained the ability to reject donor‐strain skin allografts, but were incapable of rejecting the original cardiac allograft, or a second donor‐strain cardiac allograft. Further, differentiated effector cells were incapable of mediating rejection following adoptive transfer into mice bearing long‐term allografts, suggesting that regulatory cell function, rather than effector cell deletion was responsible for long‐term graft acceptance. Collectively, these data demonstrate that inductive CD40–CD40L blockade does not result in the deletion of graft‐reactive T cells, but induces the maintenance of these cells in a quiescent precursor state. They further point to a tissue specificity of this hyporesponsiveness, suggesting that not all donor alloantigen‐reactive cells are subject to this regulation.

Contrasting alloreactive CD4+ and CD8+ T cells: There's more to it than MHC restriction

Surface expression of CD4 or CD8 is commonly used to identify T‐cell subsets that recognize antigen presented by class II MHC or class I MHC, respectively. This holds true for T cells that respond to allogeneic MHC molecules that are directly recognized as foreign, as well as peptides from allogeneic MHC molecules that are indirectly presented by self MHC molecules. CD4 or CD8 expression was initially believed to define cytokine secreting helper T cells or cytotoxic cells, respectively. However, this association of phenotype and function is not absolute, in that CD4+ cells may possess lytic activity and CD8+ cells secrete cytokines, notably IFNγ. Recently, additional fundamental differences in the immunobiology of these T‐cell subsets have been identified. These include differences in costimulatory requirements, cytokine responsiveness, cytokine production, cell survival, and the maintenance of memory. This review will survey these differences, emphasizing alloreactive T‐cell responses as well as relevant observations that have been made in other systems.