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Autoimmune hepatitis (AIH) is a rare chronic inflammatory liver disease characterized by the presence of autoantibodies, including those targeting O-phosphoseryl-tRNA:selenocysteine-tRNA synthase (SepSecS), also known as soluble liver antigen (SLA). Anti-SepSecS [...]

Efficient identification of human monoclonal antibodies targeting specific antigenic sites is pivotal for advancing vaccines and immunotherapies against infectious diseases and cancer. Existing screening techniques, however, limit our ability to discover monoclonal antibodies with desired specificity. In this study, we introduce a novel method, blocking of binding (BoB) enzyme‐linked immunoassay (ELISA), enabling the detection of high‐avidity human antibodies directed to defined epitopes. Leveraging BoB‐ELISA, we analyzed the antibody response to known epitopes of influenza A hemagglutinin (HA) in the serum of vaccinated donors. Our findings revealed that serum antibodies targeting head epitopes were immunodominant, whereas antibodies against the stem epitope, although subdominant, were highly prevalent. Extending our analysis across multiple HA strains, we examined the cross‐reactive antibody response targeting the stem epitope. Importantly, employing BoB‐ELISA we identified donors harboring potent heterosubtypic antibodies targeting the HA stem. B‐cell clonal analysis of these donors revealed three novel, genealogically independent monoclonal antibodies with broad cross‐reactivity to multiple HAs. In summary, we demonstrated that BoB‐ELISA is a sensitive technique for measuring B‐cell epitope immunogenicity, enabling the identification of novel monoclonal antibodies with implications for enhanced vaccine development and immunotherapies. [ABSTRACT FROM AUTHOR]

Author(s): Antonino Cassotta [sup.1] [sup.2] , Vincent Mikol [sup.3] , Thomas Bertrand [sup.3] , Stéphanie Pouzieux [sup.3] , Josiane Le Parc [sup.3] , Paul Ferrari [sup.3] , Jacques Dumas [sup.3] [...], Natalizumab (NZM), a humanized monoclonal IgG4 antibody to [alpha]4 integrins, is used to treat patients with relapsing-remitting multiple sclerosis (MS).sup.1,2, but in about 6% of the cases persistent neutralizing anti-drug antibodies (ADAs) are induced leading to therapy discontinuation.sup.3,4. To understand the basis of the ADA response and the mechanism of ADA-mediated neutralization, we performed an in-depth analysis of the B and T cell responses in two patients. By characterizing a large panel of NZM-specific monoclonal antibodies, we found that, in both patients, the response was polyclonal and targeted different epitopes of the NZM idiotype. The neutralizing activity was acquired through somatic mutations and correlated with a slow dissociation rate, a finding that was supported by structural data. Interestingly, in both patients, the analysis of the CD4.sup.+ T cell response, combined with mass spectrometry-based peptidomics, revealed a single immunodominant T cell epitope spanning the FR2-CDR2 region of the NZM light chain. Moreover, a CDR2-modified version of NZM was not recognized by T cells, while retaining binding to [alpha]4 integrins. Collectively, our integrated analysis identifies the basis of T-B collaboration that leads to ADA-mediated therapeutic resistance and delineates an approach to design novel deimmunized antibodies for autoimmune disease and cancer treatment. An analysis of T cell specificities sufficient to sustain a neutralizing anti-drug antibody response offers a potential approach for enhancing the clinical efficacy of a therapeutic humanized antibody.

Author(s): Daniela Latorre [sup.1] [sup.2] , Ulf Kallweit [sup.3] [sup.4] , Eric Armentani [sup.1] , Mathilde Foglierini [sup.1] [sup.5] , Federico Mele [sup.1] , Antonino Cassotta [sup.1] [sup.2] , Sandra [...], Narcolepsy is a chronic sleep disorder caused by the loss of neurons that produce hypocretin. The close association with HLA-DQB1*06:02, evidence for immune dysregulation and increased incidence upon influenza vaccination together suggest that this disorder has an autoimmune origin. However, there is little evidence of autoreactive lymphocytes in patients with narcolepsy. Here we used sensitive cellular screens and detected hypocretin-specific CD4.sup.+ T cells in all 19 patients that we tested; T cells specific for tribbles homologue 2--another self-antigen of hypocretin neurons--were found in 8 out of 13 patients. Autoreactive CD4.sup.+ T cells were polyclonal, targeted multiple epitopes, were restricted primarily by HLA-DR and did not cross-react with influenza antigens. Hypocretin-specific CD8.sup.+ T cells were also detected in the blood and cerebrospinal fluid of several patients with narcolepsy. Autoreactive clonotypes were serially detected in the blood of the same--and even of different--patients, but not in healthy control individuals. These findings solidify the autoimmune aetiology of narcolepsy and provide a basis for rapid diagnosis and treatment of this disease.The detection of hypocretin-specific autoreactive CD4.sup.+ and CD8.sup.+ T cells in patients with narcolepsy reveals the autoimmune aetiology of this disorder.

Mitophagy, a central process guarding mitochondrial quality, is commonly impaired in human diseases such as Parkinson's disease, but its impact in adaptive immunity remains unclear. The differentiation and survival of memory CD8+ T cells rely on oxidative metabolism, a process that requires robust mitochondrial quality control. Here, we found that Parkinson's disease patients have a reduced frequency of CD8+ memory T cells compared with healthy donors and failed to form memory T cells upon vaccination against COVID-19, highlighting the importance of mitochondrial quality control for memory CD8+ T cell formation. We further uncovered that regulators of mitophagy, including Parkin and NIX, were up-regulated in response to interleukin-15 (IL-15) for supporting memory T cell formation. Mechanistically, Parkin suppressed VDAC1-dependent apoptosis in memory T cells. In contrast, NIX expression in T cells counteracted ferroptosis by preventing metabolic dysfunction resulting from impaired mitophagy. Together, our results indicate that the mitophagy machinery orchestrates survival and metabolic dynamics required for memory T cell formation, as well as highlight a deficit in T cell–mediated antiviral responses in Parkinson's disease patients. Editor's summary: During an immune response, T cells rely on mitochondria to support their evolving metabolic demands. Maintenance of mitochondrial health requires removal of damaged mitochondria via mitophagy, which can occur through PINK1/Parkin- or BNIP3L/NIX-mediated pathways. Using peripheral blood from patients with Parkinson's disease and LCMV-infected mice, Franco et al. examined the function of mitochondrial quality control in memory T cell responses. Patients with Parkinson's disease, which is associated with impairments in mitophagy, had fewer CD8+ memory T cells and impaired SARS-CoV-2–specific responses after vaccination. In LCMV-infected mice, Parkin suppressed VDAC1-dependent apoptosis of memory T cells, whereas NIX restrained ferroptosis by preventing metabolic dysfunction. These results demonstrate that key pathways of mitophagy support memory T cell responses by limiting distinct forms of cell death. —Claire Olingy [ABSTRACT FROM AUTHOR]

T follicular helper (TFH) cells play an essential role in promoting B cell responses and antibody affinity maturation in germinal centers (GC). A subset of memory CD4+ T cells expressing the chemokine receptor CXCR5 has been described in human blood as phenotypically and clonally related to GC TFH cells. However, the antigen specificity and relationship of these circulating TFH (cTFH) cells with other memory CD4+ T cells remain poorly defined. Combining antigenic stimulation and T cell receptor (TCR) Vβ sequencing, we found T cells specific to tetanus toxoid (TT), influenza vaccine (Flu), or Candida albicans (C.alb) in both cTFH and non‐cTFH subsets, although with different frequencies and effector functions. Interestingly, cTFH and non‐cTFH cells specific for C.alb or TT had a largely overlapping TCR Vβ repertoire while the repertoire of Flu‐specific cTFH and non‐cTFH cells was distinct. Furthermore, Flu‐specific but not C.alb‐specific PD‐1+ cTFH cells had a "GC TFH‐like" phenotype, with overexpression of IL21, CXCL13, and BCL6. Longitudinal analysis of serial blood donations showed that Flu‐specific cTFH and non‐cTFH cells persisted as stable repertoires for years. Collectively, our study provides insights on the relationship of cTFH with non‐cTFH cells and on the heterogeneity and persistence of antigen‐specific human cTFH cells. [ABSTRACT FROM AUTHOR]

Enterobacteriaceae are a large family of Gram‐negative bacteria that includes both commensals and opportunistic pathogens. The latter can cause severe nosocomial infections, with outbreaks of multi‐antibiotics resistant strains, thus being a major public health threat. In this study, we report that Enterobacteriaceae‐reactive memory Th cells were highly enriched in a CCR6+CXCR3+ Th1*/17 cell subset and produced IFN‐γ, IL‐17A, and IL‐22. This T cell subset was severely reduced in septic patients with K. pneumoniae bloodstream infection who also selectively lacked circulating K. pneumonie‐reactive T cells. By combining heterologous antigenic stimulation, single cell cloning and TCR Vβ sequencing, we demonstrate that a large fraction of memory Th cell clones was broadly cross‐reactive to several Enterobacteriaceae species. These cross‐reactive Th cell clones were expanded in vivo and a large fraction of them recognized the conserved outer membrane protein A antigen. Interestingly, Enterobacteriaceae broadly cross‐reactive T cells were also prominent among in vitro primed naïve T cells. Collectively, these data point to the existence of immunodominant T cell epitopes shared among different Enterobacteriaceae species and targeted by cross‐reactive T cells that are readily found in the pre‐immune repertoire and are clonally expanded in the memory repertoire. [ABSTRACT FROM AUTHOR]

In mice, the ability of naive T (TN) cells to mount an effector response correlates with TCR sensitivity for self-derived Ags, which can be quantified indirectly by measuring surface expression levels of CD5. Equivalent findings have not been reported previously in humans. We identified two discrete subsets of human CD8+ TN cells, defined by the absence or presence of the chemokine receptor CXCR3. The more abundant CXCR3+ TN cell subset displayed an effector-like transcriptional profile and expressed TCRs with physicochemical characteristics indicative of enhanced interactions with peptide--HLA class I Ags. Moreover, CXCR3+ TN cells frequently produced IL-2 and TNF in response to nonspecific activation directly ex vivo and differentiated readily into Ag-specific effector cells in vitro. Comparative analyses further revealed that human CXCR3+ TN cells were transcriptionally equivalent to murine CXCR3+ TN cells, which expressed high levels of CD5. These findings provide support for the notion that effector differentiation is shaped by heterogeneity in the preimmune repertoire of human CD8+ T cells. [ABSTRACT FROM AUTHOR]

Identifying activated T lymphocytes and differentiating antigen-specific from bystander T cells is crucial for understanding adaptive immune responses. This study investigates the efficacy of activation-induced markers (AIMs) in distinguishing these cell populations. We measured the expression of commonly used AIMs (CD25, CD38, CD40L, CD69, CD137, HLA-DR, ICOS, and OX40) in an in vitro T-cell activation system and evaluated their sensitivity, specificity, and positive predictive value. We demonstrated that individual AIMs, while specific in detecting activated CD4 + T cells, poorly discriminate between antigen-specific and bystander activation, as assessed by a discriminative capacity (DC) score we developed. Our analysis revealed that dual AIM combinations significantly enhanced the ability to distinguish antigen-specific from bystander-activated T cells, achieving DC scores above 90%. These combinations also improved positive predictive value and specificity with a modest reduction in sensitivity. The CD25 hi /ICOS hi combination emerged as the most efficient, with an average sensitivity of 84.35%, specificity of 99.7%, and DC score of 90.12%. Validation through T-cell cloning and antigen re-stimulation confirmed the robustness of our predictions. This study provides a practical framework for researchers to optimize strategies for identifying and isolating antigen-specific human CD4 + T lymphocytes and studying their phenotype, function, and T-cell receptor repertoire., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)

Mitophagy, a central process guarding mitochondrial quality, is commonly impaired in human diseases such as Parkinson's disease, but its impact in adaptive immunity remains unclear. The differentiation and survival of memory CD8 + T cells rely on oxidative metabolism, a process that requires robust mitochondrial quality control. Here, we found that Parkinson's disease patients have a reduced frequency of CD8 + memory T cells compared with healthy donors and failed to form memory T cells upon vaccination against COVID-19, highlighting the importance of mitochondrial quality control for memory CD8 + T cell formation. We further uncovered that regulators of mitophagy, including Parkin and NIX, were up-regulated in response to interleukin-15 (IL-15) for supporting memory T cell formation. Mechanistically, Parkin suppressed VDAC1-dependent apoptosis in memory T cells. In contrast, NIX expression in T cells counteracted ferroptosis by preventing metabolic dysfunction resulting from impaired mitophagy. Together, our results indicate that the mitophagy machinery orchestrates survival and metabolic dynamics required for memory T cell formation, as well as highlight a deficit in T cell-mediated antiviral responses in Parkinson's disease patients.

Memory B cells (MBCs) generate rapid antibody responses upon secondary encounter with a pathogen. Here, we investigated the kinetics, avidity and cross-reactivity of serum antibodies and MBCs in 155 SARS-CoV-2 infected and vaccinated individuals over a 16-month timeframe. SARS-CoV-2-specific MBCs and serum antibodies reached steady-state titers with comparable kinetics in infected and vaccinated individuals. Whereas MBCs of infected individuals targeted both pre- and postfusion Spike (S), most vaccine-elicited MBCs were specific for prefusion S, consistent with the use of prefusion-stabilized S in mRNA vaccines. Furthermore, a large fraction of MBCs recognizing postfusion S cross-reacted with human betacoronaviruses. The avidity of MBC-derived and serum antibodies increased over time resulting in enhanced resilience to viral escape by SARS-CoV-2 variants, including Omicron BA.1 and BA.2 sub-lineages, albeit only partially for BA.4 and BA.5 sublineages. Overall, the maturation of high-affinity and broadly-reactive MBCs provides the basis for effective recall responses to future SARS-CoV-2 variants.

Enterobacteriaceae are a large family of Gram-negative bacteria that includes both commensals and opportunistic pathogens. The latter can cause severe nosocomial infections, with outbreaks of multi-antibiotics resistant strains, thus being a major public health threat. In this study, we report that Enterobacteriaceae-reactive memory Th cells were highly enriched in a CCR6 + CXCR3 + Th1*/17 cell subset and produced IFN-γ, IL-17A, and IL-22. This T cell subset was severely reduced in septic patients with K. pneumoniae bloodstream infection who also selectively lacked circulating K. pneumonie-reactive T cells. By combining heterologous antigenic stimulation, single cell cloning and TCR Vβ sequencing, we demonstrate that a large fraction of memory Th cell clones was broadly cross-reactive to several Enterobacteriaceae species. These cross-reactive Th cell clones were expanded in vivo and a large fraction of them recognized the conserved outer membrane protein A antigen. Interestingly, Enterobacteriaceae broadly cross-reactive T cells were also prominent among in vitro primed naïve T cells. Collectively, these data point to the existence of immunodominant T cell epitopes shared among different Enterobacteriaceae species and targeted by cross-reactive T cells that are readily found in the pre-immune repertoire and are clonally expanded in the memory repertoire., (© 2020 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

The wide range of effector and memory T cells is instrumental for immune regulation and tailored mechanisms of protection against pathogens. Here, we will focus on human CD4 T cells and discuss T-cell plasticity and intraclonal diversification in the context of a progressive and selective model of CD4 T-cell differentiation., (Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.)