Cellular & Molecular Immunology

Antigen-specific regulation of T cell response is crucial for limiting hyperimmune response. In this study, we discovered that antibodies specific to the antigen peptide-MHC class II complex are produced during helper T cell responses to various antigens. These antibodies specifically inhibited T cell receptor (TCR) recognition of MHC class II molecules presenting specific antigen peptide. We have termed these antibodies immuneinduced TCR-like antibodies (iTabs). Immunization with peptides containing flanking residues induced iTabs, whereas immunization with peptides lacking flanking residues did not. Furthermore, immunization of iTab-inducible peptide as well as iTab treatment suppressed autoimmune disease development. Our findings provide a strategy for suppressing antigen-specific helper T cell responses using specific peptides, potentially controlling autoimmune diseases.

Since Helicobacter pylori (H. pylori) resistance to antibiotic regimens is increased, vaccination is becoming an increasingly important alternative therapy to control H. pylori infection. UreB, FlaA, AlpB, SabA, and HpaA proteins of H. pylori were previously proved to be used as candidate vaccine antigens. Here, we developed an engineered antigen based on a recombinant chimeric protein containing a structural scaffold from UreB and B cell epitopes from FlaA, AlpB, SabA, and HpaA. The multi-epitope chimeric antigen, named MECU, could generate a broadly reactive antibody response including antigen-specific antibodies and neutralizing antibodies against H. pylori urease and adhesins. Moreover, therapeutic immunization with MECU could reduce H. pylori colonization in the stomach and protect the stomach in BALB/c mice. This study not only provides a promising immunotherapy to control H. pylori infection, but also offers a reference for antigen engineering against other pathogens.

It is generally believed that the main source of antibodies is B lymphocytes. In this study, our results first revealed that B cell-deficient mice could not produce antibodies specific for TI-Ags; however, mice with B cell deficiency could produce TD-Ag-specific antibodies, although antibody production was delayed compared with that in BALB/c mice after primary TD-Ag challenge. Subsequently, we identified that mouse lung epithelial cells could produce and secrete Ig, including IgM, IgA or IgG, which could display TD-Ag-specific antibody activity. Notably, the production of TD-Ag-specific antibodies by lung epithelial cells was found to be dependent on CD4+ T cells but not CD8+ T cells. Our findings indicate for the first time that B cells are not the only source of TD-Ag-specific antibodies but are essential for rapid TD-Ag-specific antibody production by non-B cells. This discovery may reveal a new mechanism for the production of specific antibodies.

The WHO has declared SARS-CoV-2 outbreak a public health emergency of international concern. However, to date, there was hardly any study in characterizing the immune responses, especially adaptive immune responses to SARS-CoV-2 infection. In this study, we collected blood from COVID-19 patients who have recently become virus-free and therefore were discharged, and analyzed their SARS-CoV-2-specific antibody and T cell responses. We observed SARS-CoV-2-specific humoral and cellular immunity in the patients. Both were detected in newly discharged patients, suggesting both participate in immune-mediated protection to viral infection. However, follow-up patients (2 weeks post discharge) exhibited high titers of IgG antibodies, but with low levels of virus-specific T cells, suggesting that they may enter a quiescent state. Our work has thus provided a basis for further analysis of protective immunity to SARS-CoV-2, and understanding the pathogenesis of COVID-19, especially in the severe cases. It has also implications in designing an effective vaccine to protect and treat SARS-CoV-2 infection.

The continued evolution of influenza viruses reduces the effectiveness of vaccination and antiviral drugs. The identification of novel and universal agents for influenza prophylaxis and treatment is an urgent need. We have previously described two potent single-domain antibodies (VHH), G2.3 and H1.2, which efficiently neutralize H1N1 and H5N2 influenza viruses in vivo. In this study, we modified these VHHs with Fc-fragment to enhance their antiviral activity. Reformatting of G2.3 into bivalent Fc-fusion molecule increased its in vitro neutralizing activity against H1N1 and H2N3 viruses up to 20-fold and, moreover, resulted in obtaining the ability to neutralize H5N2 and H9N2 subtypes. We demonstrated that a dose as low as 0.6 mg/kg of G2.3-Fc or H1.2-Fc administered systemically or locally before infection could protect mice from lethal challenges with both H1N1 and H5N2 viruses. Furthermore, G2.3-Fc reduced the lung viral load to an undetectable level. Both VHH-Fc showed in vivo therapeutic efficacy when delivered via systemic or local route. The findings support G2.3-Fc as a potential therapeutic agent for both prophylaxis and therapy of Group 1 influenza A infection.

Despite significant advances, the eradication of cancer remains a clinical challenge which justifies the urgent exploration of additional therapeutic strategies such as immunotherapies. Human peripheral V{gamma}9V{delta}2 T cells represent an attractive candidate subset for designing safe, feasible and effective adoptive T cell transfer-based therapies. However, following their infiltration within tumors, {gamma}{delta} T cells are exposed to various regulating constituents and signals from the tumor microenvironment (TME), which severely alter their antitumor functions. Here, we show that TGF-{beta}, whose elevated production in some solid tumors is linked to a poor prognosis, interferes with the antigenic activation of human V{gamma}9V{delta}2 T cells in vitro. This regulatory cytokine strongly impairs their cytolytic activity, which is accompanied by the induction of particular phenotypic, transcriptomic and metabolic changes. Collectively, these observations provide informations for better understanding and targeting the impact of TME components to regulate the antitumor activity of human T cell effectors.

COVID-19 is caused by a newly identified coronavirus, SARS-CoV-2, and has become a pandemic around the world. The illustration of the immune responses against SARS-CoV-2 is urgently needed for understanding the pathogenesis of the disease and its vaccine development. CD8+ T cells are critical for virus clearance and induce long lasting protection in the host. Here we identified specific HLA-A2 restricted T cell epitopes in the spike protein of SARS-CoV-2. Seven epitope peptides (n-Sp1, 2, 6, 7, 11, 13, 14) were confirmed to bind with HLA-A2 and potentially be presented by antigen presenting cells to induce host immune responses. Tetramers containing these peptides could interact with specific CD8+ T cells from convalescent COVID-19 patients, and one dominant epitope (n-Sp1) was defined. In addition, these epitopes could activate and generate epitope-specific T cells in vitro, and those activated T cells showed cytotoxicity to target cells. Meanwhile, all these epitopes exhibited high frequency of variations. Among them, n-Sp1 epitope variation 5L>F significantly decreased the proportion of specific T cell activation; n-Sp1 epitope 8L>V variant showed significantly reduced binding to HLA-A2 and decreased the proportion of n-Sp1-specific CD8+ T cell, which potentially contributes to the immune escape of SAR-CoV-2.

In sharp contrast to immune dysfunction in cancer, autoimmune diseases such as systemic lupus erythematosus (SLE) exhibit excessive immune reactions characterized by high titers of autoantibodies to HERV-K102-envelope (HERV-K102-Env) neoantigens, which are frequently found in cancer patients but seldom elicit a strong immune response. Here we aim to address whether anti-HERV-K102-Env autoantibodies in SLE can effectively eliminate autoreactive immune cells and cancer cells expressing HERV-K102-Env neoantigens. We established world-first fully human autoantibody phage display library with 3.67x108 cfu using peripheral blood mononuclear cells (PBMCs) from SLE patients. Through high-throughput screening, we identified nineteen monoclonal autoantibodies (mAbs) targeting the conserved HERV-K102 Env-TM subunit. The EC50 values of these autoantibodies binding to the HERV-K102 Env-TM subunit ranged from 0.002436 g/ml to 1.798 g/ml. Remarkably, eleven of these mAbs not only recognized the HERV-K102 Env-TM glycoprotein on the cell surface but also effectively eliminated autoreactive B, T, and natural killer (NK) cells in SLE, as well as cancer cells. Our findings provide a conceptually new immunotherapy target HERV-K102 Env-TM subunit and open the era of using natural human autoantibodies to treat autoimmune disease and cancers. Several of these monoclonal autoantibodies show promise as potential diagnostic and therapeutic agents, paving the way for innovative approaches to treating SLE and various malignancies.

In mid-2021, the SARS-CoV-2 Delta variant caused the third wave of the COVID-19 pandemic in several countries worldwide. The pivotal studies were aimed at studying changes in the efficiency of neutralizing antibodies to the spike protein. However, much less attention was paid to the T-cell response and the presentation of virus peptides by MHC-I molecules. In this study, we compared the features of the HLA-I genotype in symptomatic patients with COVID-19 in the first and third waves of the pandemic. As a result, we could identify the vanishing of carriers of the HLA-A*01:01 allele in the third wave and demonstrate the unique properties of this allele. Thus, HLA-A*01:01-binding immunoprevalent epitopes are mostly derived from ORF1ab. A set of epitopes from ORF1ab was tested, and their high immunogenicity was confirmed. Moreover, analysis of the results of single-cell phenotyping of T-cells in recovered patients showed that the predominant phenotype in HLA-A*01:01 carriers is central memory T-cells. The predominance of T-lymphocytes of this phenotype may contribute to forming long-term T-cell immunity in carriers of this allele. Our results can be the basis for highly effective vaccines based on ORF1ab peptides.

Breakdown of tolerance and abnormal activation in B cells is an important mechanism in Graves disease (GD) pathogenesis. However, the mechanism by which B cells are abnormal differentiated and activated in GD remains elusive. Here, we show that elevated BAFF expression is positively correlated with serum thyroid hormone (TH) levels in GD patients and high TH levels can induce BAFF overexpression and lead to the abnormal differentiation of B cells in mice. This BAFF overexpression can be seen in many tissues. In the spleens of mice, high TH levels induce M1 macrophages polarization, which generates BAFF overexpression. Our findings open a new perspective on the interactions between endocrine and immune system and provide insight into the involvement of thyroid hormones in the development and progression of GD.

Although vaccines have been successfully developed and approved against SARS-CoV-2, it is still valuable to perform studies on conserved antigenic sites for preventing possible pandemic-risk of other SARS-like coronavirus in the future and prevalent SARS-CoV-2 variants. By antibodies obtained from convalescent COVID-19 individuals, receptor binding domain (RBD) were identified as immunodominant neutralizing domain that efficiently elicits neutralizing antibody response with on-going affinity mature. Moreover, we succeeded to define a quantitative antigenic map of neutralizing sites within SARS-CoV-2 RBD, and found that sites S2, S3 and S4 (new-found site) are conserved sites and determined as subimmunodominant sites, putatively due to their less accessibility than SARS-CoV-2 unique sites. P10-6G3, P07-4D10 and P05-6H7, respectively targeting S2, S3 and S4, are relatively rare antibodies that also potently neutralizes SARS-CoV, and the last mAbs performing neutralization without blocking S protein binding to receptor. Further, we have tried to design some RBDs to improve the immunogenicity of conserved sites. Our studies, focusing on conserved antigenic sites of SARS-CoV-2 and SARS-CoV, provide insights for promoting development of universal SARS-like coronavirus vaccines therefore enhancing our pandemic preparedness.

Chimeric Antigen Receptor T (CAR-T) therapies have revolutionized the treatment of cancers such as relapsed and refractory B cell malignancies. However, the precise therapeutic mechanism of CAR-T action remain to be elucidated. In this study, we systematically analyzed CAR signaling via phosphotyrosine (pTyr) proteomics in CAR-T cells from both clinical patients and healthy donors. We found that CAR-T products from clinical patients displayed heightened tyrosine phosphorylation, particularly in the JAK-STAT, MAPK and TCR signaling cascades. We also identified the significantly regulated pTyr sites in primary CAR-T cells under tonic signaling or upon stimulation by antigen-presenting CD19-K562 cells. Although both CD28{zeta} and 4-1BB{zeta} CAR-T cells exhibited comparable pTyr changes, CD28{zeta} CAR-T cells displayed a more pronounced activation in the TCR signaling pathway. Additionally, comparative analysis between clinical and primary CAR-T cells suggested that CAR-T products were subject to heightened tonic signaling, which may be related to therapeutic relapse. Our findings reveal the state of clinical CAR-T products and refine the CAR-T signal transduction network, providing comprehensive insights and informed guidance for CAR-T therapies.

T cell-targeted therapies are commonly used to manage T cell hyperactivity in autoimmune disorders, graft-versus-host diseases (GVHD), and transplant rejections. However, many patients experience significant side effects or inadequate responses to current treatments, highlighting the urgent need for alternative strategies. In this study, we searched for regulators of T cells through proximity labeling with APEX2 to detect proteins interacting with CD8, a coreceptor of the T-cell receptor (TCR). This screen revealed TMX1, an ER resident transmembrane disulfide oxidoreductase, is essential for T cell cytotoxicity and NFAT, NF{kappa}B, and AP1 signaling but not cell proliferation. TMX1 deletion decreases surface TCR expression and destabilizes CD3{zeta}, a subunit of TCR complex; however, overexpression of CD3{zeta} rescues the phenotype, suggesting that TMX1 is not required for CD3{zeta} function. Mechanistically, TMX1 was found to directly engage the CxxC motif of CD3{delta}, which has been reported to be essential for proper TCR assembly and function. We hypothesize that the loss of TMX1 interaction with CD3{delta} leads to impaired TCR assembly and subsequent CD3{zeta} destabilization. These findings identify TMX1 as a novel regulator of T-cell receptor assembly and a potential target for immunosuppressive therapy.

SARS-CoV-2, which causes COVID-19 disease, is one of greatest global pandemics in history. No effective treatment is currently available for severe COVID-19 disease. One strategy for implementing cell-based immunity involves the use of chimeric antigen receptor (CAR) technology. Unlike CAR T cells, which need to be developed using primary T cells derived from COVID-19 patients with lymphopenia, clinical success of CAR NK cell immunotherapy is possible through the development of allogeneic, universal, and off-the-shelf CAR-NK cells from a third party, which will significantly broaden the application and reduce costs. Here, we develop a novel approach for the generation of CAR-NK cells for targeting SARS-CoV-2. CAR-NK cells were generated using the scFv domain of CR3022 (henceforth, CR3022-CAR-NK), a broadly neutralizing antibody for SARS-CoV-1 and SARS-CoV-2. CR3022-CAR-NK cells can specifically bind to RBD of SARS-CoV-2 and pseudotyped SARS-CoV-2 S protein, and can be activated by pseudotyped SARS-CoV-2-S viral particles in vitro. Further, CR3022-CAR-NK cells can specifically kill pseudo-SARS-CoV-2 infected target cells. Thus, off-the-shelf CR3022-CAR-NK cells may have the potential to treat patients with severe COVID-19 disease.

Regulatory T (Treg) cells are pivotal in maintaining self-tolerance and controlling immune responses. In this study, we investigated potential Treg cell epitopes in human -tubulin that were selected in silico for their promiscuous binding to class II human leukocyte antigens and full identity with antigens from enteric nematodes present in excretory-secretory products. We identified five Treg cell epitopes in -tubulin that were capable of stimulating and expanding IL-10 and TGF-{beta}-producing Foxp3+ Treg cells in peripheral blood mononuclear cells. We also proved that a peptide pool containing the identified Treg cell epitopes (TBL pool) suppressed the T cell responses elicited by different stimuli, including LPS, and class I and class II restricted T cell epitopes, as determined by intracellular cytokine staining assays. Similarly, this same peptide pool was able to suppress T cell responses in mixed lymphocyte reactions. Finally, we found that stimulation of naive CD4+ T cells with autologous monocyte-derived dendritic cells in the presence of the TBL pool promoted the differentiation of functional CD4+CD25highFoxP3+ T cells capable of suppressing the proliferation of CD3/CD28-activated T cells. -tubulin Treg cell epitopes could be useful for treating autoimmune and chronic inflammatory diseases by inducing Treg cells and, given the ubiquitous and copious expression of -tubulin, enable a general mechanism of immune homeostasis.

The COVID-19 pandemic has underscored the importance of understanding the intricate mechanisms of the humoral immune response to SARS-CoV-2. This study aimed to elucidate the diversity and specificity of antibodies generated from convalescent COVID-19 patients by isolating and characterizing monoclonal antibodies (mAbs) targeting the SARS-CoV-2 spike protein. Employing cutting-edge technologies, including single-cell analysis and fluorescence-activated cell sorting, we successfully isolated live memory B cells secreting IgG antibodies from the peripheral blood of convalescent patients. A total of 17 mAbs were generated, encompassing various heavy and light variable genes, with only a few common between patients. In vitro assays demonstrated varying degrees of inhibition against wild-type and Omicron strains, highlighting discrepancies between ACE2 competition and actual neutralization capacity. Bio-layer interferometry and in silico docking analyses revealed unique binding motifs and mechanisms of action, with notable differences in neutralization abilities based on epitope specificity. Furthermore, animal experiments using K18-hACE2 transgenic mice demonstrated the therapeutic potential of these mAbs in preventing SARS-CoV-2 infection. This study provides novel insights into the humoral immune response to SARS-CoV-2 and highlights the importance of patient-derived mAbs as therapeutic agents for COVID-19 treatment and prevention.

Antibody-dependent enhancement (ADE) has been reported in several virus infections including dengue fever virus, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronavirus infection. To study whether ADE is involved in COVID-19 infections, in vitro pseudotyped SARS-CoV-2 entry into Raji cells, K562 cells, and primary B cells mediated by plasma from recovered COVID-19 patients were employed as models. The enhancement of SARS-CoV-2 entry into cells was more commonly detected in plasma from severely-affected elderly patients with high titers of SARS-CoV-2 spike protein-specific antibodies. Cellular entry was mediated via the engagement of Fc{gamma}RII receptor through virus-cell membrane fusion, but not by endocytosis. Peptide array scanning analyses showed that antibodies which promote SARS-CoV-2 infection targeted the variable regions of the RBD domain. To further characterize the association between the spike-specific antibody and ADE, an RBD-specific monoclonal antibody (7F3) was isolated from a recovered patient, which potently inhibited SARS-Cov-2 infection of ACE-2 expressing cells and also mediated ADE in Raji cells. Site-directed mutagenesis the spike RBD domain reduced the neutralization activity of 7F3, but did not abolish its binding to the RBD domain. Structural analysis using cryo-electron microscopy (Cryo-EM) revealed that 7F3 binds to spike proteins at a shift-angled pattern with one "up" and two "down" RBDs, resulting in partial overlapping with the receptor binding motif (RBM), while a neutralizing monoclonal antibody that lacked ADE activity binds to spike proteins with three "up" RBDs, resulting in complete overlapping with RBM. Our results revealed that ADE mediated by SARS-CoV-2 spike-specific antibodies could result from binding to the receptor in slightly different pattern from antibodies mediating neutralizations. Studies on ADE using antibodies from recovered patients via cell biology and structural biology technology could be of use for developing novel therapeutic and preventive measures for control of COVID-19 infection.

B cells are important participants in the pathogenesis of rheumatoid arthritis (RA). Besides classical B cells, novel B cell subsets are continually to be identified in recent years. Natural killer-like B (NKB) cells, a newly recognized B cell subset, are proved to be actively involved in the anti-infection immunity. However, their role in RA and the potential mechanism remain elusive. Here, we showed that NKB cells were expanded dramatically in collagen-induced arthritis (CIA) mice, demonstrating dynamic changes during the disease progression. These cells promoted CD4+ effector T cell proliferation and Th17 cell differentiation in vitro, while adoptive transfer of these cells exacerbated the arthritis severity of CIA mice. RNA Sequencing revealed that NKB cells displayed distinct differential gene expression profile under RA circumstance, potential perpetuating the disease progression. Moreover, the frequencies of NKB cells were significantly increased in RA patients, positively correlated with the clinical and immunological features. After effective therapy, these cells could be recovered to normal levels. Taken together, our results preliminarily revealed the pathogenic role of NKB cells in RA by promoting Th17 proinflammatory responses. Targeting these cells might provide potential therapeutic strategies for this persistent disease.

Invariant chain (Ii) is traditionally known as the dedicated MHCII chaperone. Recent reports have broadened our understanding about various tasks that Ii plays including its physiological role in MHCI cross-presentation. Ii bound MHCI via the MHCII scaffolding CLIP peptide may facilitate MHCI trafficking to the endosomal pathway. The sorting function of Ii depends on two leucine-based sorting signals present in the cytoplasmic tail that acts as binding sites for the adaptor proteins AP-1/AP-2. Here we increased the Ii cross-presentation potency by replacing these with an AP3 motif resulting an efficient transport of Ii from TGN to late endosomes. We also replaced the CLIP region of li with a therapeutically relevant peptide, MART-1. We found the Ii AP3mutant-MART1 construct was capable of loading MHCI and stimulate specific T-cell response more efficiently than the wild type counterpart. The results show that Ii with an AP3 binding sorting motif carrying peptide epitope(s) can promote efficient antigen presentation to cytotoxic T cells (CTLs) independent of the ER located classical MHCI peptide loading machinery.

Group 2 innate lymphoid cells (ILC2) are emerging as a critical player in type 2 immunity at barrier sites in response to microbial infections and allergen exposures. Although their classical activators are known to be host epithelial-derived alarmin cytokines IL-33, IL-25 or TSLP, it remains elusive whether ILC2 cells can be activated by directly sensing microbial ligands via pattern-recognition receptors such as toll-like receptors (TLRs). Here we report that toll-like receptor 4 (TLR4) is a potent activating receptor of human ILC2. We found that among many microbial ligands examined, lipopolysaccharides (LPS) from multiple species of Gram-negative bacteria, was found to potently stimulate human, but not murine ILC2, to proliferate and produce massive amounts of type 2 effector cytokines IL-4, IL-5, and IL-13. LPS-activated ILC2 also had greatly enhanced the CD40 ligand (CD154) expression and were able to promote the proliferation and antibody production of human B cells in culture. In a humanized mouse model, LPS activated the adoptively transferred human ILC2 in mouse lungs. Both NF-kB and JAK pathways, but not the IL-33-ST2 pathway, were required for LPS to activate human ILC2. RNA-seq data further revealed that LPS induced a large set of genes overlapped significantly with those induced by IL-33. Collectively, these findings support a non-classical mode of activating human ILC2 cells via the LPS-TLR4 signaling axis. Thus, targeting TLR4 signaling pathway might be developed as a new approach by modulating ILC2 activation in treating various type 2 immunity-associated diseases.