In this study, we show that IFN-αβ can have a direct role in linking innate and adaptive responses by providing the “third signal” needed by naive CD8 T cells responding to Ag and costimulatory ligands. Stimulation of CD8 T cells in the absence of a third signal leads to proliferation, but clonal expansion is limited by poor survival and effector functions do not develop. We show that IFN-αβ can provide the third signal directly to CD8 T cells via a STAT4-dependent pathway to stimulate survival, development of cytolytic function, and production of IFN-γ. Provision of the third signal by either IFN-αβ or IL-12 results in regulation of the expression of a number of genes, including several that encode proteins critical for effector function.
Stimulation of an effective in vitro or in vivo response by naive CD8 T cells requires three signals: TCR engagement, costimulation/IL-2, and a third signal that can be provided by IL-12. In addition to being required for acquisition of cytolytic function, IL-12 is required for optimal IL-2-dependent proliferation and clonal expansion. In experiments examining in vitro stimulation of naive CD8 T cells, IL-12 is shown to stimulate expression of the IL-2R α-chain (CD25) to much higher levels than are reached in response to just TCR and costimulation and/or IL-2. In addition, high CD25 expression is substantially prolonged in the presence of IL-12. As a consequence, the cells proliferate more effectively in response to low levels of IL-2. Examination of adoptively transferred TCR transgenic CD8 T cells responding to peptide Ag confirmed that IL-12 up-regulates CD25 in vivo, even when B7-mediated costimulation is largely blocked. TCR- and IL-2-dependent proliferation of CD8 T cells from mice deficient in CD25 was also found to increase in the presence of IL-12, indicating that CD25 up-regulation is not the only mechanism by which IL-12 increases clonal expansion of the cells. IL-2 and IL-12 both act to increase expression of both CD25 and the IL-12R, thus providing positive cross-regulation of receptor expression. These results suggest that when cross-priming dendritic cells present class I/Ag and costimulatory ligands, and produce IL-12, naive CD8 T cells will begin to produce IL-2 and both receptors will be optimally up-regulated to insure that an effective response is generated.
When used as therapy for hematopoietic malignancies, allogeneic BM transplantation (BMT) relies on the graftversus-leukemia (GVL) effect to eradicate residual tumor cells through immunologic mechanisms. However, graft-versus-host disease (GVHD), which is initiated by alloreactive donor T cells that recognize mismatched major and/or minor histocompatibility antigens and cause severe damage to hematopoietic and epithelial tissues, is a potentially lethal complication of allogeneic BMT. To enhance the therapeutic potential of BMT, we sought to find therapeutic targets that could inhibit GVHD while preserving GVL and immune responses to infectious agents. We show here that T cell responses triggered in mice by either Listeria monocytogenes or administration of antigen and adjuvant were relatively well preserved in the absence of PKC isoform θ (PKCθ), a key regulator of TCR signaling. In contrast, PKCθ was required for alloreactivity and GVHD induction. Furthermore, absence of PKCθ raised the threshold for T cell activation, which selectively affected alloresponses. Most importantly, PKCθ-deficient T cells retained the ability to respond to virus infection and to induce GVL effect after BMT. These findings suggest PKCθ is a potentially unique therapeutic target required for GVHD induction but not for GVL or protective responses to infectious agents.
Clonal expansion of T cells requires cell division and survival during the proliferative phase of the response. Naive murine CD8 T cells responding to Ag and costimulation undergo an abortive response characterized by impaired clonal expansion, failure to develop effector functions, and long-term tolerance. A third signal provided by IL-12 is required for full expansion, activation, and establishment of memory. The enhanced survival, and thus clonal expansion, supported by IL-12 is not due to increased Bcl-2 or Bcl-xL expression; both are maximally activated by signals 1 and 2. In contrast, Bcl-3, recently shown to enhance survival when ectopically expressed in T cells, is increased only when IL-12 is present. Furthermore, examination of Bcl-3-deficient CD8 T cells demonstrates that the increased survival caused by IL-12 depends upon Bcl-3. The time courses of expression suggest that Bcl-2 and Bcl-xL promote survival early in the response, whereas Bcl-3 acts later in the response.
Protein kinase C- Theta (PKC𝛉) is known to be a key mediator of NF-κB activation downstream of TCR signaling. However, recent in vivo studies with PKC𝛉−/− mice have generated conflicting results regarding its role in T cell activation. To elucidate the role of PKC𝛉 in the activation of CD8 T cells, we generated ovalbumin (ova)-specific PKC𝛉−/− OT1 mice. Stimulation of PKC𝛉−/− OT1 CD8 T cells with microspheres coupled to H2Kb-ova demonstrated a critical function for PKC𝛉 in TCR-induced NF-κB activation and proliferation in vitro. In contrast, ova-induced proliferation and CTL generation in vivo was virtually the same as WT cells. In addition, PKC𝛉−/− CD8 T cells stimulated in vitro with ova-pulsed DC proliferated vigorously and to a similar extent as WT cells. Remarkably, comparable levels of NF-κB were activated in WT and PKC𝛉−/− T cells after stimulation with DC. Proliferation of PKC𝛉−/− cells is severely reduced when DC deficient for B7-1/2 are used to stimulate the cells. Costimulation provided by CD27L also plays a role, as demonstrated by the use of a specific blocking antibody. In contrast to the response against antigen, GVHD responses are severely impaired in the absence of PKC𝛉. These results suggest that impaired TCR/antigen-induced NF-κB activation in PKC𝛉−/− CD8 T cells does not have a significant impact on T cell proliferation when other non-TCR signaling pathways are able to induce NF-κB and T cell activation.
2458 Poster Board II-435 Introduction: When used as therapy for hematopoietic malignances, allogeneic hematopoietic stem cell transplantation (HCT) relies on the graft-versus-leukemia (GVL) effect to eradicate residual tumor cells through immunologic mechanisms. However, graft-versus-host-disease (GVHD) is a potentially lethal complication of allogeneic BMT. Thus, inhibition of GVHD, while preserving GVL and protective responses against infectious agents, can enhance the therapeutic potential of BMT. GVHD is initiated by alloreactive donor T cells that recognize mismatched major and/or minor histocompatibility antigens and cause severe damage to hematopoietic and epithelial tissues. Protein kinase C theta isoform (PKCθ) is crucial in TCR signaling and regulates the nature of lymphocyte-specific effector responses. The aim of this study was to investigate the significance of PKCθ in donor T-cell-mediated GVHD, anti-leukemia, and antiviral infection. Methods: Our results were validated using clinically relevant murine bone marrow transplantation (BMT) models. GVHD was measured by recipient survival and clinical signs such as body weight loss. Leukemia/lymphoma was induced by intravenous injection of a luciferase expressing A20 clone (a B lymphoma cell line derived from BALB/c mice). Tumor invasion was quantitated using the Xenogen IVIS-200 bioluminescence imaging system. Murine cytomegalovirus (MCMV) infection was induced by intraperitoneally injecting MCMV in the recipients after GVHD was established. Results: Using 3 distinct mouse models of allogeneic BMT, we found that T cells lacking PKCθ are unable to undergo robust expansion and cause damage to recipient hematopoietic or epithelial tissues, demonstrating that an essential requirement for PKCθ in alloreactivity and GVHD development. In contrast, PKCθ-/- T cells retain the ability to respond to virus infection post-BMT. Mechanistically, absence of PKCθ raises the threshold for T cell activation that selectively impacts alloresponses, but T cell responses triggered by infection or following administration of antigen plus microbial adjuvant are relatively well preserved in the absence of PKCθ Furthermore, we evaluated the role of PKCθ in GVHD and GVL responses, and found that PKCθ-/- T cells have at least a 10-fold reduction in the ability to induce GVHD but only a ∼2.5-fold reduction in GVL compared to WT T cells. Thus, absence of PKCθ impacts GVHD responses more significantly than GVL responses. Conclusion: These findings validate PKCθ as a potentially unique therapeutic target that is required for GVHD induction but not for GVL or protective responses to infectious agents in allogeneic BMT. Disclosures: No relevant conflicts of interest to declare.
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