* 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.