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ObjectiveRheumatoid arthritis (RA) is characterized by circulating anti–cyclic citrullinated peptide (CCP) autoantibodies (ACPAs), resulting in inflammation of the joints and other organs. We have established novel assays to assess immune cell subpopulations, including citrullinated antigen–specific (CAS) autoreactive B and T lymphocytes, in patients with RA.Methods and ResultsWe found that activated CD25+ T cells were markedly increased in patients with RA compared to healthy controls. Novel combinations of major histocompatibility complex class II citrulline epitope tetramers were developed, which enabled robust detection of CAS T cells and showed increases of CAS‐naive T helper cells, Th1.17 cells, CAS total circulating T follicular helper (cTfh) cells, and cTfh1 cells in ACPA+ patients with RA. In addition, an innovative assay using dual labeling with CCP–biotin probes allowed for reproducible identification of primary CAS B cells after enrichment with advantages over existing detection methods. Furthermore, patient‐derived immune cells were successfully expanded. Primary RA B cells were successfully cultured on novel feeder cell lines, whereas T cells were expanded ex vivo in the presence of interleukin‐2 and citrullinated peptides, and subsequent alterations in cell frequencies were assessed.ConclusionNovel assays were established to reliably detect CAS T and B cells in patients with RA, and specific CAS‐naive T helper cells, Th1.17 cells, cTfh cells, and cTfh1 cells were observed more frequently in RA. Based on these results, new coculture systems of disease‐relevant cells are developed to simulate human secondary lymphoid tissues ex vivo. This technology will serve as a platform to identify therapies that modulate disease‐specific immune cells.

Long coronavirus disease (COVID) is a heterogeneous clinical condition of uncertain etiology triggered by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we used ultrasensitive approaches to profile the immune system and the plasma proteome in healthy convalescent individuals and individuals with long COVID, spanning geographically independent cohorts from Sweden and the United Kingdom. Symptomatic disease was not consistently associated with quantitative differences in immune cell lineage composition or antiviral T cell immunity. Healthy convalescent individuals nonetheless exhibited higher titers of neutralizing antibodies against SARS-CoV-2 than individuals with long COVID, and extensive phenotypic analyses revealed a subtle increase in the expression of some co-inhibitory receptors, most notably PD-1 and TIM-3, among SARS-CoV-2 nonspike-specific CD8+ T cells in individuals with long COVID. We further identified a shared plasma biomarker signature of disease linking breathlessness with apoptotic inflammatory networks centered on various proteins, including CCL3, CD40, IKBKG, IL-18 and IRAK1, and dysregulated pathways associated with cell cycle progression, lung injury and platelet activation, which could potentially inform the diagnosis and treatment of long COVID. Gao and colleagues explore soluble biomarkers, immune cell subsets, viral antibody responses and plasma proteomics to better define and understand long COVID in two independent cohorts.

Pharmacodynamic assessment of T-cell-based cancer immunotherapies often focus on detecting rare circulating T-cell populations. The therapy-induced immune cells in blood-derived clinical samples are often present in very low frequencies and with the currently available T-cell analytical assays, amplification of the cells of interest prior to analysis is often required. Current approaches aiming to enrich antigen-specific T cells from human Peripheral Blood Mononuclear Cells (PBMCs) depend on in vitro culturing in presence of their cognate peptides and cytokines. In the present work, we improved a standard, publicly available protocol for T-cell immune analyses based on the in vitro expansion of T cells. We used PBMCs from healthy subjects and well-described viral antigens as a model system for optimizing the experimental procedures and conditions. Using the standard protocol, we first demonstrated significant enrichment of antigen-specific T cells, even when their starting frequency ex vivo was low. Importantly, this amplification occurred with high specificity, with no or neglectable enrichment of irrelevant T-cell clones being observed in the cultures. Testing of modified culturing timelines suggested that the protocol can be adjusted accordingly to allow for greater cell yield with strong preservation of the functionality of antigen-specific T cells. Overall, our work has led to the refinement of a standard protocol for in vitro stimulation of antigen-specific T cells and highlighted its reliability and reproducibility. We envision that the optimized protocol could be applied for longitudinal monitoring of rare blood-circulating T cells in scenarios with limited sample material.