* Corresponding author at:
Department of Dermatology, Second Xiangya Hospital, Central South University, Hunan Key Laboratory of Medical Epigenomics, Changsha, Hunan, China
Email:chriswu1010@126.com(Haijing Wu)
Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing, Jiangsu, China
Email:qianlu5860@pumcderm.cams.cn(Qianjin Lu)
These authors contributed equally to this work and share first authorship.
Abstract:Regulatory T (Treg) cells play a crucial role in immune tolerance, and numerous studies have focused on the molecular mechanisms of CD4+ Treg cells, whereas the role and relevant mechanisms of CD8+ Treg cells remain unclear. Ly49 is located at murine NK and T cells, and plays a similar role to human KIRs in regulation of immune response. In this paper, we have explored the effect of AIM2 deficiency on the homeostasis of CD8+Ly49+ regulatory T (Treg) cells in AIM2 knockout (KO) mouse model. We have performed KLH immunization model for AIM2 KO mice and found enhanced function of suppressive CD8+ Treg cells in the absence of AIM2, indicating AIM2 dampens the homeostasis of murine inhibitory CD8+Treg cells.
Keywords:CD8+T, Ly49, AIM2, Autoimmune disease, Regulatory T cell
Autoimmune diseases are characterized with the failure of immune tolerance, and regulatory T (Treg) cells are required for immune tolerance, such as FOXP3+CD4+ and killer cell immunoglobulin-like receptor (KIR)+FOXP3-CD8+Treg cells. Intensive studies have focused on the molecular mechanisms of CD4+ Treg cells, whereas the function and relevant mechanisms of CD8+Ly49+ Treg cells remain investigated. CD8+Ly49+ Treg cells are considered to have immunosurveillance and immunosuppressive effects during the process of thymic negative selection. They are able to blunt autoimmunity without compromising systemic immunity[1]. However, the specific origin of these CD8+Ly49+Treg cells and how they take part in the natural progression of autoimmunity remain poorly defined. To date, the best demonstrated role of CD8+ Treg cells is to inhibit GC reaction. The enhanced GC reaction and autoantibody production exist in many animal models with defective CD8+ Treg cells[2]. Besides, the CD8+Ly49+ Treg cells have reported in various settings, such as colitis, hepatitis, arthritis, diabetes, viral infection, tumor, atherogenesis and organ transplantation[2]. Ly49 plays a similar role to human killer cell immunoglobulin-like receptors (KIRs) in regulating immune response via MHC-Ia, but varies in structures. Ly49 family consists of inhibitory subsets including Ly49A, B, C, E, F, G, I, J, Q and activating subsets like Ly49D, H[3]. It could regulate immunosurveillance of NK cells and cytotoxic T cells (CTLs) against tumor, infection and autoimmunity[4]. Further, even though there are some evidence about the existence of human CD8+ Treg subset, they remain to be firmly established because the Ly49 is a mouse-specific gene cluster without human homologs at sequence level[2].
Absent in Melanoma 2 (AIM2) serves as a cytoplasmic sensor of double-stranded DNA (dsDNA) and participates in the assembly of inflammasome[5]. It plays vital roles in innate and adaptive immune responses with inflammasome-dependent/independent manners in diverse autoimmune diseases including systemic lupus erythematosus[6], psoriasis[7], rheumatoid arthritis[8], primary Sjogren’s syndrome[9], ankylosing spondylitis[10], and cancers[11-13]. Our group previously demonstrated that AIM2 has played an inflammasome-independent role in the differentiation of lupus B cells via regulating Blimp1 and Bcl6[6]. The expression of AIM2 in classic CD4+FOXP3+ Treg cells was elevated to suppress autoimmunity and favoring the function of Treg cells through attenuating Akt/mTOR pathway and affecting immune metabolism, and its expression could be induced by TGF-β[14].
Besides, transcription factors Aiolos (coded by Ikzf3 ) and Helios (coded by Ikzf2 ) belonging to the IKAROS family, participate in the regulation of lymphoid lineage commitment and maintenance of lymphocyte homeostasis. In human FOXP3+ Treg cells, the interaction of Aiolos and FOXP3 silenced the expression ofIL-2 to induce the differentiation of functional Treg cells[15]. Helios is mainly expressed in T-cells precursors and Treg cells, and maintains the suppressive activity of CD4+FOXP3+ and CD8+CD44+CD122+Ly49+Treg cells[16], which dampens overactivated immune responses to alleviate tissue damage in autoimmune diseases. As reported previously, reduced expression level of Helios and Aiolos contributed to the deficiency of CD8+ Treg cells in mouse model lack ofTgfbr2, and Eomes required for homeostasis of murine CD8+FOXP3- Treg cells[17]. However, to the best of our knowledge, the roles of AIM2, Helios and Aiolos in murine CD8+Ly49+ Treg cells remain elusive.
Keyhole Limpet Hemocyanin (KLH), a large protein molecule with high immunogenicity, induces immune activation. In this study, we carried out KLH immunization in AIM2 knockout (KO) and C57BL/6J wild type (WT) mice to explore the frequency of CD8+ Treg cells. The flow chart of murine experiment was shown in Figure 1A . It has been revealed that compared with WT mice, the gene expression of regulatory CD8+ T cell marker-CD122 and inhibitory Ly49 receptors, including Ly49B, C and I, was elevated in splenic CD8+ T cells of AIM2 KO mice on the 10th day of KLH immunization (Figure 1B ), whereas the gene expression of activating Ly49 receptors containing Ly49D and Ly49H was stable (Figure 1C ). These alterations indicated the immunosuppressive state of KLH-immunized AIM2 KO mice. Notably, the expression levels of Helios and Aiolos were slightly elevated in CD8+ T cells of splenocytes (Figure 1D ), revealing that they might promote Ly49 expression in murine CD8+ T cells of splenocytes.
Besides, as RNA-seq reported, splenic CD8+ T cells of KLH-immunized AIM2 KO mice (n = 3) feature with upregulated expression of KIR-related immunosuppressive genes compared with WT mice (n = 3) (Figure 2A ). These screened KIR-related differential expressed genes were correlated to activation of innate and adaptive immune response, which was confirmed by enrichment analysis of pathways (Figure 2B ). Splenic CD8+ T cells of another group of KLH-immunized AIM2 KO and WT mice (KO/WT, n = 5) were applied to validate the results of RNA-seq, and consistent with the findings of transcriptome sequencing. Activating killer cell lectin-like receptor (Klr)-i1 in murine CD8+ T cells was remarkably downregulated, in contrast, inhibitory receptors, such as Klra2, were significantly upregulated (Figure 2C ), demonstrating the deficiency of AIM2 promotes the expression of immunosuppressive receptors in murine CD8+ T cells. The primers for quantitative reverse transcription PCR (qRT-PCR) were listed inSupplemental Table 1 . Indeed, the sample size of AIM2 KO mice should be enlarged to further demonstrate this conclusion in mouse models.
To sum up, AIM2 dampens the homeostasis of murine inhibitory CD8+ Treg cells, and further research on specific molecule mechanisms of regulation remains ongoing.