Background and Aims: Refractory celiac disease type II (RCDII) is characterized by a clonally expanded aberrant cell population in the small intestine. The role of other tissue-resident immune subsets in RCDII is unknown. Here, we characterized CD8 and CD4 T cells in RCDII duodenum at the single-cell level and in situ. Methods: We applied mass cytometry on CD45⁺ duodenal cells derived from intestinal biopsies (n = 23) and blood samples (n = 20) from RCDII patients and controls. Additionally, we analyzed intestinal biopsies from celiac disease (n = 11) and RCDI (n = 2) patients. We performed single-cell RNA-sequencing on CD45⁺ duodenal cells derived from a RCDII patient, immunofluorescence staining for in situ analysis and flow cytometry for phenotyping of RCDII aberrant and CD8 T cells. Results: Compared to healthy controls, we observed that CD27⁺PD-1⁺ memory CD8αβ cells and CD4 T regulatories (Tregs) were more abundant in RCDII duodenum (CD8 ∗∗0.0029; CD4 ∗∗∗0.0001). The CD27⁺PD-1⁺ memory CD8αβ cells expressed the tissue-resident marker CD69, immunoregulatory markers (TIGIT, HAVCR2, TNFRSF9), NKG2A, were enriched for activated pathways and displayed cytotoxic gene signatures (NKG7, PRF1, GZMA). The absence of CD103 accords with their localization in the lamina propria as determined by in situ analysis. The CD25⁺FoxP3⁺CD27⁺CD127^dim/- CD4 Tregs expressed IL1R2 and IL32 and costimulatory molecules (TNFSRS4, ICOS and TNFRSF18) and resided in the lamina propria as well. Flow cytometry confirmed the presence of the inhibitory receptor NKG2A on expanded duodenal CD8 T cells and HLA-E, the ligand for NKG2A, on expanded aberrant cells. Conclusion: RCDII is characterized by the simultaneous presence of an activated CD27⁺PD-1⁺ memory CD8αβ T cell subset and CD4 Tregs, suggesting that checkpoint blockade with anti-NKG2A/PD-1 and/or anticytotoxic T lymphocyte antigen 4 may be an attractive treatment option.

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Background
Perianal fistulas are a debilitating complication of Crohn’s disease (CD). Due to unknown reasons, CD-associated fistulas are in general more difficult to treat than cryptoglandular fistulas (non-CD-associated). Understanding the immune cell landscape is a first step towards the development of more effective therapies for CD-associated fistulas. In this work, we characterized the composition and spatial localization of disease-associated immune cells in both types of perianal fistulas by high-dimensional analyses.

Methods
We applied single-cell mass cytometry (scMC), spectral flow cytometry (SFC), and imaging mass cytometry (IMC) to profile the immune compartment in CD-associated perianal fistulas and cryptoglandular fistulas. An exploratory cohort (CD fistula, n = 10; non-CD fistula, n = 5) was analyzed by scMC to unravel disease-associated immune cell types. SFC was performed on a second fistula cohort (CD, n = 10; non-CD, n = 11) to comprehensively phenotype disease-associated T helper (Th) cells. IMC was used on a third cohort (CD, n = 5) to investigate the spatial distribution/interaction of relevant immune cell subsets.

Results
Our analyses revealed that activated HLA-DR+CD38+ effector CD4+ T cells with a Th1/17 phenotype were significantly enriched in CD-associated compared with cryptoglandular fistulas. These cells, displaying features of proliferation, regulation, and differentiation, were also present in blood, and colocalized with other CD4+ T cells, CCR6+ B cells, and macrophages in the fistula tracts.

Conclusions
Overall, proliferating activated HLA-DR+CD38+ effector Th1/17 cells distinguish CD-associated from cryptoglandular perianal fistulas and are a promising biomarker in blood to discriminate between these 2 fistula types. Targeting HLA-DR and CD38-expressing CD4+ T cells may offer a potential new therapeutic strategy for CD-related fistulas.@en

Rationale: Chronic inflammation plays an important role in alveolar tissue damage in emphysema, but the underlying immune alterations and cellular interactions are incompletely understood. Objectives: To explore disease-specific pulmonary immune cell alterations and cellular interactions in emphysema. Methods: We used single-cell mass cytometry (CyTOF) to compare the immune compartment in alveolar tissue from 15 patients with severe emphysema and 5 control subjects. Imaging mass cytometry (IMC) was applied to identify altered cell-cell interactions in alveolar tissue from patients with emphysema (n = 12) compared with control subjects (n = 8). Measurements and Main Results: We observed higher percentages of central memory CD4 T cells in combination with lower proportions of effector memory CD4 T cells in emphysema. In addition, proportions of cytotoxic central memory CD8 T cells and CD127+CD27+CD69- T cells were higher in emphysema, the latter potentially reflecting an influx of circulating lymphocytes into the lungs. Central memory CD8 T cells, isolated from alveolar tissue from patients with emphysema, exhibited an IFN-γ response upon anti-CD3 and anti-CD28 activation. Proportions of CD1c+ dendritic cells, expressing migratory and costimulatory markers, were higher in emphysema. Importantly, IMC enabled us to visualize increased spatial colocalization of CD1c+ dendritic cells and CD8 T cells in emphysema in situ. Conclusions: Using CyTOF, we characterized the alterations of the immune cell signature in alveolar tissue from patients with chronic obstructive pulmonary disease stage III or IV emphysema versus control lung tissue. These data contribute to a better understanding of the pathogenesis of emphysema and highlight the feasibility of interrogating the immune cell signature using CyTOF and IMC in human lung tissue. Clinical trial registered with www.clinicaltrials.gov (NCT04918706).

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