Jean Villard

The proinflammatory cytokines IL-2, IL-15 and IL-21 modulate the repertoire of mature human natural killer cell receptors

Natural killer (NK) cells play a crucial role in the immune response to micro-organisms and tumours. Recent evidence suggests that NK cells also regulate the adaptive T-cell response and that it might be possible to exploit this ability to eliminate autoreactive T cells in autoimmune disease and alloreactive T cells in transplantation. Mature NK cells consist of a highly diverse population of cells that expresses different receptors to facilitate recognition of diseased cells and possibly pathogens themselves. Ex vivo culture of NK cells with cytokines such as IL-2 and IL-15 is an approach that permits significant expansion of the NK cell subpopulations, which are likely to have potent antitumour, antiviral, or immunomodulatory effects in autoimmunity. Our data indicate that the addition of IL-21 has a synergistic effect by increasing the numbers of NK cells on a large scale. IL-2 and IL-15 may induce the expression of killer cell immunoglobulin-like receptors (KIRs) in KIR-negative populations, the c-lectin receptor NKG2D and the natural cytotoxic receptor NKp44. The addition of IL-21 to IL-15 or IL-2 can modify the pattern of the KIR receptors and inhibit NKp44 expression by reducing the expression of the adaptor DAP-12. IL-21 also preserved the production of interferon-γ and enhanced the cytotoxic properties of NK cells. Our findings indicate that the proinflammatory cytokines IL-2, IL-15 and IL-21 can modify the peripheral repertoire of NK cells. These properties may be used to endow subpopulations of NK cells with specific phenotypes, which may be used in ex vivo cellular immunotherapy strategies.

Successful treatment of recurrent focal segmental glomerulosclerosis after kidney transplantation by plasmapheresis and rituximab

A 22‐year‐old patient whose primary kidney disease was focal segmental glomerulosclerosis (FSGS) developed severe recurrence of proteinuria (up to 57 g/24 h) immediately after a haploidentic living donor kidney transplantation despite pre‐operative plasmapheresis. The immunosuppressive treatment consisted of tacrolimus, mycophenolate mofetil, basiliximab and steroids. He underwent 10 plasmapheresis sessions in the first 3‐week post‐transplantation. In addition, he received 2 i.v. doses of rituximab (RTX) 600 mg (375 mg/m2) on days 7 and 15. Proteinuria decreased below nephrotic range at day 14 and serum creatinine returned progressively to normal values. A short course of oral ciclophosphamide (100 mg/j) was administrated between days 22 and 40 and three additional plasmapheresis sessions on days 34, 39 and 49. This strategy allowed obtaining sustained full remission of the nephrotic syndrome (NS) and excellent graft function, which persists over 2 years after transplantation. No notable adverse events related to RTX or plasmapheresis were observed. This case suggests that RTX associated with plasmapheresis may be an effective treatment of recurrent NS because of FSGS.

De novo anti-HLA antibody after pandemic H1N1 and seasonal influenza immunization in kidney transplant recipients

In solid organ transplanted patients, annual influenza immunization is strongly recommended because of morbidity and mortality of influenza infections. In 2009, the rapid spread of a novel H1N1 influenza A virus led to the accelerated development of novel pandemic influenza vaccines. In Switzerland, the recipients received one dose of seasonal influenza and two doses of AS03‐adjuvanted H1N1 vaccines. This situation provided a unique opportunity to analyze the influence of novel adjuvanted influenza vaccines on the production of de novo anti‐HLA antibodies. We prospectively followed two independent cohorts including 92 and 59 kidney‐transplanted patients, assessing their anti‐HLA antibodies before, 6 weeks and 6 months after vaccination. Sixteen of 92 (17.3%) and 7 of 59 (11.9%) patients developed anti‐HLA antibodies. These antibodies, detected using the single antigen beads technology, were mostly at low levels and included both donor‐specific and non‐donor‐specific antibodies. In 2 of the 20 patients who were followed at 6 months, clinical events possibly related to de novo anti‐HLA antibodies were observed. In conclusion, multiple doses of influenza vaccine may lead to the production of anti‐HLA antibodies in a significant proportion of kidney transplant recipients. The long‐term clinical significance of these results remains to be addressed.

Donor-specific antibody levels and three generations of crossmatches to predict antibody-mediated rejection in kidney transplantation.

Background. This study evaluated the prognostic impact of pretransplant donor-specific anti-human leukocyte antigen antibodies (DSA) detected by single-antigen beads and compared the three generations of crossmatch (XM) tests in kidney transplantation. Methods. Thirty-seven T-cell complement-dependent cytotoxicity crossmatch (CXM) negative living donor kidney recipients with a retrospectively positive antihuman leukocyte antigen antibody screening assay were included. A single-antigen bead test, a flow cytometry XM, and a Luminex XM (LXM) were retrospectively performed, and the results were correlated with the occurrence of antibody-mediated rejections (AMRs) and graft function. Results. We found that (1) pretransplant DSA against class I (DSA-I), but not against class II, are predictive for AMR, resulting in a sensitivity of 75% and a specificity of 90% at a level of 900 mean fluorescence intensity (MFI); (2) with increasing strength of DSA-I, the sensitivity for AMR is decreasing to 50% and the specificity is increasing to 100% at 5200 MFI; (3) the LXM for class I, but not for class II, provides a higher accuracy than the flow cytometry XM and the B-cell CXM. The specificity of all XMs is increased greatly in combination with DSA-I values more than or equal to 900 MFI. Conclusions. In sensitized recipients, the best prediction of AMR and consecutively reduced graft function is delivered by DSA-I alone at high strength or by DSA-I at low strength in combination with the LXM or CXM.

Lessons from the bare lymphocyte syndrome: molecular mechanisms regulating MHC class II expression

Major histocompatibility complex class II (MHCII) molecules drive the development, activation and homeostasis of CD4* T-helper cells. They play a central role in key processes of the adaptive immune system, such as the generation of T-cell-mediated immune responses, the regulation of antibody production and the development and maintenance of tol erance. It is thus not surprising that the absence of MHCII expression results in a severe primary immunodeficiency disease (the bare lymphocyte syndrome (BLS)). The genetic defects responsible for BLS do not lie within the MHCII locus, but in genes encoding transcription factors required for MHCII expression. A great deal of our current knowledge about the mechanisms regulating expression of MHCII genes has been derived from the study of BLS. Four different MHCII regulatory genes have been identified. These genes encode RFXANK, RFXS, RFXAP and CIITA. The first three are subunits of RFX, a ubiquitously expressed factor that binds to the promoters of all MHCII genes. RFX binds co-operatively with other factors to form a highly stable multiprotein complex referred to as the MHCII enhanceosome. This enhanceosome serves as a landing pad for the co-activator CIITA, which is recruited via protein-protein interactions CIITA is the master control factor for MHCII expression. The highly regulated expression pattern of CIITA ultimately dictates the cell type specificity, induction and level of MHCII expression.

Eculizumab in acute recurrence of thrombotic microangiopathy after renal transplantation

Renal thrombotic microangiopathy (TMA) is a severe complication of systemic lupus erythematosus (SLE), which is associated with the presence of antiphospholipid (aPL) antibodies. In its most fulminant form, TMA leads to a rapid and irreversible end‐stage renal failure. Eculizumab, an anti‐C5 monoclonal antibody, is a novel therapy of choice for patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome. Here, we report the case of a 27‐year‐old woman, known for SLE and end‐stage renal disease due to fulminant TMA. Both aPL antibodies and antinucleosome antibodies were positive. The patient underwent a living‐related kidney transplantation with immediate production of urine. Although serum creatinine was remaining high, a graft biopsy, performed on day 6, demonstrated a TMA recurrence. Despite a treatment with plasma exchange, the situation got worse and dialysis was started. Eculizumab treatment was subsequently administered and renal function improved rapidly. Three months after transplantation, serum creatinine was at 100 μmol/L, without proteinuria. This case illustrates the benefit of eculizumab therapy in a fulminant recurrence of TMA after kidney transplantation, resistant to classical therapy.

Natural killer cell receptor repertoire and their ligands, and the risk of CMV infection after kidney transplantation.

Cytomegalovirus (CMV) infection is the most common viral complication after solid organ transplantation (SOT). Whilst current immunosuppression is known to impair antiviral‐specific T‐cell immunity in SOT, a potential role for natural killer (NK) cells not affected by immunosuppressive therapy remains to be determined. To address this, we compared the genotype of the NK immunoglobulin‐like receptor (KIR) genes and their HLA cognate ligands to the rate of CMV infection in 196 kidney transplant recipients. We have shown that the absence of the HLA‐C ligand for inhibitory KIR and the presence of activating KIR genes in the recipients were both associated with a lower rate of CMV infection after transplantation. In a cohort of 17 recipients with acute CMV infection, NK cells were phenotyped over a period of time after diagnosis by their expression profile of C‐type lectin receptors and capacity to secrete IFN‐γ. The increased expression of the activating C‐type lectin receptors NKG2C and NKG2D was paralleled by the decreased IFN‐γ secretion during the early phase of CMV infection. In conclusion, our findings suggest that KIR/HLA genotype and expression of NKG2C and NKG2D might play a significant role in regulating NK cell function and anti‐CMV immunity after kidney transplantation.

Neural progenitors derived from human embryonic stem cells are targeted by allogeneic T and natural killer cells.

Neural progenitor cells (NPC) of foetal origin or derived from human embryonic stem cells (HESC) have the potential to differentiate into mature neurons after transplantation into the central nervous system, opening the possibility of cell therapy for neurodegenerative disorders. In most cases, the transplanted NPC are genetically unrelated to the recipient, leading to potential rejection of the transplanted cells. Very few data provide reliable information as to the potential immune response of allogeneic neural progenitors derived from HESC. In this study, we analyzed in vitro the allogeneic immune response of T lymphocytes and natural killer (NK) cells to NPC derived from HESC or of foetal origin. We demonstrate that NPC induce T‐cell stimulation and a strong NK cytotoxic response. NK‐cell activity is unrelated to MHC‐I expression but driven by the activating NKG2D receptor. Cyclosporine and dexamethasone previously used in clinical studies with foetal NPC did not only fail to prevent NK alloreactivity but strongly inhibited the terminal maturation from NPC into mature neurons. We conclude that allogenic transplantation of NPC in the central nervous system will most likely require an immunosuppressive regimen targeting allogenic T and NK cells, whereas possible interference with the differentiation of NPC needs to be carefully evaluated.

A Functionally Essential Domain of RFX5 Mediates Activation of Major Histocompatibility Complex Class II Promoters by Promoting Cooperative Binding between RFX and NF-Y

ABSTRACT Major histocompatibility complex class II (MHC-II) molecules occupy a pivotal position in the adaptive immune system, and correct regulation of their expression is therefore of critical importance for the control of the immune response. Several regulatory factors essential for the transcription of MHC-II genes have been identified by elucidation of the molecular defects responsible for MHC-II deficiency, a hereditary immunodeficiency disease characterized by regulatory defects abrogating MHC-II expression. Three of these factors, RFX5, RFXAP, and RFXANK, combine to form the RFX complex, a regulatory protein that binds to the X box DNA sequence present in all MHC-II promoters. In this study we have undertaken a dissection of the structure and function of RFX5, the largest subunit of the RFX complex. The results define two distinct domains serving two different essential functions. A highly conserved N-terminal region of RFX5 is required for its association with RFXANK and RFXAP, for assembly of the RFX complex in vivo and in vitro, and for binding of this complex to its X box target site in the MHC-II promoter. This N-terminal region is, however, not sufficient for activation of MHC-II expression. This requires an additional domain within the C-terminal region of RFX5. This C-terminal domain mediates cooperative binding between the RFX complex and NF-Y, a transcription factor binding to the Y box sequence of MHC-II promoters. This provides direct evidence that RFX5-mediated cooperative binding between RFX and NF-Y plays an essential role in the transcriptional activation of MHC-II genes.

Long-term insulin-independence after allogeneic islet transplantation for type 1 diabetes: over the 10-year mark

Results of islet of Langerhans transplantation have markedly improved in recent years, but most patients still lose insulin independence in the long‐term. We report herein the longest (over 11 years) case of insulin independence after allogeneic islet transplantation. The subject had a 27‐year history of type 1 diabetes and received a single islet‐after‐kidney graft of 8800 islet equivalents (IEQ)/kg, pooled from 2 donors. Insulin was discontinued by 3 months posttransplant and the patient has remained off insulin ever since. Yearly follow‐up studies have revealed normal metabolic control, including normal oral glucose tolerance test (OGTT). Reasons for success may involve choice of immunosuppression, low metabolic demand and low immune responsiveness as suggested by an excellent HLA matching and a high count of circulating regulatory T cells. This observation is so far an exceptional case, but clearly demonstrates the validity of the concept that long‐term insulin independence after allogeneic islet transplantation is an achievable target.