Breakthrough COVID-19 After Tixagevimab/Cilgavimab Among Patients With Systemic Autoimmune Rheumatic Diseases.

OBJECTIVE: To determine the incidence and baseline factors associated with breakthrough coronavirus disease 2019 (COVID-19) after preexposure prophylaxis (PrEP) with tixagevimab/cilgavimab among patients with systemic autoimmune rheumatic diseases (SARDs). METHODS: We performed a retrospective cohort study among patients with SARDs who received tixagevimab/cilgavimab between January 2, 2022, and November 16, 2022. The primary outcome was breakthrough COVID-19 after tixagevimab/cilgavimab. We performed multivariable Cox regression models adjusted for baseline factors to identify risk factors for breakthrough COVID-19. RESULTS: We identified 444 patients with SARDs who received tixagevimab/cilgavimab (mean age 62.0 years, 78.2% female). There were 83 (18.7%) breakthrough COVID-19 cases (incidence rate 31.5/1000 person-months, 95% CI 24.70-38.24), 7 (1.6%) hospitalizations, and 1 (0.2%) death. Older age was inversely associated with breakthrough COVID-19 (adjusted hazard ratio [aHR] 0.86/10 years, 95% CI 0.75-0.99). Higher baseline spike antibody levels were associated with lower risk of breakthrough COVID-19 (aHR 0.42, 95% CI 0.18-0.99 for spike antibody levels > 200 vs < 0.4 units). CD20 inhibitor users had a similar risk of breakthrough COVID-19 (aHR 1.05, 95% CI 0.44-2.49) compared to conventional synthetic disease-modifying antirheumatic drug (DMARD) users. CONCLUSION: We found that patients with SARDs had frequent breakthrough COVID-19, but the proportion experiencing severe COVID-19 was low. DMARD type, including CD20 inhibitors, did not significantly affect risk of breakthrough COVID-19. Evidence of prior humoral immunity was protective against breakthrough infection, highlighting the continued need for a multimodal approach to prevent severe COVID-19 as novel PrEP therapies are being developed.

Clonally expanded CD38hi cytotoxic CD8 T cells define the T cell infiltrate in checkpoint inhibitor-associated arthritis.

Immune checkpoint inhibitor (ICI) therapies used to treat cancer, such as anti-PD-1 antibodies, can induce autoimmune conditions in some individuals. The T cell mechanisms mediating such iatrogenic autoimmunity and their overlap with spontaneous autoimmune diseases remain unclear. Here, we compared T cells from the joints of 20 patients with an inflammatory arthritis induced by ICI therapy (ICI-arthritis) with two archetypal autoimmune arthritides, rheumatoid arthritis (RA) and psoriatic arthritis (PsA). Single-cell transcriptomic and antigen receptor repertoire analyses highlighted clonal expansion of an activated effector CD8 T cell population in the joints and blood of patients with ICI-arthritis. These cells were identified as CD38hiCD127- CD8 T cells and were uniquely enriched in ICI-arthritis joints compared with RA and PsA and also displayed an elevated interferon signature. In vitro, type I interferon induced CD8 T cells to acquire the ICI-associated CD38hi phenotype and enhanced cytotoxic function. In a cohort of patients with advanced melanoma, ICI therapy markedly expanded circulating CD38hiCD127- T cells, which were frequently bound by the therapeutic anti-PD-1 drug. In patients with ICI-arthritis, drug-bound CD8 T cells in circulation showed marked clonal overlap with drug-bound CD8 T cells from synovial fluid. These results suggest that ICI therapy directly targets CD8 T cells in patients who develop ICI-arthritis and induces an autoimmune pathology that is distinct from prototypical spontaneous autoimmune arthritides.

Clonal associations of lymphocyte subsets and functional states revealed by single cell antigen receptor profiling of T and B cells in rheumatoid arthritis synovium.

Rheumatoid arthritis (RA) is an autoimmune disease initiated by antigen-specific T cells and B cells, which promote synovial inflammation through a complex set of interactions with innate immune and stromal cells. To better understand the phenotypes and clonal relationships of synovial T and B cells, we performed single-cell RNA and repertoire sequencing on paired synovial tissue and peripheral blood samples from 12 donors with seropositive RA ranging from early to chronic disease. Paired transcriptomic-repertoire analyses highlighted 3 clonally distinct CD4 T cells populations that were enriched in RA synovium: T peripheral helper (Tph) and T follicular helper (Tfh) cells, CCL5+ T cells, and T regulatory cells (Tregs). Among these cells, Tph cells showed a unique transcriptomic signature of recent T cell receptor (TCR) activation, and clonally expanded Tph cells expressed an elevated transcriptomic effector signature compared to non-expanded Tph cells. CD8 T cells showed higher oligoclonality than CD4 T cells, and the largest CD8 T cell clones in synovium were highly enriched in GZMK+ cells. TCR analyses revealed CD8 T cells with likely viral-reactive TCRs distributed across transcriptomic clusters and definitively identified MAIT cells in synovium, which showed transcriptomic features of TCR activation. Among B cells, non-naive B cells including age-associated B cells (ABC), NR4A1+ activated B cells, and plasma cells, were enriched in synovium and had higher somatic hypermutation rates compared to blood B cells. Synovial B cells demonstrated substantial clonal expansion, with ABC, memory, and activated B cells clonally linked to synovial plasma cells. Together, these results reveal clonal relationships between functionally distinct lymphocyte populations that infiltrate RA synovium.

Humoral and cellular immune responses in persons with rheumatoid arthritis after a third dose of mRNA COVID-19 vaccine.

OBJECTIVE: Disease-modifying anti-rheumatic drugs (DMARDs) that treat rheumatoid arthritis (RA) may reduce immune responses to COVID-19 vaccination. We compared humoral and cell-mediated immunity before and after a 3rd dose of mRNA COVID vaccine in RA subjects. METHODS: RA patients that received 2 doses of mRNA vaccine enrolled in an observational study in 2021 before receiving a 3rd dose. Subjects self-reported holding or continuing DMARDs. Blood samples were collected pre- and 4 weeks after the 3rd dose. 50 healthy controls provided blood samples. Humoral response was measured with in-house ELISA assays for anti-Spike IgG (anti-S) and anti-receptor binding domain IgG (anti-RBD). T cell activation was measured after stimulation with SARS-CoV-2 peptide. Spearman's correlations assessed the relationship between anti-S, anti-RBD, and frequencies of activated T cells. RESULTS: Among 60 subjects, mean age was 63 years and 88% were female. 57% of subjects held at least 1 DMARD around the 3rd dose. 43% (anti-S) and 62% (anti-RBD) had a normal humoral response at week 4, defined as ELISA within 1 standard deviation of the healthy control mean. No differences in antibody levels were observed based on holding DMARDs. Median frequency of activated CD4 T cells was significantly greater post- vs. pre-3rd dose. Changes in antibody levels did not correlate with change in frequency of activated CD4 T cells. CONCLUSION: Virus-specific IgG levels significantly increased in RA subjects using DMARDs after completing the primary vaccine series, though fewer than two-thirds achieved a humoral response like healthy controls. Humoral and cellular changes were not correlated.

Granzyme K+ CD8 T cells form a core population in inflamed human tissue.

T cell-derived pro-inflammatory cytokines are a major driver of rheumatoid arthritis (RA) pathogenesis. Although these cytokines have traditionally been attributed to CD4 T cells, we have found that CD8 T cells are notably abundant in synovium and make more interferon (IFN)-γ and nearly as much tumor necrosis factor (TNF) as their CD4 T cell counterparts. Furthermore, using unbiased high-dimensional single-cell RNA-seq and flow cytometric data, we found that the vast majority of synovial tissue and synovial fluid CD8 T cells belong to an effector CD8 T cell population characterized by high expression of granzyme K (GzmK) and low expression of granzyme B (GzmB) and perforin. Functional experiments demonstrate that these GzmK+ GzmB+ CD8 T cells are major cytokine producers with low cytotoxic potential. Using T cell receptor repertoire data, we found that CD8 GzmK+ GzmB+ T cells are clonally expanded in synovial tissues and maintain their granzyme expression and overall cell state in blood, suggesting that they are enriched in tissue but also circulate. Using GzmK and GzmB signatures, we found that GzmK-expressing CD8 T cells were also the major CD8 T cell population in the gut, kidney, and coronavirus disease 2019 (COVID-19) bronchoalveolar lavage fluid, suggesting that they form a core population of tissue-associated T cells across diseases and human tissues. We term this population tissue-enriched expressing GzmK or TteK CD8 cells. Armed to produce cytokines in response to both antigen-dependent and antigen-independent stimuli, CD8 TteK cells have the potential to drive inflammation.

Cross-tissue, single-cell stromal atlas identifies shared pathological fibroblast phenotypes in four chronic inflammatory diseases.

BACKGROUND: Pro-inflammatory fibroblasts are critical for pathogenesis in rheumatoid arthritis, inflammatory bowel disease, interstitial lung disease, and Sjögren's syndrome and represent a novel therapeutic target for chronic inflammatory disease. However, the heterogeneity of fibroblast phenotypes, exacerbated by the lack of a common cross-tissue taxonomy, has limited our understanding of which pathways are shared by multiple diseases. METHODS: We profiled fibroblasts derived from inflamed and non-inflamed synovium, intestine, lungs, and salivary glands from affected individuals with single-cell RNA sequencing. We integrated all fibroblasts into a multi-tissue atlas to characterize shared and tissue-specific phenotypes. FINDINGS: Two shared clusters, CXCL10+CCL19+ immune-interacting and SPARC+COL3A1+ vascular-interacting fibroblasts, were expanded in all inflamed tissues and mapped to dermal analogs in a public atopic dermatitis atlas. We confirmed these human pro-inflammatory fibroblasts in animal models of lung, joint, and intestinal inflammation. CONCLUSIONS: This work represents a thorough investigation into fibroblasts across organ systems, individual donors, and disease states that reveals shared pathogenic activation states across four chronic inflammatory diseases. FUNDING: Grant from F. Hoffmann-La Roche (Roche) AG.

SLAMF7 engagement superactivates macrophages in acute and chronic inflammation.

Macrophages regulate protective immune responses to infectious microbes, but aberrant macrophage activation frequently drives pathological inflammation. To identify regulators of vigorous macrophage activation, we analyzed RNA-seq data from synovial macrophages and identified SLAMF7 as a receptor associated with a superactivated macrophage state in rheumatoid arthritis. We implicated IFN-γ as a key regulator of SLAMF7 expression and engaging SLAMF7 drove a strong wave of inflammatory cytokine expression. Induction of TNF-α after SLAMF7 engagement amplified inflammation through an autocrine signaling loop. We observed SLAMF7-induced gene programs not only in macrophages from rheumatoid arthritis patients but also in gut macrophages from patients with active Crohn's disease and in lung macrophages from patients with severe COVID-19. This suggests a central role for SLAMF7 in macrophage superactivation with broad implications in human disease pathology.

Th17 reprogramming of T cells in systemic juvenile idiopathic arthritis.

Systemic juvenile idiopathic arthritis (sJIA) begins with fever, rash, and high-grade systemic inflammation but commonly progresses to a persistent afebrile arthritis. The basis for this transition is unknown. To evaluate a role for lymphocyte polarization, we characterized T cells from patients with acute and chronic sJIA using flow cytometry, mass cytometry, and RNA sequencing. Acute and chronic sJIA each featured an expanded population of activated Tregs uncommon in healthy controls or in children with nonsystemic JIA. In acute sJIA, Tregs expressed IL-17A and a gene expression signature reflecting Th17 polarization. In chronic sJIA, the Th17 transcriptional signature was identified in T effector cells (Teffs), although expression of IL-17A at the protein level remained rare. Th17 polarization was abrogated in patients responding to IL-1 blockade. These findings identify evolving Th17 polarization in sJIA that begins in Tregs and progresses to Teffs, likely reflecting the impact of the cytokine milieu and consistent with a biphasic model of disease pathogenesis. The results support T cells as a potential treatment target in sJIA.

Allele-specific expression changes dynamically during T cell activation in HLA and other autoimmune loci.

Genetic studies have revealed that autoimmune susceptibility variants are over-represented in memory CD4+ T cell regulatory elements1-3. Understanding how genetic variation affects gene expression in different T cell physiological states is essential for deciphering genetic mechanisms of autoimmunity4,5. Here, we characterized the dynamics of genetic regulatory effects at eight time points during memory CD4+ T cell activation with high-depth RNA-seq in healthy individuals. We discovered widespread, dynamic allele-specific expression across the genome, where the balance of alleles changes over time. These genes were enriched fourfold within autoimmune loci. We found pervasive dynamic regulatory effects within six HLA genes. HLA-DQB1 alleles had one of three distinct transcriptional regulatory programs. Using CRISPR-Cas9 genomic editing we demonstrated that a promoter variant is causal for T cell-specific control of HLA-DQB1 expression. Our study shows that genetic variation in cis-regulatory elements affects gene expression in a manner dependent on lymphocyte activation status, contributing to the interindividual complexity of immune responses.

SerpinB1 controls encephalitogenic T helper cells in neuroinflammation.

SerpinB1, a protease inhibitor and neutrophil survival factor, was recently linked with IL-17-expressing T cells. Here, we show that serpinB1 (Sb1) is dramatically induced in a subset of effector CD4 cells in experimental autoimmune encephalomyelitis (EAE). Despite normal T cell priming, Sb1-/- mice are resistant to EAE with a paucity of T helper (TH) cells that produce two or more of the cytokines, IFNγ, GM-CSF, and IL-17. These multiple cytokine-producing CD4 cells proliferate extremely rapidly; highly express the cytolytic granule proteins perforin-A, granzyme C (GzmC), and GzmA and surface receptors IL-23R, IL-7Rα, and IL-1R1; and can be identified by the surface marker CXCR6. In Sb1-/- mice, CXCR6+ TH cells are generated but fail to expand due to enhanced granule protease-mediated mitochondrial damage leading to suicidal cell death. Finally, anti-CXCR6 antibody treatment, like Sb1 deletion, dramatically reverts EAE, strongly indicating that the CXCR6+ T cells are the drivers of encephalitis.

Publisher Correction: The immune cell landscape in kidneys of patients with lupus nephritis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The immune cell landscape in kidneys of patients with lupus nephritis.

Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.

Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry.

To define the cell populations that drive joint inflammation in rheumatoid arthritis (RA), we applied single-cell RNA sequencing (scRNA-seq), mass cytometry, bulk RNA sequencing (RNA-seq) and flow cytometry to T cells, B cells, monocytes, and fibroblasts from 51 samples of synovial tissue from patients with RA or osteoarthritis (OA). Utilizing an integrated strategy based on canonical correlation analysis of 5,265 scRNA-seq profiles, we identified 18 unique cell populations. Combining mass cytometry and transcriptomics revealed cell states expanded in RA synovia: THY1(CD90)+HLA-DRAhi sublining fibroblasts, IL1B+ pro-inflammatory monocytes, ITGAX+TBX21+ autoimmune-associated B cells and PDCD1+ peripheral helper T (TPH) cells and follicular helper T (TFH) cells. We defined distinct subsets of CD8+ T cells characterized by GZMK+, GZMB+, and GNLY+ phenotypes. We mapped inflammatory mediators to their source cell populations; for example, we attributed IL6 expression to THY1+HLA-DRAhi fibroblasts and IL1B production to pro-inflammatory monocytes. These populations are potentially key mediators of RA pathogenesis.

Plateletpheresis-associated lymphopenia in frequent platelet donors.

More than 1 million apheresis platelet collections are performed annually in the United States. After 2 healthy plateletpheresis donors were incidentally found to have low CD4+ T-lymphocyte counts, we investigated whether plateletpheresis causes lymphopenia. We conducted a cross-sectional single-center study of platelet donors undergoing plateletpheresis with the Trima Accel, which removes leukocytes continuously with its leukoreduction system chamber. We recruited 3 groups of platelet donors based on the total number of plateletpheresis sessions in the prior 365 days: 1 or 2, 3 to 19, or 20 to 24. CD4+ T-lymphocyte counts were <200 cells per microliter in 0/20, 2/20, and 6/20 donors, respectively (P = .019), and CD8+ T-lymphocyte counts were low in 0/20, 4/20, and 11/20 donors, respectively (P < .001). The leukoreduction system chamber's lymphocyte-extraction efficiency was ∼15% to 20% for all groups. Immunophenotyping showed decreases in naive CD4+ T-lymphocyte and T helper 17 (Th17) cell percentages, increases in CD4+ and CD8+ effector memory, Th1, and regulatory T cell percentages, and stable naive CD8+ and Th2 percentages across groups. T-cell receptor repertoire analyses showed similar clonal diversity in all groups. Donor screening questionnaires supported the good health of the donors, who tested negative at each donation for multiple pathogens, including HIV. Frequent plateletpheresis utilizing a leukoreduction system chamber is associated with CD4+ and CD8+ T-cell lymphopenia in healthy platelet donors. The mechanism may be repeated extraction of these cells during plateletpheresis. The cytopenias do not appear to be harmful.

Natural killer cell licensing in mice with inducible expression of MHC class I.

Mouse natural killer (NK) cells acquire effector function by an education process termed "licensing" mediated by inhibitory Ly49 receptors which recognize self-MHC class I. Ly49 receptors can bind to MHC class I on targets (in trans) and also to MHC class I on the NK-cell surface (in cis). Which of these interactions regulates NK-cell licensing is not yet clear. Moreover, there are no clear phenotypic differences between licensed and unlicensed NK cells, perhaps because of the previously limited ability to study NK cells with synchronized licensing. Here, we produced MHC class I-deficient mice with inducible MHC class I consisting of a single-chain trimer (SCT), ovalbumin peptide-ß2 microgloblin-H2K(b) (SCT-K(b)). Only NK cells with a Ly49 receptor with specificity for SCT-K(b) were licensed after MHC class I induction. NK cells were localized consistently in red pulp of the spleen during induced NK-cell licensing, and there were no differences in maturation or activation markers on recently licensed NK cells. Although MHC class I-deficient NK cells were licensed in hosts following SCT-K(b) induction, NK cells were not licensed after induced SCT-K(b) expression on NK cells themselves in MHC class I-deficient hosts. Furthermore, hematopoietic cells with induced SCT-K(b) licensed NK cells more efficiently than stromal cells. These data indicate that trans interaction with MHC class I on hematopoietic cells regulates NK-cell licensing, which is not associated with other obvious phenotypic changes.

Inositol tetrakisphosphate limits NK cell effector functions by controlling PI3K signaling.

Natural killer (NK) cells have important functions in cancer immunosurveillance, BM allograft rejection, fighting infections, tissue homeostasis, and reproduction. NK cell-based therapies are promising treatments for blood cancers. Overcoming their currently limited efficacy requires a better understanding of the molecular mechanisms controlling NK cell development and dampening their effector functions. NK cells recognize the loss of self-antigens or up-regulation of stress-induced ligands on pathogen-infected or tumor cells through invariant NK cell receptors (NKRs), and then kill such stressed cells. Two second-messenger pathways downstream of NKRs are required for NK cell maturation and effector responses: PIP(3) generation by PI3K and generation of diacylglycerol and IP(3) by phospholipase-Cγ (PLCγ). In the present study, we identify a novel role for the phosphorylated IP(3) metabolite inositol (1,3,4,5)tetrakisphosphate (IP(4)) in NK cells. IP(4) promotes NK cell terminal differentiation and acquisition of a mature NKR repertoire. However, in mature NK cells, IP(4) limits NKR-induced IFNγ secretion, granule exocytosis, and target-cell killing, in part by inhibiting the PIP(3) effector-kinase Akt. This identifies IP(4) as an important novel regulator of NK cell development and function and expands our understanding of the therapeutically important mechanisms dampening NK cell responses. Our results further suggest that PI3K regulation by soluble IP(4) is a broadly important signaling paradigm.

Effects of MHC class I alleles on licensing of Ly49A+ NK cells.

NK cells are innate immune lymphocytes that can react to cells lacking self-MHC class I. However, NK cells that cannot engage self-MHC through an inhibitory receptor are resistant to stimulation through their activation receptors. To become licensed (i.e., functionally competent to be triggered through its activation receptors), an NK cell must engage host MHC class I via a MHC class I-specific inhibitory receptor, such as a member of the murine Ly49 family. To explore potential determinants of NK cell licensing on a single Ly49 receptor, we have investigated the relative licensing impacts of the b, d, k, q, r, and s H2 haplotypes on Ly49A(+) NK cells. The results indicate that licensing is essentially analog but is saturated by moderate-binding MHC class I ligands. Interestingly, licensing exhibited a strong inverse correlation with a measure of cis engagement of Ly49A. Finally, licensing of Ly49A(+) NK cells was found to be less sensitive to MHC class I engagement than Ly49A-mediated effector inhibition, suggesting that licensing establishes a margin of safety against NK cell autoreactivity.