Andrew L. Lobashevsky

Peritransplant tolerance induction in macaques: early events reflecting the unique synergy between immunotoxin and deoxyspergualin.

BACKGROUNDnDay of transplant T cell depletion with anti-CD3 immunotoxin or F(Ab)2 immunotoxin induces stable tolerance to renal allografts in rhesus monkeys given 15-deoxyspergualin (DSG), a NF-kappaB inhibitor that suppresses proinflammatory cytokine (PC) production. Because PC and NF-kappaB are involved in dendritic cell (DC) maturation, we asked if impaired DC maturation and Th2-type cytokine deviation might be related to the synergistic effect of DSG in this novel model.nnnMETHODSnImmunosuppression was initiated 4 hr before transplanting a major histocompatibility complex mismatched renal allograft. Some groups received a supplemental 5-day course of cyclosporine A or DSG or a 15-day course of DSG. Peripheral lymph nodes were sequentially examined for presence of mature DC. In vitro effects of DSG on PC-induced maturation of DC were also examined.nnnRESULTSnAllografts survived without rejection in 87% of recipients given immunotoxin or F(Ab)2 immunotoxin with DSG x 15 days, in 50% with DSG x 5 days, and 0% with cyclosporine A. The longest DSG survivors are >1000 days with normal graft function and tolerance validated, including acceptance of challenge second donor kidneys without treatment. DSG-treated recipients were unique in developing polarized Th2-type plasma cytokines. In DSG recipients, mature DC were significantly reduced in day +5 lymph node biopsies, with complete repopulation by 30 days. In vitro studies verified an inhibitory effect of DSG on DC maturation.nnnCONCLUSIONSnThe study suggests DSG arrests DC maturation. The unusual synergy of immunotoxin and DSG apparently involves coincidental reduction in lymph node T cell mass and mature DC, a transient circumstance favoring development of stable tolerance.

Preclinical studies of allograft tolerance in rhesus monkeys: a novel anti-CD3-immunotoxin given peritransplant with donor bone marrow induces operational tolerance to kidney allografts

A major challenge in clinical transplantation today is to design a practical and effective protocol for tolerance induction compatible with cadaver organ transplantation. A preclinical rhesus monkey kidney allograft model using immediate peritransplant anti-CD3 immunotoxin (anti-CD3-IT) and donor bone marrow (DBM) is shown here to induce operational tolerance with prolonged graft survival in the absence of chronic immunosuppressive drugs. Bone marrow harvested from the kidney donor was depleted of mature alloantigen-presenting cells and T cells by removing DR(bright) cells and CD3(bright) cells, respectively. In outbred, major histocompatibility complex-incompatible donor-recipient pairs with high pretransplant mixed lymphocyte response and cytotoxic T lymphocyte precursor activity, four of six allografts survived for periods of 120 days to >1.5 years. Graft acceptance after peritransplant treatment followed robust elimination of both peripheral blood T cells and lymph node T cells. In most recipients given anti-CD3-IT and DBM infusion, anti-donor immunoglobulin G responses were completely inhibited. Microchimerism was observed in all recipients studied, including those not given DBM, but levels of microchimerism did not correlate with graft survival. Anti-CD3-IT induction in combination with modified DBM protocols such as the depletion of mature T cells and DR(bright) antigen-presenting cells may offer new opportunities to improve clinical tolerance protocols beyond those attempted in the clinic to date. Overall, these results with anti-CD3-IT show promise for development of cadaver transplant tolerance induction.

Retraction: Peritransplant tolerance induction with anti-CD3-immunotoxin: a matter of proinflammatory cytokine control.

BACKGROUND Tolerance is gaining momentum as an approach to reduce lifelong immunosuppressive therapy while improving transplant longevity. Anti-CD3 immunotoxin (IT), FN18-CRM9, has potential to induce tolerance owing to its exceptional ability to deplete sessile lymph node T cells. However, if initiated at the time of transplantation, alpha-CD3-IT alone elicits a proinflammatory cytokine response, precluding establishment of tolerance. METHODS Four groups of rhesus monkeys received kidney allografts and immunosuppression. Three groups received alpha-CD3-IT alone or alpha-CD3-IT supplemented with 15-deoxyspergualin (DSG) and/or methylprednisolone (MP). One group received alpha-CD3-monoclonal antibody with DSG and MP. Cytokines were measured by enzyme-linked immunosorbent assay. RESULTS Supplementing peritransplant alpha-CD3-IT treatment with a brief course of DSG and MP promoted rejection-free kidney allograft acceptance in 75% of macaques followed for up to 550 days. Among those given alpha-CD3-IT alone or with MP, none were long-term survivors. Tolerance developed after alpha-CD3-IT, DSG, and MP treatment, but not when the unconjugated a-CD3 monoclonal antibody was substituted for IT. Systemic production of proinflammatory cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha induced after peritransplant alpha-CD3-IT was prevented only in animals given DSG. Despite high levels of interleukin (IL)-12 in the first month after transplant, tolerant recipients exhibited IL-12 resistance, as evidenced by baseline plasma levels of IFN-gamma but elevated IL-4. DSG was shown to inhibit IL-12-driven IFN-gamma production by a mechanism associated with inhibition of nuclear factor kappa-B. CONCLUSIONS In this model, peritransplant induction of tolerance is promoted by efficient elimination of sessile lymph node T cells and control of the proinflammatory IFN-gamma response by a mechanism that appears to involve resistance to IL-12.

The strength of donor-specific antibody is a more reliable predictor of antibody-mediated rejection than flow cytometry crossmatch analysis in desensitized kidney recipients.

Mujtaba MA, Goggins W, Lobashevsky A, Sharfuddin AA, Yaqub MS, Mishler DP, Brahmi Z, Higgins N, Milgrom MM, Diez A, Taber T. The strength of donor‐specific antibody is a more reliable predictor of antibody‐mediated rejection than flow cytometry crossmatch analysis in desensitized kidney recipients.u2028Clin Transplant 2011: 25: E96–E102.

Analysis of anti-HLA antibodies in sensitized kidney transplant candidates subjected to desensitization with intravenous immunoglobulin and rituximab.

Background Preexisting donor-specific antibodies against human leukocyte antigens are major risk factors for acute antibody-mediated and chronic rejection of kidney transplant grafts. Immunomodulation (desensitization) protocols may reduce antibody concentration and improve the success of transplant. We investigated the effect of desensitization with intravenous immunoglobulin and rituximab on the antibody profile in highly sensitized kidney transplant candidates. Methods In 31 transplant candidates (calculated panel-reactive antibody [cPRA], 34%–99%), desensitization included intravenous immunoglobulin on days 0 and 30 and a single dose of rituximab on day 15. Anti–human leukocyte antigen antibodies were analyzed before and after desensitization. Results Reduction of cPRA from 25% to 50% was noted for anti–class I (5 patients, within 20–60 days) and anti–class II (3 patients, within 10–20 days) antibodies. After initial reduction of cPRA, the cPRA increased within 120 days. In 24 patients, decrease in mean fluorescence intensity of antibodies by more than 50% was noted at follow-up, but there was no reduction of cPRA. Rebound occurred in 65% patients for anti–class I antibodies at 350 days and anti–class II antibodies at 101 to 200 days. Probability of rebound effect was higher in patients with mean fluorescence intensity of more than 10,700 before desensitization, anti–class II antibodies, and history of previous transplant. Conclusions The desensitization protocol had limited efficacy in highly sensitized kidney transplant candidate because of the short period with antibody reduction and high frequency of rebound effect.

Subtypes of immunoglobulin (Ig)-G antibodies against donor class II HLA and cross-match results in three kidney transplant candidates

Preexisting donor-specific antibodies (DSA) play a critical role in the success of solid-organ transplantation. Cross-match (CM) between donor lymphocytes and recipient serum is a pivotal methodology for detecting these antibodies. Luminex platform based solid-phase methodology for anti-human leukocyte antigen (HLA) antibody analysis has revolutionized the approach to antibody detection and HLA specificity identification. In this study, we have reported three cases of successful living donor kidney transplantations performed against strongly positive B lymphocyte flow cytometry (FC) CM owing to highly reactive DSA directed to HLA class II. IgG solid-phase subtype analysis showed that more than 50% of these antibodies were represented by non-complement binding IgG2/IgG4 subtypes. These findings account for antibody mediated rejection (AMR) free long-term post-transplant course in these patients despite, the high level of DSA. Thus, we conclude that routine application of single HLA-coated beads (SAB) IgG subtype assay may provide new insights regarding transplantation or desensitization of patients presenting with negative B-cell complement dependent cytotoxic (CDC) and positive FC CM.

Prospective monitoring of donor-specific anti-HLA antibodies after intestine/multivisceral transplantation: Significance of De novo antibodies

Background Presence of circulating donor-specific antibodies (DSA) may be associated with worse clinical outcomes after intestine/multivisceral transplantation. Methods In 79 intestine/multivisceral recipients, sera were prospectively screened for DSA by Luminex Single antigen test at 1, 3, 6, 9, 12, 18, 24, and 36 months after transplantation. Standard immunosuppression included thymoglobulin-rituximab induction and tacrolimus-prednisone maintenance. C4d staining was performed retrospectively on biopsies in patients that developed acute rejection (AR). Results Twenty-two (28%) patients developed de novo DSA at a median posttransplant period of 3 (1-36) months. De novo DSA were observed in 10 of 40 liver-including and 12 of 39 liver-excluding transplants (P = 0.57). Occurrence of AR was slightly higher in patients with de novo DSA (45% vs 33%, respectively; P = 0.41). Similarly, chronic rejection (14% vs 5%; P = 0.21) and graft loss due to AR (18% vs 7%; P = 0.14) were numerically higher in patients with de novo DSA. Only 35% patients experiencing AR had circulating de novo DSA at the time of AR. Antibody-mediated rejection was diagnosed in 6 patients based on C4d staining, of these 2 patients had circulating de novo DSA at the time of biopsy. Conclusions De novo DSA formation, particularly early in the posttransplant course may be associated with trends toward worse outcomes. However, its significance in the pathophysiology of AR remains uncertain. Studies focusing mechanisms of DSA-related graft injury and intragraft DSA detection might provide further insight into this issue.

Impact of Positive Flow Cytometry Crossmatch on Outcomes of Intestinal/Multivisceral Transplantation: Role Anti-IL-2 Receptor Antibody

Background Positive crossmatch may be associated with an increased risk of acute rejection (AR) and worse overall outcomes after intestinal/multivisceral (MV) transplantation. However, the evidence from published studies in this setting is sparse and contradictory. This study reports the impact of positive flow cytometry crossmatch on clinical outcomes after intestinal/MV transplantation and the use of anti–interleukin (IL)-2 receptor antibody as a maintenance immunosuppressant. Methods Records of all intestinal/MV transplants from 2003 to 2010 were reviewed. Flow cytometry was used to evaluate T- and B-cell crossmatch status. Standard immunosuppression included rabbit anti-thymocyte globulin–rituximab induction with tacrolimus and steroid maintenance. From 2008 onwards (second era), monthly anti-IL-2 receptor antibody was added to the maintenance immunosuppression in patients receiving liver-excluding transplants. Results Of 131 intestinal/MV transplants, 27 (21%) had a positive crossmatch. Positive crossmatch was not associated with an increased incidence of AR and graft loss (30% and 37% vs. 29% and 47%; P=0.94 and 0.35, respectively). This effect was maintained in liver-excluding transplants. Overall rate of AR decreased from 39% to 22% in the second era. In liver-excluding transplants, there was a significant decrease in AR from 75% to 44% with the use of anti-IL-2 receptor antibody therapy. Conclusions With rabbit anti-thymocyte globulin–rituximab induction, positive crossmatch status is not associated with worse outcomes after intestinal/MV transplantation. Use of anti-IL-2 receptor antibody as a part of maintenance immunosuppression may be beneficial in liver-excluding transplants.

Crossmatch-positive liver transplantation in patients receiving thymoglobulin-rituximab induction.

Background Positive crossmatch (CM) in liver transplantation (LT) is associated with worse outcomes. Role of induction immunosuppression in this setting remains to be studied. Methods One thousand consecutive LT patients receiving rabbit antithymocyte globulin±rituximab induction were studied. Pretransplantation sera of 55 CM-positive (CM+) patients were tested for C1q-fixing donor-specific antibodies (DSA). Diagnosis of antibody-mediated rejection required presence of diffuse vascular C4d expression on liver biopsies. Results CM was positive in 112 (11%) recipients. Antibody-mediated rejection was observed in 3 (0.03%) patients, whereas acute cellular rejection (ACR) occurred in 31 (3%) patients. CM+ status was associated with a higher incidence of ACR (9% in CM+ vs. 2% in CM-negative [CM−]; P<0.01) and chronic rejection (4% in CM+ vs. 1% in CM−; P<0.01). Graft survival was slightly lower in CM+ patients (at 1 year; 85% in CM+ vs. 89% in CM−; P=0.26). Patients with autoimmune hepatitis, primary sclerosing cholangitis, and primary biliary cirrhosis as a group had a tendency toward CM+ status as well as developing ACR. Upon multivariate analysis, CM status was the strongest predictor of ACR (B=1.14; P=0.02). Only half of CM+ patients harbored C1q-fixing DSA. Presence of C1q-fixing DSA was not associated with increased incidence of ACR. Conclusions In LT, CM+ status is associated with an increased incidence of acute rejection, chronic rejection, and slightly worse graft survival. With the use of rabbit antithymocyte globulin±rituximab induction, overall low rejection rates can be achieved in CM+ LT.

Effect of desensitization in solid organ transplant recipients depends on some cytokines genes polymorphism.

Desensitization (DS) is widely used to decrease PRA in solid organs transplant candidates (TC). Various numbers of cycles of DS are required to reduce or eliminate donor specific antibodies (DSA). The goal of this study was to investigate if there was a correlation between polymorphism (PM) of some cytokine genes and intensity of DS required to make the recipient/donor cross match compatible. Thirty-one TCs were included in the study. Antibody specificity, percent of reactive antibodies (PRA) and serum concentration of cytokines were analyzed using the LUMINEX platform. PCR-SSP method was used for IL-1alpha, IL-1beta, IL-1R, IL-1Ralpha, IL-4Ralpha, IL-12, IFNgamma, TGFbeta1, TNFalpha, IL-2, IL-4, IL-6 and IL-10 gene PM analysis. Significant relationship between PM of genes encoding IL-4Ralpha, IFNgamma and IL-12 (p70) and susceptibility to DS was demonstrated (p=0.04, p=0.01 and p=0.05 respectively). Correlation between elevated serum level of IL-12 (p70) and A/A or C/A genotype at -1188 position was found in resistant to DS TCs (p=0.015). These results indicate that analysis PM of genes encoding IL-4R, IFNgamma and IL-12 enables to define the DS strategy in TCs more accurately regarding the number of plasmapheresis (PP) cycles and dose of intravenous immunoglobulin (IVIG).