Rapid thymectomy of NSG mice to analyze the role of native and grafted thymi in humanized mice

Borsotti

Danzl

et al. 2019

Xenotransplantation

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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.

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Reduced positive selection of a human TCR in a swine thymus using a humanized mouse model for xenotolerance induction

Borsotti

Danzl

et al. 2019

Xenotransplantation

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“…Cells were then cryopreserved as described [29]. Four-to eight-week old mice were thymectomized [30] and allowed to recover for two weeks, then sublethally irradiated (1.0 Gy) using an RS-2000 X ray irradiator (Rad Source Technologies, Inc., Suwanee, GA). Human fetal thymus fragments of about 1-3 mm 3 were implanted beneath the kidney capsule and 2 × 10 5 human FL CD34 + cells were injected intravenously.…”

Section: Methodsmentioning

confidence: 99%

Interleukin-15 Receptor α Chain Knockout NOD-SCID mice have reduced numbers of NK cells, develop thymic lymphomas and fail to engraft human hematopoietic cells

Vecchione

et al. 2021

Preprint

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NOD-SCID IL2rγc null (NSG) mice are widely used to model human immune cell development because they are more permissive for human hematopoietic cell engraftment and reconstitution than NOD-SCID mice. While increased human reconstitution in the blood of NSG mice has been attributed to the absence of mouse NK cells, deletion of the common gamma chain (γc) limits development of lymphoid tissue inducer cells and precludes development of normal secondary lymphoid structures. The disorganized lymphoid tissue leads to compromised human T cell-B cell interactions and results in variable human immune cell function in human immune system (HIS) NSG compared to NOD-SCID mice. We attempted to remove mouse NK cells from NOD-SCID mice while retaining other γc-dependent cytokine responses by targeted disruption of the mouse genomic IL15RA locus with CRISPR/Cas9. IL15Rα is required for the development, function and survival of NK cells. NOD-SCID IL15Rα-/- mice showed reductions in NK cells and NK cell function. However, NOD-SCID IL15Rα-/- mice demonstrated accelerated thymic lymphomagenesis and showed earlier mortality compared to NOD-SCID mice. This result suggests that mouse NK cells are important to delay lymphoma development in NOD-SCID mice. We transplanted thymectomized NOD-SCID IL15Rα-/- mice with human fetal liver CD34+ cells and thymus to determine if these mice supported engraftment and development of a transplanted HIS. Surprisingly, we found that peripheral human engraftment was inferior (mean 0.05% of lymphocytes) to that in both NOD-SCID (mean, 10.5% of lymphocytes) and NSG (mean, 54% of lymphocytes) mice. These results indicate that NOD-SCID IL15Rα-/- mice are not permissive for human CD34+ cell engraftment.

“…Mice were surgically thymectomized as described [ 30 ] and allowed to recover for at least two weeks, then irradiated with 1 Gy total body irradiation (TBI) by an X-Ray irradiator (RS-2000, Rad Source Technologies, Inc., Suwanee, GA). Mice were then transplanted with 2-5x10 5 lentivirally-transduced CD34 + cells intratibially.…”

Section: Methodsmentioning

confidence: 99%

Negative selection of human T cells recognizing a naturally-expressed tissue-restricted antigen in the human thymus

Madley

Journal of Translational Autoimmunity

Danzl

et al. 2020

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During T cell development in mice, thymic negative selection deletes cells with the potential to recognize and react to self-antigens. In human T cell-dependent autoimmune diseases such as Type 1 diabetes, multiple sclerosis, and rheumatoid arthritis, T cells reactive to autoantigens are thought to escape negative selection, traffic to the periphery and attack self-tissues. However, physiological thymic negative selection of autoreactive human T cells has not been previously studied. We now describe a human T-cell receptor-transgenic humanized mouse model that permits the study of autoreactive T-cell development in a human thymus. Our studies demonstrate that thymocytes expressing the autoreactive Clone 5 TCR, which recognizes insulin B:9–23 presented by HLA-DQ8, are efficiently negatively selected at the double and single positive stage in human immune systems derived from HLA-DQ8 + HSCs. In the absence of hematopoietic expression of the HLA restriction element, negative selection of Clone 5 is less efficient and restricted to the single positive stage. To our knowledge, these data provide the first demonstration of negative selection of human T cells recognizing a naturally-expressed tissue-restricted antigen. Intrathymic antigen presenting cells are required to delete less mature thymocytes, while presentation by medullary thymic epithelial cells may be sufficient to delete more mature single positive cells. These observations set the stage for investigation of putative defects in negative selection in human autoimmune diseases.