Allan D. Kirk

Identification of a B cell signature associated with renal transplant tolerance in humans

Establishing long-term allograft acceptance without the requirement for continuous immunosuppression, a condition known as allograft tolerance, is a highly desirable therapeutic goal in solid organ transplantation. Determining which recipients would benefit from withdrawal or minimization of immunosuppression would be greatly facilitated by biomarkers predictive of tolerance. In this study, we identified the largest reported cohort to our knowledge of tolerant renal transplant recipients, as defined by stable graft function and receiving no immunosuppression for more than 1 year, and compared their gene expression profiles and peripheral blood lymphocyte subsets with those of subjects with stable graft function who are receiving immunosuppressive drugs as well as healthy controls. In addition to being associated with clinical and phenotypic parameters, renal allograft tolerance was strongly associated with a B cell signature using several assays. Tolerant subjects showed increased expression of multiple B cell differentiation genes, and a set of just 3 of these genes distinguished tolerant from nontolerant recipients in a unique test set of samples. This B cell signature was associated with upregulation of CD20 mRNA in urine sediment cells and elevated numbers of peripheral blood naive and transitional B cells in tolerant participants compared with those receiving immunosuppression. These results point to a critical role for B cells in regulating alloimmunity and provide a candidate set of genes for wider-scale screening of renal transplant recipients.

Immunocompetent T-cells with a memory-like phenotype are the dominant cell type following antibody-mediated T-cell depletion

T‐cell depletion facilitates reduced immunosuppression following organ transplantation and has been suggested to be pro‐tolerant. However, the characteristics of post‐depletional T cells have not been evaluated as they relate to tolerance induction. We therefore studied patients undergoing profound T‐cell depletion with alemtuzumab or rabbit anti‐thymocyte globulin following renal transplantation, evaluating the phenotype and functional characteristics of their residual cells. Naïve T cells and T cells with potential regulatory function (CD4+CD25+) were not prevalent following aggressive depletion. Rather, post‐depletion T cells were of a single phenotype (CD3+CD4+CD45RA‐CD62L‐CCR7‐) consistent with depletion‐resistant effector memory T cells that expanded in the first month and were uniquely prevalent at the time of rejection. These cells were resistant to steroids, deoxyspergualin or sirolimus in vitro, but were calcineurin‐inhibitor sensitive. These data demonstrate that therapeutic depletion begets a limited population of functional memory‐like T cells that are easily suppressed with certain immunosuppressants, but cannot be considered uniquely pro‐tolerant.

results From A Human Renal Allograft Tolerance Trial Evaluating The Humanized Cd52-specific Monoclonal Antibody Alemtuzumab (campath-1h)

Background. Profound T‐cell depletion before allotransplantation with gradual posttransplant T‐cell repopulation induces a state of donor‐specific immune hyporesponsiveness or tolerance in some animal models. Alemtuzumab (Campath‐1H, Millennium Pharmaceuticals, Cambridge, MA) is a humanized CD52‐specific monoclonal antibody that produces profound T‐cell depletion in humans and reduces the need for maintenance immunosuppression after renal transplantation. We therefore performed a study to determine if pretransplant T‐cell depletion with alemtuzumab would induce tolerance in human renal allografts and to evaluate the nature of the alloimmune response in the setting of T‐cell depletion. Methods. Seven nonsensitized recipients of livingdonor kidneys were treated perioperatively with alemtuzumab and followed postoperatively without maintenance immunosuppression. Patients were evaluated clinically by peripheral flow cytometry, protocol biopsies evaluated immunohistochemically, and real‐time polymerase chain reaction‐based transcriptional analysis. Results. Lymphocyte depletion was profound in the periphery and secondary lymphoid tissues. All patients developed reversible rejection episodes within the first month that were characterized by predominantly monocytic (not lymphocytic) infiltrates with only rare T cells in the peripheral blood or allograft. These episodes were responsive to treatment with steroids or sirolimus or both. After therapy, patients remained rejection‐free on reduced immunosuppression, generally monotherapy sirolimus, despite the recovery of lymphocytes to normal levels. Conclusions. T‐cell depletion alone does not induce tolerance in humans. These data underscore a prominent role for early responding monocytes in human allograft rejection.

Association of cytokine polymorphic inheritance and in vitro cytokine production in anti-CD3/CD28-stimulated peripheral blood lymphocytes.

Background. Genetic variations in cytokine genes are thought to regulate cytokine protein production. However, studies using T cell mitogens have not always demonstrated a significant relationship between cytokine polymorphisms and in vitro protein production. Furthermore, the functional consequence of a polymorphism at position −330 in the IL-2 gene has not been described. We associated in vitro protein production with cytokine gene polymorphic genotypes after costimulation of cultured peripheral blood lymphocytes. Methods. PBL were isolated from forty healthy volunteers. Cytokine protein production was assessed by enzyme-linked immunosorbent assay. Polymorphisms in interleukin- (IL) 2, IL-6, IL-10, tumor necrosis factor (TNF-&agr;), tumor growth factor (TGF-&bgr;), and interferon (IFN-&ggr;) were determined by polymerase chain reaction (PCR). Results. Statistical difference between protein production and cytokine polymorphic variants in the IL-10, IFN-&ggr;, and TNF-&agr; genes was not evident after 48-hour stimulation with concanavalin-A. In contrast, after anti-CD3/CD28 stimulation significant differences (P <0.05) were found among high and low producers for IL-2, IL-6, and among high, intermediate, and low producers for IFN-&ggr;, and IL-10. Augmented levels of IL-2 in individuals that were homozygous for the polymorphic IL-2 allele were due to an early and sustained enhancement of IL-2 production. No association was found among TNF-&agr; and TGF-&bgr; genotypes and protein production. Conclusion. Polymorphisms in IL-2, IL-6, IL-10, and IFN-&ggr; genes are associated with their protein production after anti-CD3/CD28 stimulation. The profound effect of the IL-2 gene polymorphism in homozygous individuals may serve as a marker for those that could mount the most vigorous allo- or autoimmune responses, or perhaps become tolerant more easily.

Ethnicity greatly influences cytokine gene polymorphism distribution.

Polymorphisms in the regulatory regions of cytokine genes are associated with high and low cytokine production and may modulate the magnitude of alloimmune responses following transplantation. Ethnicity influences allograft half‐life and the incidence of acute and chronic rejection. We have questioned whether ethnic‐based differences in renal allograft survival could be due in part to inheritance of cytokine polymorphisms. To address that question, we studied the inheritance patterns for polymorphisms in several cytokine genes (IL‐2, IL‐6, IL‐10, TNF‐α, TGF‐β, and IFN‐γ) within an ethnically diverse study population comprised of 216 Whites, 58 Blacks, 25 Hispanics, and 31 Asians. Polymorphisms were determined by allele‐specific polymerase chain reaction and restriction fragment length analysis. We found striking differences in the distribution of cytokine polymorphisms among ethnic populations. Specifically, significant differences existed between Blacks and both Whites and Asians in the distribution of the polymorphic alleles for IL‐2. Blacks, Hispanics and Asians demonstrated marked differences in the inheritance of IL‐6 alleles and IL‐10 genotypes that result in high expression when compared with Whites. Those of Asian descent exhibited an increase in IFN‐γ genotypes that result in low expression as compared to Whites. In contrast, we did not find significant ethnic‐based differences in the inheritance of polymorphic alleles for TNF‐α. Our results show that the inheritance of certain cytokine gene polymorphisms is strongly associated with ethnicity. These differences may contribute to the apparent influence of ethnicity on allograft outcome.

The Banff 2007 working classification of skin-containing composite tissue allograft pathology.

Composite tissue allotransplantation (CTA) is a recently introduced option for limb replacement and reconstruction of tissue defects. As with other allografts, CTA can undergo immune‐mediated rejection; therefore standardized criteria are required for characterizing and reporting severity and types of rejection. This article documents the conclusions of a symposium on CTA rejection held at the Ninth Banff Conference on Allograft Pathology in La‐Coruňa, Spain, on 26 June 2007, and proposes a working classification, the Banff CTA‐07, for the categorization of CTA rejection. This classification was derived from a consensus discussion session attended by the first authors of three published classification systems, pathologists and researchers from international centers where clinical CTA has been performed. It was open to all attendees to the Banff conference. To the extent possible, the format followed the established National Institutes of Health (NIH) guidelines on Consensus Development Programs. By consensus, the defining features to diagnose acute skin rejection include inflammatory cell infiltration with involvement of epidermis and/or adnexal structures, epithelial apoptosis, dyskeratosis and necrosis. Five grades of severity of rejection are defined. This classification refines proposed schemas, represents international consensus on this topic, and establishes a working collective classification system for CTA reporting of rejection in skin‐containing CTAs.

The effect of soluble complement receptor type 1 on hyperacute rejection of porcine xenografts

The use of xenografts (Xgs) from distantly related species to relieve the increasing shortage of organs for clinical transplantation is prevented by the occurrence of hyperacute rejection (HAR). This process, in which C activation plays a central role, cannot be inhibited with currently available immunosuppressants. In two clinically relevant xenotransplantation models, this study evaluated the effect of C inhibition using recombinant soluble complement receptor type 1 (sCR1) on HAR. In an ex vivo model in which porcine cardiac Xgs were perfused with human blood, cardiac function ceased within 34 min when the perfusate blood was untreated (n = 3). When the perfusate blood was treated with sCR1 (300 micrograms/ml), cardiac Xg function was maintained for up to 4 hr (n = 3). Immunohistologic examination of these Xgs demonstrated deposition of C3b/iC3b and C3d in Xgs perfused with untreated human blood but only C3d deposition in those Xgs perfused with sCR1-treated human blood. These findings are consistent with the cofactor activity of sCR1 for factor I-mediated degradation of deposited C3b/iC3b to C3d. Treatment with sCR1 also prevented the histopathologic changes of HAR observed when untreated blood was used as the perfusate. In an in vivo pig-to-primate heterotopic cardiac xenotransplantation model, in which porcine Xgs transplanted into untreated cynomolgus monkey recipients underwent HAR in 1 hr or less (n = 3), a single intravenous bolus of sCR1 (15 mg/kg) administered to the recipient immediately before Xg reperfusion markedly inhibited total and alternative pathway serum C activity and prolonged Xg survival to between 48 and 90 hr (n = 5). These studies confirm the important role of C activation in HAR of porcine cardiac Xgs by primates and indicate that sCR1 may be a useful agent for xenotransplantation.

Alefacept promotes co-stimulation blockade based allograft survival in nonhuman primates

Memory T cells promote allograft rejection particularly in co-stimulation blockade–based immunosuppressive regimens. Here we show that the CD2-specific fusion protein alefacept (lymphocyte function–associated antigen-3–Ig; LFA -3–Ig) selectively eliminates memory T cells and, when combined with a co-stimulation blockade–based regimen using cytotoxic T lymphocyte antigen-4 (CTLA-4)-Ig, a CD80- and CD86-specific fusion protein, prevents renal allograft rejection and alloantibody formation in nonhuman primates. These results support the immediate translation of a regimen for the prevention of allograft rejection without the use of calcineurin inhibitors, steroids or pan–T cell depletion.

Dissociation of depletional induction and posttransplant lymphoproliferative disease in kidney recipients treated with alemtuzumab.

Transplant patients are at the risk for posttransplant lymphoproliferative disease (PTLD), a virally‐driven malignancy. Induction with the depleting antibody preparations Thymoglobulin and OKT3 is associated with PTLD suggesting that the T‐cell depletion increases PTLD risk. We therefore studied 59 560 kidney recipients from the Organ Procurement and Transplantation Network/United Network for Organ Sharing (OPTN/UNOS) database for a relationship between induction agent use and PTLD. Two agents with comparable T‐cell depletional effects, alemtuzumab and Thymoglobulin, were compared to nondepletional induction agents or no induction. The overall incidence of PTLD was 0.46% and differed significantly by induction strategy (p < 0.01): without induction (0.43%), basiliximab (0.38%), daclizumab (0.33%), Thymoglobulin (0.67%) and alemtuzumab (0.37%). Thymoglobulin was associated with significantly increased PTLD risk (p = 0.0025), but alemtuzumab (p = 0.74), basiliximab (p = 0.33) and daclizumab, which trended toward a protective effect (p = 0.06), were not. Alemtuzumab and Thymoglobulin treated patients did not differ in any established parameter affecting PTLD risk although alemtuzumab is known to have a more pronounced B‐cell depleting effect. Interestingly, maintenance therapy with an mTOR inhibitor was strongly associated with PTLD (0.71%, p < 0.0001). Thus, depletional induction is not an independent risk factor for PTLD. Rather, maintenance drug selection or perhaps the balance between B‐ and T‐cell depletion may be more relevant determinants of PTLD risk.

Effect of Alemtuzumab (CAMPATH 1-H) in patients with inclusion-body myositis

Sporadic inclusion-body myositis (sIBM) is the most common disabling, adult-onset, inflammatory myopathy histologically characterized by intense inflammation and vacuolar degeneration. In spite of T cell-mediated cytotoxicity and persistent, clonally expanded and antigen-driven endomysial T cells, the disease is resistant to immunotherapies. Alemtuzumab is a humanized monoclonal antibody that causes an immediate depletion or severe reduction of peripheral blood lymphocytes, lasting at least 6 months. We designed a proof-of-principle study to examine if one series of Alemtuzumab infusions in sIBM patients depletes not only peripheral blood lymphocytes but also endomysial T cells and alters the natural course of the disease. Thirteen sIBM patients with established 12-month natural history data received 0.3 mg/kg/day Alemtuzumab for 4 days. The study was powered to capture ≥10% increase strength 6 months after treatment. The primary end-point was disease stabilization compared to natural history, assessed by bi-monthly Quantitative Muscle Strength Testing and Medical Research Council strength measurements. Lymphocytes and T cell subsets were monitored concurrently in the blood and the repeated muscle biopsies. Alterations in the mRNA expression of inflammatory, stressor and degeneration-associated molecules were examined in the repeated biopsies. During a 12-month observation period, the patients’ total strength had declined by a mean of 14.9% based on Quantitative Muscle Strength Testing. Six months after therapy, the overall decline was only 1.9% (P < 0.002), corresponding to a 13% differential gain. Among those patients, four improved by a mean of 10% and six reported improved performance of daily activities. The benefit was more evident by the Medical Research Council scales, which demonstrated a decline in the total scores by 13.8% during the observation period but an improvement by 11.4% (P < 0.001) after 6 months, reaching the level of strength recorded 12 months earlier. Depletion of peripheral blood lymphocytes, including the naive and memory CD8+ cells, was noted 2 weeks after treatment and persisted up to 6 months. The effector CD45RA+CD62L cells, however, started to increase 2 months after therapy and peaked by the 4th month. Repeated muscle biopsies showed reduction of CD3 lymphocytes by a mean of 50% (P < 0.008), most prominent in the improved patients, and reduced mRNA expression of stressor molecules Fas, Mip-1a and αB-crystallin; the mRNA of desmin, a regeneration-associated molecule, increased. This proof-of-principle study provides insights into the pathogenesis of inclusion-body myositis and concludes that in sIBM one series of Alemtuzumab infusions can slow down disease progression up to 6 months, improve the strength of some patients, and reduce endomysial inflammation and stressor molecules. These encouraging results, the first in sIBM, warrant a future study with repeated infusions (Clinical Trials. Gov NCT00079768).