Donor CD4 T-cell Research Articles

CD4 Donor Lymphocyte Infusion Can Cause Conversion of Chimerism Without GVHD by Inducing Immune Responses Targeting Minor Histocompatibility Antigens in HLA Class II.

Under non-inflammatory conditions HLA class II is predominantly expressed on hematopoietic cells. Therefore, donor CD4 T-cells after allogeneic stem cell transplantation (alloSCT) may mediate graft-vs.-leukemia reactivity without graft-vs.-host disease (GVHD). We analyzed immune responses in four patients converting from mixed to full donor chimerism without developing GVHD upon purified CD4 donor lymphocyte infusion (DLI) from their HLA-identical sibling donor after T-cell depleted alloSCT. In vivo activated T-cells were clonally isolated after CD4 DLI. Of the alloreactive T-cell clones, 96% were CD4 positive, illustrating the dominant role of CD4 T-cells in the immune responses. We identified 9 minor histocompatibility antigens (MiHA) as targets for alloreactivity, of which 8 were novel HLA class II restricted MiHA. In all patients, MiHA specific CD4 T-cells were found that were capable to lyse hematopoietic cells and to recognize normal and malignant cells. No GVHD was induced in these patients. Skin fibroblasts forced to express HLA class II, were recognized by only two MiHA specific CD4 T-cell clones. Of the 7 clones that failed to recognize fibroblasts, two targeted MiHA were encoded by genes not expressed in fibroblasts, presentation of one MiHA was dependent on HLA-DO, which is absent in fibroblasts, and T-cells recognizing the remaining 4 MiHA had an avidity that was apparently too low to recognize fibroblasts, despite clear recognition of hematopoietic cells. In conclusion, purified CD4 DLI from HLA-identical sibling donors can induce conversion from mixed to full donor chimerism with graft-vs.-malignancy reactivity, but without GVHD, by targeting HLA class II restricted MiHA.

Germinal center humoral autoimmunity independently mediates progression of allograft vasculopathy

The development of humoral autoimmunity following organ transplantation is increasingly recognised, but of uncertain significance. We examine whether autoimmunity contributes independently to allograft rejection.In a MHC class II-mismatched murine model of chronic humoral rejection, we report that effector antinuclear autoantibody responses were initiated upon graft-versus-host allorecognition of recipient B cells by donor CD4 T-cells transferred within heart allografts. Consequently, grafts were rejected more rapidly, and with markedly augmented autoantibody responses, upon transplantation of hearts from donors previously primed against recipient. Nevertheless, rejection was dependent upon recipient T follicular helper (TFH) cell differentiation and provision of cognate (peptide-specific) help for maintenance as long-lived GC reactions, which diversified to encompass responses against vimentin autoantigen. Heart grafts transplanted into stable donor/recipient mixed haematopoietic chimeras, or from parental strain donors into F1 recipients (neither of which can trigger host adaptive alloimmune responses), nevertheless provoked GC autoimmunity and were rejected chronically, with rejection similarly dependent upon host TFH cell differentiation.Thus, autoantibody responses contribute independently of host adaptive alloimmunity to graft rejection, but require host TFH cell differentiation to maintain long-lived GC responses. The demonstration that one population of helper CD4 T-cells initiates humoral autoimmunity, but that a second population of TFH cells is required for its maintenance as a GC reaction, has important implications for how autoimmune-related phenomena manifest.

A Novel Standardized Quantitative Suppression Assay Reveals a Diversity of Human Immune-Regulatory Cell Potency

Immunologic tolerance is a critical homeostatic function to protect self from auto-immunity. Various immune-regulatory cells, including regulatory T cell (Treg), myeloid derived suppressor cell (MDSC), tolerogenic dendritic cell, and mesenchymal stromal cell (MSC) are responsible for orchestrating this tolerance. Immune-regulatory cells play a central role in the pathophysiology of cancer immunity, autoimmune disease, and graft versus host disease and can be used in adoptive cell therapy. There is therefore a need for a standardized method to evaluate the suppressive function of these heterogeneous cell populations. We developed an in vitro standardized quantitative suppression assay utilizing the suppressor cell line KARPAS-299 as a standard by which to compare suppressive potency of human immune-regulatory cell populations.Peripheral CD4 T-cells, used as targets in all assays, were isolated from healthy donors (n=30) using an automated cell separator or by flow sorting. After labeling with Cell Tracer Violet (CTV), CD4 T-cells were co-cultured with immune-regulatory cells such as autologous Treg, autologous or third party MDSC (CD11b+CD14+), third party bone marrow derived MSC, and primary leukemia cells, or with the reference KARPAS-299 cells. After stimulating with CD3/CD28 beads, CD154 activation of CD4 T-cells was measured at 16 hours, and CD4 T-cell proliferation was measured by CTV dilution within the viable cell population at 72 hours. Suppressive capacities of immune-regulatory cell types were represented as KARPAS-299 suppressor units (KSU), calculated using the equation b/a, where b is the percentage of suppression in the presence of a given immune-regulatory cell, and a is the percentage of suppression in the presence of KARPAS-299.KARPAS-299 cells reproducibly suppressed healthy donor CD4 T-cells at suppressor: responder ratio of 4:1 for both CD154 activation (percentage of suppression 46.2±18.8%) and proliferation (51.0±20.7%). Immune-regulatory cells showed diverse suppressive capacities: autologous MDSC (63.2±24.5%), allogeneic MDSC (55.8±21.8%), third party MSC (51.26±23.67%), and autologous Treg, (8.7±10.4%). After standardization with KSU, MDSC and MSC showed significantly higher suppressive capacity compared to Treg (MDSC 1.18±0.6 KSU, MSC 1.3±1.11 KSU, Treg 0.15±0.3 KSU: p<0.0001). This standardized assay was also applicable to other types of proliferating targets, such as flow-sorted conventional T cells (Tcon: CD4+CD127highCD25low: 2.73±1.78 KSU with autologous MDSC, 1.6±0.75 KSU with third party MSC) and CD8 T-cells (1.49±0.96 KSU with autologous MDSC, 1.08±0.77 with third party MSC). We validated the method as a potency assay of MSC products, and showed inter-individual differences in MSC. Finally, we used the assay to demonstrate a modest suppressive capacity of acute myeloid leukemia blasts (0.35±0.28 KSU).This method provides a platform for standardizing suppressor function to facilitate comparison between laboratories and for use in cell product release assay. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Stat3 Activation in Donor CD4 T-Cells Is Critical for Acute Gvhd

Abstract 1906Stat3-deficient (Stat3KO) T-cells reportedly are less proliferative due to accelerated apoptosis in the presence of IL-6, and have suppressed IL-2Ra expression. There are conflicting mouse model results concerning Stat3 in graft-versus-host disease (GVHD). Previously, we demonstrated IL-21 signaling is essential for GVHD. As Stat3 is a downstream signaling molecule, clarifying its role in GVHD T-cells is important.After co-transplantation of bone marrow and wild-type or Stat3KO CD4+ T-cells, recipients of the latter developed attenuated GVHD with longer survival than recipients of the former, and their splenic CD4+ T-cells exhibited a profound proliferation defect in the mixed lymphocyte reaction (MLR). In contrast to IL-21R-deficient CD4+ T-cells, pre-transplantation Stat3KO CD4+ T-cells showed similar MLR impairment, suggesting an intrinsic defect. Consistently, proliferation in low-dose anti-CD3 stimulation was defective, but MLR apoptosis was not increased. Neutralizing antibody against IL-2, but not IL-6R, suppressed MLR wild-type CD4 T-cell proliferation, suggesting Stat3KO CD4+ T-cells' MLR apoptosis might be attributed to lack of IL-6 contribution.Pallandre et al. reported Stat3 ablation in CD4 exacerbated GVHD, speculating Stat3 depletion suppressed regulatory T-cell function, leading to accelerated GVHD. Lu et al. analyzed T-cell phosphorylated proteins during allo-activation, demonstrating Stat3 and Erk1/2 are important for this and GVHD. Radojcic et al. showed Stat3KO T-cells cannot induce chronic cutaneous GVHD because of repressed CD4 proliferation and loss of Th17 differentiation. Reasons for differing conclusions are unknown. One possibility is enhanced innate immunity induced by Stat3-siRNA-transduced stem and progenitor cells, consistent with several reports. Our use of Stat3KO T-cells overcame this problem. The latter two reports are consistent with our results. To our knowledge, we report the first direct evidence Stat3 activation in donor CD4+ T-cells is critical for acute GVHD. Durant et al. delineated detailed gene regulation in Stat3KO CD4+ T-cells, and one of their conclusions—Stat3 is important for CD4+ T-cell proliferation—is consistent with our results. Disclosures:No relevant conflicts of interest to declare.

Donor CD4 T Cells Contribute to Cardiac Allograft Vasculopathy by Providing Help for Autoantibody Production

The development of autoantibody after heart transplantation is increasingly associated with poor graft outcome, but what triggers its development and whether it has a direct causative role in graft rejection is not clear. Here, we study the development of antinuclear autoantibody in an established mouse model of heart allograft vasculopathy. Humoral vascular changes, including endothelial complement staining, were present in bm12 heart grafts, explanted 50 days after transplantation. Alloantibody was not detectable, but long-lasting autoantibody responses developed in C57BL/6 recipients from the third week after transplantation. No autoantibody was generated if donor CD4 T cells were depleted before heart graft retrieval or in recipients that lacked B-cell major histocompatibility complex class II expression, indicating that humoral autoimmunity is a consequence of donor CD4 T-cell allorecognition of the major histocompatibility complex class II complex on recipient autoreactive B cells. An effector role for autoantibody in graft rejection was confirmed by abrogation of humoral vascular rejection, and attenuation of vasculopathy, in B-cell deficient recipients and by development of vascular obliteration and accelerated rejection in recipients primed for autoantibody before transplantation. Passenger CD4 T cells within heart transplants can contribute to allograft vasculopathy by providing help to recipient B cells for autoantibody generation.