Long noncoding RNAs (lncRNA) are versatile cell- and context-specific regulators of immune cell responses. As such, they may be ideal for precise targeting of T cell-driven disorders; however, lncRNA regulation of pathogenic T cell function in vivo remains poorly understood. We recently identified a novel T cell-specific lncRNA (ReLoT/LINC00402) as a biomarker for acute graft-versus-host disease (aGVHD) and regulator of allogeneic (allo) T cell responses (Peltier et al., Sci. Trans. Med., 2021). Here, we confirmed ReLoT's cell-intrinsic in vivo role as a regulator of pathologic Rorγt+ T cell differentiation and function in the context of aGVHD.

To assess ReLoT's function, we first developed an in vivo overexpression system using lentivirus-transduced congenic hematopoietic stem cells (HSCs) adoptively transferred into C57BL/6 (B6) mice. Of note, the murine LINC00402 ortholog, Gm34199, is expressed ~500-fold lower than its human counterpart and is poorly annotated thereby precluding knockout approaches. ReLoT overexpression did not affect HSC engraftment, thymic development, or peripheral T cell subsets. In an MHC-mismatched (B6→BALB/c) aGVHD model, mice receiving ReLoT-overexpressing allo-T cells along with wild-type (WT) B6 T cell depleted bone marrow showed reduced mortality (p=0.027) and clinical scores relative to vector control T cell recipients.

To validate these results, we established a Rosa26 knock-in T cell conditional overexpression B6 mouse. Thymic T cell development and spleen T cell subsets were also not altered in this knock-in system, but mortality was reduced in allogeneic BALB/c recipients of knock-in ReLoT overexpressing donor T cells (p=0.007). This phenotype was model-independent because ReLoT overexpressing donor allo-T cells improved overall survival (p=0.003), aGVHD clinical score (p=0.005), and weight loss (p=0.03) in MHC-matched minor histocompatibility antigen-mismatched C3H.sw recipients. Histopathological analysis of aGVHD target organs in BALB/c recipients of knock-in ReLoT overexpressing T cells revealed reduced GI tract damage, especially in the small intestines (p=0.0002).

Mechanistically, ReLoT overexpression did not alter in vitro proliferation, Il-2 production, or activation marker expression following either non-specific or allo-antigen-driven T cell activation. Therefore, we next analyzed the in vivo accumulation of donor T cell subsets known to either promote or inhibit aGVHD. In the spleen, there was no difference in CD3+ donor T cells nor in donor naïve, central memory, effector memory, Th1/Tc1, Th2/Tc2, or Treg cells. By contrast, accumulation of aGVHD-promoting donor Th17/Tc17-like cells (i.e., CD4+ Rorγt+ or CD8+ Rorγt+ T cells, respectively) decreased in ReLoT overexpressing allo-T cell recipients (p=0.004 CD4+, p=0.004 CD8+). In vitro Th17 polarization was also impaired (p=0.007), suggesting a direct effect.

In the small intestinal epithelium, Th17/Tc17-like cells were reduced (p=0.003 CD4+, p=0.02 CD8+), but in contrast to the spleen, helper CD4+ donor T cell accumulation in the small intestinal epithelium was also reduced (p=0.011) suggesting that donor helper T cell trafficking to the GI tract may be impaired. Consistent with this, spleen CD4+ T cells co-expressing Rorgt+ with either Cxcr3 or Ccr6, which are Th17-associated chemokine receptors known to aggravate allo-T cell-driven GI aGVHD, were reduced in ReLoT overexpressing recipients (p=0.0011 and 0.0047, respectively). However, in the small intestinal epithelium, only CD4+ cells co-expressing Cxcr3 (p=0.005) with Rorγt+ were reduced. In addition, serum levels of Cxcl9 and Cxcl10, which are Cxcr3 ligands, were decreased (p=0.03 each), likely further contributing to impaired GI tract accumulation of ReLoT overexpressing donor helper T cells.

To assess the cytotoxic potential of allogeneic ReLoT overexpressing intestinal epithelial helper T cells, we measured granzyme B and perforin expression, which are critical mediators of allo-T cytotoxicity. The co-expression of Rorγt with both granzyme B (p=0.002) and perforin (p=0.002) was reduced in CD4+ T cells. Altogether, our results are the first to demonstrate regulation of pathogenic Rorγt+ T cells by a lncRNA in vivo, and they suggest ReLoT improves aGVHD by impairing Th17 polarization, GI tract homing, and cytotoxic potential. Therefore, enhancing ReLoT expression or function may be novel strategies for mitigating aGVHD and other Rorγt+ T cell-driven disorders.

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2025
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