Thomas M. Ellis

T cell infiltration of the prostate induced by androgen withdrawal in patients with prostate cancer

Manipulations capable of breaking host tolerance to induce tissue-specific T cell-mediated inflammation are of central importance to tumor immunotherapy and our understanding of autoimmunity. We demonstrate that androgen ablative therapy induces profuse T cell infiltration of benign glands and tumors in human prostates. T cell infiltration is readily apparent after 7–28 days of therapy and is comprised predominantly of a response by CD4+ T cells and comparatively fewer CD8+ T cells. Also, T cells within the treated prostate exhibit restricted TCR Vβ gene usage, consistent with a local oligoclonal response. Recruitment/activation of antigen-presenting cells in treated prostate tissues may contribute to local T cell activation. The induction of T cell infiltration in prostate tissues treated with androgen ablation may have implications for the immunotherapeutic treatment of prostate cancer as well as other hormone-sensitive malignancies, including breast carcinoma.

Diagnosis and management of antibody-mediated rejection: Current status and novel approaches

Advances in multimodal immunotherapy have significantly reduced acute rejection rates and substantially improved 1‐year graft survival following renal transplantation. However, long‐term (10‐year) survival rates have stagnated over the past decade. Recent studies indicate that antibody‐mediated rejection (ABMR) is among the most important barriers to improving long‐term outcomes. Improved understanding of the roles of acute and chronic ABMR has evolved in recent years following major progress in the technical ability to detect and quantify recipient anti‐HLA antibody production. Additionally, new knowledge of the immunobiology of B cells and plasma cells that pertains to allograft rejection and tolerance has emerged. Still, questions regarding the classification of ABMR, the precision of diagnostic approaches, and the efficacy of various strategies for managing affected patients abound. This review article provides an overview of current thinking and research surrounding the pathophysiology and diagnosis of ABMR, ABMR‐related outcomes, ABMR prevention and treatment, as well as possible future directions in treatment.

C1q Binding Activity of De Novo Donor-specific HLA Antibodies in Renal Transplant Recipients With and Without Antibody-mediated Rejection.

Background Complement fixation by donor-specific HLA antibodies (DSA) is a primary mechanism for antibody-mediated damage of organ allografts. Using a recently developed kit that measures C1q binding to distinguish complement fixing and nonfixing antibodies, studies showed that C1q + DSAs have a higher risk of rejection and graft loss compared to C1q-DSA. The objective of this study was to assess the ability of the C1q-binding assay to identify clinically significant de novo DSA in renal transplant recipients and to define the properties of DSA that confer C1q binding ability. Methods The DSA-positive sera from 34 kidney recipients, 19 with biopsy-proven antibody-mediated rejection (AMR) + and 15 who were AMR−, were assayed in C1q-binding assays (C1q Screen; One Lambda, Inc. Canoga Park, CA). The correlation between C1q-binding activity, presence of AMR, DSA mean fluorescence intensity (MFI) values, and immunoglobulin G isotype was determined. Results Fifty-three percent (10/19) of sera from AMR+ patients had C1q + DSA, whereas only 13% (2/15) of sera from AMR− patients contained C1q + DSA. C1q + DSA exhibited significantly higher MFI values regardless of whether they were from AMR+ or AMR− patients (16,118 ± 6698 vs 6429 ± 4003; P < 0.0001). C1q + DSA converted to C1q − when diluted to a comparable MFI level as the C1q − DSA from AMR− patients, and some C1q − antibodies converted to C1q + when concentrated to MFI levels comparable to those observed for AMR+/C1q + sera. Conclusions The C1q binding activity by de novo DSA in patients with AMR largely reflects differences in antibody strength. The C1q assay does not appear to distinguish functionally distinct DSA with clinical significance.

Comparison of O-6-methylguanine DNA methyltransferase (MGMT) mRNA levels in Mer+ and Mer- human tumor cell lines containing the MGMT gene by the polymerase chain reaction technique.

O-6-Alkylguanine is a mutagenic and carcinogenic DNA lesion induced by a variety of alkylating agents, including the chloroethylnitrosoureas. The lesion is repaired by the alkyl-accepting suicide enzyme O-6-methylguanine DNA methyltransferase (MGMT). Approximately 25% of cell lines derived from human tumors are phenotypically deficient in this enzyme and are described as Mer-. Recent cloning of the human MGMT cDNA (Tano, K.; Shiota, S.; Collier, J.; Foote, R.S.; Mitra, S. Proc. Natl. Acad. Sci. USA 87:686-690; 1990) has allowed for a more detailed analysis of the basis of the Mer- phenotype in human Mer- tumor cell lines. Using the polymerase chain reaction (PCR) technique, an MGMT cDNA probe based on the published sequence was generated. The probe and the PCR technique were then used to analyze the presence and expression of the MGMT gene in two Mer+ and four Mer- lines, including one SV40-transformed Mer- line and three Mer- human tumor cell lines. The data demonstrate that while all six cell lines contained a relatively nonamplified, nonrearranged MGMT gene, Mer- lines contained levels of MGMT mRNA detectable only by PCR analysis. Of the three Mer- tumor cell lines examined, two (COLO 320 HSR, A1235) contained MGMT mRNA levels that were four to five orders of magnitude lower than that of the prototype Mer+ tumor line (HT-29), while one (BE) contained no consistently detectable MGMT mRNA. These results suggest that in the human Mer- tumor lines tested, the Mer- phenotype was mediated by a severe reduction in MGMT mRNA levels, despite the presence of the MGMT gene.

A phase I clinical trial of recombinant interleukin-2 by periodic 24-hour intravenous infusions.

Recombinant interleukin-2 (rIL-2) (NSC# 600664; Hoffmann-La Roche, Inc., Nutley, NJ) was studied in a phase I clinical trial in 33 patients with advanced, measureable cancer of the colon or malignant melanoma, Eastern Cooperative Oncology Group (ECOG) performance status O-1, and no prior chemotherapy or radiotherapy. The goal of the study was to identify a dose and schedule of IL-2 to generate maximal immune modulation with tolerable toxicity. Such a regimen might allow the addition of other treatment modalities and/or prolonged treatment duration in later trials. Each patient received IL-2 as a continuous 24-hour infusion once weekly for 4 weeks and then twice weekly for 4 weeks. Five treatment groups received from 10(3) U/m2 to 3 x 10(7) U/m2 per 24-hour infusion. The maximal tolerated dose was 3 x 10(7) U/m2/d twice weekly. Patients treated twice weekly at 1 x 10(7) and 3 x 10(7) U/m2/d had immune modulation in terms of lymphocytosis, eosinophilia, increased natural killer (NK) activity, and elevated numbers of peripheral blood mononuclear cells expressing CD16, OKT10/Leu-17, and Leu-19 surface markers. Endogenous generation of peripheral blood lymphokine-activated killer (LAK) activity was demonstrated by lysis of NK-resistant Daudi targets, in patients treated at 3 x 10(7) U/m2/d. Biochemical and hematological abnormalities were moderate and reversible. Clinical toxicity included hypotension, myalgia, arthralgia, stomatitis, fever, fatigue, nausea, headache, chills, diarrhea, and oliguria at high doses. Cardiovascular toxicity was tolerable for most patients and reversed after IL-2 was stopped. Two of six melanoma patients at 3 x 10(7) U/m2/d achieved partial responses by the end of the eighth week. This IL-2 schedule appears to produce potentially clinically useful immune enhancement with tolerable toxicity.

Antithymocyte Globulin is Associated with a Lower Incidence of De Novo Donor-Specific Antibodies in Moderately Sensitized Renal Transplant Recipients

Background Recent evidence suggests that de novo donor-specific antibodies (dnDSA) are associated with antibody-mediated rejection (ABMR) and graft failure after kidney transplantation. The effects of induction immunosuppression on dnDSA are unknown. Methods The study population comprised 114 consecutive moderately sensitized (positive DSA and negative flow crossmatch) recipients who received deceased donor renal transplants between December 2009 and November 2011. Patients were divided into two groups based on induction immunosuppression: antithymocyte globulin (ATG) (n=85) or basiliximab (n=29) and were followed up for 36 months. Results Patients in the ATG group received a mean dose of 4.98 mg/kg±7.9 mg/kg, had a significantly higher PRA, and received more plasmapheresis and IVIG at the time of transplant. The incidence of dnDSA (P=0.02, HR=0.33, 95% CI 0.09–1.24) and ABMR (P=0.001, HR=0.9, 95% CI 0.04–0.87) was significantly lower in the ATG group. In multivariate regression analyses, ATG induction was the single most important variable associated with both ABMR and dnDSA. Conclusions In moderately sensitized deceased donor renal transplant recipients, induction with ATG is associated with a reduction in the occurrence of dnDSA and ABMR when compared with basiliximab.

Pretransplant donor‐specific anti‐HLA antibodies as predictors of early allograft rejection in ABO‐compatible liver transplantation

The significance of preexisting donor‐specific HLA antibodies (HLA‐DSAs) for liver allograft function is unclear. Our previous studies have shown that humoral alloreactivity frequently accompanies acute cellular rejection (ACR). In the present study, we set out to determine whether pretransplant HLA‐DSAs correlate with clinically significant ACR in the first 90 days after transplantation and, if so, to determine their predictive values. Class I HLA‐DSAs and class II HLA‐DSAs were determined by single‐antigen bead flow cytometry for 113 consecutive adult transplants. A statistical analysis was performed for data from 109 consecutive patients with graft survival greater than or equal to 90 days. All patients who developed biochemical graft dysfunction underwent liver biopsy for hematoxylin‐eosin and complement component 4d staining. Cox proportional hazards models and associated hazard ratios revealed a significant association of pretransplant HLA‐DSAs with clinically significant ACR: this association started with a mean fluorescence intensity (MFI) as low as 300 for both class I (hazard ratio = 2.7, P  < 0.01) and class II (hazard ratio = 6.0, P  < 0.01). Pretransplant HLA‐DSAs were associated with an increased risk of ACR: P  < 0.01 for class I (42% versus 18%), P  < 0.001 for class II (37% versus 7%), and P  < 0.001 for either class I or II (36% versus 3%). Class I or II HLA‐DSAs with an MFI ≥ 1000 had the best positive predictive value for clinically significant ACR at 46%, whereas class I or II HLA‐DSAs with an MFI ≥ 300 had the best negative predictive value at 97.1%. Although our study was based on consecutive patients, it was limited by the relatively low number of single‐center subjects. In conclusion, the present study indicates that pretransplant HLA‐DSAs, even at low levels of allosensitization, correlate with the risk of clinically significant ACR. Our findings suggest that anti–human leukocyte antigen antibodies could serve as donor‐specific markers of immunoreactivity to the liver graft. Liver Transpl 19:1132‐1141, 2013.

Increased C4d in post-reperfusion biopsies and increased donor specific antibodies at one-week post transplant are risk factors for acute rejection in mild to moderately sensitized kidney transplant recipients

In order to define the intensity of immunosuppression, we examined risk factors for acute rejection in desensitization protocols that use baseline donor specific antibody levels measured as mean fluorescence intensity (MFImax). The study included 146 patients transplanted with a negative flow crossmatch and a mean follow-up of 18 months with the majority (83%) followed for at least 1 year. At the time of transplant, mean calculated panel reactive antibody and MFImax ranged from 10.3% to 57.2%, and 262 to 1691, respectively, between low and high-risk protocols. Mean MFImax increased significantly from transplant to one-week and one-year. The incidence of acute rejection (mean 1.65 months) as a combination of clinical and subclinical rejection was 32% including 14% cellular, 12% antibody-mediated and 6% mixed rejection. In regression analyses, only C4d staining in post-reperfusion biopsies (hazard ratio 3.3, confidence interval 1.71 to 6.45) and increased donor specific antibodies at 1 week post-transplant were significant predictors of rejection. A rise in MFImax by 500 was associated with a 2.8-fold risk of rejection. Thus, C4d staining in post-reperfusion biopsies and an early rise in donor specific antibodies after transplantation are risk factors for rejection in moderately sensitized patients.

Current outcomes of chronic active antibody mediated rejection – A large single center retrospective review using the updated BANFF 2013 criteria

BACKGROUND The updated BANFF 2013 criteria has enabled a more standardized and complete serologic and histopathologic diagnosis of chronic active antibody mediated rejection (cAMR). Little data exists on the outcomes of cAMR since the initiation of this updated criteria. METHODS 123 consecutive patients with biopsy proven cAMR (BANFF 2013) between 2006 and 2012 were identified. RESULTS Patients identified with cAMR were followed for a median of 9.5 (2.7-20.3) years after transplant and 4.3 (0-8.8) years after cAMR. Ninety-four (76%) recipients lost their grafts with a median survival of 1.9 years after diagnosis with cAMR. Mean C4d and allograft glomerulopathy scores were 2.6 ± 0.7 and 2.2 ± 0.8, respectively. 53.2% had class II DSA, 32.2% had both class I and II, and 14.5% had class I DSA only. Chronicity score >8 (HR 2.9, 95% CI 1-8.4, p=0.05), DSA >2500 MFI (HR 2.8, 95% CI 1.1-6.8, p=0.03), Scr >3mg/dL (HR 3.2, 95% CI 1.6-6.3, p=0.001) and UPC >1g/g (HR 2.5, 95% CI 1.4-4.5, p=0.003) were associated with a higher risk of graft loss. CONCLUSIONS cAMR was associated with poor graft survival after diagnosis. Improved therapies and earlier detection strategies are likely needed to improve outcomes of cAMR in kidney transplant recipients.

Interpretation of HLA single antigen bead assays

The introduction of single antigen bead (SAB) assays for detection and quantitation of HLA antibodies has improved our ability to identify and manage allosensitized transplant candidates and recipients and to improve organ allocation, and was critical to the creation of national paired kidney exchanges. The principal limitations of the technology have been detailed in the literature and include artifacts resulting in non-specific background, variability, lack of standardization, and interpretive challenges. Accurate interpretation of SAB assays requires consideration of a number of factors, including identification of epitope reactivity patterns, mean fluorescence intensity (MFI) values, patient history, and appreciation of individual bead and assay nuances. The MFI value provides an estimate of relative HLA antibody levels although limited by saturation and epitope distribution effects. A better understanding of SAB assays and MFI values will be necessary to ensure appropriate application of these assays clinically and a higher quality of antibody data used in support of published clinical studies.