Dominant protection from HLA-linked autoimmunity by antigen-specific regulatory T cells

Susceptibility and protection against human autoimmune diseases, including type I diabetes, multiple sclerosis, and Goodpasture disease, is associated with particular human leukocyte antigen (HLA) alleles. However, the mechanisms underpinning such HLA-mediated effects on self-tolerance remain unclear. Here we investigate the molecular mechanism of Goodpasture disease, an HLA-linked autoimmune renal disorder characterized by an immunodominant CD4+ T-cell self-epitope derived from the α3 chain of type IV collagen (α3135–145)1,2,3,4. While HLA-DR15 confers a markedly increased disease risk, the protective HLA-DR1 allele is dominantly protective in trans with HLA-DR15 (ref. 2). We show that autoreactive α3135–145-specific T cells expand in patients with Goodpasture disease and, in α3135–145-immunized HLA-DR15 transgenic mice, α3135–145-specific T cells infiltrate the kidney and mice develop Goodpasture disease. HLA-DR15 and HLA-DR1 exhibit distinct peptide repertoires and binding preferences and present the α3135–145 epitope in different binding registers. HLA-DR15-α3135–145 tetramer+ T cells in HLA-DR15 transgenic mice exhibit a conventional T-cell phenotype (Tconv) that secretes pro-inflammatory cytokines. In contrast, HLA-DR1-α3135–145 tetramer+ T cells in HLA-DR1 and HLA-DR15/DR1 transgenic mice are predominantly CD4+Foxp3+ regulatory T cells (Treg cells) expressing tolerogenic cytokines. HLA-DR1-induced Treg cells confer resistance to disease in HLA-DR15/DR1 transgenic mice. HLA-DR15+ and HLA-DR1+ healthy human donors display altered α3135–145-specific T-cell antigen receptor usage, HLA-DR15-α3135–145 tetramer+ Foxp3− Tconv and HLA-DR1-α3135–145 tetramer+ Foxp3+CD25hiCD127lo Treg dominant phenotypes. Moreover, patients with Goodpasture disease display a clonally expanded α3135–145-specific CD4+ T-cell repertoire. Accordingly, we provide a mechanistic basis for the dominantly protective effect of HLA in autoimmune disease, whereby HLA polymorphism shapes the relative abundance of self-epitope specific Treg cells that leads to protection or causation of autoimmunity

Intrarenal Single-Cell Sequencing of Hepatitis B Virus Associated Membranous Nephropathy

To date, the pathogenesis of hepatitis B virus (HBV)-associated membranous nephropathy (MN) remains elusive. This study aimed to decipher the etiopathogenesis of HBV-associated MN by performing single-cell RNA sequencing (scRNA-seq) of kidney biopsy specimens from a patient with HBV-associated MN and two healthy individuals. We generated 4,114 intrarenal single-cell transcriptomes from the HBV-associated MN patient by scRNA-seq. Compared to healthy individuals, podocytes in the HBV-associated MN patient showed an increased expression of extracellular matrix formation-related genes, including HSPA5, CTGF, and EDIL3. Kidney endothelial cells (ECs) in the HBV-associated MN were enriched in inflammatory pathways, including NF-kappa B signaling, IL-17 signaling, TNF signaling and NOD-like receptor signaling. Gene ontology (GO) functional enrichment analysis and Gene Set Variation Analysis (GSVA) further revealed that differentially expressed genes (DEGs) of ECs from the HBV-associated MN patients were enriched in apoptotic signaling pathway, response to cytokine and leukocyte cell-cell adhesion. The up-regulated DEGs in glomerular ECs of HBV-associated MN patients were involved in biological processes such as viral gene expression, and protein targeting to endoplasmic reticulum. We further verified that the overexpressed genes in ECs from HBV-associated MN were mainly enriched in regulation of protein targeting to endoplasmic reticulum, exocytosis, viral gene expression, IL-6 and IL-1 secretion when compared with anti-phospholipase A2 receptor (PLA2R)-positive idiopathic membranous nephropathy (IMN). The receptor-ligand crosstalk analysis revealed potential interactions between endothelial cells and other cells in HBV-associated-MN. These results offer new insight into the pathogenesis of HBV-associated MN and may identify new therapeutic targets for HBV-associated MN

Treg Enhancing Therapies to Treat Autoimmune Diseases

Regulatory T cells (Tregs) are a small yet critical subset of CD4+ T cells, which have the role of maintaining immune homeostasis by, for example, regulating self-tolerance, tumor immunity, anti-microbial resistance, allergy and transplantation rejection. The suppressive mechanisms by which Tregs function are varied and pleiotropic. The ability of Tregs to maintain self-tolerance means they are critical for the control and prevention of autoimmune diseases. Irregularities in Treg function and number can result in loss of tolerance and autoimmune disease. Restoring immune homeostasis and tolerance through the promotion, activation or delivery of Tregs has emerged as a focus for therapies aimed at curing or controlling autoimmune diseases. Such therapies have focused on the Treg cell subset by using drugs to suppress T effector cells and promote Tregs. Other approaches have trialed inducing tolerance by administering the autoantigen via direct administration, by transient expression using a DNA vector, or by antigen-specific nanoparticles. More recently, cell-based therapies have been developed as an approach to directly or indirectly enhance Treg cell specificity, function and number. This can be achieved indirectly by transfer of tolerogenic dendritic cells, which have the potential to expand antigen-specific Treg cells. Treg cells can be directly administered to treat autoimmune disease by way of polyclonal Tregs or Tregs transduced with a receptor with high affinity for the target autoantigen, such as a high affinity T cell receptor (TCR) or a chimeric antigen receptor (CAR). This review will discuss the strategies being developed to redirect autoimmune responses to a state of immune tolerance, with the aim of the prevention or amelioration of autoimmune disease

Cytokine concentrations in stimulated splenocyte supernatant for mice receiving PBS or FL before and during sensitization to the nephritogenic antigen in the anti-GBM disease model.

<p>Data presented as mean±SEM, except for IL-10, presented as median (range) as data is non-parametric. IL-6, IL-12p70 and TNF were not detected.</p><p>Cytokine concentrations in stimulated splenocyte supernatant for mice receiving PBS or FL before and during sensitization to the nephritogenic antigen in the anti-GBM disease model.</p

FL therapy expands pDCs but increases delayed type hypersensitivity four days after sensitization to sheep globulin.

<p>(A) Experimental design. (B, C) Total spleen and LN cell number 4 and 10 days after sensitization, respectively. (D, E) Proportion of pDCs in the spleen and LN 4 and 10 days post-sensitization to sheep globulin respectively. (F, G) Footpad swelling in mice 4 and 10 days post-sensitization to sheep globulin, respectively. Black bars represent PBS treated mice. White bars represent FL treated mice. n = 4 per group. *P&lt;0.05, **P&lt;0.01.</p

FL administered before accelerated autologous phase anti-GBM disease did not protect against nephritis, but altered systemic immunity.

<p>(A) Proteinuria (dotted line represents measured level in non-nephritic WT mice, n = 4). (B) Serum urea (dotted line represents measured level in non-nephritic WT mice, n = 4). (C) Proportions of glomeruli with crescents and (D) glomerular segmental necrosis. (E) Representative images of glomeruli from PBS and FL treated mice taken at high power (400x, PAS stain). (F) Total spleen and LN cell number. (G) Proportion of CD11c+ cells from leukocytes in spleen and LN. (H) Proportion of pDCs in spleen and LN. (I) Serum mouse anti-sheep IgG antibody levels. Black bars represent PBS treated mice. White bars represent FL treated mice. OD, optical density. n = 6 per group. *P&lt;0.05.</p

FL administered throughout accelerated autologous phase anti-GBM disease reduced mouse survival.

<p>(A) Experimental design. (B) Survival following the induction of nephritis. Black bars represent PBS treated mice. White bars represent FL treated mice. n = 9 per group. *P&lt;0.05.</p

FL therapy throughout accelerated anti-GBM disease did not protect mice from nephritis.

<p>(A) Experimental design. (B) Proteinuria. (C) Percentage of glomerular crescents and (D) glomeruli with segmental necrosis. (E) Representative images of glomeruli from PBS and FL treated mice taken at high power (400x, PAS stain). (F–H) gcs, periglomerular and interstitial hpf CD11c+ immunofluorescence assessed by fluorescent microscopy of renal sections. (I) Representative images of CD11c+ immunofluorescent staining within the interstitial, glomerular and periglomerular regions. Black bars represent PBS treated mice. White bars represent FL treated mice. AU, arbitrary units; gcs, glomerular cross section; hpf, high-powered field. n = 7 per group. *P&lt;0.05.</p

The effects of 10 days of intraperitoneal FL on DC and Treg populations.

<p>(A) Experimental design, where FL or PBS was administered intraperitoneally to mice for daily for 10 days. (B) Total spleen and pooled LN cell number. (C) Representative FACS plots showing proportions of pDCs (CD11c+PDCA-1+ cells) in the spleen, gating on all CD11c+ leukocytes. (D) Proportions of CD11c+ cells in the spleen and LN. (E) Proportions of pDCs in the spleen and LN. (F) Representative FACS plots showing Tregs in the spleen, gating on lymphocytes, staining for CD4+ and foxp3+ cells. (G) Proportion of Tregs in the spleen and LN. Black bars represent PBS treated mice. White bars represent FL treated mice. n = 4 per group. *P&lt;0.05, **P&lt;0.01, ***P&lt;0.001.</p

Intrarenal leukocytes in mice treated with PBS or FL throughout anti-GBM disease until day 9.

<p>c/gcs, cells per glomerular cross section; c/hpf, cells per high power field.</p><p>*P = 0.02.</p><p>Intrarenal leukocytes in mice treated with PBS or FL throughout anti-GBM disease until day 9.</p