B cells in operational tolerance.

Transplantation is currently the therapy of choice for endstage organ failure even though it requires long-term immunosuppresive therapy, with its numerous side effects, for acceptance of the transplanted organ. In rare cases however, patients develop operational tolerance, that is, graft survival without immunosuppression. Studies conducted on these patients reveal genetic, phenotypic, and functional signatures. They provide a better understanding of the immunological mechanisms involved in operational tolerance and define biomarkers that could be used to adapt immunosuppressive treatment to the individual, safely reduce immunosuppression doses, and ideally and safely guide immunosuppression withdrawal. This review summarizes studies that suggest a role for B cells as biomarkers of operational tolerance and discusses the use of B cells as a predictive tool for immunologic risk.

Decrease of blood anti-α1,3 Galactose Abs levels in multiple sclerosis (MS) and clinically isolated syndrome (CIS) patients.

The etiology of multiple sclerosis (MS) remains elusive. Among the possible causes, the increase of anti-Neu5Gc antibodies during EBV primo-infection of Infectious mononucleosis (IMN) may damage the integrity of the blood-brain barrier facilitating the transfer of EBV-infected B cells and anti-EBV T cell clones in the brain. We investigated the change in titers of anti-Neu5Gc and anti-α1,3 Galactose antibodies in 49 IMN, in 76 MS, and 73 clinically isolated syndrome (CIS) patients, as well as age/gender-matched healthy individuals. Anti-Gal and anti-Neu5Gc are significantly increased during IMN (p=0.02 and p<1.10-4 respectively), but not in acute CMV primo-infection. We show that, whereas there was no change in anti-Neu5Gc in MS/CIS, the two populations exhibit a significant decrease in anti-Gal (combined p=2.7.10-3), in contrast with patients with non-MS/CIS central nervous system pathologies. Since anti-Gal result from an immunization against α1,3 Gal, lacking in humans but produced in the gut, our data suggest that CIS and MS patients have an altered microbiota or an altered response to this microbiotic epitope.

Renal Operational Tolerance Is Associated With a Defect of Blood Tfh Cells That Exhibit Impaired B Cell Help.

Renal operationally tolerant patients (TOL) display a defect in B cell differentiation, with a deficiency in plasma cells. Recently described, T follicular helper (Tfh) cells play a critical role in B cell differentiation. We analyzed blood Tfh subsets in TOL and transplanted patients with stable graft function under immunosuppression (STA). We observed a reduced proportion of blood activated and highly functional Tfh subsets in TOL, without affecting Tfh absolute numbers. Functionally, Tfh cells from TOL displayed a modified gene expression profile, failed to produce interleukin-21, and were unable to induce IgG production by naive B cells. This Tfh defect is linked to a low incidence of postgraft de novo donor-specific antibody (dnDSA) immunization, suggesting that the lack of Tfh cells in TOL may induce a protolerogenic environment with reduced risk of developing dnDSA. Finally, we showed that elevated Tfh in STA precedes the occurrence of dnDSA during an alloresponse. These data provide new insights into the mechanisms of antibody response in operational tolerance. Disrupted homeostasis and impaired Tfh function in TOL could lead to a reduced risk of developing dnDSA and suggest a predictive role of blood Tfh cells on the occurrence of dnDSA in transplant recipients.

Failure of Calcineurin Inhibitor (Tacrolimus) Weaning Randomized Trial in Long-Term Stable Kidney Transplant Recipients.

Long-term renal transplant outcome is limited by side effects of immunosuppressive drugs, particularly calcineurin inhibitor (CNI). We assumed that some patients selected for a "low immunological risk of rejection" could be eligible and benefit from a CNI weaning strategy. We designed a prospective, randomized, multicenter, double-blind placebo-controlled clinical study (Eudract: 2010-019574-33) to analyze the benefit-risk ratio of tacrolimus weaning on highly selected patients (≥4 years of transplantation, normal histology, stable graft function, no anti-HLA immunization). The primary endpoint was improvement of renal function. Fifty-two patients were scheduled in each treatment arm, placebo compared to the CNI maintenance arm. Only 10 patients were eligible and randomized. Five patients were assigned to the placebo arm and five were assigned to the tacrolimus maintenance arm. In the tacrolimus maintenance arm, all patients maintained stable graft function and no immunological events occurred. Contrastingly, in the placebo arm, all five patients had to reintroduce a full dose of tacrolimus since three of them presented an acute rejection episode (one humoral, one mixed, and one borderline) and two displayed anti-HLA antibodies without histological lesion (one donor-specific antibodies [DSA] and one non-DSA). Clearly, tacrolimus withdrawal must be avoided even in long-term highly selective stable kidney recipients.

Cell Therapy for Parkinson's Disease: A Translational Approach to Assess the Role of Local and Systemic Immunosuppression.

Neural transplantation is a promising therapeutic approach for neurodegenerative diseases; however, many patients receiving intracerebral fetal allografts exhibit signs of immunization to donor antigens that could compromise the graft. In this context, we intracerebrally transplanted mesencephalic pig xenografts into primates to identify a suitable strategy to enable long-term cell survival, maturation, and differentiation. Parkinsonian primates received WT or CTLA4-Ig transgenic porcine xenografts and different durations of peripheral immunosuppression to test whether systemic plus graft-mediated local immunosuppression might avoid rejection. A striking recovery of spontaneous locomotion was observed in primates receiving systemic plus local immunosuppression for 6 mo. Recovery was associated with restoration of dopaminergic activity detected both by positron emission tomography imaging and histological examination. Local infiltration by T cells and CD80/86+ microglial cells expressing indoleamine 2,3-dioxigenase were observed only in CTLA4-Ig recipients. Results suggest that in this primate neurotransplantation model, peripheral immunosuppression is indispensable to achieve the long-term survival of porcine neuronal xenografts that is required to study the beneficial immunomodulatory effect of local blockade of T cell costimulation.

Bortezomib, C1-inhibitor and plasma exchange do not prolong the survival of multi-transgenic GalT-KO pig kidney xenografts in baboons.

Galactosyl-transferase KO (GalT-KO) pigs represent a potential solution to xenograft rejection, particularly in the context of additional genetic modifications. We have performed life supporting kidney xenotransplantation into baboons utilizing GalT-KO pigs transgenic for human CD55/CD59/CD39/HT. Baboons received tacrolimus, mycophenolate mofetil, corticosteroids and recombinant human C1 inhibitor combined with cyclophosphamide or bortezomib with or without 2-3 plasma exchanges. One baboon received a control GalT-KO xenograft with the latter immunosuppression. All immunosuppressed baboons rejected the xenografts between days 9 and 15 with signs of acute humoral rejection, in contrast to untreated controls (n = 2) that lost their grafts on days 3 and 4. Immunofluorescence analyses showed deposition of IgM, C3, C5b-9 in rejected grafts, without C4d staining, indicating classical complement pathway blockade but alternate pathway activation. Moreover, rejected organs exhibited predominantly monocyte/macrophage infiltration with minimal lymphocyte representation. None of the recipients showed any signs of porcine endogenous retrovirus transmission but some showed evidence of porcine cytomegalovirus (PCMV) replication within the xenografts. Our work indicates that the addition of bortezomib and plasma exchange to the immunosuppressive regimen did not significantly prolong the survival of multi-transgenic GalT-KO renal xenografts. Non-Gal antibodies, the alternative complement pathway, innate mechanisms with monocyte activation and PCMV replication may have contributed to rejection.

Early corticosteroid avoidance in kidney transplant recipients receiving ATG-F induction: 5-year actual results of a prospective and randomized study.

One hundred ninety-seven patients received anti-T-lymphocyte globulins Fresenius, mycophenolate mofetil and delayed cyclosporine, and were randomized to ≥6-month corticosteroids (+CS; n=99) or no CS (-CS; n=98). One- and five-year actual graft survival (censored for death) was 93.2% and 86.4% in the +CS group versus 94.9% and 89.8% in the -CS group (5-year follow-up, p=0.487). Freedom from clinical rejection was 86.9% and 81.8% versus 74.5% and 74.5% (p=0.144), respectively, at 1 and 5 years; 5-year freedom from biopsy-proven rejection was 88.9% versus 83.7% (p=0.227). More late first rejections occurred in the +CS group. Significantly lower 5-year graft survival in patients experiencing rejection was observed for +CS (55.6% vs. 92.0%; p=0.005) with 8/18 versus 2/25 graft losses. Renal function at 5 years was stable and comparable (median serum creatinine, 159 vs. 145 µmol/L; creatinine clearance, 53.5 vs. 56.6 mL/min). More +CS patients developed diabetes, dyslipidemia and malignancies. Rejections in -CS patients occurred early after transplantation and did not impair long-term renal function. In patients receiving CS, rejections occurred later and with a higher risk for subsequent graft failure. A similar and not inferior 5-year efficacy profile and a reduced morbidity were observed in CS-free patients compared to patients who received CS for at least 6 months.

Unique B cell differentiation profile in tolerant kidney transplant patients.

Operationally tolerant patients (TOL) display a higher number of blood B cells and transcriptional B cell signature. As they rarely develop an allo-immune response, they could display an abnormal B cell differentiation. We used an in vitro culture system to explore T-dependent differentiation of B cells into plasma cells. B cell phenotype, apoptosis, proliferation, cytokine, immunoglobulin production and markers of differentiation were followed in blood of these patients. Tolerant recipients show a higher frequency of CD20(+) CD24(hi) CD38(hi) transitional and CD20(+) CD38(lo) CD24(lo) naïve B cells compared to patients with stable graft function, correlating with a decreased frequency of CD20(-) CD38(+) CD138(+) differentiated plasma cells, suggestive of abnormal B cell differentiation. B cells from TOL proliferate normally but produce more IL-10. In addition, B cells from tolerant recipients exhibit a defective expression of factors of the end step of differentiation into plasma cells and show a higher propensity for cell death apoptosis compared to patients with stable graft function. This in vitro profile is consistent with down-regulation of B cell differentiation genes and anti-apoptotic B cell genes in these patients in vivo. These data suggest that a balance between B cells producing IL-10 and a deficiency in plasma cells may encourage an environment favorable to the tolerance maintenance.

Pretransplant sensitization against angiotensin II type 1 receptor is a risk factor for acute rejection and graft loss.

The angiotensin II type 1 receptor (AT1R) is an emerging target of functional non-HLA antibodies (Ab). We examined the potential of determining the degree of presensitization against AT1R as a risk factor for graft survival and acute rejection (AR). The study included 599 kidney recipients between 1998 and 2007. Serum samples were analyzed in a blinded fashion for anti-AT1R antibodies (AT1R-Abs) using a quantitative solid-phase assay. A threshold of AT1R-Ab levels was statistically determined at 10 U based on the time to graft failure. An extended Cox model determined risk factors for occurrence of graft failure and a first AR episode. AT1R-Abs >10 U were detected in 283 patients (47.2%) before transplantation. Patients who had a level of AT1R-Abs >10 U had a 2.6-fold higher risk of graft failure from 3 years posttransplantation onwards (p = 0.0005) and a 1.9-fold higher risk of experiencing an AR episode within the first 4 months of transplantation (p = 0.0393). Antibody-mediated rejection (AMR) accounted for 1/3 of AR, whereby 71.4% of them were associated with >10 U of pretransplant AT1R-Abs. Pretransplant anti-AT1R-Abs are an independent risk factor for long-term graft loss in association with a higher risk of early AR episodes.

The Tribbles-1 protein in humans: roles and functions in health and disease.

This review describes the key role of the serine-threonine kinase like protein Tribbles-1 in health as well as in diverse human pathologies. Tribbles-1 is a homolog protein of the Drosophila Tribbles. In Drosophila, the Tribbles protein is involved in the cell-cycle progression during mitosis and in mammals initial data showed TRIB1 to be involved in cell proliferation. In mammals, TRIB1 lacks a catalytic domain and thus acts as an adaptor protein by interacting with several partners. The activity of TRIB1 seems to be very specific to the environment and the cells type in which it is expressed, and a role for this molecule has been mainly described in several pathological states including various cancers such as acute myeloid leukemia and ovarian cancer. Further evidence has also linked TRIB1 to the control of plasmalipid homeostasis thus indicating the role of this molecule as a risk factor for myocardial infarction. Finally, TRIB1 is shown to be up-regulated during inflammatory events such as chronic inflammation of atherosclerotic arteries or chronic antibody-mediated rejection of transplanted organs. Here we provide a review of the current state of the scientific literature for TRIB1, highlighting its role in diverse pathologies and inflammatory states. A better understanding of the role of this protein as both a target as well as a biological marker in diseases should drive the development of new therapeutic strategies.

PARVG gene polymorphism and operational renal allograft tolerance.

A unique blood transcriptional profile of 49 genes has been previously highlighted that may be used to distinguish drug-free operationally tolerant kidney recipients (TOL) from other kidney recipients with contrasted clinical situations and healthy volunteers. The aim of this study was to investigate whether the pattern of these 49 genes could be influenced by genetic polymorphisms located in the corresponding genomic sequences and whether some of these single nucleotide polymorphisms (SNPs) could be associated with clinical status of kidney transplant recipients. In this study, 1152 candidate tag SNPs spanning these genes were genotyped using a Golden Gate Illlumina assay in a sample of 164 kidney transplant recipients, including 11 operationally tolerant patients, 134 patients with stable graft function, 19 with proven signs of chronic rejection, and 27 healthy volunteers. The gene expression and clinical status were studied according to the different SNPs. Among the genes demonstrating expression difference between TOL compared with CR&STA patients, PARVG, which is a member of a family of actin-binding proteins associated with focal contacts, stands out with 2 SNPs, (rs139144 and rs5764592) explaining about 20% of the gene expression variability. Linkage disequilibrium analysis of these 2 SNPs showed the rs139144GG genotype was associated with decreased PARVG expression and was numerically more frequent in TOL (60%) than in CR&STA (28%) patients (P = .068). These preliminary results, which should be confirmed in a larger population, open new perspective of regulation pathways and hypothesis in operational tolerance mechanism.

The natural history of clinical operational tolerance after kidney transplantation through twenty-seven cases.

We report here on a European cohort of 27 kidney transplant recipients displaying operational tolerance, compared to two cohorts of matched kidney transplant recipients under immunosuppression and patients who stopped immunosuppressive drugs and presented with rejection. We report that a lower proportion of operationally tolerant patients received induction therapy (52% without induction therapy vs. 78.3%[p = 0.0455] and 96.7%[p = 0.0001], respectively), a difference likely due to the higher proportion (18.5%) of HLA matched recipients in the tolerant cohort. These patients were also significantly older at the time of transplantation (p = 0.0211) and immunosuppression withdrawal (p = 0.0002) than recipients who rejected their graft after weaning. Finally, these patients were at lower risk of infectious disease. Among the 27 patients defined as operationally tolerant at the time of inclusion, 19 still display stable graft function (mean 9 ± 4 years after transplantation) whereas 30% presented slow deterioration of graft function. Six of these patients tested positive for pre-graft anti-HLA antibodies. Biopsy histology studies revealed an active immunologically driven mechanism for half of them, associated with DSA in the absence of C4d. This study suggests that operational tolerance can persist as a robust phenomenon, although eventual graft loss does occur in some patients, particularly in the setting of donor-specific alloantibody.

BAFF and BAFF-R levels are associated with risk of long-term kidney graft dysfunction and development of donor-specific antibodies.

There are lines of evidence that B cells may play a role in transplantation. B cell activating factor, BAFF, is a homotrimer that has been shown to play a role in B cell survival, maturation and activation. To date, little is known of the role of BAFF and its receptors in transplantation. We analyzed the level of BAFF mRNA and its soluble protein, as well as transcripts coding for its receptors, BAFF-R, TACI and BCMA, in the blood of 143 patients with stable kidney transplant function 5 years or more posttransplantation. Three endpoints were analyzed: the time to renal dysfunction, the time to appearance of anti-HLA antibodies and the time to development of donor-specific antibodies. We established threshold values for BAFF and BAFF-R and showed that (1) stable patients with high BAFF-R levels had a higher risk of developing graft dysfunction, (2) patients with lower levels of BAFF transcripts or a higher level of soluble BAFF had a significantly higher risk of developing donor-specific antibodies. These data suggest that BAFF constitutes a risk factor for renal graft dysfunction and development of donor-specific antibodies. They also suggest that agents targeting BAFF-R interactions may offer new therapeutic opportunities in transplantation.

A need for biomarkers of operational tolerance in liver and kidney transplantation.

Both kidney and particularly liver recipients can occasionally discontinue all immunosuppressive drugs without undergoing rejection. These patients, who maintain stable graft function off immunosuppressive drugs without clinically significant detrimental immune responses and/or immune deficits, are conventionally termed operationally tolerant and offer a unique paradigm of tolerance in humans. The immune characterization of operationally tolerant transplant recipients has recently received substantial attention. Operationally tolerant patients might exhibit a signature of tolerance that could potentially be useful to select recipients amenable to drug minimization or withdrawal. Furthermore, elucidation of the molecular pathways associated with the operational tolerance phenotype could provide novel targets for therapy. Particular emphasis has been placed on the use of blood samples and high-throughput transcriptional profiling techniques. In liver transplantation, natural killer related transcripts seem to be the most robust markers of operational tolerance, whereas enrichment in B cell-related gene expression markers has been consistently found in blood samples from operationally tolerant kidney recipients, suggesting that different mechanisms operate in the two situations. In this minireview, we summarize the main achievements of recently published reports focused on the identification of transcriptional markers of operational tolerance, we highlight their mechanistic and clinical implications and describe their methodological limitations.

SMILE silencing and PMA activation gene networks in HeLa cells: comparison with kidney transplantation gene networks.

Recent findings indicated that the SMILE gene may be involved in kidney graft operational tolerance in human. This gene was found to be up-regulated in blood from patients with a well functioning kidney transplant in the absence of immunosuppression compared to other transplanted recipients with clinically different status. A microarray study of SMILE knock-down and phorbol 12-myristate 13-acetate (PMA) activation in HeLa cells was herein compared to our earlier analysis based on microarray data of kidney allograft tolerance and rejection in humans and in a rat model of allograft transplantation to determine possible new genes and gene networks involved in kidney transplantation. The nearest neighbors at the intersection of the SMILE knock-down network with the human tolerance/rejection networks are shown to be NPHS1 and ARRB2, the former (Nephrin) being involved in kidney podocyte function, and the decrease of the latter (Arrestin ß2) being recently shown to be involved in monocyte activation during acute kidney allograft rejection in rat. Moreover, another one of the neighbors at the intersection of SMILE network and tolerance/rejection networks is XBP-1, that we report previously to be increased, at a transcript level, after ER stress in SMILE silenced cells. Finally, in this study, we also show that topological properties (both local and global) of joint SMILE knock-down network-tolerance/rejection networks and joint PMA activation network-tolerance/rejection networks in rat and human are essentially different, likely due to the inherent nature of the gene SMILE and the mitogen PMA, that do not act the same way on genes and do not interfere the same way on networks. We also show that interestingly SMILE networks contain more feed-forward loop (FFL) motifs and thus SMILE calls for a more fine-tuned genetic regulation.

Xenotransplantation of galactosyl-transferase knockout, CD55, CD59, CD39, and fucosyl-transferase transgenic pig kidneys into baboons.

Galactosyl-transferase knockout (GT-KO) pigs represent the latest major progress to reduce immune reactions in xenotransplantation. However, their organs are still subject to rapid humoral rejection involving complement activation requiring the ongoing development of further genetic modifications in the pig. In a pig-to-baboon renal transplantation setting, we have used donor pigs that are not only GT-KO, but also transgenic for human CD55 (hCD55), hCD59, hCD39, and fucosyl-transferase (hHT). We studied kidney xenograft survival, physiological and immunologic parameters, xenogeneic rejection characteristics, as well as viral transmission aspects among two groups of baboons: control animals (n = 2), versus those (n = 4) treated with a cocktail of cyclophosphamide, tacrolimus, mycophenolate mofetil, steroids, and a recombinant human C1 inhibitor. Whereas control animals showed clear acute humoral rejection at around day 4, the treated animals showed moderately improved graft survival with rejection at around 2 weeks posttransplantation. Biopsies showed signs of acute vascular rejection (interstitial hemorrhage, glomerular thrombi, and acute tubular necrosis) as well as immunoglobulin (Ig)M and complement deposition in the glomerular and peritubular capillaries. The low level of preformed non-Gal-α1.3Gal IgM detected prior to transplantation increased at 6 days posttransplantation, whereas induced IgG appeared after day 6. No porcine endogenous retrovirus (PERV) transmission was detected in any transplanted baboon. Thus, surprisingly, organs from the GT-KO, hCD55, hCD59, hCD39, and hHT transgenic donors did not appear to convey significant protection against baboon anti-pig antibodies and complement activation, which obviously continue to be significant factors under a suboptimal immunosuppression regimen. The association, timing, and doses of immunosuppressive drugs remain critical. They will have to be optimized to achieve longer graft survivals.

Posttransplant donor-specific anti-HLA antibodies negatively impact pancreas transplantation outcome.

During a 9-year follow-up, 167 consecutive pancreas transplant recipients (152 simultaneous pancreas-kidney [SPK]) were followed for the detection of posttransplant anti-HLA antibodies. Forty patients (24%) developed anti-HLA antibodies, 26 (65%) had donor-specific antibodies (DSA; 61% anticlass 2) and 14 (35%) non-DSA (78.6% anticlass 1). More rejection episodes were observed in patients with positive anti-HLA antibodies than in patients without antibodies (42.5% vs. 11%; p = 0.001), with the highest incidence observed in DSA patients (53.8%). More severe rejections (according to rescue therapy) were observed in DSA patients compared to non-DSA (p < 0.05) or to negative patients (p < 0.001). Contrasting with the kidney, pancreas graft survival did not differ between patients with or without anti-HLA antibodies. On the contrary, pancreas and kidney survivals were significantly lower in DSA positive patients (75% for both organs) as compared to non-DSA positive patients (100% for pancreas and 92% for kidney) or to HLA-negative patients (91% for pancreas and 89% for kidney). Nontechnical pancreas and kidney graft failures were significantly higher in positive than in negative anti-HLA patients (32.5% vs. 11%; p < 0.01). Occurrence of posttransplant DSA was an independent risk factor for both pancreas and kidney survival (HR 3.2; p = 0.039) in diabetic transplant recipients.

Comparison of transcriptional and blood cell-phenotypic markers between operationally tolerant liver and kidney recipients.

A proportion of transplant recipients can spontaneously accept their grafts in the absence of immunosuppression (operational tolerance). Previous studies identified blood transcriptional and cell-phenotypic markers characteristic of either liver or kidney tolerant recipients. However, the small number of patients analyzed and the use of different transcriptional platforms hampered data interpretation. In this study we directly compared samples from kidney and liver tolerant recipients in order to identify potential similarities in immune-related parameters. Liver and kidney tolerant recipients differed in blood expression and B-cell immunophenotypic patterns and no significant overlaps were detectable between them. Whereas some recipients coincided in specific NK-related transcripts, this observation was not reproducible in all cohorts analyzed. Our results reveal that certain immune features, but not others, are consistently present across all cohorts of operationally tolerant recipients. This provides a set of reproducible biomarkers that should be explored in future large-scale immunomonitoring trials.

Long-term allograft tolerance is characterized by the accumulation of B cells exhibiting an inhibited profile.

Numerous reports have highlighted the central role of regulatory T cells in long-term allograft tolerance, but few studies have investigated the B-cell aspect. We analyzed the B-cell response in a rat model of long-term cardiac allograft tolerance induced by a short-term immunosuppression. We observed that tolerated allografts are infiltrated by numerous B cells organized in germinal centers that are strongly regulated in their IgG alloantibody response. Moreover, alloantibodies from tolerant recipients exhibit a deviation toward a Th2 isotype and do not activate in vitro donor-type endothelial cells in a pro-inflammatory way but maintained expression of cytoprotective molecules. Interestingly, this inhibition of the B-cell response is characterized by the progressive accumulation in the graft and in the blood of B cells blocked at the IgM to IgG switch recombination process and overexpressing BANK-1 and the inhibitory receptor Fcgr2b. Importantly, B cells from tolerant recipients are able to transfer allograft tolerance. Taken together, these results demonstrate a strong regulation of the alloantibody response in tolerant recipients and the accumulation of B cells exhibiting an inhibited and regulatory profile. These mechanisms of regulation of the B-cell response could be instrumental to develop new strategies to promote tolerance.