Clinical & Translational Immunology

Background. Persistent SARS-CoV-2 antigen is a leading hypothesis for post-acute sequelae of SARS-CoV-2 infection (PASC), commonly known as Long COVID. Impaired Fc-mediated antiviral antibody function could contribute to inefficient clearance of infected cells and viral antigen despite preserved antibody levels. Therefore, we tested whether PASC is associated with altered Fc{gamma} receptor-mediated antibody function and differences in IgG1 Fc fucosylation, a major regulator of Fc effector function. Methods. Serum samples from RECOVER-Adult participants with prior SARS-CoV-2 infection (99 PASC, 139 non-PASC) were analyzed for anti-SARS-CoV-2 spike receptor-binding domain (RBD) IgG and subclasses, Fc{gamma}RIIIa- and Fc{gamma}RIIa-mediated reporter activity as surrogate measures of antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), anti-RBD IgG1 Fc glycosylation, and circulating fucose-modifying enzymes. Results. Despite higher anti-RBD IgG and IgG1 abundance, PASC sera showed lower ADCC- and ADCP-associated Fc{gamma}R reporter activity, including after normalization for antibody abundance. Anti-RBD IgG1 showed greater fucosylation in PASC samples (30 PASC, 34 non-PASC). Serum FUCA2 was lower in PASC samples (24 PASC, 34 non-PASC), whereas FUCA1 and soluble CD16a levels did not differ between the two groups. Conclusion. PASC is associated with impaired antiviral IgG Fc function, increased IgG1 Fc fucosylation, and reduced circulating FUCA2, identifying a glycoimmune axis that may contribute to impaired Fc-dependent antigen clearance and represents a potentially targetable pathway in Long COVID.

APECED (Autoimmune PolyEndocrinopathy Candidiasis Ectodermal Dystrophy) is a rare syndrome of multi-organ autoimmunity driven by the presence of self-reactive T cells and autoantibodies caused by mutations in the gene Autoimmune regulator (AIRE). Compared to the well-defined role of AIRE in establishing and maintaining T cell central tolerance, less is known about how AIRE deficiency impacts B cell phenotypes that may contribute to a breakdown in peripheral B cell tolerance. Here we analyzed serum and peripheral blood cells from APECED patients and healthy donors using autoantibody profiling, proteomics, flow cytometry, scRNAseq based subset analysis, BCRseq, and autoantigen binding assays finding significant changes to the APECED B cell compartment. We show that while Naive and Transitional B cells are reduced, alternatively activated B cell subsets are expanded in APECED patients including IgM CD27+ and class switched Atypical B cells which exhibit BCR chain features prone to autoreactivity. Antibodies derived from either APECED or Healthy class switched atypical B cells bind autoantigens at significantly higher rates than control IgG B cells. Serum autoantibodies and proteomics highlight APECED common and patient variable changes. These results together show that APECED causes a compositional shift towards subsets that may promote broken B cell tolerance and autoimmunity.

In the developing human placenta, three subtypes of trophoblast cells, cytotrophoblasts (CTBs), extravillous trophoblasts (EVTs), and syncytiotrophoblasts (STBs), mediate critical functions essential for a successful pregnancy. CTBs constitute the stem/progenitor compartment and differentiate into STBs and EVTs within the floating and anchoring villi, respectively. STBs establish the maternal-fetal exchange interface and secrets human chorionic gonadotropin (hCG), a hormone vital for the maintenance of early pregnancy. EVTs anchor the maternal endometrium and invade the uterine tissue to remodel maternal cells, supporting implantation and progression of pregnancy. In this study, we used human trophoblast stem cells (hTSCs) as a model system and performed quantitative, label-free liquid chromatography-tandem Mass Spectrometry (LC-MS/MS) to profile proteome and phosphoproteome in TSC stem state (analogous to undifferentiated CTBs) and following their differentiation to STBs and EVTs. Through a multiomics approach, we integrated our proteomics data with global gene expression profiles to correlate cell-type specific gene and protein expression during human trophoblast development. We also identified global phosphoproteome and analyzed kinases that are specifically active in hTSC stem state, as well as in differentiated STBs and EVTs. We experimentally validated specific kinases, such as BUB1B, PAK6, PKYMT1 and TNIK are essential for maintaining the hTSC stem-state. Additionally, atypical protein kinase C isoforms PKC{zeta} is essential for STB development, while PTK2B, SRC, TRIO and LYN are important for EVT development. Our findings highlight key kinases uniquely required for specific stages of trophoblast development during human placentation and suggest that pharmacological inhibition of these kinases could negatively impact the placentation process during pregnancy.

Extravillous trophoblasts (EVTs) are a trophoblast subpopulation critical for feto-maternal tolerance during early pregnancy, primarily using HLA-G to exert immunomodulatory effect, and possessing N-glycomic profiles distinct from other trophoblast subpopulations. However, whether the N-glycosylation confers distinct immunological properties to EVTs remains poorly understood. To investigate this, we employed JEG-3, a human choriocarcinoma cell line having the capacity to produce pregnancy-related hormones and expressing both HLA-C and HLA-G resembling placental EVTs, as an in vitro EVT model, alongside cell line JAR which exhibits villous trophoblast phenotypes distinct from JEG-3. Both cell lines were treated with kifunensine or swainsonine, inhibitors of -mannosidases, to remodel their N-glycosylation patterns. This led to significant remodelling of their N-glycomic profiles, with JEG-3 cells showing an increased level of polylactosamine chains and decreased levels of -2,6-sialylation and core -1,6-fucosylation. Western blot analysis showed that inhibiting -mannosidases altered only the composition of N-glycans on cell-surface HLA-G, without affecting the overall abundance of cell-surface HLA-G. In kifunensine-treated JEG-3 cells that predominantly express oligomannose type N-glycans, an intracellular accumulation of unfolded HLA-G fragments, increased hCG secretion, and down-regulations of EVT markers GATA3 and KRT7 were observed compared to untreated control, while swainsonine treatment did not impact N-glycan expression. Cytotoxicity assays using NK-92 as effector cells showed that the de-sialylation of JEG-3 by neuraminidase treatment led to increased NK-92 mediated killing. JEG-3 cell sustained its EVT immunological properties through generating bioequivalent N-glycans, exemplified by NK-92 cells pre-conditioned with used culture media of kifunensine-treated JEG-3 cells displaying reduced cytotoxicity toward NK-sensitive lymphoblast cell line K562, an effect not observed with swainsonine-treated JEG-3 cells. This model suggests that EVTs immunological properties are dependent on specific N-glycomic profiles that are maintained by unique N-glycosylation homeostasis, and overall improves our understanding of how EVTs maintain their immunomodulatory effect at the maternal-fetal interface.

BackgroundReactive oxygen species (ROS) overproduction, inflammatory cytokines, and plasma mediators disrupt alveolar tight junction integrity and perpetuate oxidative injury in acute respiratory distress syndrome (ARDS). Biological heterogeneity of ARDS, reflected by hyper- and hypoinflammatory subphenotypes with distinct plasma mediator profiles, may contribute to heterogeneity of treatment effect. Stem cell-derived EVs have been shown to exert regenerative and immunomodulatory effects by horizontal transfer of proteins, lipids and genetic material to injured target cells. This study investigated whether extracellular vesicles derived from bone marrow (MSC-EVs) and adipose tissue (ASC-EVs) mesenchymal stem cells restore barrier integrity and redox homeostasis, and whether efficacy differs across subphenotypes. MethodsBronchial epithelial cells (BEAS-2B), microvascular endothelial cells (HMEC-1), and small airway epithelial cells (SAEC) were challenged with plasma from patients with hyperinflammatory or hypoinflammatory ARDS subphenotypes, including those treated with venovenous ECMO. MSC-EVs or ASC-EVs were added concomitantly with plasma exposure or six hours later, at a dose of 5,000 particles/cell. After 24 h, the expression of actin and tight junction proteins, zonula occludens (ZO)-1, occludin, and vascular endothelial (VE)-cadherin, was evaluated by immunofluorescence. Oxidative stress parameters, antioxidant defenses, and antioxidant enzyme activities, were measured. ResultsPlasma from patients with the hyperinflammatory ARDS subphenotype induced marked disruption of ZO-1, occludin, and VE-cadherin expression, together with actin cytoskeletal rearrangement, across BEAS-2B, HMEC-1, and SAEC cells. Barrier disruption was accompanied by increased oxidative stress markers and altered antioxidant enzyme activity. Both MSC-EVs and ASC-EVs restored tight junction protein expression and cytoskeletal organization when administered either concurrently with ARDS plasma or 6 hours after exposure, and both effectively modulated redox parameters. Delayed administration 6 hours after injury produced greater effects for several oxidative stress endpoints, particularly in cells challenged with hypoinflammatory plasma. ConclusionARDS-associated plasma mediators induce subphenotype-dependent impairment of epithelial-endothelial barrier integrity through oxidative mechanisms. Both MSC-EVs and ASC-EVs mitigate this injury, with efficacy that varies according to ARDS subphenotype and timing of administration.

BackgroundStrategies to define human monocytes by flow cytometry vary considerably across studies. Recently, toll-like receptor 2 (TLR2) has been proposed as a marker to identify "all monocytes" in human peripheral blood. However, the TLR2-defined monocytes also contained a previously ignored TLR2posCD14dim/negCD16neg population, which we termed the unclassified subset (UCS). MethodsPeripheral blood mononuclear cells (PBMCs) from healthy donors and patients with multiple myeloma (MM) or monoclonal gammopathy of undetermined significance (MGUS) were analyzed by multiparameter flow cytometry using TLR2pos gating. PBMCs from additional healthy donors were analyzed to characterize the UCS population, including the impact of using either TLR2pos or a negative selection-based gating strategy. ResultsThe TLR2pos CD14dim/neg CD16neg UCS population was present in healthy controls, MGUS, and MM patients. The UCS expressed the monocyte-macrophage scavenger receptor CD163 and was significantly reduced in MM patients compared to healthy donors (P<0.002). Further phenotypic characterization in healthy blood donors revealed that approximately 80% of UCS cells expressed CD163 at levels comparable to classical monocytes, yet phenotypically resembled CD163pos dendritic cells (DCs). Importantly, gating strategies influenced the composition of the UCS: negative selection-based gating captured all DC subsets, whereas TLR2pos gating primarily included CD1cpos DCs that were highly CD163pos. ConclusionsThese findings demonstrate that circulating CD163pos CD1cpos DCs are included in the TLR2pos cell population previously described as exclusively monocytes, highlighting the impact of gating strategy on monocyte subset identification. Further, the lower level of TLR2pos CD14dim/neg CD16neg CD163pos cells in MM patients may represent decreased levels of circulating DCs that may contribute to the immune dysregulation in this disease.

Immune checkpoint inhibitor-induced myocarditis (ICIM) is a severe immune-related adverse event with heterogeneous clinical presentations and potential genetic susceptibility. Here, we established a human induced pluripotent stem cell (iPSC) line from an ICIM patient with an HLA-type distinct from previously reported line, who developed concurrent type I diabetes following ICI treatment. This line exhibited typical morphology, normal female karyotype, pluripotency, trilineage differentiation into all three germ layers, Sendai virus clearance, and no mycoplasma contamination. Given the fulminant nature and diverse clinical presentations of ICIM, expanding the repertoire of iPSC lines are critical for investigating ICIM heterogeneity and its underlying mechanisms.

BackgroundPost-acute COVID-19 syndrome (PACS) can cause persistent disability involving fatigue, post-exertional malaise, neurocognitive symptoms, and autonomic manifestations. A clinically overlapping condition has been reported following COVID-19 vaccination and termed post-acute COVID-19 vaccination syndrome (PACVS), but its underlying mechanisms remain poorly understood. We investigated whether immune dysregulation, autoreactivity, antigen persistence, and viral reactivation--mechanisms implicated in PACS--are also present in PACVS. MethodsThis cross-sectional study included 28 PACS patients, 29 PACVS patients, and 16 convalescent controls (CCG). Clinical manifestations were assessed using ME/CFS-related symptom questionnaires and the Bell Score. T-cell phenotypes and T cells reactive to SARS-CoV-2 Spike, {beta}1/{beta}2-adrenergic receptor, and M3/M4 muscarinic receptor antigens were analyzed by multicolor flow cytometry. Circulating Spike protein and GPCR autoantibodies were quantified by ELISA, and EBV load by qPCR. ResultsPACS and PACVS showed substantial clinical overlap, although several symptoms were more severe in PACS. GPCR autoantibody profiles were comparable between patient groups, while {beta}2-adrenergic receptor autoantibodies were increased compared with CCG. Only three of 57 conventional immunophenotyping parameters differed between PACS and PACVS in unadjusted analyses, whereas both groups showed multiple alterations compared with CCG. Circulating Spike protein was detected in both patient groups, with the highest prevalence in PACVS. {beta}1/{beta}2- and M3/M4-reactive CD4+ T cells were increased, whereas Spike-specific CD4+ T cells were reduced compared with CCG. EBV reactivation was rare. ConclusionsPACS and PACVS exhibit substantial clinical and immunological overlap, including GPCR-directed autoreactivity and altered receptor-reactive T-cell responses. These shared immune-mediated features suggest overlapping pathophysiological mechanisms and warrant investigation of whether therapeutic strategies currently explored for PACS may also be relevant to PACVS.

Background: Reactivation of herpes simplex viruses (HSV) can occur in the early post-allogeneic hematopoietic cell transplant (aHCT) period despite antiviral prophylaxis. Few studies have assessed HSV infection in the modern era, in which acyclovir/valacyclovir is recommended for up to 1 year post aHCT. We evaluated the incidence and management of breakthrough HSV during the first 100 days post-aHCT over two decades. Methods: Patients who received their first aHCT at Fred Hutchinson Cancer Center between 2002-2022 were reviewed for breakthrough HSV infection within the first 100 days on prophylaxis (acyclovir 800 mg or valacyclovir 500 mg twice daily). Cases were identified via culture, polymerase chain reaction, and/or direct fluorescent antibody testing; clinical records were reviewed for symptoms, outcomes, and prophylaxis/treatment regimens. Refractory/resistant (R/R) infections were defined according to consensus guidelines. Results: We reviewed data from 4,357 aHCT recipients aged [&ge;]18 years, among whom 3,749 (86%) were HSV seropositive and 23 developed breakthrough HSV infection (observed probability = 0.6%). Among those who had an infection, the median time from transplant to first positive test was 46 days (IQR: 24.0-69.5). Oral and genital mucosa were the most common sites of infection. In total, 11 of 23 (47.8%) patients with breakthrough HSV developed R/R infection. Conclusions: Breakthrough HSV infections are rare in the first 100 days after aHCT among patients receiving antiviral prophylaxis. Refractory/resistant infections were uncommon but represented almost half of breakthrough cases. Our findings highlight the sustained effectiveness of universal prophylaxis in the early post-transplant period.

Background Sepsis is the primary driver of mortality in intensive care. Personalized medicine requires an accurate understanding of the syndrome, and plasma proteomics is increasingly used to characterize its molecular alterations. Comorbidities, demographics and postoperative sterile inflammation complicate this task, and proteins associated with them may be mistaken as sepsis-specific. Methods We compared 343 sepsis patients with two independent perioperative control cohorts (n = 73 and n = 75) sampled before and after surgery. Plasma was analyzed by mass spectrometry, and analyzed using linear models adjusted for age, sex and cohort-specific comorbidities. Propensity score matching was used to characterize renal comorbidities and Elastic Net classifiers were trained per cohort and time point. Results We show that renal comorbidities had the strongest impact on the plasma proteome and affected sepsis-associated proteins including Cystatin-C and Beta-2-microglobulin. Eleven proteins were robustly associated with sepsis, among them established acute phase proteins and the less well characterized Beta-1,4-galactosyltransferase 1. Feature importance analysis confirmed this core set and identified further proteins that contributed in a cohort- and time point-specific manner. Classifiers performed very well except against control patients at day 3 after surgery, when sterile inflammation peaked. Conclusions Sepsis, comorbidities and sterile inflammation may alter the same acute phase and renal proteins, differing in magnitude rather than in the type of response. We disentangled these influences and report a robust set of sepsis-associated proteins together with confounder-associated proteins, providing a basis for future studies and the development of diagnostic and therapeutic strategies.

Several vaccines against respiratory syncytial virus (RSV) have been commercialized recently. Their immediate immunogenicity in various conditions of immunosuppression, including patients receiving allogeneic hematopoietic stem cell transplantation (HSCT), have been reported in 2025 by different teams, including ours. In immunocompromised patients, the duration of vaccinal humoral protection as well as the relevance of seasonal revaccination is still unknown. By analyzing the kinetics of humoral responses after a single dose of bivalent RSVpreF vaccine over 12 months in 71 HSCT recipients, we show that annual revaccination benefits mostly those primarily vaccinated within the early 18 months after HSCT. We also provide a first evaluation of vaccinal clinical effectiveness in HSCT patients.

Immune thrombocytopenia purpura (ITP) is an autoimmune disorder characterized by a reduction in circulating platelet levels, primarily due to generation of autoantibodies to platelet surface antigens followed by their spleen macrophage-mediated clearance. Emerging evidence implicates neuraminidase (sialidase) enzymes including neuraminidase 1 (NEU1) in platelet clearance and ITP severity; however, the underlying cellular mechanisms remain unknown. Using tissue-specific NEU1 knockout mouse models, we studied the contribution of platelet and macrophage NEU1 to ITP pathogenesis and evaluated whether pharmacological inhibition of NEU1 could preserve platelet counts in a murine ITP model. Constitutive and macrophage-specific, but not platelet-specific, NEU1 knockout mice showed a protection against reduction of platelet counts in the passive ITP model suggesting that macrophage, but not platelet, NEU1 promotes platelet clearance. Genetic deletion or pharmacological blockade of macrophage NEU1 also reduced platelet phagocytosis by cultured macrophages in vitro. The selective NEU1 inhibitor CG33301 protected mice against anti-CD41a antibody-induced thrombocytopenia and showed a higher efficacy compared to pan neuraminidase inhibitor oseltamivir phosphate. Our results demonstrate that the macrophage pool of NEU1 plays a central role in platelet clearance by splenocytes during ITP by activating their phagocytosis and suggest that selective NEU1 inhibition may be a promising therapeutic strategy for this disease.

Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable therapeutic outcomes in hematological cancers. However, broader clinical use has uncovered substantial challenges arising from intrinsic properties of both T cells and tumor tissues. As the functional phenotype of CAR T cells is affected by the CAR molecular architecture, optimizing CAR constructs continues to be a critical and ongoing task. Here, we present a practical workflow for scalable screening of CAR variants in primary T cells using fitness-guided design and mRNA electroporation. Using a CD19-targeted second-generation CAR, we built a library of point mutants that focused mutagenesis on hinge and costimulatory domains. Amino acid substitutions were prioritized using the sequence-based zero-shot fitness predictor to enrich evolutionarily tolerated variants. From 340 designed variants, we electroporated mRNA encoding 85 constructs into primary human CD8+ T cells and quantified cytotoxicity against CD19-positive Nalm6 cells. Twenty-four variants reproducibly exceeded wild-type cytotoxicity across three runs, and three hits were selected for lentiviral validation. One of the selected variants showed significantly improved cytotoxicity despite lower expression frequency and exhibited higher CD62L within CAR-positive cells, suggesting enhanced intrinsic function with a less differentiated phenotype. This approach enables scalable, rapid discovery of improved CAR domain variants directly in primary T cells.

The human T cell repertoire is generated through recombination of a multitude of T cell receptor gene segments, yielding a complex array of T cell clones. T cell receptor beta (TRB) V gene segment defined, T cell clonal frequency when organized in correspondence with the respective V gene segment positions on the TRB genomic locus yields a periodic, undulating curve in the spatial domain. Using the genomic distance from the TRB-D1 segment to the TRB-V1-29 segments, spectral analysis was performed utilizing Lomb-Scargle periodogram (modified Fourier analysis) to obtain spectral power curves quantifying the TRB V clonal frequencies from 12 allogeneic stem cell transplant donors (baseline) and recipients (<100 days or b/w 100-365) using a variety of analytic software. Patients either underwent HLA matched related bone marrow or HLA matched unrelated blood stem cell transplantation, using PT-CY for the former and ATG for the latter. Spectral Power curves revealed dominant spectral peaks at wavelengths ranging from 113-335 millicycles/kb in the 12 donors, with consistent frequency domain spectral patterns, supporting similar use of V segments across healthy individuals. PT-CY recipients had power spectra closely corresponding to the respective donors, however the ATG recipients had relatively dispersed spectra, with spectral centroid shifted towards higher frequencies compared to donors and a reciprocal decline in the low frequency indices. Spectral power was concentrated in the <3 kb and 3-12 kb wavelengths in both groups, consistent with the periodicity observed in the relative V gene segment usage across the repertoire. The analyses reported here demonstrate that the healthy SCT donors have a spectral signature occupying short to intermediate wavelegnths in the frequency domain, PT CY recipients recover with donor-like spectra on the average, however ATG recipients tend to shift towards higher frequencies. These findings are consistent with a normal organized distribution of TRB V segment usage in healthy individuals, with PT-CY preserving this organization more consistently than ATG. Spectral (Fourier) analysis of TRB sequencing data provides a repertoire wide summary of T cell clonal distribution.

Abstract Objective Fungal infections can represent opportunistic infections leading to the diagnosis of immunocompromise. Specific inborn errors of immunity are associated with aspergillus but it is unclear how sensitive aspergillus is for the subsequent diagnosis of those inborn errors of immunity. Methods The patient registry USIDNET was utilized to extract clinical conditions and laboratory values from patients with candida and aspergillus diagnosis codes. We restricted the diagnoses to deep infections and then identified the associated clinical conditions along with potential settings, such as bone marrow transplant. Results We identified 41 patients with aspergillus as a diagnosis code. The types of diagnoses associated with aspergillus included T cell disorders, antibody disorders, as well as the anticipated myeloid disorders. Bone marrow transplant was identified as a potential predisposing condition. 64. Patients were identified with candida as a diagnosis code. Similarly, T cell disorders, antibody disorders, and myeloid disorders were found. Conclusions Candida and aspergillus are generally considered sentinel infections for myeloid disorders and T cell disorders. However, we found these infections associated with a broad range of inborn errors of immunity. This suggests that diagnostic testing should be broadened when facing patients with these infections.

Post-COVID-19 syndrome (PCS) is characterized by fatigue, neurological impairment and systemic symptoms. This heterogeneity of symptoms hinders biomarker development. Here, we profiled extracellular-vesicle (EV) surface markers in plasma and CSF from 61 participants with PCS (COVIDpost), 80 recovered controls (COVIDreco), and 10 participants with non-SARS-CoV-2 post-viral syndromes. EVs were analysed by bead-based multiplex flow cytometry using tetraspanin-directed (TSPN) and phosphatidylserine-directed lactadherin (PS) detection. Amongst 37 targets covering tetraspanins and vasculature-, immunity- and stemness-associated markers, none met a 1% false-discovery-rate threshold. However, L1-regularized logistic regression under fully nested 5x5 cross-validation identified a distributed plasma EV profile, with mean out-of-fold areas under the receiver operating characteristic curve (AUCs) of 0.788 (95% CI 0.715 - 0.852) for TSPN and 0.716 (95% CI 0.636 - 0.792) for PS detection. Across the pooled COVIDpost and COVIDreco population, EV classification scores covaried with clinical group differences, but did not track clinical severity within either cohort. These PCS-EV classification scores decreased at one-year follow-up in COVIDpost participants. Our findings identify an internally cross-validated multivariable EV surface profile associated with COVIDpost versus COVIDreco status and support independent validation and exploration of EV-based biomarkers in post-viral fatigue syndromes.

Chikungunya virus (CHIKV) infection is typically not life-threatening but may, in rare cases, affect the central nervous system (CNS). In three cohorts of confirmed CHIKV cases from Martinique (French overseas territory) and Brazil (patients aged 0--89 years, n = 245), 20 patients had CNS infection (aged 17--87 years). Autoantibodies neutralizing type I interferons (AAN-I-IFN) were found in 35% of patients (4 male and 3 female, aged 17--87 years) with encephalitis (4/15), encephalomyelitis (1/2), or myelitis (2/3), but were absent in 225 patients without CNS infection. They neutralized high concentrations (10--1,000 ng/mL) of IFN-2, IFN-8, and IFN-{omega}, and the antibodies of one patient also neutralized IFN-{beta}. All samples also neutralized the other 10 IFN- subtypes (at least 100 pg/mL). This combination of blood autoantibodies occurs in ~0.02% and 0.6% of healthy individuals under and over 70 years of age, respectively. The presence of AAN-I-IFN before CHIKV infection therefore increased the risk of CNS disease ~850-fold relative to the general population.

Glycosylphosphatidylinositol (GPI) anchors are involved in the organization of membrane microdomains that support T cell receptor (TCR) signaling. However, their role in regulating expression of TCR-related proteins and downstream functional output remains unclear. This study aimed to characterize the effects of GPI-deficiency on TCR, cluster of differentiation 3 (CD3), and CD28 expression as well as interleukin-2 (IL-2) production using a GPI-deficient Jurkat T cell line (S12). Flow cytometry confirmed the complete loss of GPI anchors and GPI-anchored proteins (GPI-APs) in the S12 cell line. Compared to GPI-producing parental Jurkat, S12 had significantly higher expression of CD3 and TCR{beta} while CD28 had similar expression. IL-2 production by S12 was assessed following stimulation with anti-CD3/anti-CD28 beads and following stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Neither S12 nor parental Jurkat produced detectable IL-2 in response to anti-CD3/anti-CD28 bead-mediated stimulation. Both parental Jurkat and S12 produced IL-2 following PMA/ionomycin-mediated stimulation. No significant difference in IL-2 production was observed between S12 and parental Jurkat following PMA/ionomycin-mediated stimulation. These findings demonstrate that GPI-deficiency influences surface receptor expression but does not significantly impair downstream IL-2 production under PMA/ionomycin stimulation. This finding suggests that GPI anchors and GPI-APs contribute to proximal signaling organization but are not required for cytokine production when downstream pathways are directly activated.

ObjectiveIn the first year after onset of the autoimmune disease RA (RA), 40-60% do not achieve remission on conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs). To understand how autoreactive T cells may contribute to unstable or non-remission, we studied CD4+ T cells, including those recognising citrullinated (Cit) vimentin in participants with RA. MethodsTwo cohorts of drug-naive new-onset participants with RA were treated with csDMARDs. Disease activity score (DAS28-CRP) and peripheral blood (PB) mononuclear cells were collected longitudinally. In HLA-DR-shared epitope+ cohort 1 (n=21), T cells were assessed with a 17-marker spectral flow panel, incorporating HLA-DRB1*04:01/01:01- VimentinCit6459-71 or HLA-DRB1*04:04-VimentinCit7166-78 tetramers. Changes in T cell subsets over time were assessed in remitting and non-remitting participants using a generalized linear mixed model with a negative binomial distribution. In cohort 2 (n=26), the transcriptome of disaggregated synovial tissue (ST) and PB CD4+ T cells were analysed at baseline, and ST biopsy spatial proteomics at baseline and 6 months. ResultsCD4CXCR5-PD1+ peripheral helper T cells (Tph), including Cit-vimentin-reactive Tph, CD4CCR7+CXCR5-PD1+ stem-like Tph and TIGIT+PD1+ Tph were increased with moderate/high DAS28-CRP at any time point. Remission outcome was associated with low CD4 follicular helper T cell (Tfh) and Cit-vimentin-reactive Tfh. In non-remitting participants, Tph/fh infiltrated germinal-centre-like ST aggregates. This decreased in remission. Circulating TIGIT+ Tph genes reflected B lymphoid activation and lymph node egress, while in ST they reflected local differentiation. ConclusionPersistently high circulating TIGIT+ Tph and Tfh, including Cit-vimentin specificities, reflecting antigen-presenting B-cell interactions, are associated with reduced response to csDMARDs in recent-onset RA. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSO_LITph and Tfh cells interacting with B cells are implicated in active ACPA+ RA C_LIO_LICitrullinated (Cit)-vimentin is an important neutrophil extracellular trap-derived target of ACPA C_LI What this study addsO_LIHigh circulating TIGIT+ Tph, Cit-vimentin-autoreactive Tph and Tfh associate with failure to reach remission on conventional synthetic DMARDs within the first year in early HLA-DR shared-epitope+ RA C_LIO_LITph/Tfh and adjacent regulatory T cells surround follicular B-cells in lymphoid aggregates in synovial tissue in non-remission C_LIO_LICirculating TIGIT+ Tph bear a transcriptional signature of lymphoid tissue expansion and lymph node egress as compared to functional differentiation and antigen experience in synovial tissue C_LI How this study might affect research, practice or policyO_LIWhen a remission target is not achieved in HLA-DR shared-epitope+ RA patients during the first year of treatment, high circulating TIGIT+ Tph implicate autoreactive T-B-lymphoid expansion in synovial tissue. C_LI

BACKGROUND Twin pregnancies have a two- to three-fold higher risk of preeclampsia (PE), but early prediction remains poor because current biomarkers and prediction models are largely derived from singleton pregnancies. Defining the biological changes that precede PE in twins may facilitate the development of more effective risk assessment approaches. OBJECTIVE We aimed to characterize maternal plasma protein changes preceding PE in twin pregnancies and to explore whether early circulating proteins could serve as improved risk stratification for twin PE. STUDY DESIGN We performed a nested case-control study within a prospective twin pregnancy cohort at Peking University Third Hospital. A total of 84 women with twin pregnancies were included, comprising 40 pregnancies complicated by PE and 44 normotensive controls. Longitudinal maternal plasma samples were collected in the first, second, and third trimesters. In the discovery cohort, maternal plasma proteins were profiled by mass spectrometry to identify trimester-specific protein changes associated with PE. Candidate proteins were then validated in an independent validation set. We further assessed the functional relevance of selected proteins in trophoblast and endothelial cell models and constructed prediction models integrating biomarkers with maternal clinical characteristics. RESULTS Twin pregnancies complicated by PE showed distinct trimester-dependent protein alterations, with first- and second-trimester changes mainly involving immune regulation and glycolipid metabolism, and later changes enriched in cardiovascular-related pathways. Several proteins demonstrated early discriminatory patterns, including increased annexin A3, chitotriosidase-1, and ferritin light chain (FTL), together with decreased annexin A2 in the first trimester, whereas the conventional singleton markers (sFlt-1 and PAPP-A) did not differ. ELISA validation of these candidate biomarkers yielded an AUC of 0.93 for twin preeclampsia prediction; integration with standard maternal clinical factors further improved discriminatory performance to an AUC of 0.97. Accordingly, a multivariable model combining annexin A2, annexin A3, chitotriosidase-1, FTL, and maternal clinical factors achieved strong internal performance for early discrimination of PE in twin pregnancies (AUC, 0.97; 95% CI, 0.93-1.00). Moreover, annexin A2 knockdown impaired trophoblast migration and invasion and reduced inflammatory cytokine secretion in endothelial cells, supporting a role in placental development and maternal vascular adaptation. CONCLUSION Twin pregnancies that later develop PE exhibit early and progressive maternal plasma protein alterations that can be detected before clinical disease onset. These findings identify candidate biomarkers and biologically relevant pathways that may help explain why PE in twins differs from singleton disease and may support future twin-specific risk stratification strategies. These results require external validation before clinical application.