Tissue-resident memory T cells in human kidney transplants have alloreactive potential.

The extent to which tissue-resident memory T (TRM) cells in transplanted organs possess alloreactivity is uncertain. This study investigates the alloreactive potential of TRM cells in kidney explants from 4 patients who experienced severe acute rejection leading to graft loss. Alloreactive T cell receptor (TCR) clones were identified in pretransplant blood samples through mixed lymphocyte reactions, followed by single-cell RNA and TCR sequencing of the proliferated recipient T cells. Subsequently, these TCR clones were traced in the TRM cells of kidney explants, which were also subjected to single-cell RNA and TCR sequencing. The proportion of recipient-derived TRM cells expressing an alloreactive TCR in the 4 kidney explants varied from 0% to 9%. Notably, these alloreactive TCRs were predominantly found among CD4+ and CD8+ TRM cells with an effector phenotype. Intriguingly, these clones were present not only in recipient-derived TRM cells but also in donor-derived TRM cells, constituting up to 4% of the donor population, suggesting the presence of self-reactive TRM cells. Overall, our study demonstrates that T cells with alloreactive potential present in the peripheral blood prior to transplantation can infiltrate the kidney transplant and adopt a TRM phenotype.

The balance between memory and regulatory cell populations in kidney transplant recipients with operational tolerance.

Donor-reactive memory cells represent a barrier to long-term kidney graft survival. A better understanding of regulatory mechanisms that counterbalance alloreactive memory responses may help to identify patients with operational tolerance. This prospective study investigated the equilibrium between memory T-cell subsets and regulatory T or B cells (Tregs, Bregs) in peripheral blood of kidney transplant recipients with operational tolerance (N = 8), chronic rejection (N = 8), and different immunosuppressive treatment regimens (N = 81). Patients on hemodialysis and healthy individuals served as controls (N = 50). In addition, the expression of Treg- and Breg-associated molecule genes was analyzed. Patients with chronic rejection showed a disrupted memory T-cell composition with a significantly higher frequency of circulating CD8+ terminally differentiated effector memory (TEMRA) T cells than patients with operational tolerance, patients on hemodialysis, or healthy controls (P < 0.001). Low frequency of CD8+ TEMRA and high frequency of Tregs and transitional Bregs were found in operationally tolerant patients. Consequently, operationally tolerant patients showed, as compared to all other transplant recipients with different immunosuppressive regiments, the lowest ratios between CD8+ TEMRA T cells and Tregs or Bregs (for both P < 0.001). Moreover, a specific peripheral blood transcription pattern was found in operationally tolerant patients with an increased expression of Breg- and Treg-associated genes CD22 and FoxP3 and a decreased FcγRIIA/FcγRIIB transcript ratio (for all P < 0.001). In conclusion, monitoring the balance between circulating CD8+ TEMRA T cells and regulatory cell subsets and their transcripts may help to distinguish transplant recipients with operational tolerance from recipients at risk of graft loss.

Effect of lethal total body irradiation on bone marrow chimerism, acute graft-versus-host disease, and tumor engraftment in mouse models: impact of different radiation techniques using low- and high-energy X-rays.

PURPOSE: Effects of X­ray energy levels used for myeloablative lethal total body irradiation (TBI) delivery prior to bone marrow transplantation (BMT) in preclinical mouse models were examined. MATERIALS AND METHODS: In mouse models, single-fraction myeloablative TBI at a lethal dose was delivered using two different X­ray devices, either low (160 kV cabinet irradiator) or high energy (6 MV linear accelerator), before semi-allogeneic hematopoietic stem-cell transplantation (HSCT) to ensure bone marrow (BM) chimerism, graft-versus-host disease (GVHD), and tumor engraftment. Recipient mice were clinically followed for 80 days after bone marrow transplantation (BMT). Flow cytometry was performed to assess donor chimerism and tumor engraftment in recipient mice. RESULTS: Both X­ray irradiation techniques delivered a 10 Gy single fraction of TBI, presented a lethal effect, and could allow near-complete early donor chimerism on day 13. However, low-energy irradiation increased T cells' alloreactivity compared to high-energy irradiation, leading to clinical consequences for GVHD and tumor engraftment outcomes. The alloreactive effect differences might be attributed to the distinction in inflammatory status of irradiated recipients at donor cell infusion (D0). Delaying donor cell administration (D1 after lethal TBI) attenuated T cells' alloreactivity and clinical outcomes in GVHD mouse models. CONCLUSION: Different X­ray irradiation modalities condition T cell alloreactivity in experimental semi-allogeneic BMT. Low-energy X­ray irradiator induces a post-TBI inflammatory burst and exacerbates alloreactive reactions. This technical and biological information should be considered in interpreting GVHD/ graft-versus-leukemia effect results in mice experimental models of BMT.

TCR Sequencing in Mouse Models of Allorecognition Unveils the Features of Directly and Indirectly Activated Clonotypes.

Allorecognition is known to involve a large number of lymphocytes carrying diverse T-cell receptor repertoire. Thus, one way to understand allorecognition and rejection mechanisms is via high-throughput sequencing of T-cell receptors. In this study, in order to explore and systematize the properties of the alloreactive T-cell receptor repertoire, we modeled direct and indirect allorecognition pathways using material from inbred mice in vitro and in vivo. Decoding of the obtained T-cell receptor genes using high-throughput sequencing revealed some features of the alloreactive repertoires. Thus, alloreactive T-cell receptor repertoires were characterized by specific V-gene usage patterns, changes in CDR3 loop length, and some amino acid occurrence probabilities in the CDR3 loop. Particularly pronounced changes were observed for directly alloreactive clonotypes. We also revealed a clustering of directly and indirectly alloreactive clonotypes by their ability to bind a single antigen; amino acid patterns of the CDR3 loop of alloreactive clonotypes; and the presence in alloreactive repertoires of clonotypes also associated with infectious, autoimmune, and tumor diseases. The obtained results were determined by the modeling of the simplified allorecognition reaction in inbred mice in which stimulation was performed with a single MHCII molecule. We suppose that the decomposition of the diverse alloreactive TCR repertoire observed in humans with transplants into such simple reactions will help to find alloreactive repertoire features; e.g., a dominant clonotype or V-gene usage pattern, which may be targeted to correct the entire rejection reaction in patients. In this work, we propose several technical ways for such decomposition analysis, including separate modeling of the indirect alloreaction pathway and clustering of alloreactive clonotypes according to their ability to bind a single antigen, among others.

[Medication adherence in renal transplantation: Evaluation, predictive factors and impact on humoral alloreactivity]. / Adhésion médicamenteuse en greffe rénale : évaluation, facteurs prédictifs et impact sur l'allo-réactivité humorale.

OBJECTIVES: The aims of this study were to assess medication adherence to immunosuppressive treatment in kidney transplanted patients, to identify predictive factors of medication non-adherence and to analyse its impact on the development of Donor Specific Antibodies (DSA) de novo, biomarkers of rejection in transplant recipients. METHODS: A cross-sectional single-centre study was conducted to assess medication adherence to immunosuppressive treatment with the BAASIS (Basel Assessment of Adherence Scale for Immunosuppressives) self-report questionnaire. Univariate and multivariate analyses were performed to determine non-adherence predictive factors and its role in the development of DSA de novo. RESULTS: A total of 212 renal transplanted patients completed the BAASIS questionnaire: 36,3 % were non-adherent to their immunosuppressive treatment. Patient's age and taking azathioprine were independent predictors of non-adherence and "married or living together" family status was a protective factor in the multivariate analysis. Medication non-adherence was associated with DSA de novo development in the multivariate model and it multiplied their risk of development by 3. CONCLUSIONS: This study, which detected a large proportion of patients who did not adhere to immunosuppressive treatment, highlighted non-adherence predictors and showed the association between non-adherence and development of DSA de novo. In case of non-adherent behavior, it is crucial to set up a personalised support for patients with a multidisciplinary approach of therapeutic education, in which the clinical pharmacist has a role.

Elucidation of CRISPR-Cas9 application in novel cellular immunotherapy.

Novel cellular immunotherapy with engineered T cells has improved cancer treatment and established therapeutic promises to prevent tumor formation in clinical studies. Due to certain restrictions and difficulties, CAR and TCR T-cells therapies were inadequate at points. CRISPR Cas9 genome-editing tool has significant potential for these two cell-based therapies. As a specialized gene-editing technique, CRISPR Cas9 is used to repair genetic alternations with minimal damage. It is used as an adjunct to immunotherapy to stimulate a more robust immune response. CRISPR has long outpaced other target-specific genome editing methods such as ZFNs and TALEN because of its high efficiency, competence in targeting, and stable operating conditions. CRISPR can overcome the two major drawbacks of universal CAR T cells: allorejection and graft-vs-host disease. TCR-based T cell treatment can reduce inappropriate binding between endogenous and transgenic TCR, resulting in a reduction of severe toxicity. The CAR and TCR T based cell therapies uphold an excellent future for tumor malignancies. This article has elucidated the administration of CRISPR Cas9 in novel cellular immunotherapy, CAR, and TCR T cell therapy. However, this article did not fail to observe this technology's ethical concerns, limitations, and challenges. Furthermore, the article compares CRISPR-mediated allogeneic CAR T cell to TCR-T cell therapy.

No detectable alloreactive transcriptional responses under standard sample preparation conditions during donor-multiplexed single-cell RNA sequencing of peripheral blood mononuclear cells.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) provides high-dimensional measurements of transcript counts in individual cells. However, high assay costs and artifacts associated with analyzing samples across multiple sequencing runs limit the study of large numbers of samples. Sample multiplexing technologies such as MULTI-seq and antibody hashing using single-cell multiplexing kit (SCMK) reagents (BD Biosciences) use sample-specific sequence tags to enable individual samples to be sequenced in a pooled format, markedly lowering per-sample processing and sequencing costs while minimizing technical artifacts. Critically, however, pooling samples could introduce new artifacts, partially negating the benefits of sample multiplexing. In particular, no study to date has evaluated whether pooling peripheral blood mononuclear cells (PBMCs) from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30 min co-incubation at 4 °C) results in significant changes in gene expression resulting from alloreactivity (i.e., response to non-self). The ability to demonstrate minimal to no alloreactivity is crucial to avoid confounded data analyses, particularly for cross-sectional studies evaluating changes in immunologic gene signatures. RESULTS: Here, we applied the 10x Genomics scRNA-seq platform to MULTI-seq and/or SCMK-labeled PBMCs from a single donor with and without pooling with PBMCs from unrelated donors for 30 min at 4 °C. We did not detect any alloreactivity signal between mixed and unmixed PBMCs across a variety of metrics, including alloreactivity marker gene expression in CD4+ T cells, cell type proportion shifts, and global gene expression profile comparisons using Gene Set Enrichment Analysis and Jensen-Shannon Divergence. These results were additionally mirrored in publicly-available scRNA-seq data generated using a similar experimental design. Moreover, we identified confounding gene expression signatures linked to PBMC preparation method (e.g., Trima apheresis), as well as SCMK sample classification biases against activated CD4+ T cells which were recapitulated in two other SCMK-incorporating scRNA-seq datasets. CONCLUSIONS: We demonstrate that (i) mixing PBMCs from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30 min co-incubation at 4 °C) does not cause an allogeneic response, and (ii) that Trima apheresis and PBMC sample multiplexing using SCMK reagents can introduce undesirable technical artifacts into scRNA-seq data. Collectively, these observations establish important benchmarks for future cross-sectional immunological scRNA-seq experiments.

Thymic stromal lymphopoietin levels after allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) is an immunoregulatory, Th2-polarizing cytokine produced by epithelial cells. We hypothesized that TSLP affects immune reconstitution after hematopoietic stem cell transplantation (HSCT) leading to increased alloreactivity. METHODS: We measured plasma TSLP by ELISA in 38 patients and assessed the immune reconstitution by flow cytometry. RESULTS: TSLP levels rose after initiation of the conditioning to peak at day +21 after HSCT (p = .03), where TSLP levels correlated with counts of neutrophils (rho = 0.36, p = .04), monocytes (rho = 0.58, p = .006), and lymphocytes (rho = 0.59, p = .02). Overall absolute TSLP levels were not associated with acute or chronic graft-vs-host disease (a/cGvHD). However, patients mounting a sustained increase in TSLP levels at day +90 had a higher risk of cGvHD compared to patients who had returned to pre-conditioning levels at that stage (cumulative incidence: 77% vs. 38%, p = .01). CONCLUSION: In conclusion, this study suggests a role of TSLP in immune reconstitution and alloreactivity post-HSCT. lymphopoietin (TSLP) is an immunoregulatory, Th2-polarizing cytokine produced by epithelial cells. We hypothesized that TSLP affects immune reconstitution after hematopoietic stem cell transplantation (HSCT) leading to increased alloreactivity. We measured plasma TSLP by ELISA in 38 patients and assessed the immune reconstitution by flow cytometry.

Variances in Antiviral Memory T-Cell Repertoire of CD45RA- and CD62L-Depleted Lymphocyte Products Reflect the Need of Individual T-Cell Selection Strategies to Reduce the Risk of GvHD while Preserving Antiviral Immunity in Adoptive T-Cell Therapy.

INTRODUCTION: Viral infections and reactivations still remain a cause of morbidity and mortality after hematopoietic stem cell transplantation due to immunodeficiency and immunosuppression. Transfer of unmanipulated donor-derived lymphocytes (DLI) represents a promising strategy for improving cellular immunity but carries the risk of graft versus host disease (GvHD). Depleting alloreactive naïve T cells (TN) from DLIs was implemented to reduce the risk of GvHD induction while preserving antiviral memory T-cell activity. Here, we compared two TN depletion strategies via CD45RA and CD62L expression and investigated the presence of antiviral memory T cells against human adenovirus (AdV) and Epstein-Barr virus (EBV) in the depleted fractions in relation to their functional and immunophenotypic characteristics. METHODS: T-cell responses against ppEBV_EBNA1, ppEBV_Consensus and ppAdV_Hexon within TN-depleted (CD45RA-/CD62L-) and TN-enriched (CD45RA+/CD62L+) fractions were quantified by interferon-gamma (IFN-γ) ELISpot assay after short- and long-term in vitro stimulation. T-cell frequencies and immunophenotypic composition were assessed in all fractions by flow cytometry. Moreover, alloimmune T-cell responses were evaluated by mixed lymphocyte reaction. RESULTS: According to differences in the phenotype composition, antigen-specific T-cell responses in CD45RA- fraction were up to 2 times higher than those in the CD62L- fraction, with the highest increase (up to 4-fold) observed after 7 days for ppEBV_EBNA1-specific T cells. The CD4+ effector memory T cells (TEM) were mainly responsible for EBV_EBNA1- and AdV_Hexon-specific T-cell responses, whereas the main functionally active T cells against ppEBV_Consensus were CD8+ central memory T cells (TCM) and TEM. Moreover, comparison of both depletion strategies indicated that alloreactivity in CD45RA- was lower than that in CD62L- fraction. CONCLUSION: Taken together, our results indicate that CD45RA depletion is a more suitable strategy for generating TN-depleted products consisting of memory T cells against ppEBV_EBNA1 and ppAdV_Hexon than CD62L in terms of depletion effectiveness, T-cell functionality and alloreactivity. To maximally exploit the beneficial effects mediated by antiviral memory T cells in TN-depleted products, depletion methods should be selected individually according to phenotype composition and CD4/CD8 antigen restriction. TN-depleted DLIs may improve the clinical outcome in terms of infections, GvHD, and disease relapse if selection of pathogen-specific donor T cells is not available.

Genetic Bias, Diversity Indices, Physiochemical Properties and CDR3 Motifs Divide Auto-Reactive from Allo-Reactive T-Cell Repertoires.

The distinct properties of allo-reactive T-cell repertoires are not well understood. To investigate whether auto-reactive and allo-reactive T-cell repertoires encoded distinct properties, we used dextramer enumeration, enrichment, single-cell T-cell receptor (TCR) sequencing and multiparameter analysis. We found auto-reactive and allo-reactive T-cells differed in mean ex vivo frequency which was antigen dependent. Allo-reactive T-cells showed clear differences in TCR architecture, with enriched usage of specific T-cell receptor variable (TRBJ) genes and broader use of T-cell receptor variable joining (TRBJ) genes. Auto-reactive T-cell repertoires exhibited complementary determining regions three (CDR3) lengths using a Gaussian distribution whereas allo-reactive T-cell repertoires exhibited distorted patterns in CDR3 length. CDR3 loops from allo-reactive T-cells showed distinct physical-chemical properties, tending to encode loops that were more acidic in charge. Allo-reactive T-cell repertoires differed in diversity metrics, tending to show increased overall diversity and increased homogeneity between repertoires. Motif analysis of CDR3 loops showed allo-reactive T-cell repertoires differed in motif preference which included broader motif use. Collectively, these data conclude that allo-reactive T-cell repertoires are indeed different to auto-reactive repertoires and provide tangible metrics for further investigations and validation. Given that the antigens studied here are overexpressed on multiple cancers and that allo-reactive TCRs often show increased ligand affinity, this new TCR bank also has translational potential for adoptive cell therapy, soluble TCR-based therapy and rational TCR design.

How an alloreactive T-cell receptor achieves peptide and MHC specificity.

T-cell receptor (TCR) allorecognition is often presumed to be relatively nonspecific, attributable to either a TCR focus on exposed major histocompatibility complex (MHC) polymorphisms or the degenerate recognition of allopeptides. However, paradoxically, alloreactivity can proceed with high peptide and MHC specificity. Although the underlying mechanisms remain unclear, the existence of highly specific alloreactive TCRs has led to their use as immunotherapeutics that can circumvent central tolerance and limit graft-versus-host disease. Here, we show how an alloreactive TCR achieves peptide and MHC specificity. The HCV1406 TCR was cloned from T cells that expanded when a hepatitis C virus (HCV)-infected HLA-A2- individual received an HLA-A2+ liver allograft. HCV1406 was subsequently shown to recognize the HCV nonstructural protein 3 (NS3):1406-1415 epitope with high specificity when presented by HLA-A2. We show that NS3/HLA-A2 recognition by the HCV1406 TCR is critically dependent on features unique to both the allo-MHC and the NS3 epitope. We also find cooperativity between structural mimicry and a crucial peptide "hot spot" and demonstrate its role, along with the MHC, in directing the specificity of allorecognition. Our results help explain the paradox of specificity in alloreactive TCRs and have implications for their use in immunotherapy and related efforts to manipulate TCR recognition, as well as alloreactivity in general.

Allograft rejection is associated with development of functional IgE specific for donor MHC antigens.

BACKGROUND: Donor-specific antibodies of the IgG isotype are measured routinely for diagnostic purposes in renal transplant recipients and are associated with antibody-mediated rejection and long-term graft loss. OBJECTIVE: This study aimed to investigate whether MHC-specific antibodies of the IgE isotype are induced during allograft rejection. METHODS: Anti-MHC/HLA IgE levels were measured in sera of mice grafted with skin or heart transplants from various donor strains and in sera of kidney transplant patients with high levels of HLA IgG. Mediator release was triggered in vitro by stimulating basophils that were coated with murine or human IgE-positive serum, respectively, with specific recombinant MHC/HLA antigens. Kidney tissue samples obtained from organ donors were analyzed by using flow cytometry for cells expressing the high-affinity receptor for IgE (FcεRI). RESULTS: Donor MHC class I- and MHC class II-specific IgE was found on acute rejection of skin and heart grafts in several murine strain combinations, as well as during chronic antibody-mediated heart graft rejection. Anti-HLA IgE, including donor HLA class I and II specificities, was identified in a group of sensitized transplant recipients. Murine and human anti-MHC/HLA IgE triggered mediator release in coated basophils on stimulation with specific MHC/HLA antigens. HLA-specific IgE was not linked to atopy, and allergen-specific IgE present in allergic patients did not cross-react with HLA antigens. FcεRI+ cells were found in the human renal cortex and medulla and provide targets for HLA-specific IgE. CONCLUSION: These results demonstrate that MHC/HLA-specific IgE develops during an alloresponse and is functional in mediating effector mechanisms.

Donor-specific antibodies in liver transplantation.

Despite unique immunoregulatory properties pointing toward tolerance, the liver allograft can be negatively impacted by humoral alloreactivity, with immediate as well as long-term harmful consequences. With regard to the unmet need of long-term outcomes improvement after liver transplantation, donor-specific antibodies have recently been the matter of intense study in this context. We review here recent advances regarding the understanding of the impact of preformed as well as de novo anti-human leukocyte antigen donor-specific antibodies in liver transplantation and discuss potential strategies to overcome this problem.

Next generation sequencing based assessment of the alloreactive T cell receptor repertoire in kidney transplant patients during rejection: a prospective cohort study.

BACKGROUND: Kidney transplantation is the optimal treatment in end stage renal disease but the allograft survival is still hampered by immune reactions against the allograft. This process is driven by the recognition of allogenic antigens presented to T-cells and their unique T-cell receptor (TCR) via the major histocompatibility complex (MHC), which triggers a complex immune response potentially leading to graft injury. Although the immune system and kidney transplantation have been studied extensively, the subtlety of alloreactive immune responses has impeded sensitive detection at an early stage. Next generation sequencing of the TCR enables us to monitor alloreactive T-cell populations and might thus allow the detection of early rejection events. METHODS/DESIGN: This is a prospective cohort study designed to sequentially evaluate the alloreactive T cell repertoire after kidney transplantation. The TCR repertoire of patients who developed biopsy confirmed acute T cell mediated rejection (TCMR) will be compared to patients without rejection. To track the alloreactive subsets we will perform a mixed lymphocyte reaction between kidney donor and recipient before transplantation and define the alloreactive TCR repertoire by next generation sequencing of the complementary determining region 3 (CDR3) of the T cell receptor beta chain. After initial clonotype assembly from sequencing reads, TCR repertoire diversity and clonal expansion of T cells of kidney transplant recipients in periphery and kidney biopsy will be analyzed for changes after transplantation, during, prior or after a rejection. The goal of this study is to describe changes of overall T cell repertoire diversity, clonality in kidney transplant recipients, define and track alloreactive T cells in the posttransplant course and decipher patterns of expanded alloreactive T cells in acute cellular rejection to find an alternative monitoring to invasive and delayed diagnostic procedures. DISCUSSION: Changes of the T cell repertoire and tracking of alloreactive T cell clones after combined bone marrow and kidney transplant has proven to be of potential use to monitor the donor directed alloresponse. The dynamics of the donor specific T cells in regular kidney transplant recipients in rejection still rests elusive and can give further insights in human alloresponse. TRIAL REGISTRATION: Clinicaltrials.gov: NCT03422224 , registered February 5th 2018.

Higher CD45RA+ Regulatory T Cells in the Graft Improves Outcome in Younger Patients Undergoing T Cell-Replete Haploidentical Transplantation: Where Donor Age Matters.

To understand the phenomenon of early alloreactivity (EA) in younger children undergoing post-transplantation cyclophosphamide (PTCy)-based haploidentical transplantation, we studied the graft composition and the immune reconstitution in 32 consecutive patients (aged 2 to 25 years) undergoing PTCy and T cell costimulation blockade based peripheral blood stem cell transplantation with emphasis on CD45RA+ subset of regulatory T cells (Tregs). All but 1 engrafted, and 14 patients experienced EA (acute graft-versus-host disease grades II to IV, n = 8; and post-transplantation hemophagocytic syndrome, n = 6) with a cumulative incidence of 43.7%; 42% developed mild chronic graft-versus-host disease. The overall survival was 70.2% with a nonrelapse mortality of 16.8% at a median of 19 months. Age < 10 years, donor age > 45 years, and poor recovery of Tregs correlated with EA. Not Tregs but higher CD45RA+ Tregs in the graft was associated with less EA (11.7% versus 32.5%, P = .0001). Higher donor age correlated with a lower CD45RA+ Tregs in the graft (P = .01). However, only higher CD45RA+ Treg percentage in the graft favorably impacted EA as well as nonrelapse mortality and overall survival. Our study demonstrates a critical role for CD45RA+ Tregs in determining EA and outcome after PTCy-based haploidentical peripheral blood stem cell transplantation, and the age-related physiologic decline in this population might be responsible for adverse impact of donor age.

Generation of alloreactivity-reduced donor lymphocyte products retaining memory function by fully automatic depletion of CD45RA-positive cells.

BACKGROUND AIMS: For patients needing allogeneic stem cell transplantation but lacking a major histocompatibility complex (MHC)-matched donor, haplo-identical (family) donors may be an alternative. Stringent T-cell depletion required in these cases to avoid lethal graft-versus-host disease (GVHD) can delay immune reconstitution, thus impairing defense against virus reactivation and attenuating graft-versus-leukemia (GVL) activity. Several groups reported that GVHD is caused by cells residing within the naive (CD45RA+) T-cell compartment and proposed use of CD45RA-depleted donor lymphocyte infusion (DLI) to accelerate immune reconstitution. We developed and tested the performance of a CD45RA depletion module for the automatic cell-processing device CliniMACS Prodigy and investigated quality attributes of the generated products. METHODS: Unstimulated apheresis products from random volunteer donors were depleted of CD45RA+ cells on CliniMACS Prodigy, using Good Manufacturing Practice (GMP)-compliant reagents and methods throughout. Using phenotypic and functional in vitro assays, we assessed the cellular constitution of CD45RA-depleted products, including T-cell subset analyses, immunological memory function and allo-reactivity. RESULTS: Selections were technically uneventful and proceeded automatically with minimal hands-on time beyond tubing set installation. Products were near-qualitatively CD45RA+ depleted, that is, largely devoid of CD45RA+ T cells but also of almost all B and natural killer cells. Naive and effector as well as γ/δ T cells were greatly reduced. The CD4:CD8 ratio was fivefold increased. Mixed lymphocyte reaction assays of the product against third-party leukocytes revealed reduced allo-reactivity compared to starting material. Anti-pathogen responses were retained. DISCUSSION: The novel, closed, fully GMP-compatible process on Prodigy generates highly CD45RA-depleted cellular products predicted to be clinically meaningfully depleted of GvH reactivity.

Transplantation tolerance and its outcome during infections and inflammation.

Much progress has been made toward understanding the mechanistic basis of transplantation tolerance in experimental models, which implicates clonal deletion of alloreactive T and B cells, induction of cell-intrinsic hyporesponsiveness, and dominant regulatory cells mediating infectious tolerance and linked suppression. Despite encouraging success in the laboratory, achieving tolerance in the clinic remains challenging, although the basis for these challenges is beginning to be understood. Heterologous memory alloreactive T cells generated by infections prior to transplantation have been shown to be a critical barrier to tolerance induction. Furthermore, infections at the time of transplantation and tolerance induction provide a pro-inflammatory milieu that alters the stability and function of regulatory T cells as well as the activation requirements and differentiation of effector T cells. Thus, infections can result in enhanced alloreactivity, resistance to tolerance induction, and destabilization of the established tolerance state. We speculate that these experimental findings have relevance to the clinic, where infections have been associated with allograft rejection and may be a causal event precipitating the loss of grafts after long periods of stable operational tolerance. Understanding the mechanisms by which infections prevent and destabilize tolerance can lead to therapies that promote stable life-long tolerance in transplant recipients.

Posttransplant peripheral blood donor-specific interferon-γ enzyme-linked immune spot assay differentiates risk of subclinical rejection and de novo donor-specific alloantibodies in kidney transplant recipients.

Noninvasive diagnosis of kidney allograft inflammation in transplant recipients with stable graft function (subclinical rejection) could permit more effective therapy and prevent later development of de novo anti-donor HLA antibodies and/or graft dysfunction. Here we tested whether quantifying posttransplant donor-specific alloreactive T-cells by IFN-γ ELISPOT assay noninvasively detects subclinical T-cell mediated rejection and/or predicts development of anti-donor HLA antibodies. Using an initial cross-sectional cohort of 60 kidney transplant patients with six-month surveillance biopsies, we found that negative donor-specific IFN-γ ELISPOT assays accurately ruled out the presence of subclinical T-cell mediated rejection. These results were validated using a distinct prospective cohort of 101 patients where donor-specific IFN-γ ELISPOT results at both three- and six-months posttransplant significantly differentiated patients with subclinical T-cell mediated rejection at six months, independent of other clinical variables (odds ratio 0.072, 95% confidence interval 0.008-0.653). The posttransplant donor-specific IFN-γ ELISPOT results independently associated with subsequent development of significant anti-donor HLA antibodies (0.085, 0.008-0.862) and with significantly worse two-year function (estimated glomerular filtration rate) compared to patients with a negative test. Thus, posttransplant immune monitoring by donor-specific IFN-γ ELISPOT can assess risk for developing subclinical T-cell mediated rejection and anti-donor HLA antibodies, potentially limiting the need for surveillance biopsies. Our study provides a guide for individualizing immunosuppression to improve posttransplant outcomes.

Infectious pathogens may trigger specific allo-HLA reactivity via multiple mechanisms.

Transplant recipients can be sensitized against allo-HLA antigens by previous transplantation, blood transfusion, or pregnancy. While there is growing awareness that multiple components of the immune system can act as effectors of the alloresponse, the role of infectious pathogen exposure in triggering sensitization and allograft rejection has remained a matter of much debate. Here, we describe that exposure to pathogens may enhance the immune response to allogeneic HLA antigens via different pathways. The potential role of allo-HLA cross-reactivity of virus-specific memory T cells, activation of innate immunity leading to a more efficient induction of the adaptive alloimmune response by antigen-presenting cells, and bystander activation of existing memory B cell activation will be discussed in this review.

Diabetic kidney transplant recipients: Impaired infection control and increased alloreactivity.

BACKGROUND: Post-transplantation diabetes mellitus (PTDM) has been associated with inferior patient and allograft outcomes. However, previous studies did not identify differences in infection control and alloreactivity. METHODS: We studied 449 kidney transplant recipients (KTRs) between 2005 and 2013. Fifty (11.1%) KTRs were diagnosed with PTDM and 60 (13.4%) KTRs with pre-existing diabetes. Samples were collected pretransplantation, at +1, +2, +3 months post-transplantation. CMV specific and alloreactive T cells were quantified by interferon-γ Elispot assay. Lymphocyte subpopulations were quantified by flow cytometry. RESULTS: Upon multivariate analysis, age, obesity, and the use of tacrolimus increased the risk of PTDM (P<.05). KTRs with pre-existing diabetes/PTDM showed higher rates of sepsis (P<.01). Total CD3+ and CD4+ T cell counts were significantly lower in KTRs with PTDM/pre-existing diabetes post-transplantation (P<.05). No differences were observed for CMV-specific T cells between any group (P>.05). KTRs developing PTDM showed increased frequencies of alloreactive T-cells post-transplantation (P<.05). CONCLUSIONS: Our results suggest higher rates of infection in KTRs with pre-existing diabetes/PTDM that may be attributed to impaired overall immunity. Higher frequencies of alloreactive T cells contribute to inferior long-term outcomes. As acute rejection, but not pre-existing diabetes/PTDM, was associated with inferior allograft survival and function, maintaining adequate immunosuppression to prevent rejection seems important.