Jidong Zhang

Homeostatic proliferation is a barrier to transplantation tolerance

Despite the ease of inhibiting immune responses by blockade of T-cell costimulation in naive rodent models, it is difficult to suppress those responses in animals with memory cells. Studies demonstrating the importance of alloreactive T-cell deletion during tolerance induction have promoted use of peritransplant T-cell-depleting therapies in clinical trials. But potentially complicating wide-scale, nonspecific T-cell depletion is the finding that extensive T-cell proliferation can occur under conditions of lymphopenia. This process, termed homeostatic proliferation, may induce acquisition of functional memory T cells. Here, using clinically relevant mouse models of peripheral T-cell depletion, we show that residual nondepleted T cells undergo substantial homeostatic expansion. In this setting, costimulatory blockade neither significantly suppresses homeostatic proliferation nor prevents allograft rejection. In addition, T cells that have completed homeostatic proliferation show dominant resistance to tolerance when adoptively transferred into wild-type recipients, consistent with known properties of memory cells in vivo. These findings establish the importance of homeostatic proliferation in clinically relevant settings, demonstrate the barrier that homeostatic proliferation can present to the induction of transplantation tolerance, and have important implications for transplantation protocols that use partial or complete peripheral T-cell depletion.

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.

Distinct IL-2 Receptor Signaling Pattern in CD4+CD25 + Regulatory T Cells

Despite expression of the high-affinity IL-2R, CD4+CD25+ regulatory T cells (Tregs) are hypoproliferative upon IL-2R stimulation in vitro. However the mechanisms by which CD4+CD25+ T cells respond to IL-2 signals are undefined. In this report, we examine the cellular and molecular responses of CD4+CD25+ Tregs to IL-2. IL-2R stimulation results in a G1 cell cycle arrest, cellular enlargement and increased cellular survival of CD4+CD25+ T cells. We find a distinct pattern of IL-2R signaling in which the Janus kinase/STAT pathway remains intact, whereas IL-2 does not activate downstream targets of phosphatidylinositol 3-kinase. Negative regulation of phosphatidylinositol 3-kinase signaling and IL-2-mediated proliferation of CD4+CD25+ T cells is inversely associated with expression of the phosphatase and tensin homologue deleted on chromosome 10, PTEN.

Allograft rejection mediated by memory T cells is resistant to regulation

Alloreactive memory T cells may be refractory to many of the tolerance-inducing strategies that are effective against naive T cells and thus present a significant barrier to long-term allograft survival. Because CD4+CD25+ regulatory T cells (Tregs) are critical elements of many approaches to successful induction/maintenance of transplantation tolerance, we used MHC class I and II alloreactive TCR-transgenic models to explore the ability of antigen-specific Tregs to control antigen-specific memory T cell responses. Upon coadoptive transfer into RAG-1−/− mice, we found that Tregs effectively suppressed the ability of naive T cells to reject skin grafts, but neither antigen-unprimed nor antigen-primed Tregs suppressed rejection by memory T cells. Interestingly, different mechanisms appeared to be active in the ability of Tregs to control naive T cell-mediated graft rejection in the class II versus class I alloreactive models. In the former case, we observed decreased early expansion of effector cells in lymphoid tissue. In contrast, in the class I model, an effect of Tregs on early proliferation and expansion was not observed. However, at a late time point, significant differences in cell numbers were seen, suggesting effects on responding T cell survival. Overall, these data indicate that the relative resistance of both CD4+ and CD8+ alloreactive memory T cells to regulation may mediate resistance to tolerance induction seen in hosts with preexisting alloantigen-specific immunity and further indicate the multiplicity of mechanisms by which Tregs may control alloimmune responses in vivo.

Mechanisms Underlying Blockade of Allograft Acceptance by TLR Ligands

Immune activation via TLRs is known to prevent transplantation tolerance in multiple animal models. To investigate the mechanisms underlying this barrier to tolerance induction, we used complementary murine models of skin and cardiac transplantation in which prolonged allograft acceptance is either spontaneous or pharmacologically induced with anti-CD154 mAb and rapamycin. In each model, we found that prolonged allograft survival requires the presence of natural CD4+Foxp3+ T regulatory cells (Tregs), and that the TLR9 ligand CpG prevents graft acceptance both by interfering with natural Treg function and by promoting the differentiation of Th1 effector T cells in vivo. We further demonstrate that although Th17 cells differentiate from naive alloreactive T cells, these cells do not arise from natural Tregs in either CpG-treated or untreated graft recipients. Finally, we show that CpG impairs natural Treg suppressor capability and prevents Treg-dependent allograft acceptance in an IL-6-independent fashion. Our data therefore suggest that TLR signals do not prevent prolonged graft acceptance by directing natural Tregs into the Th17 lineage or by using other IL-6-dependent mechanisms. Instead, graft destruction results from the ability of CpG to drive Th1 differentiation and interfere with immunoregulation established by alloreactive natural CD4+Foxp3+ Tregs.

A reassessment of soluble urokinase-type plasminogen activator receptor in glomerular disease

It has been suggested that soluble urokinase receptor (suPAR) is a causative circulating factor for and a biomarker of focal and segmental glomerulosclerosis (FSGS). Here we undertook validation of these assumptions in both mouse and human models. Injection of recombinant suPAR in wild-type mice did not induce proteinuria within 24 hours. Moreover, a disease phenotype was not seen in an inducible transgenic mouse model that maintained elevated suPAR concentrations for 6 weeks. Plasma and urine suPAR concentrations were evaluated as clinical biomarkers in 241 patients with glomerular disease from the prospective, longitudinal multi-center observational NEPTUNE cohort. The serum suPAR concentration at baseline inversely correlated with estimated glomerular filtration rate (eGFR) and the urine suPAR/creatinine ratio positively correlated with the urine protein/creatinine ratio. After adjusting for eGFR and urine protein, neither the serum nor urine suPAR level was an independent predictor of FSGS histopathology. A multivariable mixed-effects model of longitudinal data evaluated the association between the change in serum suPAR concentration from baseline with eGFR. After adjusting for baseline suPAR concentration, age, gender, proteinuria and time, the change in suPAR from baseline was associated with eGFR, but this association was not different for patients with FSGS as compared to other diagnoses. Thus, these results do not support a pathological role for suPAR in FSGS.

Crk1/2-dependent signaling is necessary for podocyte foot process spreading in mouse models of glomerular disease

The morphology of healthy podocyte foot processes is necessary for maintaining the characteristics of the kidney filtration barrier. In most forms of glomerular disease, abnormal filter barrier function results when podocytes undergo foot process spreading and retraction by remodeling their cytoskeletal architecture and intercellular junctions during a process known as effacement. The cell adhesion protein nephrin is necessary for establishing the morphology of the kidney podocyte in development by transducing from the specialized podocyte intercellular junction phosphorylation-mediated signals that regulate cytoskeletal dynamics. The present studies extend our understanding of nephrin function by showing that nephrin activation in cultured podocytes induced actin dynamics necessary for lamellipodial protrusion. This process required a PI3K-, Cas-, and Crk1/2-dependent signaling mechanism distinct from the previously described nephrin-Nck1/2 pathway necessary for assembly and polymerization of actin filaments. Our present findings also support the hypothesis that mechanisms governing lamellipodial protrusion in culture are similar to those used in vivo during foot process effacement in a subset of glomerular diseases. In mice, podocyte-specific deletion of Crk1/2 prevented foot process effacement in one model of podocyte injury and attenuated foot process effacement and associated proteinuria in a delayed fashion in a second model. In humans, focal adhesion kinase and Cas phosphorylation - markers of focal adhesion complex-mediated Crk-dependent signaling - was induced in minimal change disease and membranous nephropathy, but not focal segmental glomerulosclerosis. Together, these observations suggest that activation of a Cas-Crk1/2-dependent complex is necessary for foot process effacement observed in distinct subsets of human glomerular diseases.

MyD88 Plays a Critical T Cell-Intrinsic Role in Supporting CD8 T Cell Expansion during Acute Lymphocytic Choriomeningitis Virus Infection

During acute lymphocytic choriomeningitis virus (LCMV) infection, CD8 T cells rapidly expand and differentiate into effectors that are required for viral clearance. The accumulation of activated T cells is greatly reduced in mice lacking the adaptor molecule MyD88. Although MyD88 has generally been considered to indirectly regulate adaptive immune responses by controlling inflammatory cytokine production and Ag presentation in innate immune cells, in this study, we identify an unappreciated cell-intrinsic role for MyD88 in LCMV-specific CD8 T cells. Using reciprocal adoptive transfer models and bone marrow chimeras, we show that Myd88−/− CD8 T cells are defective in their clonal expansion in response to LCMV infection, independent of their environment. Furthermore, we show that while MyD88 is dispensable for initial activation and division of LCMV-specific CD8 T cells during the early stages of viral infection, MyD88-dependent signals are critical for supporting their survival and sustained accumulation.

Podocyte-Specific Deletion of Myh9 Encoding Nonmuscle Myosin Heavy Chain 2A Predisposes Mice to Glomerulopathy

ABSTRACT Genome-wide association studies linked single-nucleotide polymorphisms (SNPs) at the MYH9 locus to chronic kidney disease among African-Americans, particularly glomerular diseases such as HIV nephropathy and idiopathic focal and segmental glomerulosclerosis (FSGS). However, these MYH9 SNPs are intronic, and despite extensive sequencing, a causal variant remains elusive. To investigate the role of MYH9 in kidney disease, we selectively deleted Myh9 from mouse podocytes and found that mutant C57BL/6 mice did not develop renal insufficiency or proteinuria compared to control littermates, even when the mice were aged for 9 months. To explain the surprisingly normal phenotype, we considered genetic redundancy with the paralog Myh10 in podocytes, but we found that Myh10 was not expressed in podocytes in Myh9-deficient or control mice. We tested whether Myh9 podocyte deletion predisposed mice to glomerulopathy in response to injury by doxorubicin hydrochloride (Adriamycin), and we found that Myh9 podocyte-deleted mice developed proteinuria and glomerulosclerosis, while control mice were resistant. In summary, Myh9 podocyte deletion in C57BL/6 mice results in susceptibility to experimental doxorubicin hydrochloride glomerulopathy. We review evidence that MYH9 dysfunction in humans results in similar susceptibility and place our data, the first examination of Myh9 kidney disease in experimental animals, in the context of recent findings in human kidney disease, including the role of APOL1.

Regulation of T Cell Activation and Tolerance by Phospholipase Cγ-1-Dependent Integrin Avidity Modulation

Ag receptor engagement without costimulation induces a tolerant state in CD4+ T cells termed anergy. Anergic CD4+ T cells are primarily characterized by the inability to produce IL-2, but the biochemical basis for this functional defect is not completely understood. We demonstrate that primary CD4+ T cells anergized by costimulatory blockade exhibit impaired TCR-coupled phospholipase C (PLC)γ-1 activation. This defect is associated with the marked reduction of multiple downstream signaling events required for IL-2 transcription, including mobilization of intracellular Ca2+ and activation of the mitogen-activated protein kinase cascade. We also found that primary anergic CD4+ T cells fail entirely to modulate their integrin binding avidity in response to TCR stimulation. Integrin avidity modulation is required for full T cell activation and effector function, and as we show in this study, is completely dependent upon PLCγ-1 activity. Finally, analogs that mimic the actions of diacylglycerol and inositol 1,4,5-triphosphate, the immediate products of PLCγ-1 activity, restored integrin avidity modulation and IL-2 production by anergic T cells. Thus, deficient coupling of PLCγ-1 to the TCR appears to be a central biochemical defect that could potentially account for the failure of multiple functional responses in primary anergic CD4+ T cells.