Geoff Yu Zhang

Foxp3-Transduced Polyclonal Regulatory T Cells Protect against Chronic Renal Injury from Adriamycin

Chronic proteinuric renal injury is a major cause of ESRD. Adriamycin nephropathy is a murine model of chronic proteinuric renal disease whereby chemical injury is followed by immune and structural changes that mimic human disease. Foxp3 is a gene that induces a regulatory T cell (Treg) phenotype. It was hypothesized that Foxp3-transduced Treg could protect against renal injury in Adriamycin nephropathy. CD4+ T cells were transduced with either a Foxp3-containing retrovirus or a control retrovirus. Foxp3-transduced T cells had a regulatory phenotype by functional and phenotypic assays. Adoptive transfer of Foxp3-transduced T cells protected against renal injury. Urinary protein excretion and serum creatinine were reduced (P<0.05), and there was significantly less glomerulosclerosis, tubular damage, and interstitial infiltrates (P<0.01). It is concluded that Foxp3-transduced Treg cells may have a therapeutic role in protecting against immune injury and disease progression in chronic proteinuric renal disease.

Depletion of γδ T Cells Exacerbates Murine Adriamycin Nephropathy

It has been reported that the presence of gammadelta T cells in kidney is associated with kidney damage in human IgA nephropathy and in rat models of chronic renal injury, including Adriamycin nephropathy (AN), but the functional role of gammadelta T cells in this setting is unknown. This study examined the functional role of gammadelta T cells in tissue injury in a murine model of AN. Murine AN was induced in BALB/c mice by a single injection of Adriamycin. gammadelta T cells as a proportion of CD3(+) T cells were significantly increased in AN kidneys (16.8 +/- 3.9%) but not in lymph nodes (1.3 +/- 0.8%; P < 0.001). The proportion of gammadelta T cells in AN kidney correlated positively with serum creatinine and glomerular sclerosis. The Vgammadelta T cell receptor (TCR) repertoire in kidney showed expansion of a subset of cells that expressed Vgamma6/Vdelta1 genes and that used canonical TCR Vgamma6/Vdelta1 sequences in the CDR3 region of the TCR. gammadelta T cells that were sorted from the kidneys expressed TGF-beta but not IL-4, IL-10, or IFN-gamma. gammadelta T cells also expressed the activating receptor NKG2D and the NKG2D adaptor molecule DAP12. RAE-1, a ligand of NKG2D, was upregulated in AN kidney. Depletion of gammadelta T cells using anti-TCR gammadelta antibody resulted in worsening of serum creatinine, glomerulosclerosis, and interstitial inflammation. These studies indicate the involvement of the gammadelta T cell in innate recognition and regulation of inflammation in AN.

Absence of MyD88 Signaling Induces Donor-Specific Kidney Allograft Tolerance

Toll-like receptors (TLRs) play a fundamental role in innate immunity and provide a link between innate and adaptive responses to an allograft; however, whether the development of acute and chronic allograft rejection requires TLR signaling is unknown. Here, we studied TLR signaling in a fully MHC-mismatched, life-sustaining murine model of kidney allograft rejection. Mice deficient in the TLR adaptor protein MyD88 developed donor antigen-specific tolerance, which protected them from both acute and chronic allograft rejection and increased their survival after transplantation compared with wild-type controls. Administration of an anti-CD25 antibody to MyD88-deficient recipients depleted CD4(+)CD25(+)FoxP3(+) cells and broke tolerance. In addition, defective development of Th17 immune responses to alloantigen both in vitro and in vivo occurred, resulting in an increased ratio of Tregs to Th17 effectors. Thus, MyD88 deficiency was associated with an altered balance of Tregs over Th17 cells, promoting tolerance instead of rejection. This study provides evidence that targeting innate immunity may be a clinically relevant strategy to facilitate transplantation tolerance.

IL-2/IL-2Ab Complexes Induce Regulatory T Cell Expansion and Protect against Proteinuric CKD

Regulatory T cells (Tregs) help protect against autoimmune renal injury. The use of agonist antibodies and antibody/cytokine combinations to expand Tregs in vivo may have therapeutic potential for renal disease. Here, we investigated the effects of administering IL-2/IL-2Ab complexes in mice with adriamycin nephropathy, a model of proteinuric kidney disease that resembles human focal segmental glomerulosclerosis. Injecting IL-2/IL-2Ab complexes before or, to a lesser extent, after induction of disease promoted expansion of Tregs. Furthermore, administration of this complex was renoprotective, evidenced by improved renal function, maintenance of body weight, less histologic injury, and reduced inflammation. IL-2/IL-2Ab reduced serum IL-6 and renal expression of IL-6 and IL-17 but enhanced expression of IL-10 and Foxp3 in the spleen. In vitro, the addition of IL-2/IL-2Ab complexes induced rapid STAT-5 phosphorylation in CD4 T cells. In summary, these data suggest that inducing the expansion of Tregs by administering IL-2/IL-2Ab complexes is a possible strategy to treat renal disease.

CD8+ Regulatory T Cells Induced by T Cell Vaccination Protect Against Autoimmune Nephritis

Autoreactive T cells play a pivotal role in the pathogenesis of autoimmune kidney disease. T cell vaccination (TCV) may limit autoimmune disease and induce CD8+ regulatory T cells (Tregs). We used Heymann nephritis (HN), a rat model of human membranous nephritis, to study the effects of TCV on autoimmune kidney disease. We harvested CD4+ T cells from renal tubular antigen (Fx1A) -immunized rats and activated these cells in vitro to express the MHC Class Ib molecule Qa-1. Vaccination of Lewis rats with these autoreactive Fx1A-induced T cells protected against HN, whereas control-primed T cells did not. Rats that underwent TCV had lower levels of proteinuria and serum creatinine and significantly less glomerulosclerosis, tubular damage, and interstitial infiltrates. Furthermore, these rats expressed less IFN-γ and IL-6 in splenocytes, whereas the numbers of Tregs and the expression of Foxp3 were unchanged. In vitro cytotoxicity assays showed CD8+ T cell-mediated elimination of Qa-1-expressing CD4+ T cells. In vivo, TCV abrogated the increase in Qa-1-expressing CXCR5+ TFH cells observed in HN compared with controls. Taken together, these results suggest that TCV protects against autoimmune kidney disease by targeting Qa-1-expressing autoreactive CD4+ cells.

Infiltrating Foxp3(+) regulatory T cells from spontaneously tolerant kidney allografts demonstrate donor-specific tolerance.

Foxp3+ regulatory T cells (Tregs) have an essential role in immune and allograft tolerance. However, in both kidney and liver transplantation in humans, FOXP3+ Tregs have been associated with clinical rejection. Therefore, the role and function of graft infiltrating Tregs have been of great interest. In the studies outlined, we demonstrated that Foxp3+ Tregs were expanded in tolerant kidney allografts and in draining lymph nodes in the DBA/2 (H‐2d) to C57BL/6 (H‐2b) mouse spontaneous kidney allograft tolerance model. Kidney allograft tolerance was abrogated after deletion of Foxp3+ Tregs in DEpletion of REGulatory T cells (DEREG) mice. Kidney allograft infiltrating Foxp3+ Tregs (K‐Tregs) expressed elevated levels of TGF‐β, IL‐10, interferon gamma (IFN‐γ), the transcriptional repressor B lymphocyte‐induced maturation protein‐1 (Blimp‐1) and chemokine receptor 3 (Cxcr3). These K‐Tregs had the capacity to transfer dominant tolerance and demonstrate donor alloantigen‐specific tolerance to skin allografts. This study demonstrated the crucial role, potency and specificity of graft infiltrating Foxp3+ Tregs in the maintenance of spontaneously induced kidney allograft tolerance.

Regulatory T cells participate in CD39‐mediated protection from renal injury

CD39 is an ecto‐enzyme that degrades extracellular nucleotides, such as ATP, and is highly expressed on by the vasculature and circulating cells including Foxp3+ regulatory T (Treg) cells. To study the role of purinergic regulation in renal disease, we used the adriamycin nephropathy (AN) mouse model of chronic renal injury, using human CD39‐transgenic (hCD39Tg) and wild‐type (WT) BALB/c mice. Effects of CD39 expression by Treg cells were assessed in AN by adoptive transfer of CD4+CD25+ and CD4+CD25− T cells isolated from hCD39Tg and WT mice. hCD39Tg mice were protected from renal injury in AN with decreased urinary protein and serum creatinine, and significantly less renal injury compared with WT mice. While WT CD25+ and hCD39Tg CD25− T cells conferred some protection against AN, hCD39Tg CD25+ Treg cells offered greater protection. In vitro studies showed direct pro‐apoptotic effects of ATP on renal tubular cells. In conclusion, hCD39 expressed by circulating leukocytes and intrinsic renal cells limits innate AN injury. Specifically, CD39 expression by Treg cells contributes to its protective role in renal injury. These findings suggest that extracellular nucleotides mediate AN kidney injury and that CD39, expressed by Treg cells and other cells, is protective in this model.

Foxp3 as a marker of tolerance induction versus rejection.

Purpose of reviewFoxp3 is the transcription factor that induces the regulatory T cell phenotype. This review will examine issues around Foxp3 induction and function as well as clinical data on tolerance and rejection. Recent findingsRecent findings have included identification of the signals that drive naive T lymphocytes to express Foxp3 in the thymus and the signals peripherally that induce non-Foxp3 expressing T cells to express FOXP3. Further, the identification of the downstream targets of Foxp3 both by analysis of Foxp3 expressing cells and by analysis of gene promoters that bind Foxp3 has provided new insights into its function. Whereas Foxp3 T regulatory cells (Tregs) are associated with tolerance in a variety of animal transplant models, the human data show expansion of Foxp3 Tregs associated with rejection, though Tregs are also found in transplants in patients with mixed chimerism-induced tolerance. Further, there is a significant difference in the effect of the different immunosuppressive medications on Treg function and expansion that may be important in developing strategies to enhance Tregs in human trials. ConclusionFoxp3 CD4 T cells are frequently associated with rejection; however, this does not preclude their protective role and importance in tolerance induction.

Long-Term Cardiac Allograft Survival across an MHC Mismatch after “Pruning” of Alloreactive CD4 T Cells

Specific tolerance to allografts has been achieved by a variety of means. We have previously shown that ex vivo removal of dividing CD4+ T cells from an MLR or “pruning” delays skin allograft rejection. We tested pruning of alloreactive T cells as a strategy for retaining a broad T cell repertoire while removing alloreactive T cells in a model of cardiac allograft transplant. Using CFSE staining of responder BALB/c cells with stimulator C57BL/6 cells in an MLR, SCID mice were reconstituted with either dividing (D) or nondividing (ND) CD4+ T cells derived from an MLR and then challenged with heterotopic cardiac allografts. Mice reconstituted with D CD4+ T cells rejected cardiac allografts from the stimulator strain with a median survival time (MST) of 29 days, while mice reconstituted with ND CD4+ T cells maintained allografts from the stimulator strain (MST of >100 days) while rejecting third-party allografts (B10.BR) (MST = 11 days). ELISPOT assays demonstrate donor-specific hyporesponsiveness of the ND CD4+ T cells. TCR β-chain V region (TRBV) repertoire analysis demonstrates clonal expansion within both rejecting D cardiac allografts and ND cardiac allografts surviving for the long-term. Histology showed greater allograft infiltration by the D CD4+ T cells. The surviving ND cardiac allografts demonstrated reduced cellular infiltration and reduced incidence of allograft vasculopathy, but with the development of chronic fibrosis. Thus, pruning of alloreactive T cells allows long-term-specific cardiac allograft survival while retaining the ability to reject third-party allografts.

Development and function of Foxp3+ regulatory T cells

Regulatory T cells (Tregs) have been recognized as having a major role in maintaining peripheral tolerance and preventing and limiting autoimmune and chronic inflammatory diseases. Tregs derive from the thymus and also develop peripherally. In this review, we discuss recent progress in our understanding of the basic mechanisms involved in Treg development and function in protecting against autoimmunity in the periphery, including thymic selection, peripheral induction and the many mechanisms of Treg suppression. Specifically in kidney disease, Tregs have been shown to play a role in limiting injury and may potentially have a therapeutic role.