In humans receiving intestinal transplantation (ITx), long-term multilineage blood chimerism often develops. Donor T cell macrochimerism (>4%) frequently occurs without graft-versus-host disease ...(GVHD) and is associated with reduced rejection. Here we demonstrate that patients with macrochimerism had high graft-versus-host (GvH) to host-versus-graft (HvG) T cell clonal ratios in their allografts. These GvH clones entered the circulation, where their peak levels were associated with declines in HvG clones early after transplant, suggesting that GvH reactions may contribute to chimerism and control HvG responses without causing GVHD. Consistently, donor-derived T cells, including GvH clones, and CD34.sup.+ hematopoietic stem and progenitor cells (HSPCs) were simultaneously detected in the recipients' BM more than 100 days after transplant. Individual GvH clones appeared in ileal mucosa or PBMCs before detection in recipient BM, consistent with an intestinal mucosal origin, where donor GvH-reactive T cells expanded early upon entry of recipient APCs into the graft. These results, combined with cytotoxic single-cell transcriptional profiles of donor T cells in recipient BM, suggest that tissue-resident GvH-reactive donor T cells migrated into the recipient circulation and BM, where they destroyed recipient hematopoietic cells through cytolytic effector functions and promoted engraftment of graft-derived HSPCs that maintain chimerism. These mechanisms suggest an approach to achieving intestinal allograft tolerance.\
We evaluated the role of the thymus in development of multi-organ autoimmunity in human immune system (HIS) mice. T cells were essential for disease development and the same T cell clones with ...varying phenotypes infiltrated multiple tissues. De novo-generated hematopoietic stem cell (HSC)-derived T cells were the major disease drivers, though thymocytes pre-existing in grafted human thymi contributed if not first depleted. HIS mice with a native mouse thymus developed disease earlier than thymectomized mice with a thymocyte-depleted human thymus graft. Defective structure in the native mouse thymus was associated with impaired negative selection of thymocytes expressing a transgenic TCR recognizing a self-antigen. Disease developed without direct recognition of antigens on recipient mouse MHC. While human thymus grafts had normal structure and negative selection, failure to tolerize human T cells recognizing mouse antigens presented on HLA molecules may explain eventual disease development. These new insights have implications for human autoimmunity and suggest methods of avoiding autoimmunity in next-generation HIS mice.
•T cells are essential for development of multi-organ autoimmunity in humanized mice.•Macrophages and De novo-generated T cells were the major disease drivers.•Defective structure and impaired selection of thymocytes in the NSG mouse thymus.•Lower regulatory T cells in native mouse thymus compared to grafted human thymus.•Disease developed without direct recognition of antigens on recipient mouse MHC.
Background
Tolerance‐inducing approaches to xenotransplantation would be optimal and may be necessary for long‐term survival of transplanted pig organs in human patients. The ideal approach would ...generate donor‐specific unresponsiveness to the pig organ without suppressing the patient's normal immune function. Porcine thymus transplantation has shown efficacy in promoting xenotolerance in humanized mice and large animal models. However, murine studies demonstrate that T cells selected in a swine thymus are positively selected only by swine thymic epithelial cells, and therefore, cells expressing human HLA‐restricted TCRs may not be selected efficiently in a transplanted pig thymus. This may lead to suboptimal patient immune function.
Methods
To assess human thymocyte selection in a pig thymus, we used a TCR transgenic humanized mouse model to study positive selection of cells expressing the MART1 TCR, a well‐characterized human HLA‐A2‐restricted TCR, in a grafted pig thymus.
Results
Positive selection of T cells expressing the MART1 TCR was inefficient in both a non‐selecting human HLA‐A2– or swine thymus compared with an HLA‐A2+ thymus. Additionally, CD8 MART1 TCRbright T cells were detected in the spleens of mice transplanted with HLA‐A2+ thymi but were significantly reduced in the spleens of mice transplanted with swine or HLA‐A2– thymi. Correction added on October 15, 2019, after first online publication: The missing superscript values +, –, and bright have been included in the Results section.
Conclusions
Positive selection of cells expressing a human‐restricted TCR in a transplanted pig thymus is inefficient, suggesting that modifications to improve positive selection of cells expressing human‐restricted TCRs in a pig thymus may be necessary to support development of a protective human T‐cell pool in future patients.
Abstract
Autoimmune hepatitis (AIH) is an enigmatic disease in which loss of tolerance towards self-antigens is suspected among the causes; animal models are limited, however. TRAF6 is an important ...signaling adaptor protein that plays a critical role in T cell tolerance by regulating medullary thymic epithelial cell (mTEC) development. mTECs in turn regulate central tolerance by ectopically expressing self-antigens and eliminating autoreactive T cells during development. In this report, conditional knockout mice lacking TRAF6 expression in thymic epithelial cells (TRAF6ΔTEC) were generated using FoxN1-cre transgenic mice (FoxN1 expression is confined to mTECs). TRAF6 deletion inhibited mTEC development and the production of mature mTECs in the thymus. This correlated with spontaneous liver-specific autoimmunity and pathognomonic features of chronic autoimmune hepatitis including interphase and lobular hepatitis, T cell and plasma cell infiltration, elevated serum transaminases and IgG, and production of anti-nuclear autoantibodies against the livers of TRAF6ΔTEC animals. A significant increase in fibrosis was also detected in TRAF6ΔTEC mice compared to littermate controls. Our data indicate that mTECs play an intrinsic role in central tolerance, and their depletion leads to AIH. This model of spontaneous AIH represents an exciting new tool to investigate autoantigen-specific T and B cell responses and regulatory mechanisms underlying the disease.
CD4+ regulatory T (Treg) cells control adaptive immune responses and promote self-tolerance. Various humanized mouse models have been developed in efforts to reproduce and study a human immune ...system. However, in models that require T cell differentiation in the recipient murine thymus, only low numbers of T cells populate the peripheral immune systems. T cells are positively selected by mouse MHC and therefore do not function well in an HLA-restricted manner. In contrast, cotransplantation of human fetal thymus/liver and i.v. injection of CD34+ cells from the same donor achieves multilineage human lymphohematopoietic reconstitution, including dendritic cells (DCs) and formation of secondary lymphoid organs, in NOD/SCID mice. Strong antigen-specific immune responses and homeostatic expansion of human T cells that is dependent on peripheral human APCs occurs. We now demonstrate that FoxP3+ Helios+ “natural” Tregs develop normally in human fetal thymic grafts and are present in peripheral blood, spleen and lymph nodes of these humanized mice. Humanized mice exhibit normal reversal of CD45 isoform expression in association with thymic egress, post-thymic “naïve” to “activated” phenotypic conversion, and suppressive function. These studies demonstrate the utility of this humanized mouse model for the study of human Treg ontogeny, immunobiology and therapy.
We investigated human T-cell repertoire formation using high throughput TCRβ CDR3 sequencing in immunodeficient mice receiving human hematopoietic stem cells (HSCs) and human thymus grafts. Replicate ...humanized mice generated diverse and highly divergent repertoires. Repertoire narrowing and increased CDR3β sharing was observed during thymocyte selection. While hydrophobicity analysis implicated self-peptides in positive selection of the overall repertoire, positive selection favored shorter shared sequences that had reduced hydrophobicity at positions 6 and 7 of CDR3βs, suggesting weaker interactions with self-peptides than unshared sequences, possibly allowing escape from negative selection. Sharing was similar between autologous and allogeneic thymi and occurred between different cell subsets. Shared sequences were enriched for allo-crossreactive CDR3βs and for Type 1 diabetes-associated autoreactive CDR3βs. Single-cell TCR-sequencing showed increased sharing of CDR3αs compared to CDR3βs between mice. Our data collectively implicate preferential positive selection for shared human CDR3βs that are highly cross-reactive. While previous studies suggested a role for recombination bias in producing "public" sequences in mice, our study is the first to demonstrate a role for thymic selection. Our results implicate positive selection for promiscuous TCRβ sequences that likely evade negative selection, due to their low affinity for self-ligands, in the abundance of "public" human TCRβ sequences.
Medullary thymic epithelial cells establish self-tolerance in the thymus by elimination or functional inactivation of auto-reactive T cells. Loss of self-tolerance results in development of ...autoimmune symptoms such as inflammatory cell infiltration and tissue destruction. EFS/Sin is an adapter protein that is highly and specifically expressed in the medullary thymic epithelial cell (mTEC) pool. Absence of EFS/Sin causes disorganized medullary architecture, impaired steady-state proliferation and decreased numbers of terminally differentiated mTECs. The non-hematopoietic cells of the EFS/Sin deficient animals were causally linked to production of an activated T cell pool in bone marrow transfer experiments. Consistent with these data mTEC-mediated negative selection is impaired in the EFS/Sin-/- thymi. Defective establishment of central tolerance correlates with the development of multi-organ lymphocyte infiltration and autoantibody production in aged EFS/Sin deficient mice. EFS/Sin is phosphorylated downstream of keratinocyte growth factor (KGF) stimulation and is required for KGF-stimulated expansion of mature UEA+mTECs in vivo. These studies identify the adapter function of EFS/Sin as an important regulator of mTEC development and maturation. Absence of EFS/Sin leads to defective mTEC-mediated elimination of auto-reactive T cells in the thymus, and development of autoimmunity.
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