Probes for CXCL9

Glucocorticoids remain a cornerstone of therapeutic regimes for autoimmune and chronic inflammatory diseases, for example, in different forms of crescentic glomerulonephritis because of their rapid anti-inflammatory effects, low cost, and wide availability. Despite their routine use for decades, the underlying cellular mechanisms by which steroids exert their therapeutic effects need to be fully elucidated.Here, we demonstrate that high-dose steroid treatment rapidly reduced the number of proinflammatory CXCR3+ CD4+ T cells in the kidney by combining high-dimensional single-cell and morphological analyses of kidney biopsies from patients with antineutrophil cytoplasmic antibody (ANCA)-associated crescentic glomerulonephritis. Using an experimental model of crescentic glomerulonephritis, we show that the steroid-induced decrease in renal CD4+ T cells is a consequence of reduced T-cell recruitment, which is associated with an ameliorated disease course. Mechanistic in vivo and in vitro studies revealed that steroids act directly on renal tissue cells, such as tubular epithelial cells, but not on T cells, which resulted in an abolished renal expression of CXCL9 and CXCL10, as well as in the prevention of CXCR3+ CD4+ T-cell recruitment to the inflamed kidneys. Thus, we identified the CXCL9/10-CXCR3 axis as a previously unrecognized cellular and molecular target of glucocorticoids providing protection from immune-mediated pathology.

Immune checkpoint blockade is therapeutically successful for many patients across multiple cancer types. However, immune-related adverse events (irAE) frequently occur and can sometimes be life threatening. It is critical to understand the immunologic mechanisms of irAEs with the goal of finding novel treatment targets. Herein, we report our analysis of tissues from patients with irAE dermatitis using multiparameter immunofluorescence (IF), spatial transcriptomics, and RNA in situ hybridization (RISH). Skin psoriasis cases were studied as a comparison, as a known Th17-driven disease, and colitis was investigated as a comparison. IF analysis revealed that CD4+ and CD8+ tissue-resident memory T (TRM) cells were preferentially expanded in the inflamed portion of skin in cutaneous irAEs compared with healthy skin controls. Spatial transcriptomics allowed us to focus on areas containing TRM cells to discern functional phenotype and revealed expression of Th1-associated genes in irAEs, compared with Th17-asociated genes in psoriasis. Expression of PD-1, CTLA-4, LAG-3, and other inhibitory receptors was observed in irAE cases. RISH technology combined with IF confirmed expression of IFNγ, CXCL9, CXCL10, and TNFα in irAE dermatitis, as well as IFNγ within TRM cells specifically. The Th1-skewed phenotype was confirmed in irAE colitis cases compared with healthy colon.

Vitiligo is an autoimmune skin disease characterized by the destruction of melanocytes by autoreactive CD8+ T cells. Melanocyte destruction in active vitiligo is mediated by CD8+ T cells but why white patches in stable disease persist is poorly understood. The interaction between immune cells, melanocytes, and keratinocytes in situ in human skin has been difficult to study due to the lack of proper tools. We combine non-invasive multiphoton microscopy (MPM) imaging and single-cell RNA sequencing (scRNA-seq) to identify subpopulations of keratinocytes in stable vitiligo patients. We show that, as compared to non-lesional skin, these keratinocytes are enriched in lesional vitiligo skin and shift their energy utilization towards oxidative phosphorylation. Systematic investigation of cell-cell communication networks show that this small population of keratinocyte secrete CXCL9 and CXCL10 to potentially drive vitiligo persistence. Pseudotemporal dynamics analyses predict an alternative differentiation trajectory that generates this new population of keratinocytes in vitiligo skin. Further MPM imaging of patients undergoing punch grafting treatment showed that keratinocytes favoring oxidative phosphorylation persist in non-responders but normalize in responders. In summary, we couple advanced imaging with transcriptomics and bioinformatics to discover cell-cell communication networks and keratinocyte cell states that can perpetuate inflammation and prevent repigmentation.

Following injury in the central nervous system, a population of astrocytes occupy the lesion site, form glial bridges and facilitate axon regeneration. These astrocytes originate primarily from resident astrocytes or NG2+ oligodendrocyte progenitor cells. However, the extent to which these cell types give rise to the lesion-filling astrocytes, and whether the astrocytes derived from different cell types contribute similarly to optic nerve regeneration remain unclear. Here we examine the distribution of astrocytes and NG2+ cells in an optic nerve crush model. We show that optic nerve astrocytes partially fill the injury site over time after a crush injury. Viral mediated expression of a growth-promoting factor, ciliary neurotrophic factor (CNTF), in retinal ganglion cells (RGCs) promotes axon regeneration without altering the lesion size or the degree of lesion-filling GFAP+ cells. Strikingly, using inducible NG2CreER driver mice, we found that CNTF overexpression in RGCs increases the occupancy of NG2+ cell-derived astrocytes in the optic nerve lesion. An EdU pulse-chase experiment shows that the increase in NG2 cell-derived astrocytes is not due to an increase in cell proliferation. Lastly, we performed RNA-sequencing on the injured optic nerve and reveal that CNTF overexpression in RGCs results in significant changes in the expression of distinct genes, including those that encode chemokines, growth factor receptors, and immune cell modulators. Even though CNTF-induced axon regeneration has long been recognized, this is the first evidence of this procedure affecting glial cell fate at the optic nerve crush site. We discuss possible implication of these results for axon regeneration.