Peter T. Jindra

HLA Class I Antibody-Mediated Endothelial Cell Proliferation via the mTOR Pathway

Anti-HLA Abs have been shown to contribute to the process of transplant vasculopathy by binding to HLA class I molecules expressed by the endothelial and smooth muscle cells of the graft and transducing intracellular signals that elicit cell proliferation. The aim of this study was to determine the role of mammalian target of rapamycin (mTOR) in HLA class I-induced endothelial cell proliferation and to explore in depth the relationship between mTOR complexes and their downstream targets following ligation of HLA class I molecules by anti-HLA Abs. We used small interfering RNA technology to abrogate mTOR, rapamycin-insensitive companion of mTOR (rictor), or regulatory associated protein of mTOR (raptor) to study the function of these gene products to activate proteins involved in MHC class I-induced cell proliferation and survival. Knockdown of mTOR inhibited class I-mediated phosphorylation of proteins downstream of mTOR complex 1 and mTOR complex 2. Furthermore, knockdown of mTOR, rictor, or raptor blocked HLA class I-induced endothelial cell proliferation. Long-term pretreatment with the mTOR inhibitor rapamycin significantly blocked both mTOR-raptor and mTOR-rictor complex formation. Interestingly, rapamycin also blocked class I-induced Akt phosphorylation at Ser473 and Bcl-2 expression. These results support the role of anti-HLA Abs in the process of transplant vasculopathy and suggest that exposure of the graft endothelium to anti-HLA Abs may promote proliferation through the mTOR pathway.

Phosphorylated S6 Ribosomal Protein: A Novel Biomarker of Antibody‐Mediated Rejection in Heart Allografts

We tested the hypothesis that phosphorylation of S6 ribosomal protein (S6RP), a downstream target of the PI3K/Akt/mTOR pathway, is a biomarker of antibody‐mediated rejection (AMR) in heart allografts. Primary cultures of human aortic and microvascular endothelial cells (EC) were treated with anti‐HLA class I and class II antibodies (Ab) and cell lysates were studied for phosphorylation of S6 ribosmal protein at Serine235/236 (p‐S6RP). Treatment of cultured EC with anti‐class I and class II Ab stimulated S6RP phosphorylation. Immunohistochemical techniques were used to detect the level of p‐S6RP in endomyocardial biopsies (n = 131) from 46 heart transplant recipients and the results were correlated with histopathological diagnosis of rejection, C4d staining, production of posttransplant anti‐HLA Ab and clinical outcome. Increased phosphorylation of S6RP in endomyocardial biopsies was significantly associated with the diagnosis of AMR (p < 0.0001). No significant association between acute cellular rejection (ACR) and p‐S6RP was observed. C4d staining was positively associated with both AMR and p‐S6RP. Posttransplant anti‐HLA class II Ab production was also significantly associated with a positive p‐S6RP status in cardiac biopsies. These results indicate that p‐S6RP is a useful biomarker for the diagnosis of AMR.

Anti-MHC Class I Antibody Activation of Proliferation and Survival Signaling in Murine Cardiac Allografts

Anti-MHC class I alloantibodies have been implicated in the process of acute and chronic rejection because these Abs can bind to endothelial cells and transduce signals leading to the activation of cell survival and proliferation pathways. To characterize the role of the MHC class I-signaling pathway in the pathogenesis of Ab-mediated rejection, we developed a mouse vascularized heterotopic cardiac allograft model in which B6.RAG1 KO hosts (H-2Kb/Db) received a fully MHC-incompatible BALB/c (H-2Kd/Dd) heart transplant and were passively transfused with anti-donor MHC class I Ab. We demonstrate that cardiac allografts of mice treated with anti-MHC class I Abs show characteristic features of Ab-mediated rejection including microvascular changes accompanied by C4d deposition. Phosphoproteomic analysis of signaling molecules involved in the MHC class I cell proliferation and survival pathways were elevated in anti-class I-treated mice compared with the isotype control-treated group. Pairwise correlations, hierarchical clustering, and multidimensional scaling algorithms were used to dissect the class I-signaling pathway in vivo. Treatment with anti-H-2Kd Ab was highly correlated with the activation of Akt and p70S6Kinase (S6K). When measuring distance as a marker of interrelatedness, multidimensional scaling analysis revealed a close association between members of the mammalian target of rapamycin pathway including mammalian target of rapamycin, S6K, and S6 ribosomal protein. These results provide the first analysis of the interrelationships between these signaling molecules in vivo that reflects our knowledge of the signaling pathway derived from in vitro experiments.

Costimulation-dependent expression of microRNA-214 increases the ability of T cells to proliferate by targeting Pten.

T cell activation requires signaling through the TCR and costimulatory molecules, such as CD28. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally and are also known to be involved in lymphocyte development and function. In this paper, we set out to examine potential roles of miRNAs in T cell activation, using genome-wide expression profiling to identify miRNAs differentially regulated following T cell activation. One of the miRNAs upregulated after T cell activation, miR-214, was predicted to be capable of targeting Pten based on bioinformatics and reports suggesting that it targets Pten in ovarian tumor cells. Upregulation of miR-214 in T cells inversely correlated with levels of phosphatase and tensin homolog deleted on chromosome 10. In vivo, transcripts containing the 3′ untranslated region of Pten, including the miR-214 target sequence, were negatively regulated after T cell activation, and forced expression of miR-214 in T cells led to increased proliferation after stimulation. Blocking CD28 signaling in vivo prevented miR-214 upregulation in alloreactive T cells. Stimulation of T cells through the TCR alone was not sufficient to result in upregulation of miR-214. Thus, costimulation-dependent upregulation of miR-214 promotes T cell activation by targeting the negative regulator Pten. Thus, the requirement for T cell costimulation is, in part, related to its ability to regulate expression of miRNAs that control T cell activation.

Anti-HLA antibodies can induce endothelial cell survival or proliferation depending on their concentration.

Patients exhibiting a humoral immune response to the transplanted organ are at increased risk of antibody-mediated rejection and development of transplant vasculopathy. Historically, antibodies were thought to elicit transplant rejection through complement mediated damage of the endothelium of the graft. More recently, studies from our laboratory and others have shown that antibody ligation of class I molecules on the surface of endothelial cells transduces signals resulting in functional changes including expression of cell survival proteins and cell proliferation. The intracellular events initiated by antibody ligation are dependent upon the degree of molecular aggregation and influenced by the concentration of the antibody and level of human leukocyte antigen (HLA) expression. Herein we describe our recent findings on the effect of molecular aggregation on the class I signaling pathway in human endothelial cells.

RNA interference elucidates the role of focal adhesion kinase in HLA class I-mediated focal adhesion complex formation and proliferation in human endothelial cells.

Ligation of class I molecules by anti-HLA Ab stimulates an intracellular signaling cascade resulting in endothelial cell (EC) survival and proliferation, and has been implicated in the process of chronic allograft rejection and transplant-associated vasculopathy. In this study, we used small interfering RNA blockade of focal adhesion kinase (FAK) protein to determine its role in class I-mediated organization of the actin cytoskeleton, cell survival, and cell proliferation in primary cultures of human aortic EC. Knockdown of FAK appreciably inhibited class I-mediated phosphorylation of Src at Tyr418, p85 PI3K, and Akt at both Thr308 and Ser473 sites. FAK knockdown also reduced class I-mediated phosphorylation of paxillin at Try118 and blocked class I-induced paxillin assembly into focal contacts. FAK small interfering RNA completely abrogated class I-mediated formation of actin stress fibers. Interestingly, FAK knockdown did not modify fibroblast growth factor receptor expression induced by class I ligation. However, FAK knockdown blocked HLA class I-stimulated cell cycle proliferation in the presence and absence of basic fibroblast growth factor. This study shows that FAK plays a critical role in class I-induced cell proliferation, cell survival, and focal adhesion assembly in EC and may promote the development of transplant-associated vasculopathy.

MHC class I and integrin ligation induce ERK activation via an mTORC2-dependent pathway.

The aim of this study was to characterize the interaction between mTOR and ERK in primary endothelial cells (EC) following MHC class I and integrin ligation. Ligation of MHC class I molecules or integrins on the surface of EC leads to phosphorylation of ERK at Thr202/Tyr204. We utilized small interfering RNA (siRNA) blockade of mTOR and proteins involved in mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) to define a relationship between mTOR and ERK following MHC class I signaling. We found mTORC2 was responsible for MHC class I and integrin induced phosphorylation of ERK at Thr202/Tyr204. We corroborated these results demonstrating that long-term exposure to rapamycin also inhibited ERK pathway activation in response to MHC class I signaling. Our results demonstrate, for the first time, that engagement of either MHC class I or integrin on the surface of EC leads to ERK activation through an mTORC2-dependent pathway.

Glomerular mRNA expression of prothrombotic and antithrombotic factors in renal transplants with thrombotic microangiopathy.

Background Thrombotic microangiopathy (TMA) in renal transplants (rTx-TMA) is a serious complication and is usually either recurrent TMA (RecTMA) due to humoral rejection (HR-TMA) or due to calcineurin inhibitor toxicity (CNI-TMA). Although the triggers are known, our knowledge about the thrombogenic transcriptome changes in the microvessels is rudimentary. Methods We examined the expression of several prothrombotic and antithrombotic genes in 25 biopsies with rTx-TMA (6 RecTMA, 9 HR-TMA, and 10 CNI-TMA) and 8 controls. RNA from microdissected glomeruli of paraffin-embedded tissue was isolated and mRNA transcripts were quantified with real-time polymerase chain reaction after preamplification. Results were correlated with clinicopathologic parameters. Results Glomerular mRNA expression of KLF2, KLF4, and tPA was lower and that of PAI-1 was higher in rTx-TMA than in the controls. Glomerular mRNA expression of KLF2 and KLF4 correlated with that of tPA and inversely with that of PAI-1 in rTx-TMA. The mRNA expression of complement regulators CD46 and CD59 were higher in rTx-TMA than in the controls. Only in HR-TMA were glomerular ADAMTS13 and CD55 down-regulated. Conclusions The glomerular capillary bed seems to contribute to all subtypes of rTx-TMA by down-regulation of the endothelial transcription factors KLF2 and KLF4, indicating dedifferentiation with subsequent up-regulation of PAI-1 and down-regulation of tPA, resulting in inhibition of local fibrinolysis. Decreased glomerular expression of ADAMTS13 and CD55 could be an additional pathway toward microthrombosis exclusively in HR-TMA.

Tolerance to MHC class II disparate allografts through genetic modification of bone marrow.

Induction of molecular chimerism through genetic modification of bone marrow is a powerful tool for the induction of tolerance. Here, we demonstrate for the first time that expression of an allogeneic MHC class II gene in autologous bone marrow cells, resulting in a state of molecular chimerism, induces tolerance to MHC class II mismatched skin grafts, a stringent test of transplant tolerance. Reconstitution of recipients with syngeneic bone marrow transduced with retrovirus encoding H-2I-Ab (I-Ab) resulted the long-term expression of the retroviral gene product on the surface of MHC class II-expressing bone marrow-derived cell types. Mechanistically, tolerance was maintained by the presence of regulatory T cells, which prevented proliferation and cytokine production by alloreactive host T cells. Thus, the introduction of MHC class II genes into bone marrow-derived cells through genetic engineering results in tolerance. These results have the potential to extend the clinical applicability of molecular chimerism for tolerance induction.

Induction of transplantation tolerance to fully mismatched cardiac allografts by T cell mediated delivery of alloantigen

Induction of transplantation tolerance has the potential to allow for allograft acceptance without the need for life-long immunosuppression. Here we describe a novel approach that uses delivery of alloantigen by mature T cells to induce tolerance to fully allogeneic cardiac grafts. Adoptive transfer of mature alloantigen-expressing T cells into myeloablatively conditioned mice results in long-term acceptance of fully allogeneic heart transplants without evidence of chronic rejection. Since myeloablative conditioning is clinically undesirable we further demonstrated that adoptive transfer of mature alloantigen-expressing T cells alone into mice receiving non-myeloablative conditioning resulted in long-term acceptance of fully allogeneic heart allografts with minimal evidence of chronic rejection. Mechanistically, tolerance induction involved both deletion of donor-reactive host T cells and the development of regulatory T cells. Thus, delivery of alloantigen by mature T cells induces tolerance to fully allogeneic organ allografts in non-myeloablatively conditioned recipients, representing a novel approach for tolerance induction in transplantation.