Microrna-31 Promotes T-Cell Pathogenicity in Chronic Graft-Versus-Host Disease

MicroRNAs (miRs) are short, non-coding RNAs that repress gene expression at post-transcriptional level. Certain miRs play critical roles on T-cell responses in inflammation and autoimmunity. To understand the biology of miRs in T-cell allo-responses, we measured expression profiles of miRs in donor T cells in allogeneic vs. syngeneic recipients following bone marrow transplantation (BMT). We found miR-31 was dramatically up-regulated in T cells under alloantigen-driven vs. homeostatic proliferation in vivo(Fig. A). MiR-31 was shown to regulate autoimmunity and cancer. However, its roles in regulating T-cell response remain largely undetermined. Recent study shows miR-31 represses peripheral Treg differentiation in EAE development. In contrast, miR-31 inhibits CD8 T-cell function in anti-virus response. We hypothesize that miR-31 is required for CD4 T-cell pathogenicity in chronic graft-versus-host disease (cGVHD), but is dispensable for CD8 T-cell cytolytic function in graft-versus-leukemia (GVL) activity.

To study how miR-31 regulates T-cell allo-response, we used mice with miR-31 conditional knock out (KO) in T cells (miR-31^fl/fl CD4Cre). No differences were observed in the baseline composition of naïve WT (miR-31^fl/fl) and miR-31 KO splenocytes. After being transferred into allogeneic recipients, KO T cells had impaired ability to proliferate, reflected by fewer divided (% CFSE^low) cells and reduced donor T-cell expansion in recipient spleens (Fig. B). To further evaluate how miR-31 contributes to T-cell pathogenicity in cGVHD, we used a murine model of BMT (B6 → BALB/c), in which low dose spleonocyes can induce scleroderma manifestation. Significant reduction of clinical scores (Fig. C), including improved skin scores and body weight loss, were found in the recipients of miR-31 KO grafts compared to those with WT grafts. This finding was validated in another haploidentical BMT model of scleroderma (B6 → B6D2F1), indicating miR-31 is required for T-cell to induce cGVHD. Consistently, the pathological damage was dramatically reduced in skin, gut and lung in the recipients of miR-31 KO grafts (Fig. D).

Mechanistically, miR-31 deficiency in donor T cells resulted in significantly reduced numbers of CD4 T cells that produce IFNγ, IL-4/5 or IL-17 in recipient spleen and mesenteric lymph nodes. In absence of miR-31, fewer effector and central memory cells were generated from donor CD4 T cells. Further, T cells that are capable of localizing to B-cell follicles (CXCR5⁺PD-1^hi) were significantlyreduced in spleen (Fig. E) and peripheral lymph nodes in recipients of miR-31 KO grafts. Interestingly, among these cells, more foxp3⁺ follicular T-regulatory (Tfr) but fewer foxp3^- follicular T-helper (Tfh) cells were observed in the recipients of KO grafts (Fig. F), indicating miR-31 contributes to the unbalance of Tfr and Tfh cells during cGVHD development. Consistently with the role of miR-31 in regulating Tfr and Tfh cells, we found Blimp1 is the predicted targets of miR-31, which is critically involved in the homeostasis of Tfr and Tfh cells. On donor B-cell compartment, lower levels of CD86 and MHC-II expression were observed in the recipients of miR-31 KO grafts. In addition, germinal center B cells and plasma cells were also reduced in those recipients, indicating miR-31 indirectly regulates B-cell activation and differentiation through controlling T-cell pathogenicity.

For translational purpose, we evaluated how miR-31 impacts T cell-mediated GVL effect. Using B6 → BDF1 BMT model, we consistently found attenuated skin manifestation in recipients of KO T cells. Importantly, in the ongoing experiment, miR-31 KO T cells were capable of controlling P815(mastocytoma) growth as efficiently as WT T cells (Fig. G), suggesting miR-31 may be dispensable for T cells to mediating GVL activity after allogeneic BMT.

Taken together, the current work reveals that miR-31 plays pleotropic roles in enhancing CD4 T-cell pathogenicity in cGVHD. MiR-31 represents a promising therapeutic target for the control of cGVHD while sparing GVL effects after allogeneic hematopoietic stem cell transplantation.

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