Immunosuppression withdrawal in living-donor renal transplant recipients following induction with antithymocyte globulin and rituximab: Results of a prospective clinical trial.

Durable tolerance in kidney transplant recipients remains an important but elusive goal. We hypothesized that adding B cell depletion to T cell depletion would generate an immune milieu postreconstitution dominated by immature transitional B cells, favoring tolerance. The Immune Tolerance Network ITN039ST Research Study of ATG and Rituximab in Renal Transplantation was a prospective multicenter pilot study of live donor kidney transplant recipients who received induction with rabbit antithymocyte globulin and rituximab and initiated immunosuppression (IS) withdrawal (ISW) at 26 weeks. The primary endpoint was freedom from rejection at 52 weeks post-ISW. Six of the 10 subjects successfully completed ISW. Of these 6 subjects, 4 restarted immunosuppressive medications due to acute rejection or recurrent disease, 1 remains IS-free for over 9 years, and 1 was lost to follow-up after being IS-free for 42 weeks. There were no cases of patient or graft loss. CD19+ B cell frequencies returned to predepletion levels by 26 weeks posttransplant; immunoglobulin D+CD27--naïve B cells predominated. In contrast, memory cells dominated the repopulation of the T cell compartment. A regimen of combined B and T cell depletion did not generate the tolerogenic B cell profile observed in preclinical studies and did not lead to durable tolerance in the majority of kidney transplant recipients.

Expanding Access to Organ Transplant for People Living With HIV: Can Policy Catch Up to Outcomes Data?

Advances in antiretroviral and immunosuppressive regimens have improved outcomes following solid organ transplantation in people living with HIV (PLWH). The HIV Organ Policy and Equity Act was conceived to reduce the discard of HIV-positive organs and improve access to transplant for PLWH. Nevertheless, PLWH continue to experience disproportionately low rates of transplant. This overview examines the hurdles to transplantation in PLWH with end-organ disease, the potential and realized impact of the HIV Organ Policy and Equity Act, and changes that could permit expanded access to organ transplant in this population.

Understanding heterogeneity of human bone marrow plasma cell maturation and survival pathways by single-cell analyses.

Human bone marrow (BM) plasma cells are heterogeneous, ranging from newly arrived antibody-secreting cells (ASCs) to long-lived plasma cells (LLPCs). We provide single-cell transcriptional resolution of 17,347 BM ASCs from five healthy adults. Fifteen clusters are identified ranging from newly minted ASCs (cluster 1) expressing MKI67 and high major histocompatibility complex (MHC) class II that progress to late clusters 5-8 through intermediate clusters 2-4. Additional ASC clusters include the following: immunoglobulin (Ig) M predominant (likely of extra-follicular origin), interferon responsive, and high mitochondrial activity. Late ASCs are distinguished by G2M checkpoints, mammalian target of rapamycin (mTOR) signaling, distinct metabolic pathways, CD38 expression, utilization of tumor necrosis factor (TNF)-receptor superfamily members, and two distinct maturation pathways involving TNF signaling through nuclear factor κB (NF-κB). This study provides a single-cell atlas and molecular roadmap of LLPC maturation trajectories essential in the BM microniche. Altogether, understanding BM ASC heterogeneity in health and disease enables development of new strategies to enhance protective ASCs and to deplete pathogenic ones.

Human Bone Marrow Plasma Cell Atlas: Maturation and Survival Pathways Unraveled by Single Cell Analyses.

Human bone marrow (BM) plasma cells are heterogeneous, ranging from newly arrived antibody-secreting cells (ASC) to long-lived plasma cells (LLPC). We provide single cell transcriptional resolution of 17,347 BM ASC from 5 healthy adults. Fifteen clusters were identified ranging from newly minted ASC (cluster 1) expressing MKI67 and high MHC Class II that progressed to late clusters 5-8 through intermediate clusters 2-4. Additional clusters included early and late IgM-predominant ASC of likely extra-follicular origin; IFN-responsive; and high mitochondrial activity ASC. Late ASCs were distinguished by differences in G2M checkpoints, MTOR signaling, distinct metabolic pathways, CD38 expression, and utilization of TNF-receptor superfamily members. They mature through two distinct paths differentiated by the degree of TNF signaling through NFKB. This study provides the first single cell resolution atlas and molecular roadmap of LLPC maturation, thereby providing insight into differentiation trajectories and molecular regulation of these essential processes in the human BM microniche. This information enables investigation of the origin of protective and pathogenic antibodies in multiple diseases and development of new strategies targeted to the enhancement or depletion of the corresponding ASC. One Sentence Summary: The single cell transcriptomic atlas of human bone marrow plasma cell heterogeneity shows maturation of class-switched early and late subsets, specific IgM and Interferon-driven clusters, and unique heterogeneity of the late subsets which encompass the long-lived plasma cells.

Infliximab Induction Lacks Efficacy and Increases BK Virus Infection in Deceased Donor Kidney Transplant Recipients: Results of the CTOT-19 Trial.

BACKGROUND: Ischemia-reperfusion (IR) of a kidney transplant (KTx) upregulates TNF α production that amplifies allograft inflammation and may negatively affect transplant outcomes. METHODS: We tested the effects of blocking TNF peri-KTx via a randomized, double-blind, placebo-controlled, 15-center, phase 2 clinical trial. A total of 225 primary transplant recipients of deceased-donor kidneys (KTx; 38.2% Black/African American, 44% White) were randomized to receive intravenous infliximab (IFX) 3 mg/kg or saline placebo (PLBO) initiated before kidney reperfusion. All patients received rabbit anti-thymocyte globulin induction and maintenance immunosuppression (IS) with tacrolimus, mycophenolate mofetil, and prednisone. The primary end point was the difference between groups in mean 24-month eGFR. RESULTS: There was no difference in the primary end point of 24-month eGFR between IFX (52.45 ml/min per 1.73 m 2 ; 95% CI, 48.38 to 56.52) versus PLBO (57.35 ml/min per 1.73 m 2 ; 95% CI, 53.18 to 61.52; P =0.1). There were no significant differences between groups in rates of delayed graft function, biopsy-proven acute rejection (BPAR), development of de novo donor-specific antibodies, or graft loss/death. Immunosuppression did not differ, and day 7 post-KTx plasma analyses showed approximately ten-fold lower TNF ( P <0.001) in IFX versus PLBO. BK viremia requiring IS change occurred more frequently in IFX (28.9%) versus PLBO (13.4%; P =0.004), with a strong trend toward higher rates of BKV nephropathy in IFX (13.3%) versus PLBO (4.9%; P =0.06). CONCLUSIONS: IFX induction therapy does not benefit recipients of kidney transplants from deceased donors on this IS regimen. Because the intervention unexpectedly increased rates of BK virus infections, our findings underscore the complexities of targeting peritransplant inflammation as a strategy to improve KTx outcomes.Clinical Trial registry name and registration number:clinicaltrials.gov (NCT02495077).

Impact of interleukin-6 on T cells in kidney transplant recipients.

Interleukin-6 (IL-6), a multifunctional proinflammatory cytokine, plays a key role in T cell activation, survival, and differentiation. Acting as a switch that induces the differentiation of naïve T cells into Th17 cells and inhibits their development into regulatory T cells, IL-6 promotes rejection and abrogates tolerance. Therapies that target IL-6 signaling include antibodies to IL-6 and the IL-6 receptor and inhibitors of janus kinases; several of these therapeutics have demonstrated robust clinical efficacy in autoimmune and inflammatory diseases. Clinical trials of IL-6 inhibition in kidney transplantation have focused primarily on its effects on B cells, plasma cells, and HLA antibodies. In this review, we summarize the impact of IL-6 on T cells in experimental models of transplant and describe the effects of IL-6 inhibition on the T cell compartment in kidney transplant recipients.

Approaches to Establishing Tolerance in Immune Mediated Diseases.

The development of rational approaches to restore immune tolerance requires an iterative approach that builds on past success and utilizes new mechanistic insights into immune-mediated pathologies. This article will review concepts that have evolved from the clinical trial experience of the Immune Tolerance Network, with an emphasis on lessons learned from the innovative mechanistic studies conducted for these trials and new strategies under development for induction of tolerance.

Treatment of immunocompromised COVID-19 patients with convalescent plasma.

Immunosuppressed patients such as solid organ transplant and hematologic malignancy patients appear to be at increased risk for morbidity and mortality due to coronavirus disease 2019 (COVID-19) caused by SARS coronavirus 2 (SARS-CoV-2). Convalescent plasma, a method of passive immunization that has been applied to prior viral pandemics, holds promise as a potential treatment for COVID-19. Immunocompromised patients may experience more benefit from convalescent plasma given underlying deficits in B and T cell immunity as well as contraindications to antiviral and immunomodulatory therapy. We describe our institutional experience with four immunosuppressed patients (two kidney transplant recipients, one lung transplant recipient, and one chronic myelogenous leukemia patient) treated with COVID-19 convalescent plasma through the Expanded Access Program (NCT04338360). All patients clinically improved after administration (two fully recovered and two discharged to skilled nursing facilities) and none experienced a transfusion reaction. We also report the characteristics of convalescent plasma product from a local blood center including positive SARS-CoV-2 IgG and negative SARS-CoV-2 PCR in all samples tested. This preliminary evidence suggest that convalescent plasma may be safe among immunosuppressed patients with COVID-19 and emphasizes the need for further data on the efficacy of convalescent plasma as either primary or adjunctive therapy for COVID-19.

The Fourth International Workshop on Clinical Transplant Tolerance.

The International Workshop on Clinical Transplant Tolerance is a biennial meeting that aims to provide an update on the progress of studies of immunosuppression minimization or withdrawal in solid organ transplantation. The Fourth International Workshop on Clinical Tolerance was held in Pittsburgh, Pennsylvania, September 5-6, 2019. This report is a summary of presentations on the status of clinical trials designed to minimize or withdraw immunosuppressive drugs in kidney, liver, and lung transplantation without subsequent evidence of rejection. All protocols had in common the use of donor or recipient cell therapy combined with organ transplantation. The workshop also included presentations of mechanistic studies designed to improve understanding of the cellular and molecular basis of tolerance and to identify potential predictors/biomarkers of tolerance. Strategies to enhance the safety of hematopoietic cell transplantation and to improve patient selection/risk stratification for clinical trials were also discussed.

Interleukin-6 blockade with tocilizumab increases Tregs and reduces T effector cytokines in renal graft inflammation: A randomized controlled trial.

Interleukin-6 (IL-6) is a proinflammatory cytokine and key regulator of Treg: T effector cell (Teff) balance. We hypothesized that IL-6 blockade with tocilizumab, a monoclonal antibody to IL-6R, would increase Tregs, dampen Teff function, and control graft inflammation. We conducted a randomized controlled clinical trial (2014-2018) of clinically stable kidney transplant recipients on calcineurin inhibitor, mycophenolate mofetil, and prednisone, with subclinical graft inflammation noted on surveillance biopsies during the first year posttransplant. Subjects received tocilizumab (8 mg/kg IV every 4 weeks; 6 doses; n = 16) or no treatment (controls; n = 14) on top of usual maintenance immunosuppression. Kidney biopsies pre- and post-treatment were analyzed using Banff criteria. Blood was analyzed for serum cytokines, Treg frequencies, and T cell effector molecule expression (IFN-γ, IL-17, granzyme B) post-stimulation ex vivo. Tocilizumab-treated subjects were more likely to show improved Banff ti-score (62.5% vs. 21.4%, p = .03), increased Treg frequency (7.1% ± 5.55% vs. 3.6% ± 1.7%, p = .0168), and a blunted Teff cytokine response compared to controls. Changes in Banff i- and t-scores were not significantly different. The treatment was relatively well tolerated with no patient deaths or graft loss. Blockade of IL-6 is a novel and promising treatment option to regulate the T cell alloimmune response in kidney transplant recipients. NCT02108600.

Single-cell RNA sequencing of Tocilizumab-treated peripheral blood mononuclear cells as an in vitro model of inflammation.

COVID-19 has posed a significant threat to global health. Early data has revealed that IL-6, a key regulatory cytokine, plays an important role in the cytokine storm of COVID-19. Multiple trials are therefore looking at the effects of Tocilizumab, an IL-6 receptor antibody that inhibits IL-6 activity, on treatment of COVID-19, with promising findings. As part of a clinical trial looking at the effects of Tocilizumab treatment on kidney transplant recipients with subclinical rejection, we performed single-cell RNA sequencing of comparing stimulated PBMCs before and after Tocilizumab treatment. We leveraged this data to create an in vitro cytokine storm model, to better understand the effects of Tocilizumab in the presence of inflammation. Tocilizumab-treated cells had reduced expression of inflammatory-mediated genes and biologic pathways, particularly amongst monocytes. These results support the hypothesis that Tocilizumab may hinder the cytokine storm of COVID-19, through a demonstration of biologic impact at the single-cell level.

The impact of belatacept on the phenotypic heterogeneity of renal T cell-mediated alloimmune response: The critical role of maintenance treatment and inflammatory load.

Belatacept offers superior long-term outcome relative to calcineurin inhibitor (CNI)-based immunosuppression. However, the higher frequency of early T cell-mediated rejection (TCMR) in belatacept-treated patients hampered the widespread adoption of costimulation blockade. Here, we applied gene expression analysis and whole-slide inflammatory cell quantification to assess the impact of belatacept on intragraft immune signature. We studied formalin-fixed, paraffin-embedded renal biopsies from 92 patients stratified by histopathologic diagnosis (TCMR, borderline changes, or normal) and immunosuppression regimen (belatacept, CNI). An interaction model was built to explore maintenance treatment-dependent expression level changes of immune response-related genes across diagnostic categories of normal, borderline changes, and TCMR. Ninety-one percent of genes overexpressed in TCMR showed significant correlation with whole section inflammatory load. There were 27 genes that had a positive association with belatacept treatment. These were mostly related to myeloid cells and innate immunity. Genes negatively associated with costimulation blockade (n = 14) could be linked to B-cell differentiation and proliferation. We concluded that expression levels of genes characteristic of TCMR are strongly interconnected with quantitative changes of the biopsy inflammatory load. Our results might suggest differential involvement of the innate immune system, and an altered B-cell engagement during TCMR in belatacept-treated patients relative to CNI-treated referents.

Clinical outcomes and serologic response in solid organ transplant recipients with COVID-19: A case series from the United States.

The coronavirus disease 2019 (COVID-19) pandemic caused by SARS coronavirus 2 (SARS-CoV-2) has caused significant morbidity and mortality for patients and stressed healthcare systems worldwide. The clinical features, disease course, and serologic response of COVID-19 among immunosuppressed patients such as solid organ transplant (SOT) recipients, who are at presumed risk for more severe disease, are not well characterized. We describe our institutional experience with COVID-19 among 10 SOT patients, including the clinical presentation, treatment modalities, and outcomes of 7 renal transplant recipients, 1 liver transplant recipient, 1 heart transplant recipient, and 1 lung transplant recipient. In addition, we report the serologic response in SOT recipients, documenting a positive IgG response in all 7 hospitalized patients. We also review the existing literature on COVID-19 in SOT recipients to consolidate the current knowledge on COVID-19 in the SOT population for the transplant community.

Single-Cell RNA Sequencing of Tocilizumab-Treated Peripheral Blood Mononuclear Cells as an in vitro Model of Inflammation.

COVID-19 has posed a significant threat to global health. Early data has revealed that IL-6, a key regulatory cytokine, plays an important role in the cytokine storm of COVID-19. Multiple trials are therefore looking at the effects of Tocilizumab, an IL-6 receptor antibody that inhibits IL-6 activity, on treatment of COVID-19, with promising findings. As part of a clinical trial looking at the effects of Tocilizumab treatment on kidney transplant recipients with subclinical rejection, we performed single-cell RNA sequencing of comparing stimulated PBMCs before and after Tocilizumab treatment. We leveraged this data to create an in vitro cytokine storm model, to better understand the effects of Tocilizumab in the presence of inflammation. Tocilizumab-treated cells had reduced expression of inflammatory-mediated genes and biologic pathways, particularly amongst monocytes. These results support the hypothesis that Tocilizumab may hinder the cytokine storm of COVID-19, through a demonstration of biologic impact at the single-cell level.

Refining the Policy for Timing of Kidney Transplant Waitlist Qualification.

BACKGROUND: Earlier qualification for the kidney transplant waitlist expedites transplant and is therefore associated with improved outcomes. U.S. Organ Procurement and Transplantation Network policies state that "measured or calculated creatinine clearance or glomerular filtration rate less than or equal to 20 mL/min" triggers waitlist time accrual. The choice of qualification method is somewhat arbitrary, and the policy implies that decline in renal function is monotonic. METHODS: (1) We used survival analysis to quantify temporal differences in waitlist qualification by applying 3 kidney-function-estimating equations (Cockcroft-Gault, Modification of Diet in Renal Disease study, Chronic Kidney Disease Epidemiology Collaboration) to serial creatinine measurements from 3 patient cohorts: 1 of waitlisted patients at a major U.S. academic center and 2 national, multicenter cohorts of chronic kidney disease patients (African American Study of Kidney Disease and Hypertension, Modification of Diet in Renal Disease). (2) Survival analysis assessed whether requiring patients to demonstrate persistently reduced renal function on 2 occasions at least 90 days apart would meaningfully change qualification order. RESULTS: On average, time to waitlist qualification would be delayed on the order of 1 to 2 years by using calculated creatinine clearance (per the Cockcroft-Gault equation). Compared with current policy, requiring demonstration of persistently reduced renal function delayed qualification by 0.6 to 2.1 years and caused 40% to 50% of patients to switch the order in which they qualify by 6 months or more. CONCLUSIONS: The kidney transplantation policies should be revised, such that timing of waitlist qualification is more standardized. We suggest that mention of using calculated creatinine clearance be dropped from the Organ Procurement and Transplantation Network policy wording and the units to quantify kidney function be changed to mL/min per 1.73 m2. Some consideration should be given to whether requiring persistently reduced renal function would better identify patients most likely to benefit from earlier waitlist qualification.

Fasting Insulin is Better Partitioned according to Family History of Type 2 Diabetes Mellitus than Post Glucose Load Insulin of Oral Glucose Tolerance Test in Young Adults.

INTRODUCTION: Hyperinsulinemia is contributed by insulin resistance, hepatic insulin uptake, insulin secretion and rate of insulin degradation. Family history of type 2 diabetes mellitus has been reported to cause hyperinsulinemia. AIM: Correlation of fasting insulin with post glucose load Oral Glucose Tolerance Test (OGTT) insulin in young adults and their partitioning according to family history of type 2 diabetes. MATERIALS AND METHODS: In this observational cross-sectional study, clinical evaluation and biochemical assays of insulin and diabetes related parameters, and secondary clinical influences on type 2 diabetes in volunteers were done for inclusion as participants (n=90) or their exclusion. Cut off levels of quantitative biochemical variables were fixed such that they included the effects of insulin resistance, but excluded other secondary clinical influences. Distribution was analysed by Shapiro-Wilk test; equality of variances by Levene's test; Log10 transformations for conversion of groups to Gaussian distribution and for equality of variances in the groups compared. When the groups compared had Gaussian distribution and there was equality of variance, parametric methods were used. Otherwise, non parametric methods were used. RESULTS: Fasting insulin was correlating significantly with 30, 60 and 120 minute OGTT insulin showing that hyperinsulinemia in the fasting state was related to hyperinsulinemia in the post glucose load states. When fasting and post glucose load OGTT insulin were partitioned into those without and with family history of type 2 diabetes, maximum difference was seen in fasting insulin (p<0.001), followed by 120 (p=0.001) and 60 (p= 0.002) minute OGTT insulin. The 30 minute insulin could not be partitioned (p=0.574). CONCLUSION: Fasting, 60 and 120 minute OGTT insulin can be partitioned according to family history of type 2 diabetes, demonstrating stratification and heterogeneity in the insulin sample. Of these, fasting insulin was better partitioned and could be used for baseline reference interval calculations.