Clinical & Translational Immunology

Polymeric immunoglobulin M (IgM) and A (IgA) play key roles in systemic and mucosal immunity, yet quantitative assessment of their polymeric forms has been hampered by the lack of robust, high-throughput assays. Polymerization of both isotypes implies incorporation of the joining chain (J chain), making direct detection of integrated J chain an attractive surrogate marker. Here, we report the generation and characterization of a novel panel of monoclonal antibodies targeting human J chain. Binding analyses revealed distinct antibody clusters with differential preferences for IgA-J and/or IgM-J. We developed sensitive ELISAs that allow reliable quantification of J-chain- containing IgM and IgA in recombinant preparations and complex biological samples such as serum and saliva. For IgM, assay performance in serum required mild dissociation of the IgM- CD5L complex, enabling accurate detection of integrated J chain. For IgA, clone 9G10 showed remarkable specificity for IgA-J, with minimal cross-reactivity to IgM. Application of these assays demonstrates that on average, 10% of circulating IgA is J-chain-containing, with proportional contributions of IgA1 and IgA2, and enables high-throughput measurement of antigen-specific polymeric IgA responses, exemplified by SARS-CoV-2 vaccination. These tools provide a long-needed platform to study polymeric antibody dynamics in health, infection, vaccination, and B-cell-driven diseases.

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.

One barrier to developing an HIV-1 cure is viral reservoirs persisting within B cell follicles of lymphatic tissues, partly due to failure of HIV-specific cytotoxic cells to express the follicular-homing receptor CXCR5. Our group explores CAR cell therapies which also express CXCR5 as a potential cure strategy for HIV. Although previous studies have mostly explored CAR T cell therapies, CAR NK cells may be an attractive alternative as they can be used in allogeneic settings and are naturally cytotoxic towards HIV-infected cells. Here, we developed a novel and innovative multiplex engineering method for rhesus macaque NK cells to create virus-specific CAR NK cells multiplexed (MP) with CAR/CXCR5/IL-15/PD-1 KO/transient-CCR7. We first evaluated MP NK cells in vitro for functionality. MP NK cells were then infused into one chronically SIV-infected rhesus macaque to observe tolerance and localization of therapeutic cells. Finally, we performed a larger primate study in which SIV-infected rhesus macaques were infused with two doses of MP NK cells to study long-term localization, safety, and efficacy. In vitro, MP NK cells were expanded to clinically relevant numbers, migrated to chemokine signaling, and secreted cytotoxic cytokines in response to SIV-Env-expressing cells. In the preliminary rhesus macaque study, the therapy caused no adverse reactions, and CAR+ NK cells localized to sites of SIV replication within the spleen and lymph nodes. In the larger primate study, two doses of MP NK cells at 1.2 x 108 cells/kg were safe and increased the levels of NK cells and CAR+ NK cells found within lymphatic tissues. Importantly, the CAR+ NK cells detected in lymph nodes were predominantly CCR7+, demonstrating the importance of CCR7 and CXCR5 in combination for migration to SIV viral reservoirs in follicles of lymphatic tissues. This study is the first to demonstrate this type of complexity and combination of engineering techniques in NK cells. With further optimization, these techniques could lead to the development of novel NK cell therapies to treat HIV and other diseases.

BackgroundSevere eosinophilic asthma (SEA), eosinophilic granulomatosis with polyangiitis (EGPA) and nasal polyposis (NP) are immune-mediated diseases characterised by eosinophilic inflammation. However, there is also increasing interest in the potential pathological roles of autoantibodies in these diseases. Understanding their B cell receptor (BCR) repertoires may provide valuable insights into disease mechanisms, and potential role of B cells in their pathology. MethodsWe conducted BCR repertoire sequencing using peripheral blood from 43 patients, comprising SEA with nasal polyps (SEA+NP), SEA without nasal polyps (SEA-NP), and EGPA, along with 16 healthy controls (HCs). ResultsCompared to HCs, patients with EGPA exhibited increased relative proportions of IgA1, IgG1, IgG2, and IgG4 subclasses. Similarly, SEA-NP patients demonstrated significantly high proportion of IgG2 sequences. Notably, the IgG4 subclass was significantly elevated across all patient groups compared to HCs. Patients receiving anti-IL-5/5R biologic treatments showed increased relative proportions of IgA2 and IgG2 subclasses compared to untreated patients. Some variation across participant groups in mean somatic hypermutation and mutation frequency was evident. 1,508 clones shared across patients, but not healthy controls, were evident though the majority showed low clonal expansion. Nevertheless, a few shared clones did show either high prevalence across patients and/or higher clonal expansion. ConclusionChanges in BCR repertoires in SEA/EGPA are consistent with a pattern of a more mature B cell component in the periphery and with the T2 inflammatory response observed in SEA and EGPA. BCR clonotypes shared across patients were evident, however, whether such clonotypes are pathological in SEA/EGPA requires further investigation.

Dimeric immunoglobulin A (dIgA) is generated from IgA monomers (mIgA) via JCHAIN-dependent polymerization. DIgA is transported across epithelial barriers by the poly Ig receptor (PIGR) and confers mucosal protection, while serum contains substantial amounts of IgA monomers. Distinct plasma cell subsets have been proposed to produce either monomeric or dimeric IgA, with bone marrow plasma cells as a primary source of mIgA. Here, we addressed whether IgA plasma cell populations segregate based on mIgA or dIgA production. Flow cytometric analysis of antibody-secreting cells from bone marrow, lymphoid and mucosal tissues revealed universal intracellular JCHAIN expression across isotypes and failed to identify a discrete JCHAIN-negative IgA plasma cell population. To detect polymeric IgA, we generated a recombinant soluble PIGR that selectively bound JCHAIN-containing dIgA in Western blot, ELISA, and flow cytometry. Soluble PIGR binding was detected in all IgA plasma cells irrespective of tissue origin, arguing against a dedicated mIgA-producing plasma cell subset incapable of dIgA formation. Ex vivo cultures and single-cell DropMap secretion assays demonstrated that bone marrow and lamina propria IgA antibody-secreting cells co-secrete mIgA and dIgA. These findings suggest that dIgA assembly and secretion are general properties of IgA plasma cells and disfavor a dedicated mIgA-producing population.

Human immunoglobulin G4 (IgG4) shapes both protective and pathogenic immunity, influencing conditions ranging from allergy and autoimmunity to cancer. However, subclass-specific targeting of memory B cells remains challenging due to limited knowledge of their phenotypic and functional heterogeneity. Here, we show that IgG4+ memory B cells are overrepresented in a niche of FCER2+BAFFR+ memory B cells, can be classified in several subtypes, have a distinct transcription factor profile and are the only memory B cell subset to express sterile IGHE transcripts. While IgG4+ B cells display a unique transcriptional profile, only BAFFR, IL5Rb and the B cell receptor were upregulated on protein level on IgG4+ cells. IgG4+ B cells have normal repertoire diversity, but a distinct germline V-gene usage, suggesting IgG4 responses are driven by specific antigens. By labeling autoreactive B cells, we confirmed that MuSK myasthenia gravis (an archetypal IgG4-mediatedautoimmune disease) patients have a normal memory B cell profile and that autoreactive IgG4+ memory B cells are extremely rare. These results highlight the unique features of IgG4+ B cells, provide insight on potential subclass-specific therapeutic targets and point towards an antigen-driven IgG4 response within a largely non-autoreactive memory B cell pool.

Numbers of ARID3a (AT-Rich Interaction Domain 3a) -expressing B lymphocytes from patients with systemic lupus erythematosus (SLE) are associated with increased disease activity. Normally, ARID3a-expressing circulating naive B cells are rare, but in SLE naive B cells dramatically increase ARID3a expression. We found that in vitro stimulation of B lymphocytes from healthy individuals with a cocktail of cytokines and agonists induced ARID3a in a subset of activated naive B cells and in IgD-CD27- double negative B cells previously associated with autoimmunity. Single cell RNA-seq of isolated naive B cells from ten SLE patients, with varying frequencies of ARID3a-expressing cells, revealed that ARID3a-associated genes included activation markers. Moreover, our data revealed the unexpected co-expression of the scavenger receptor CD68 with ARID3a, at both the transcript and protein level, in activated subsets of naive B cells. Inhibition of ARID3a in stimulated B cell cultures blocked naive B cell activation and CD68 expression. Together, these data identify ARID3a and CD68 as markers of naive B cell precursors associated with autoimmunity in SLE.

The IgG Fc chain carries a single N-linked glycan which may undergo changes. Increased agalactosylated N-glycans are associated with rheumatoid arthritis (RA) and regarded as pro-inflammatory. Dysregulated neutrophils can make important contributions to host tissue damage. In RA, immune complexes (ICs) that have precipitated onto synovial joint surfaces activate neutrophils via Fc receptors, promoting localised inflammation. We engineered recombinant human monoclonal IgG with agalactosylated or galactosylated N-glycans, generated immobilised ICs and stimulated healthy donor and RA patient blood-derived neutrophils, comparing reactive oxygen species (ROS) production as read-out of neutrophilic inflammation. Both healthy donor and RA patient neutrophils generated less ROS when stimulated with ICs made from agalactosylated IgG. Mechanistically this was due to poorer binding of agalactosylated ICs to neutrophil FcgammaRs, causing lower activation of Akt and p38 MAPK. Both are required for immobilised IC-mediated stimulation of the neutrophil NADPH oxidase. Taken together, this suggests that disease-associated, agalactosylated IgG does not in fact promote inflammation and host tissue injury, at least not by acting on neutrophils. We propose that rather than promoting inflammation, agalactosylated IgG N-glycans that accompany inflammatory disease may arise as part of a compensatory mechanism that is aimed at reducing excessive inflammation and host tissue injury.

Endometriosis (EMS) is a chronic inflammatory disease characterized by ectopic endometrial-like tissue growth and immune dysregulation. Aberrant T cell responses are implicated in EMS pathogenesis, however, functional reprogramming of T helper (Th) subsets across disease stages remain unclear. We profiled systemic and local immune mediators (cytokines/chemokines) and performed bulk RNA sequencing to comprehensively characterize Th1, Th1/17, and Th17 cell subsets from EMS patients and healthy controls. Across patient plasma, peritoneal fluid (PF), and matched eutopic and ectopic tissues, we observed systemic and local cytokine/chemokine alterations, including elevated IL-6 (plasma), FLT-3L and G-CSF (eutopic), and IL-1RA and IL-23 (p40; PF) in severe-stage EMS. Flow cytometry depicted elevated pathogenic Th17 cells in patient PF compared to matched non-pathogenic Th17 and Treg cell subsets. Additionally, circulating Th17 cells were increased in patients with mild (stages I-II) relative to severe (stages III-IV) EMS. RNA sequencing revealed extensive Th subset reprogramming in EMS, most predominately in Th17 cells (2,220 DEGs). Collectively, we reveal significant immune remodeling in EMS and highlight a distinct, aberrant Th17 cell phenotype. Results support repurposing of IL-23- and IL-17-targeted therapeutics, already effectively implemented in other chronic inflammatory diseases, to broaden therapeutic options for EMS and associated comorbidities.

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.

Interleukin-38 (IL-38) is a cytokine of the IL-1 cytokine family that promotes the resolution of inflammation. Resolution mechanisms comprise the induction or recovery of immune tolerance that is lacking in various acute and chronic inflammatory pathologies, including Graft-versus-Host Disease (GvHD). The role of IL-38 in the context of immune tolerance, its primary immune cell targets and underlying molecular mechanisms are not defined. In this study, we investigated the impact of IL-38 on human alloreactivity and in a mouse model of acute GvHD. Our data suggests that monocytes differentiating into macrophages are the main cellular target of IL-38. Specifically, IL-38 reduces antigen presentation capacity in differentiating monocytes through an IL-1 family receptor-independent mechanism, which subsequently avoids T-cell activation. In parallel, IL-38 ameliorates inflammation in allogeneic settings in human and murine GvHD models by promoting the expansion of regulatory T-cells. Our findings indicate that IL-38 promotes immune tolerance during alloreactivity by affecting myeloid cells and T-cells.

Tissue-resident memory (TRM) T-cells are increasingly recognized as key mediators of chronic autoimmune inflammation, yet their organization and functional adaptation within the eye remain poorly understood. We investigated paired vitreous body biopsies and peripheral blood T cells from patients with chronic posterior segment uveitis using multiparameter flow cytometry, antigen-specific stimulation assays, single-cell RNA sequencing, and T-cell receptor sequencing to define the intraocular tissue-adaptive immune states. Vitreous T cells were phenotypically, transcriptionally, and clonally distinct from their circulating counterparts and enriched for canonical TRM markers. However, tissue adaptation differed substantially between CD4+ and CD8+ lineages. Vitreous CD4+ T cells segregated into clonally expanded tissue-adaptive states characterized by greater CXCR6 expression, enhanced antigen-specific cytokine responses, and well-defined transcriptional profiles. In contrast, vitreous CD8+ T cells expressed higher levels of the retention-associated markers CD103 and CD49a yet maintained greater clonal and phenotypic continuity with peripheral blood T cells. Both vitreous CD4+ and CD8+ subsets exhibited a restrained effector profile associated with tissue-adaptive transcriptional programs. Our data reveal that CD4+ and CD8+ T cells in chronic uveitis assume distinct states in the vitreous microenvironment, such that the intraocular immune response relies on both localized tissue retention and active adaptation to the inflammatory niche.

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.

Introduction: Congenital cytomegalovirus (cCMV) is the most common congenital viral infection and a leading non-genetic cause of neurodevelopmental impairment. Current diagnostic methods using fetal biofluids provide limited insight into fetal pathophysiology. Extracellular vesicles (EVs) in amniotic fluid (AF) are a promising source of stable biomolecules that reflect real-time fetal physiology. This proof-of-concept study compared amniotic fluid EV (AF-EV) characteristics in fetuses with severe CMV infection with those of uninfected fetuses and aimed to develop hypotheses about fetal response to cCMV in utero. Methods: AF samples were collected from pregnancies with symptomatic CMV infection and gestational-age-matched uninfected controls (4 pairs, n=8 total). EVs were isolated and characterised by Western blotting, cryo-electron microscopy, and nanoparticle tracking analysis. Label-free quantitative proteomics identified CMV-associated changes in the AF-EV proteome. Results: CMV-infected AF showed higher vesicle levels (Hedges' g = 1.55), indicating inflammation and virus-induced changes in EV biogenesis. Proteomic analysis found 8.6% of proteins dysregulated. Upregulated proteins included haemoglobin subunits, immunoglobulin heavy chain mu, and myeloperoxidase (Hedges' g = 1.51 to 1.88), indicating haemolysis and immune activation. Eleven host proteins related to neurodevelopment, mitochondrial function, lipid metabolism, and Golgi trafficking were absent in infected cases, indicating viral disruption of host pathways. Protein enrichment analysis revealed differences in neurological, haematological, and immune pathways, aligning with severe cCMV pathology. Conclusion: This study acts as a proof-of-principle investigation of the AF-EV proteome in cCMV. Although the results highlight key protein signatures associated with severe fetal outcomes, they primarily serve to generate hypotheses and inform larger prospective studies.

Background: Plasma proteomics may identify host-response signatures in sepsis, but it is unclear whether extracellular vesicle (EV)-enriched plasma provides distinct or redundant information compared with plasma. We compared paired plasma and EV-enriched plasma proteomes in critically ill patients with sepsis and critically ill non-sepsis controls (CINS). Methods: In this prospective observational study, paired plasma and EV-enriched plasma samples were analyzed from 56 critically ill adults, including 40 patients with sepsis and 16 CINS patients. Protein abundance was quantified using liquid chromatography-tandem mass spectrometry. Analyses compared proteomic depth, protein overlap, global concordance between compartments, and differential protein abundance between CINS and sepsis. Exploratory Gene Ontology enrichment was performed as a supplementary analysis. Results: EV-enriched plasma expanded proteomic detection, identifying 2,476 filtered proteins compared with 506 in plasma. Only 386 proteins were detected in both compartments, while 2,090 were unique to EV-enriched plasma and 120 were unique to plasma. Among shared proteins, plasma and EV-enriched plasma showed modest global concordance across critically ill patients (Spearman coeff = 0.322, p = 9.19 x 10^-11), with similar findings in sepsis alone. Differential abundance analysis identified 11 sepsis-associated proteins in plasma and 22 in EV-enriched plasma. Only SAA1, SAA2, and IGFBP6 were significant in both compartments. Exploratory pathway analysis supported acute-phase and inflammatory enrichment in plasma sepsis-associated proteins, while EV-enriched signals were directionally plausible but did not meet prespecified FDR thresholds. Conclusion: Plasma and EV-enriched plasma proteomics capture related but nonredundant sepsis-associated host-response information in critically ill patients.

Cross-reactive antibodies against dengue virus are known to cause antibody-dependent enhancement (ADE) of infection or disease severity under specific conditions. In our previous study, we showed that primary immunization with the COVID-19 vaccine induces induces cross-reactive IgG causing ADE against dengue. In the present study, we investigated the influence of IgG Fc-glycosylation (analyzed by LC-MS/MS) on ADE mediated by cross-reactive IgG against dengue from IgG against SARS-CoV-2. We found a clear correlation between anti-DENV2 E IgG2 galactosylation and the ADE capacity of cross-reactive IgG against dengue in individuals vaccinated against COVID-19. IgG2 sialylation increased over time; however, it was not correlated with ADE capacity. This phenomenon was restricted to IgG2, whereas anti-DENV2 E IgG1 Fc-glycosylation remained stable after COVID-19 vaccination.

The rapid evolution of avian influenza A/H5N1, including the recent U.S. clade 2.3.4.4b outbreak, highlights its pandemic potential and the urgent need for durable, broadly protective vaccines. Given the capacity of CD8+ T cells to mediate cross-strain immunity, we investigated whether geographically distinct HLA-A33 allotypes, HLA-A*33:01 in East/Southeast Asia and HLA-A*33:03 in South Asia, differentially shape the influenza immunopeptidome and influence antiviral immunity. Antigen-presenting cells overexpressing HLA-A*33:01 or HLA-A*33:03 were transfected with single A/H5N1 antigens or infected with A/X-31 (H3N2) as a control comparison representing current seasonal influenza virus. We identified novel ligands restricted to HLA-A*33:01 (57 from A/H5N1; 55 from A/X-31) and HLA-A*33:03 (29 from A/H5N1; 45 from A/X-31). Although fewer peptides were recovered for HLA-A*33:03, a larger proportion of A/X-31-derived peptides were predicted as high-affinity binders (74%) compared with HLA-A*33:01 (61%), indicating qualitative differences in antigen presentation. To determine immunogenicity, peripheral blood lymphocytes from HLA-A*33:03-positive, A/H5N1-naive donors were stimulated with four conserved peptides: PB2GTF, PB2KTY, NPSVQ and PB1MTK. All elicited robust CD8 T cell activation despite the absence of prior A/H5N1 exposure, demonstrating cross-recognition by memory T cells primed against seasonal influenza. These findings define HLA-A33-restricted influenza epitopes and reveal allotype-specific presentation features that shape CD8+ T cell immunity. Conserved, immunogenic peptides identified here represent promising candidates for rational design of broadly cross-reactive vaccines to protect HLA-A33-expressing populations against severe A/H5N1 disease. Data are available via ProteomeXchange with identifier PXD078870. Author SummaryAvian influenza A/H5N1 continues to pose a significant pandemic threat because of its ability to infect humans and its potential to acquire sustained human-to-human transmissibility. While current influenza vaccines primarily target rapidly evolving viral surface proteins, CD8+ T cells can recognize more conserved internal viral proteins and may provide broader protection against diverse influenza strains. In this study, we investigated how two common HLA-A33 variants, which are prevalent in South, East, and Southeast Asian populations, present influenza-derived peptides to CD8+ T cells. We identified novel influenza peptides presented by HLA-A*33:01 and HLA-A*33:03. Importantly, several conserved A/H5N1-derived peptides were recognized by memory CD8+ T cells from healthy individuals with no prior exposure to A/H5N1, suggesting that previous infection with seasonal influenza viruses can generate cross-reactive immune responses. Our findings expand the current repository of influenza T cell targets and provide new insights into antiviral immunity in HLA-A33-expressing populations. The conserved and immunogenic peptides identified in this study may help guide the development of broadly protective influenza vaccines and contribute to future pandemic preparedness efforts.

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.

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.