Debbie Watson

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.

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.

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.

“Pruning” of Alloreactive CD4+ T Cells Using 5- (and 6-)Carboxyfluorescein Diacetate Succinimidyl Ester Prolongs Skin Allograft Survival

Removal of alloreactive cells by either thymic deletion or deletion/anergy in the periphery is regarded as crucial to the development of tolerance. Dyes, such as CFSE, that allow monitoring of cell division suggest that in vitro proliferation could be a used as a way of “pruning” alloreactive cells while retaining a normal immune repertoire with retention of memory to previously encountered pathogens. This would overcome the problems occurring as a result of therapies that use massive depletion of T cells to allow acceptance of organ transplants or bone marrow grafts. We therefore used a skin graft model of CD4-mediated T cell rejection across a major H-2 mismatch (C57BL/6 (H-2b) to BALB/c (H-2d) mice) to evaluate whether nondividing CD4+ T cells derived from a mixed lymphocyte culture would exhibit tolerance to a skin graft from the initial stimulator strain. We demonstrate that selective removal of dividing alloreactive CD4+ T cells resulted in marked specific prolongation of allogeneic skin graft survival, and that the nondividing CD4+ T cells retained a broad TCR repertoire and the ability to maintain memory. This novel way of depleting alloreactive T cells may serve as a useful strategy in combination with other mechanisms to achieve transplant tolerance.

Activation of the P2X7 receptor induces the rapid shedding of CD23 from human and murine B cells

Activation of the P2X7 receptor by the extracellular damage‐associated molecular pattern, adenosine 5′‐triphosphate (ATP), induces the shedding of cell surface molecules including the low‐affinity IgE receptor, CD23, from human leukocytes. A disintegrin and metalloprotease (ADAM) 10 mediates P2X7‐induced shedding of CD23 from multiple myeloma RPMI 8226 B cells; however, whether this process occurs in primary B cells is unknown. The aim of the current study was to determine whether P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells. Flow cytometric and ELISA measurements showed that ATP treatment of human and murine B cells induced the rapid shedding of CD23. Treatment of cells with the specific P2X7 antagonist, AZ10606120, near‐completely impaired ATP‐induced CD23 shedding from both human and murine B cells. ATP‐induced CD23 shedding was also impaired in B cells from P2X7 knockout mice. The absence of full‐length, functional P2X7 in the P2X7 knockout mice was confirmed by immunoblotting of splenic cells, and by flow cytometric measurements of ATP‐induced YO‐PRO‐12+ uptake into splenic B and T cells. The broad‐spectrum metalloprotease antagonist, BB‐94, and the ADAM10 antagonist, GI254023X, impaired P2X7‐induced CD23 shedding from both human and murine B cells. These data indicate that P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells and that this process may be mediated by ADAM10.

DNA vaccine encoding CD40 targeted to dendritic cells in situ prevents the development of Heymann nephritis in rats

The CD40-CD154 costimulatory pathway has been shown to be critical for both T- and B-cell activation in autoimmune disease. Here, we assessed the effects of blocking this pathway using CD40 DNA vaccine enhanced by dendritic cell targeting on the development of active Heymann nephritis, a rat model of human membranous nephropathy. DNA vaccination delivers plasmid DNA encoding the target antigen, either alone or in combination with enhancing elements, to induce both humoral and cellular immune responses. To determine whether CD40 DNA vaccine targeting the encoded CD40 directly to dendritic cells would improve the efficacy of the vaccination against self-protein CD40, we utilized a plasmid encoding a single-chain Fv antibody specific for the dendritic cell-restricted antigen-uptake receptor DEC205 (scDEC), the target gene CD40, and the adjuvant tetanus sequence p30. This vaccine plasmid was compared to a control plasmid without scDEC. Rats vaccinated with scDEC-CD40 had significantly less proteinuria and renal injury than did rats receiving scControl-CD40 and were protected from developing Heymann nephritis. Thus, CD40 DNA vaccination targeted to dendritic cells limits the development of Heymann nephritis.

Tolerance induction by removal of alloreactive T cells: in-vivo and pruning strategies

Purpose of reviewCurrent depletion strategies used in clinical transplantation can prevent acute rejection of a transplanted organ; however, they are nonspecific and are limited by their efficacy or the side effects of wide ranging cellular depletion. This review will focus on strategies that prevent rejection of allografts using specific allodepletion of the T cells that mediate rejection. Recent findingsStrategies that use either in-vivo targeting of alloreactive T cells or ex-vivo manipulation to specifically reduce the alloreactive T-cell pool have been developed. The advantage of these approaches is that they are specific, by depleting cells that cause rejection while leaving the remaining immune system intact, thereby minimizing the detrimental complications associated with standard immunosuppression. SummaryStrategies to reduce the proportion of alloreactive T cells that initiate transplant rejection are emphasized. This factor has the specific advantage of leaving the remaining T-cell repertoire intact and may therefore be used in combination with other immune-modulating and tolerance strategies.

Selective depletion of alloreactive T cells leads to long-term islet allograft survival across a major histocompatibility complex mismatch in diabetic mice.

Islet cell transplantation as a therapy for type 1 diabetes has been limited by progressive graft loss. Significant immunosuppression including T-cell ablation has been used in an attempt to limit islet rejection. Here, we show that CD3+ lymphocytes depleted of alloreactive T cells selected from a mixed lymphocyte reaction (MLR), where responder BALB/c splenocytes stained with carboxyfluorescein succinimidyl ester (CFSE) were stimulated with irradiated C57BL/6 splenocytes for 5 days, infused into diabetic immunodeficient mice are capable of restoring a broad T-cell repertoire and specifically do not reject islet transplants from the strain (C57BL/6) used in the original depletion. These mice demonstrate reconstitution with CD4+ and CD8+ T cells, the capacity to reject third-party grafts (CBA), and restoration of interferon-γ (IFN-γ) responses to third-party alloantigens. Over time, both forkhead box P3-positive (Foxp3+) T regulatory cells (Tregs) and γδ T cells expand, suggesting a role for peripheral tolerance, in addition to the initial depletion of alloreactive T cells, in long-term islet graft survival. Our results suggest that immune restoration with CD3+ lymphocytes where alloreactive T cells are removed can restore cognate immunity without islet allograft loss and recurrence of diabetes.

P2X7 antagonism using Brilliant Blue G reduces body weight loss and prolongs survival in female SOD1G93A amyotrophic lateral sclerosis mice

Background Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease characterised by the accumulation of aggregated proteins, microglia activation and motor neuron loss. The mechanisms underlying neurodegeneration and disease progression in ALS are unknown, but the ATP-gated P2X7 receptor channel is implicated in this disease. Therefore, the current study aimed to examine P2X7 in the context of neurodegeneration, and investigate whether the P2X7 antagonist, Brilliant Blue G (BBG), could alter disease progression in a murine model of ALS. Methods Human SOD1G93A transgenic mice, which normally develop ALS, were injected with BBG or saline, three times per week, from pre-onset of clinical disease (62–64 days of age) until end-stage. During the course of treatment mice were assessed for weight, clinical score and survival, and motor coordination, which was assessed by rotarod performance. Various parameters from end-stage mice were assessed as follows. Motor neuron loss and microgliosis were assessed by immunohistochemistry. Relative amounts of lumbar spinal cord SOD1 and P2X7 were quantified by immunoblotting. Serum monocyte chemoattractant protein-1 was measured by ELISA. Splenic leukocyte populations were assessed by flow cytometry. Relative expression of splenic and hepatic P2X7 mRNA was measured by quantitative real-time PCR. Lumbar spinal cord SOD1 and P2X7 were also quantified by immunoblotting in untreated female SOD1G93A mice during the course of disease. Results BBG treatment reduced body weight loss in SOD1G93A mice of combined sex, but had no effect on clinical score, survival or motor coordination. BBG treatment reduced body weight loss in female, but not male, SOD1G93A mice. BBG treatment also prolonged survival in female, but not male, SOD1G93A mice, extending the mean survival time by 4.3% in female mice compared to female mice treated with saline. BBG treatment had no effect on clinical score or motor coordination in either sex. BBG treatment had no major effect on any end-stage parameters. Total amounts of lumbar spinal cord SOD1 and P2X7 in untreated female SOD1G93A mice did not change over time. Discussion Collectively, this data suggests P2X7 may have a partial role in ALS progression in mice, but additional research is required to fully elucidate the contribution of this receptor in this disease.

Matching T-Cell Receptors Identified in Renal Biopsies and Urine at the Time of Acute Rejection in Pediatric Renal Transplant Patients

Urinary monitoring of kidney allograft function has been used for many years. More recently, molecular identification of cytotoxic T‐cell products has been used as a diagnostic tool in acute rejection. Monitoring of T‐cell infiltrates by analysis of the T‐cell receptor (TcR) gene usage has been performed on biopsies with acute and chronic rejection, but not on urine samples. The aim of this study was to identify and compare TRBV gene usage assessing the CDR3 (Complementarity Determining Region 3) length distribution and sequence in urine and biopsies of pediatric renal allograft patients at the time of acute rejection and compare them with peripheral blood. We studied four pediatric renal transplant recipients with acute cellular rejection. We identified restricted and matched TRBV CDR3 spectratypes with overexpressed TRBV families and show identical, clonally expanded TRBV CDR3 sequences in all four patients present in the urine and renal allograft. We demonstrate that urinary monitoring can detect graft‐infiltrating lymphocytes in acute rejection and may have a role in the monitoring of renal transplants.