Andrew E. Gelman

Toll-Like Receptor Ligands Directly Promote Activated CD4+ T Cell Survival

Toll-like receptor (TLR) engagement by pathogen-associated molecular patterns (PAMPs) is an important mechanism for optimal cellular immune responses. APC TLR engagement indirectly enhances activated CD4+ T cell proliferation, differentiation, and survival by promoting the up-regulation of costimulatory molecules and the secretion of proinflammatory cytokines. However, TLRs are also expressed on CD4+ T cells, suggesting that PAMPs may also act directly on activated CD4+ T cells to mediate functional responses. In this study, we show that activated mouse CD4+ T cells express TLR-3 and TLR-9 but not TLR-2 and TLR-4. Treatment of highly purified activated CD4+ T cells with the dsRNA synthetic analog poly(I:C) and CpG oligodeoxynucleotides (CpG DNA), respective ligands for TLR-3 and TLR-9, directly enhanced their survival without augmenting proliferation. In contrast, peptidoglycan and LPS, respective ligands for TLR-2 and TLR-4 had no effect. Enhanced survival mediated by either poly(I:C) or CpG DNA required NF-κB activation and was associated with Bcl-xL up-regulation. However, only CpG DNA, but not poly(I:C)-mediated effects on activated CD4+ T cells required the TLR/IL-1R domain containing adaptor molecule myeloid differentiation factor 88. Collectively, our results demonstrate that PAMPs can directly promote activated CD4+ T cell survival, suggesting that TLRs on T cells can directly modulate adaptive immune responses.

Non-hematopoietic allograft cells directly activate CD8+ T cells and trigger acute rejection : an alternative mechanism of allorecognition

Despite evidence that human non-hematopoietic cells, such as vascular endothelium, can activate allogeneic T lymphocytes in vitro, the prevailing view has been that hematopoietic antigen-presenting cells are required to trigger alloimmune responses in vivo. Here we report that mouse non-hematopoietic cells activate alloreactive CD8+ T lymphocytes in vitro and in vivo. We also show that vascularized cardiac allografts are acutely rejected via CD8+ direct allorecognition even if the alloantigen is not presented by hematopoietic professional antigen-presenting cells. Because activation of alloreactive CD8+ T cells by donor-type non-hematopoietic cells can continue for the life of the allograft, these findings present a new clinically relevant mechanism of allorecognition and should be taken into consideration when developing strategies to prevent allograft vasculopathy or to induce tolerance.

Cutting Edge: Murine Vascular Endothelium Activates and Induces the Generation of Allogeneic CD4+25+Foxp3+ Regulatory T Cells

Unlike graft-resident donor-derived hemopoietic APCs, which decrease in number over time after transplantation, vascular endothelial cells are lifelong residents of a vascularized allograft. Endothelial cells are potent APCs for allogeneic CD8+ T lymphocytes but are unable to induce proliferation of allogeneic CD4+ T lymphocytes. Although the reason for this differential response has been poorly understood, here we report that alloantigen presentation by vascular endothelium to CD4+ T lymphocytes activates and induces CD4+25+Foxp3+ regulatory T cells, which can inhibit proliferation of alloreactive T cells both in vitro and in vivo. This process occurs independently of B7.1 costimulation but is dependent on programmed death ligand 1 (B7-H1). This finding may have important implications for tolerance induction in transplantation.

A Mouse Model of Orthotopic Vascularized Aerated Lung Transplantation

Outcomes after lung transplantation are markedly inferior to those after other solid organ transplants. A better understanding of cellular and molecular mechanisms contributing to lung graft injury will be critical to improve outcomes. Advances in this field have been hampered by the lack of a mouse model of lung transplantation. Here, we report a mouse model of vascularized aerated single lung transplantation utilizing cuff techniques. We show that syngeneic grafts have normal histological appearance with minimal infiltration of T lymphocytes. Allogeneic grafts show acute cellular rejection with infiltration of T lymphocytes and recipient‐type antigen presenting cells. Our data show that we have developed a physiological model of lung transplantation in the mouse, which provides ample opportunity for the study of nonimmune and immune mechanisms that contribute to lung allograft injury.

T cell expression of MyD88 is required for resistance to Toxoplasma gondii

Resistance to Toxoplasma gondii depends on dendritic cells to recognize this pathogen and secrete IL-12, in turn promoting IFN-γ production from responding T cells. The adaptor protein, myeloid differentiation primary-response gene 88 (MyD88), is important for most Toll-like receptor (TLR) signaling, as well as IL-1R/IL-18R signals. There is considerable evidence that MyD88 is required for the innate sensing of T. gondii and IL-12 responses. Although Myd88−/− mice challenged with T. gondii have defective IL-12 and Th1 effector responses and succumb to disease, administration of IL-12 to Myd88−/− mice partially restores the Th1 response and yet fails to prolong survival. This finding suggested that MyD88 may mediate signals within T cells important for resistance to this pathogen. To evaluate the role of MyD88 in T cells under noncompetitive conditions, bone marrow chimeras were generated, in which the T cells lacked MyD88, but MyD88-dependent innate immune responses were intact. Upon challenge with T. gondii, these chimeric mice were more susceptible to disease, developing severe toxoplasmic encephalitis and succumbing within 30 days. Splenocytes and brain mononuclear cells isolated from infected chimeric mice produced less IFN-γ when cultured with a T. gondii-derived antigen. The increase in susceptibility observed was independent of signals via the IL-1R and IL-18R, suggesting a role for TLRs in MyD88-mediated T cell responses to T. gondii. These observations show that, in addition to a role for MyD88 in innate responses, T cell expression of MyD88 is necessary for prolonged resistance to a pathogen.

Cutting edge: acute lung allograft rejection is independent of secondary lymphoid organs

It is the prevailing view that adaptive immune responses are initiated in secondary lymphoid organs. Studies using alymphoplastic mice have shown that secondary lymphoid organs are essential to initiate allograft rejection of skin, heart, and small bowel. The high immunogenicity of lungs is well recognized and allograft rejection remains a major contributing factor to poor outcomes after lung transplantation. We show in this study that alloreactive T cells are initially primed within lung allografts and not in secondary lymphoid organs following transplantation. In contrast to other organs, lungs are acutely rejected in the absence of secondary lymphoid organs. Two-photon microscopy revealed that recipient T cells cluster predominantly around lung-resident, donor-derived CD11c+ cells early after engraftment. These findings demonstrate for the first time that alloimmune responses following lung transplantation are initiated in the graft itself and therefore identify a novel, potentially clinically relevant mechanism of lung allograft rejection.

Mouse Vascular Endothelium Activates CD8+ T Lymphocytes in a B7-Dependent Fashion

Despite several studies examining the contribution of allorecognition pathways to acute and chronic rejection of vascularized murine allografts, little data describing activation of alloreactive T cells by mouse vascular endothelium exist. We have used primary cultures of resting or IFN-γ-activated C57BL/6 (H-2b) vascular endothelial cells as stimulators and CD8+ T lymphocytes isolated from CBA/J (H-2k) mice as responders. Resting endothelium expressed low levels of MHC class I, which was markedly up-regulated after activation with IFN-γ. It also expressed moderate levels of CD80 at a resting state and after activation. Both resting and activated endothelium were able to induce proliferation of unprimed CD8+ T lymphocytes, with proliferation noted at earlier time points after coculture with activated endothelium. Activated endothelium was also able to induce proliferation of CD44low naive CD8+ T lymphocytes. Activated CD8+ T lymphocytes had the ability to produce IFN-γ and IL-2, acquired an effector phenotype, and showed up-regulation of the antiapoptotic protein Bcl-xL. Treatment with CTLA4-Ig led to marked reduction of T cell proliferation and a decrease in expression of Bcl-xL. Moreover, we demonstrate that nonhemopoietic cells such as vascular endothelium induce proliferation of CD8+ T lymphocytes in a B7-dependent fashion in vivo. These results suggest that vascular endothelium can act as an APC for CD8+ direct allorecognition and may, therefore, play an important role in regulating immune processes of allograft rejection.

Bcl3 prevents acute inflammatory lung injury in mice by restraining emergency granulopoiesis

Granulocytes are pivotal regulators of tissue injury. However, the transcriptional mechanisms that regulate granulopoiesis under inflammatory conditions are poorly understood. Here we show that the transcriptional coregulator B cell leukemia/lymphoma 3 (Bcl3) limits granulopoiesis under emergency (i.e., inflammatory) conditions, but not homeostatic conditions. Treatment of mouse myeloid progenitors with G-CSF--serum concentrations of which rise under inflammatory conditions--rapidly increased Bcl3 transcript accumulation in a STAT3-dependent manner. Bcl3-deficient myeloid progenitors demonstrated an enhanced capacity to proliferate and differentiate into granulocytes following G-CSF stimulation, whereas the accumulation of Bcl3 protein attenuated granulopoiesis in an NF-κB p50-dependent manner. In a clinically relevant model of transplant-mediated lung ischemia reperfusion injury, expression of Bcl3 in recipients inhibited emergency granulopoiesis and limited acute graft damage. These data demonstrate a critical role for Bcl3 in regulating emergency granulopoiesis and suggest that targeting the differentiation of myeloid progenitors may be a therapeutic strategy for preventing inflammatory lung injury.

Emergency granulopoiesis promotes neutrophil-dendritic cell encounters that prevent mouse lung allograft acceptance

The mechanisms by which innate immune signals regulate alloimmune responses remain poorly understood. In the present study, we show by intravital 2-photon microscopy direct interactions between graft-infiltrating neutrophils and donor CD11c(+) dendritic cells (DCs) within orthotopic lung allografts immediately after reperfusion. Neutrophils isolated from the airways of lung transplantation recipients stimulate donor DCs in a contact-dependent fashion to augment their production of IL-12 and expand alloantigen-specific IFN-γ(+) T cells. DC IL-12 expression is largely regulated by degranulation and induced by TNF-α associated with the neutrophil plasma membrane. Extended cold ischemic graft storage enhances G-CSF-mediated granulopoiesis and neutrophil graft infiltration, resulting in exacerbation of ischemia-reperfusion injury after lung transplantation. Ischemia reperfusion injury prevents immunosuppression-mediated acceptance of mouse lung allografts unless G-CSF-mediated granulopoiesis is inhibited. Our findings identify granulopoiesis-mediated augmentation of alloimmunity as a novel link between innate and adaptive immune responses after organ transplantation.

Vascular Endothelium Does Not Activate CD4+ Direct Allorecognition in Graft Rejection

Expression of MHC class II by donor-derived APCs has been shown to be important for allograft rejection. It remains controversial, however, whether nonhemopoietic cells, such as vascular endothelium, possess Ag-presenting capacity to activate alloreactive CD4+ T lymphocytes. This issue is important in transplantation, because, unlike hemopoietic APCs, allogeneic vascular endothelium remains present for the life of the organ. In this study we report that cytokine-activated vascular endothelial cells are poor APCs for allogeneic CD4+ T lymphocytes in vitro and in vivo despite surface expression of MHC class II. Our in vitro observations were extended to an in vivo model of allograft rejection. We have separated the allostimulatory capacity of endothelium from that of hemopoietic APCs by using bone marrow chimeras. Hearts that express MHC class II on hemopoietic APCs are acutely rejected in a mean of 7 days regardless of the expression of MHC class II on graft endothelium. Alternatively, hearts that lack MHC class II on hemopoietic APCs are acutely rejected at a significantly delayed tempo regardless of the expression of MHC class II on graft endothelium. Our data suggest that vascular endothelium does not play an important role in CD4+ direct allorecognition and thus does not contribute to the vigor of acute rejection.