doi: 10.1097/HS9.0000000000000613.
eCollection 2021 Aug.
Sphingosine-1-phosphate Receptor-1 Agonist Averts the De Novo Generation of Autoreactive T-cells in Murine Acute Graft-versus-Host Disease
Affiliations
- PMID: 34263142
- PMCID: PMC8274799
- DOI: 10.1097/HS9.0000000000000613
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Sphingosine-1-phosphate Receptor-1 Agonist Averts the De Novo Generation of Autoreactive T-cells in Murine Acute Graft-versus-Host Disease
Hemasphere.
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KRP203 inhibits alloreactive T-cell migration to the host thymus and prevents injury of the thymic medullary cell compartment. The effects of KRP203 were studied in 2 fully MHC-mismatched alloHSCT models and 1 unirradiated haploidentical model. In the fully MHC-disparate B6→Balb/c model (H-2b→H-2d; A–D), 5 × 106 TCDBM from C57BL/6 mice and 1.2 × 107 splenocytes (containing 3 × 106 donor T cells) from congenic B6.CD45.1 mice were transplanted on day 0 into lethally irradiated (800 cGy) Balb/c mice. AlloHSCT recipients received either KRP203 in 0.5% methylcellulose vehicle prophylactically from day −1 (designated [b→d] + T + KRP203) or vehicle alone ([b→d] + T + vehicle). An additional control group received TCDBM without alloreactive T cells and did not develop acute GVHD ([b→d]). In some experiments, the control cohorts did not receive vehicle. In the Balb/c→B6 model (H-2d→H-2b; F, I), thymic acute GVHD was induced by transplantation on day 0 of 7 × 106 TCDBM from Balb/c mice (Thy1.2+) and 2 × 107 purified CD3+ T cells from congenic Balb/c.Thy1.1+ mice into lethally irradiated (1000 cGy) B6 mice. Transplant groups in this [d→b] alloHSCT model were the same as in the above model. In the haploidentical setting (E, G, H), thymic acute GVHD was induced in unirradiated 8-wk-old female BDF1 recipients (CD45.2+) by injection on day 0 of 35 × 106 splenocytes from parental B6.CD45.1 donors (H-2b→H-2bd). AlloHSCT recipients received either KRP203 prophylactically from day −1 until the end of experiment (designated [b→bd] + T + KRP203) or did not receive the drug ([b→bd] + T). As non-GVHD controls, BDF1 mice received syngeneic splenocytes and no KRP203 ([bd→bd]). (A), Survival was measured in alloHSCT recipients receiving KRP203 (1 mg/kg/d) daily until day +21. Kaplan-Meier plot with Gehan-Breslow-Wilcoxon statistical analysis to compare survival curves. The data shown are from 1 experiment that is representative of a total of 3 experiments; with n=6 BM + T cells and n=7 BM +T cells + KRP203. (B), Colon histopathology was scored at day +7 in the same cohorts. One-way ANOVA; data are representative of 2 experiments. (C), Donor T-cells (H-2b-positive CD4+ and CD8+) were analyzed by flow cytometry in lymph nodes at day +6 in the same cohorts. t test; data are from 2 pooled experiments. (D), Antitumor activity. The A20 lymphoma cell line (1 × 104 luciferase+ cells) was injected into the left inguinal lymph node. Mice were then treated with KRP203 (3 mg/kg, i.p., every second day) until the end of the experiment. One representative bioluminescence image is shown for each tested group at days +14 and +24 after alloHSCT. Tumor area and signal intensity data of luc+A20 cells were obtained from 2 pooled experiments with n=3 mice per group (right panel). (E, F), Donor T-cell infiltration into recipient thymi was analyzed in the 2 alloHSCT models by flow cytometry assessment of CD45.1+ cells at day +14 and is given as absolute cell numbers and also frequencies among total thymic cells. In these experiments, mice received KRP203 (3 mg/kg, i.p., every second day) from day −1 until the end of experiment. (G), mTEChigh cells were identified as EpCAM+CD45-UEA-1+Ly51− mTEC cells that express MHCIIhigh and Aire. (H, I), Absolute cell numbers of mTEChigh and Aire+mTEChigh cells were examined at days +14 and +28 in mice without acute GVHD, mice that developed acute GVHD without or with treatment with KRP203 (3 mg/kg, i.p., every second day from day-1 until the end of experiment). Statistical analyses for pooled experiments (E, F, H, I) were done with 2-way ANOVA and Fisher’s LSD test. The graphs represent data from 2 to 4 independent experiments with 3 to 5 mice per group and experiment and statistical significance is given as *P < 0.05, **P < 0.01, ***P < 0.001. BM=bone marrow; GVHD=graft-versus-host disease; HSCT=hematopoietic stem cell transplantation; i.p.=intraperitoneal; LSD=least significant difference; MHC=major histocompatibility complex; mTEC=medullary thymic epithelial cell; TCDBM=T-cell depleted bone marrow cells.
KRP203 inhibits thymic de novo generation of autoreactive T-cells following alloHSCT. The effects of KRP203 were studied in 2 alloHSCT models. In an unirradiated haploidentical transplantation model (A), acute GVHD (H-2b→H-2bd) was induced as described in the legend to Figure 1E, G, H. AlloHSCT recipients received either KRP203 (3 mg/kg, i.p., every second day) prophylactically from day −1 until the end of the experiment (designated [b→bd] + T+ KRP203) or did not receive the drug ([b→bd] + T). As non-GVHD controls, BDF1 mice received syngeneic splenocytes and no KRP203 ([bd→bd]). Negatively selected T cells were detected among CD4-single-positive mature thymocytes (A, lower panel). Thymocytes that underwent negative selection were TCR+CD5−CCR7− and PD-1+/Helios+ or PD-1−/Helios+. Absolute cell numbers of cells marked to become negatively selected and express PD1 and Helios or Helios alone were determined. The graphs show representative data from 1 experiment with n=5 mice per group. *P < 0.05, Kruskal-Wallis, and Dunnetts‘s multiple comparison test. To study the effects of KRP203 on thymic negative selection, B6.RIP-mOVA mice were used as recipients in a transgenic murine [d→b] alloHSCT model (B–D) as described by Dertschnig et al. To this end, acute GVHD was induced at day 0 in 8 wk old, lethally irradiated (950 cGy) B6.RIP-mOVA mice (H-2b) by transfer of Thy1.1+ T-cell depleted bone marrow cells together with Thy1.2+ splenic T-cells (H-2d) from Balb/c donors (designated [d→RIP-mOVAb] + T). These recipients received either KRP203 from day −1 to +28 or were not treated. As controls without acute GVHD, KRP203 untreated mice received Balb/c-Thy1.1+ TCDBM only (designated [d→RIP-mOVAb]). On cessation of KRP203 treatment at day +28, all mice were lethally reirradiated and retransplanted in a second syngeneic HSCT with hematopoietic stem cells from CD45.1+ OT-II mice (H-2b) mixed at a 1:4 ratio with TCDBM from wild-type CD45.2+ C57BL/6 mice (H-2b). This approach generated chimeric mice (designated OT-IIb→[d→RIP-mOVAb]) as detailed in (B) and as described. (C, D), OVA-specific OT-II CD4+ T cells in transplanted chimeric mice were identified as CD4+CD45.1+ T cells in lymph nodes at day +28 later in all cohorts treated or not treated with KRP203. An untreated and nontransgenic alloHSCT recipient cohort without acute GVHD was included as positive controls that did not delete OVA-specific T cell due to the absence of the negative selector OVA on mTEChigh cells (designated OT-IIb→[d→B6b]). To authenticate that CD4+CD45.1+ cells accurately represented OT-II T-cells, the population of Vα2+Vβ5+ T cells was analyzed in lymph nodes by flow cytometry (C; 1 representative experiment is depicted). Frequencies of OT-II cells among total CD4+ CD45.1+ T cells are given in (D). The figure represents data from 3 combined experiments. *P < 0.05, Mann-Whitney U Test. GVHD=graft-versus-host disease; HSCT=hematopoietic stem cell transplantation; i.p.=intraperitoneal; mOVA=membrane-bound form of ovalbumin; mTEC=medullary thymic epithelial cell; TCDBM=T-cell depleted bone marrow cells.
References
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