Decreased IL-10(+) regulatory B cells (Bregs) in lupus nephritis patients.

OBJECTIVES: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by B cell-dependent autoantibody production. Recently, a new B-cell subset was discovered that has a regulatory capacity. The aim of this study was to analyse regulatory B cells (Bregs) in SLE patients. METHOD: Peripheral mononuclear blood cells (PBMCs) of 34 SLE patients fulfilling the American College of Rheumatology (ACR) criteria for SLE and 21 healthy controls (HC) were included. PBMCs were stained for CD19, CD24, and CD38 and analysed by flow cytometry. In vitro stimulated PBMCs with CpG and restimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin were investigated for IL-10(+) Bregs . RESULTS: The percentages of circulating CD19(+)CD24(hi)CD38(hi) cells in HC were not different those in from SLE patients. The percentages of IL-10(+) Bregs were significantly decreased in SLE patients, in particular those with lupus nephritis (LN), compared to HC. The proportion was independent of disease activity. CONCLUSIONS: This is the first study to demonstrate a decrease in IL-10-producing B cells in LN patients compared to HC, reflecting an impaired regulatory function.

Peripherally circulating CD4⁺ FOXP3⁺ CXCR3⁺ T regulatory cells correlate with renal allograft function.

Peripheral immunoregulation depends on T regulatory cell trafficking into the allograft to modulate the local alloresponse. Little is known about the relevance of trafficking receptors for Tregs after solid organ transplantation in humans. In this study, expression of the peripheral chemokine receptors CXCR3 and CCR5 on CD4⁺ FOXP3⁺ Treg cells was analysed and correlated with allograft function in renal transplant recipients. Flow cytometry analysis of peripheral blood mononuclear cells of 54 renal transplant recipients receiving a calcineurin inhibitor-based immunosuppression was performed for CD4, CD25, FOXP3, CXCR3 and CCR5 within the first 18 months post-transplantation. Correlation analysis of chemokine receptor expression and glomerular filtration rate as calculated by MDRD (eGFR) was performed. Expression of the peripheral homing receptors CXCR3 (r = 0.44, P < 0.05) and CCR5 (r = 0.45, P < 0.05) on FOXP3⁺ Tregs correlated with renal allograft function (eGFR) in patients receiving tacrolimus (n = 28), but not cyclosporine A (CsA) (n = 26). CsA but not tacrolimus reduced surface expression of CXCR3 on FOXP3⁺ Tregs in renal transplant recipients as correlated to trough levels (r = -0.42, P < 0.05). In contrast to CD4⁺ CXCR3⁺ CD25(lo) T cells, flow-sorted CD4⁺ CXCR3⁺ CD25(hi) Tregs isolated from healthy individuals did not produce IFNγ or IL-17 ex vivo and expressed high levels of GARP mRNA both at baseline as well as after TCR activation indicating functional regulatory activity. Expression of the peripheral trafficking receptors CXCR3 and CCR5 on FOXP3⁺ Tregs is associated with renal allograft function. These results suggest that Treg trafficking may also depend on the interaction of CXCR3 or CCR5 and their respective ligands.

Cyclosporin but not everolimus inhibits chemokine receptor expression on CD4+ T cell subsets circulating in the peripheral blood of renal transplant recipients.

The peripheral chemokine receptors chemokine receptor 3 (CXCR3) and CC chemokine receptor 5 (CCR5) have been reported to be associated with allograft rejection. The impact of the expression of immunosuppressive drugs on peripherally circulating CD4(+) T cell subsets after renal transplantation is unknown. Expression of CXCR3 and CCR5 was investigated by flow cytometry in 20 renal allograft recipients participating in a prospective, randomized trial (NCT00514514). Initial immunosuppression consisted of basiliximab, cyclosporin A (CsA), mycophenolate sodium and corticosteroids. After 3 months, patients were treated either with CsA, mycophenolate sodium (MPA) plus corticosteroids (n = 6), CsA and everolimus plus corticosteroids (n =8) or CsA-free (CsA(free)) receiving everolimus, MPA and corticosteroids (n = 6). After initial reduction of CD4(+) forkhead box protein 3 (FoxP3)(+) and CD4(+) CD25(hi) FoxP3(+) regulatory T cells (T(regs)) (P < 0.05; P < 0.01), 3-month post-transplant percentages of T(regs) were reconstituted in CsA(free) and CsA(lo) arms compared to CsA(reg) 12 months post transplant. Expression of CCR5 and CXCR3 on CD4(+) FoxP3(+) and CD4(+) FoxP3(-) T cells 12 months post transplant was increased in CsA(free) versus CsA(reg). Increase in CCR5(+) CXCR3(+) co-expressing CD4(+) FoxP3(-) cells between 3 and 12 months correlated negatively with the glomerular filtration rate (GFR) slope/year [modification of diet in renal disease (MDRD); r = -0.59, P < 0.01]. CsA, but not everolimus, inhibits both T(reg) development and expression of CXCR3 and CCR5 on CD4(+) T cell subsets. Increase in CCR5(+) CXCR3(+) co-expressing CD4(+) FoxP3(-) T cells is associated with early loss in allograft function.

mTOR-understanding the clinical effects.

The target of rapamycin (TOR) is a highly conserved serine/threonine kinase that controls cell growth and metabolism in response to nutrients, growth factors, cellular energy, and stress. The TOR kinase, which was originally discovered in yeast, is also expressed in human cells as mammalian TOR (mTOR). In this review, we focus on how mTOR-inducible signals function in cell protection and cell survival of effector and regulatory T cells as well as its role in endothelial cell biology. We evaluate how signaling is important for vascular endothelial cell growth, survival, and proliferation; and we consider how the function of mTOR in endothelial cells may be clinically important in the rejection process. Understanding the biology of mTOR allows clinicians to use mTOR inhibitors optimally as therapeutics following solid organ transplantation.

Immigrating progenitor cells contribute to human podocyte turnover.

Podocyte depletion is a critical event in glomerular diseases in general and in the development of focal segmental glomerulosclerosis in particular. Progenitor cell immigration is a possible mechanism of podocyte replacement for the preservation of nephron function since, with rare exception, mature podocytes are thought to be incapable of replication. We examined eight paraffin-embedded renal biopsies from six male recipients of female transplant kidneys for receiver-derived podocytes. Fluorescent in situ hybridization for the Y chromosome was combined with immunofluorescence for the podocyte marker, Wilms tumor-1 antigen. Recipient-derived podocytes were found in 4 of 8 biopsies representing 3 of the 6 patients. Overall, 5 of the 740 podocytes examined in the female-donated kidneys were male derived. Our study suggests that immigrating progenitor cells are able to replace podocytes in humans; however, the importance of this process in physiologic and pathologic conditions is unknown.

Polymorphisms in the non-coding region of the human mitochondrial genome in unrelated plateletapheresis donors.

Human mitochondrial DNA polymorphisms are unique targets to discriminate nucleated cells and platelets between donor and recipient in the setting of transplantation or transfusion. We have previously used this approach to discriminate allogeneic platelets from autologous platelets after transfusion. In the present study, we used DNA sequencing to investigate polymorphisms present in two of the hypervariable segments (HVR1 and HVR2) found within the non-coding region of the mitochondrial genome among 100 plateletapheresis donors. Alignments were made with the Cambridge Reference Sequence (CRS) for human mitochondrial DNA (mtDNA). Combining the sequencing information of HVR1 and HVR2 we could demonstrate that, of the 100 investigated mtDNA samples, none was identical to the CRS. We found a total of 2-17 polymorphisms per donor in the investigated regions, most of them were basepair substitutions (563) and insertions (151). No deletions were found. Sixty-six of the 110 detected polymorphisms were detected in more than one sample. Seven polymorphisms are newly described and have not been published in the Mitomap database. Our results demonstrate that polymerase chain reaction analysis of the many polymorphisms found in the hypervariable region of mitochondrial DNA represents a more informative target than previously described mitochondrial polymorphisms for discriminating donor-recipient cells after transfusion or transplantation.

Identifying allogeneic platelets by resolution of point mutations in mitochondrial DNA using single-stranded conformational polymorphism PCR.

BACKGROUND: The purpose of this study was to evaluate single-stranded conformational polymorphism (SSCP)-PCR utilizing two different regions of mitochondrial DNA (mtDNA) as a method to discriminate between donor platelets and recipient cells. STUDY DESIGN AND METHODS: Twenty-eight mixtures of platelets (1:1 ratio) were prepared from eight randomly selected persons to simulate donor-recipient combinations after allogeneic platelet transfusion. The mtDNA was extracted from each donor and each prepared mixture. Four primer pairs were designed to amplify two regions of mtDNA, hypervariable region (HVR) 1 and 2. An SSCP-PCR method was developed to analyze the four different amplicons. In addition, the amplified DNA samples containing HVR1 and HVR2 mtDNA of the eight persons were sequenced by using dye-terminator cycle sequencing to determine mtDNA polymorphisms. RESULTS: With four different primer pairs and SSCP-PCR, it was possible to discriminate between donor and recipient DNA in all 28 combinations. DNA sequencing confirmed that the suspected differences were localized within the amplicons examined by SSCP-PCR. CONCLUSION: SSCP-PCR analysis targeting the HVR1 and HVR2 mtDNA is a promising new method to potentially identify donor cells on the basis of mtDNA polymorphisms. The method does not require prior knowledge of sequence differences between donor and recipient and can be optimized to quantify the amount of residual transfused allogeneic platelets.