Deciphering the interplay of HPV infection, MHC-II expression, and CXCL13+ CD4+ T cell activation in oropharyngeal cancer: implications for immunotherapy.

BACKGROUND: Human papillomavirus (HPV) infection has become an important etiological driver of oropharyngeal squamous cell carcinoma (OPSCC), leading to unique tumor characteristics. However, the interplay between HPV-associated tumor cells and tumor microenvironment (TME) remains an enigma. METHODS: We performed a single-cell RNA-sequencing (scRNA-seq) on HPV-positive (HPV+) and HPV-negative (HPV‒) OPSCC tumors, each for three samples, and one normal tonsil tissue. Ex vivo validation assays including immunofluorescence staining, cell line co-culture, and flow cytometry analysis were used to test specific subtypes of HPV+ tumor cells and their communications with T cells. RESULTS: Through a comprehensive single-cell transcriptome analysis, we uncover the distinct transcriptional signatures between HPV+ and HPV‒ OPSCC. Specifically, HPV+ OPSCC tumor cells manifest an enhanced interferon response and elevated expression of the major histocompatibility complex II (MHC-II), potentially bolstering tumor recognition and immune response. Furthermore, we identify a CXCL13+CD4+ T cell subset that exhibits dual features of both follicular and pro-inflammatory helper T cells. Noteworthily, HPV+ OPSCC tumor cells embrace extensive intercellular communications with CXCL13+CD4+ T cells. Interaction with HPV+ OPSCC tumor cells amplifies CXCL13 and IFNγ release in CD4+T cells, fostering a pro-inflammatory TME. Additionally, HPV+ tumor cells expressing high MHC-II and CXCL13+CD4+ T cell prevalence are indicative of favorable overall survival rates in OPSCC patients. CONCLUSIONS: Together, our study underscores a synergistic inflammatory immune response orchestrated by highly immunogenic tumor cells and CXCL13+CD4+ T cells in HPV+ OPSCC, offering useful insights into strategy development for patient stratification and effective immunotherapy in OPSCC.

Expansion of the HSV-2-specific T cell repertoire in skin after immunotherapeutic HSV-2 vaccine.

The skin at the site of HSV-2 reactivation is enriched for HSV-2-specific T cells. To evaluate whether an immunotherapeutic vaccine could elicit skin-based memory T cells, we studied skin biopsies and HSV-2-reactive CD4+ T cells from PBMCs by T cell receptor (TCR) ß chain (TRB) sequencing before and after vaccination with a replication-incompetent whole-virus HSV-2 vaccine candidate (HSV529). The representation of HSV-2-reactive CD4+ TRB sequences from PBMCs in the skin TRB repertoire increased after the first vaccine dose. We found sustained expansion after vaccination of unique, skin-based T cell clonotypes that were not detected in HSV-2-reactive CD4+ T cells isolated from PBMCs. In one participant, a switch in immunodominance occurred with the emergence of a TCR αß pair after vaccination that was not detected in blood. This TCRαß was shown to be HSV-2 reactive by expression of a synthetic TCR in a Jurkat-based NR4A1 reporter system. The skin in areas of HSV-2 reactivation possessed an oligoclonal TRB repertoire that was distinct from the circulation. Defining the influence of therapeutic vaccination on the HSV-2-specific TRB repertoire requires tissue-based evaluation.

Transcription of HIV-1 at sites of intact latent provirus integration.

HIV-1 antiretroviral therapy is highly effective but fails to eliminate a reservoir of latent proviruses, leading to a requirement for life-long treatment. How the site of integration of authentic intact latent proviruses might impact their own or neighboring gene expression or reservoir dynamics is poorly understood. Here, we report on proviral and neighboring gene transcription at sites of intact latent HIV-1 integration in cultured T cells obtained directly from people living with HIV, as well as engineered primary T cells and cell lines. Proviral gene expression was correlated to the level of endogenous gene expression under resting but not activated conditions. Notably, latent proviral promoters were 100-10,000× less active than in productively infected cells and had little or no measurable impact on neighboring gene expression under resting or activated conditions. Thus, the site of integration has a dominant effect on the transcriptional activity of intact HIV-1 proviruses in the latent reservoir, thereby influencing cytopathic effects and proviral immune evasion.

Comprehensive immune cell spectral library for large-scale human primary T, B, and NK cell proteomics.

Although proteomics is extensively used in immune research, there is currently no publicly accessible spectral assay library for the comprehensive proteome of immune cells. This study generated spectral assay libraries for five human immune cell lines and four primary immune cells: CD4 T, CD8 T, natural killer (NK) cells, and B cells. This was achieved by utilizing data-dependent acquisition (DDA) and employing fractionated samples from over 100 µg of proteins, which was applied to acquire the highest-quality MS/MS spectral data. In addition, Data-indedendent acquisition (DIA) was used to obtain sufficient data points for analyzing proteins from 10,000 primary CD4 T, CD8 T, NK, and B cells. The immune cell spectral assay library generated included 10,544 protein groups and 127,106 peptides. The proteomic profiles of 10,000 primary human immune cells obtained from 15 healthy volunteers analyzed using DIA revealed the highest heterogeneity of B cells among other immune cell types and the similarity between CD4 T and CD8 T cells. All data and spectral library are deposited in ProteomeXchange (PXD047742).

Efgartigimod as a promising add-on therapy for myasthenic crisis: a prospective case series.

Introduction: Efgartigimod is effective and well-tolerated in patients with anti-acetylcholine receptor (AChR) antibody-positive generalized myasthenia gravis (MG). However, the therapeutic potential and the safety profile of efgartigimod in myasthenic crisis (MC) remained largely unknown. Methods: This is an observational, prospective, multicenter, real-world study to follow 2 MC patients who initiated efgartigimod as a first-line rescue therapy and 8 cases who used it as an add-on therapy. Baseline demographic features and immunotherapies were collected, and the MG-activities of daily living (MG-ADL) scale was evaluated every week since efgartigimod treatment for 8 weeks. Additionally, serum IgG and anti-AChR antibody levels and the peripheral CD4+ T lymphocytes were measured before and after one cycle of treatment. Results: Ten patients with MC were enrolled in the study, including 9 anti-AChR antibody positive and 1 anti-muscle-specific kinase (MuSK) positive. All patients were successfully weaned from the ventilation after receiving efgartigimod treatment, with a length of 10.44 ± 4.30 days. After one cycle of infusions, the MG-ADL score reduced from 15.6 ± 4.4 at the baseline to 3.4 ± 2.2, while the corticosteroid dose was tapered from 55.0 ± 20.7 mg to 26.0 ± 14.1 mg. The proportions of regulatory T cells and naïve T cells (% in CD4+ T) significantly decreased post-efgartigimod treatment (5.48 ± 1.23 vs. 6.90 ± 1.80, P=0.0313, and 34.98 ± 6.47 vs. 43.68 ± 6.54, P=0.0313, respectively). Conclusion: These findings validated the rapid action of efgartigimod in facilitating the weaning process with a good safety profile in patients with MC.

Alloreactive memory CD4 T cells promote transplant rejection by engaging DCs to induce innate inflammation and CD8 T cell priming.

Alloreactive memory T cells have been implicated as central drivers of transplant rejection. Perplexingly, innate cytokines, such as IL-6, IL-1ß, and IL-12, are also associated with rejection of organ transplants. However, the pathways of innate immune activation in allogeneic transplantation are unclear. While the role of microbial and cell death products has been previously described, we identified alloreactive memory CD4 T cells as the primary triggers of innate inflammation. Memory CD4 T cells engaged MHC II-mismatched dendritic cells (DCs), leading to the production of innate inflammatory cytokines. This innate inflammation was independent of several pattern recognition receptors and was primarily driven by TNF superfamily ligands expressed by alloreactive memory CD4 T cells. Blocking of CD40L and TNFα resulted in dampened inflammation, and mice genetically deficient in these molecules exhibited prolonged survival of cardiac allografts. Furthermore, myeloid cell and CD8 T cell infiltration into cardiac transplants was compromised in both CD40L- and TNFα-deficient recipients. Strikingly, we found that priming of naive alloreactive CD8 T cells was dependent on licensing of DCs by memory CD4 T cells. This study unravels the key mechanisms by which alloreactive memory CD4 T cells contribute to destructive pathology and transplant rejection.

A pathologically expanded, clonal lineage of IL-21-producing CD4+ T cells drives inflammatory neuropathy.

Inflammatory neuropathies, which include chronic inflammatory demyelinating polyneuropathy (CIDP) and Guillain Barré syndrome (GBS), result from autoimmune destruction of the PNS and are characterized by progressive weakness and sensory loss. CD4+ T cells play a key role in the autoimmune destruction of the PNS. Yet, key properties of pathogenic CD4+ T cells remain incompletely understood. Here, we used paired single-cell RNA-Seq (scRNA-Seq) and single-cell T cell receptor-sequencing (scTCR-Seq) of peripheral nerves from an inflammatory neuropathy mouse model to identify IL-21-expressing CD4+ T cells that were clonally expanded and multifunctional. These IL-21-expressing CD4+ T cells consisted of 2 transcriptionally distinct expanded cell populations, which expressed genes associated with T follicular helper (Tfh) and T peripheral helper (Tph) cell subsets. Remarkably, TCR clonotypes were shared between these 2 IL-21-expressing cell populations, suggesting a common lineage differentiation pathway. Finally, we demonstrated that IL-21 receptor-KO (IL-21R-KO) mice were protected from neuropathy development and had decreased immune infiltration into peripheral nerves. IL-21 signaling upregulated CXCR6, a chemokine receptor that promotes CD4+ T cell localization in peripheral nerves. Together, these findings point to IL-21 signaling, Tfh/Tph differentiation, and CXCR6-mediated cellular localization as potential therapeutic targets in inflammatory neuropathies.

Crystalline silica-induced recruitment and immuno-imbalance of CD4+ tissue resident memory T cells promote silicosis progression.

Occupational crystalline silica (CS) particle exposure leads to silicosis. The burden of CS-associated disease remains high, and treatment options are limited due to vague mechanisms. Here we show that pulmonary CD4+ tissue-resident memory T cells (TRM) accumulate in response to CS particles, mediating the pathogenesis of silicosis. The TRM cells are derived from peripheral lymphocyte recruitment and in situ expansion. Specifically, CD69+CD103+ TRM-Tregs depend more on circulating T cell replenishment. CD69 and CD103 can divide the TRM cells into functionally distinct subsets, mirroring the immuno-balance within CD4+ TRM cells. However, targeting CD103+ TRM-Tregs do not mitigate disease phenotype since the TRM subsets exert immunosuppressive but not pro-fibrotic roles. After identifying pathogenic CD69+CD103- subsets, we highlight IL-7 for their maintenance and function, that present a promising avenue for mitigating silicosis. Together, our findings highlight the distinct role of CD4+ TRM cells in mediating CS-induced fibrosis and provide potential therapeutic strategies.

Identification of Major Histocompatibility Complex Class II Epitopes From Lyme Autoantigen Apolipoprotein B-100 and Borrelia burgdorferi Mcp4 in Murine Lyme Arthritis.

BACKGROUND: During infection with the Lyme arthritis (LA) pathogen Borrelia burgdorferi, T-cell responses to both host and pathogen are dysregulated, resulting in chronic infection and frequent development of autoimmunity. METHODS: To assess CD4+ T-cell epitopes presented during development of LA, we used an unbiased, immunopeptidomics approach to characterize the major histocompatibility complex (MHC) class II immunopeptidome in B burgdorferi-infected C57BL/6 (B6) mice, which develop mild, self-limiting LA, and infected B6 Il10-/- mice, which develop severe, persistent LA at 0, 4, and 16 weeks postinfection (22-23 mice per group). RESULTS: Peptides derived from proteins involved in adaptive T- and B-cell responses and cholesterol metabolism, including human Lyme autoantigen apolipoprotein B-100 (apoB-100), were enriched in infected Il10-/- mice; whereas peptides derived from proteins involved in neutrophil extracellular net formation were enriched in infected B6 mice. Presentation of apoB-100 peptides showed evidence of epitope expansion during infection. Of several identified B burgdorferi peptides, only 1, a methyl-accepting chemotaxis protein peptide Mcp4442-462, was immunogenic. CONCLUSIONS: ApoB-100, a human Lyme autoantigen, undergoes marked epitope expansion during LA development. The paucity of immunogenic B burgdorferi epitopes supports previous findings suggesting CD4+ T-cell responses are suppressed in murine LA.

Wriggly woes: Helminths stirring up T cell trouble.

Understanding determinants of immune response variation is central to developing treatment options. Even et al. show that naive CD4+ T cell transcriptional heterogeneity is altered by helminth infection leading to impaired immune responses independent of commensals.

Cystatin F Depletion in Mycobacterium tuberculosis-Infected Macrophages Improves Cathepsin C/Granzyme B-Driven Cytotoxic Effects on HIV-Infected Cells during Coinfection.

Cystatin F (CstF) is a protease inhibitor of cysteine cathepsins, including those involved in activating the perforin/granzyme cytotoxic pathways. It is targeted at the endolysosomal pathway but can also be secreted to the extracellular milieu or endocytosed by bystander cells. CstF was shown to be significantly increased in tuberculous pleurisy, and during HIV coinfection, pleural fluids display high viral loads. In human macrophages, our previous results revealed a strong upregulation of CstF in phagocytes activated by interferon γ or after infection with Mycobacterium tuberculosis (Mtb). CstF manipulation using RNA silencing led to increased proteolytic activity of lysosomal cathepsins, improving Mtb intracellular killing. In the present work, we investigate the impact of CstF depletion in macrophages during the coinfection of Mtb-infected phagocytes with lymphocytes infected with HIV. The results indicate that decreasing the CstF released by phagocytes increases the major pro-granzyme convertase cathepsin C of cytotoxic immune cells from peripheral blood-derived lymphocytes. Consequently, an observed augmentation of the granzyme B cytolytic activity leads to a significant reduction in viral replication in HIV-infected CD4+ T-lymphocytes. Ultimately, this knowledge can be crucial for developing new therapeutic approaches to control both pathogens based on manipulating CstF.

Sculpting the tumour microenvironment by combining radiotherapy and ATR inhibition for curative-intent adjuvant immunotherapy.

The combination of radiotherapy/chemoradiotherapy and immune checkpoint blockade can result in poor outcomes in patients with locally advanced head and neck squamous cell carcinoma (HNSCC). Here, we show that combining ATR inhibition (ATRi) with radiotherapy (RT) increases the frequency of activated NKG2A+PD-1+ T cells in animal models of HNSCC. Compared with the ATRi/RT treatment regimen alone, the addition of simultaneous NKG2A and PD-L1 blockade to ATRi/RT, in the adjuvant, post-radiotherapy setting induces a robust antitumour response driven by higher infiltration and activation of cytotoxic T cells in the tumour microenvironment. The efficacy of this combination relies on CD40/CD40L costimulation and infiltration of activated, proliferating memory CD8+ and CD4+ T cells with persistent or new T cell receptor (TCR) signalling, respectively. We also observe increased richness in the TCR repertoire and emergence of numerous and large TCR clonotypes that cluster based on antigen specificity in response to NKG2A/PD-L1/ATRi/RT. Collectively, our data point towards potential combination approaches for the treatment of HNSCC.

Transcriptional and methylation outcomes of didehydro-cortistatin A use in HIV-1-infected CD4+ T cells.

Ongoing viral transcription from the reservoir of HIV-1 infected long-lived memory CD4+ T cells presents a barrier to cure and associates with poorer health outcomes for people living with HIV, including chronic immune activation and inflammation. We previously reported that didehydro-cortistatin A (dCA), an HIV-1 Tat inhibitor, blocks HIV-1 transcription. Here, we examine the impact of dCA on host immune CD4+ T-cell transcriptional and epigenetic states. We performed a comprehensive analysis of genome-wide transcriptomic and DNA methylation profiles upon long-term dCA treatment of primary human memory CD4+ T cells. dCA prompted specific transcriptional and DNA methylation changes in cell cycle, histone, interferon-response, and T-cell lineage transcription factor genes, through inhibition of both HIV-1 and Mediator kinases. These alterations establish a tolerogenic Treg/Th2 phenotype, reducing viral gene expression and mitigating inflammation in primary CD4+ T cells during HIV-1 infection. In addition, dCA suppresses the expression of lineage-defining transcription factors for Th17 and Th1 cells, critical HIV-1 targets, and reservoirs. dCA's benefits thus extend beyond viral transcription inhibition, modulating the immune cell landscape to limit HIV-1 acquisition and inflammatory environment linked to HIV infection.

The amalgam of naive CD4+ T cell transcriptional states is reconfigured by helminth infection to dampen the amplitude of the immune response.

Naive CD4+ T cells in specific pathogen-free (SPF) mice are characterized by transcriptional heterogeneity and subpopulations distinguished by the expression of quiescence, the extracellular matrix (ECM) and cytoskeleton, type I interferon (IFN-I) response, memory-like, and T cell receptor (TCR) activation genes. We demonstrate that this constitutive heterogeneity, including the presence of the IFN-I response cluster, is commensal independent insofar as being identical in germ-free and SPF mice. By contrast, Nippostrongylus brasiliensis infection altered this constitutive heterogeneity. Naive T cell-intrinsic transcriptional changes acquired during helminth infection correlated with and accounted for decreased immunization response to an unrelated antigen. These compositional and functional changes were dependent variables of helminth infection, as they disappeared at the established time point of its clearance in mice. Collectively, our results indicate that the naive T cell pool is subject to dynamic transcriptional changes in response to certain environmental cues, which in turn permutes the magnitude of the immune response.

Exploring the Potential Role of Dexmedetomidine in Reducing Postoperative Cognitive Dysfunction in Elderly Hip Fracture Patients.

BACKGROUND: Hip fractures are prevalent in the elderly; however, Postoperative Cognitive Dysfunction (POCD) is a possible complication of hip fracture surgery in elderly patients. This study examines the influence and the underlying mechanism of dexmedetomidine on POCD in elderly patients following hip fracture surgery. METHODS: The retrospective study involved elderly patients with hip fracture who were treated at the Fifth Affiliated Hospital of Xinjiang Medical University from October 2021 to August 2022. During the surgery procedures, dexmedetomidine was administrated and the peripheral blood samples were collected from the patients. Inflammatory factors were measured using Enzyme-linked immunosorbent assay (ELISA), while pyroptosis-related proteins were detected through quantitative reverse transcription PCR (RT-qPCR) and western blot. Additionally, the levels of CD4+T and CD8+T cells were assessed using flow cytometry. An aged rats hip fracture model was established to further investigate the impact of dexmedetomidine on postoperative mobility, cognition function, pyroptosis and immune cells in rats. RESULTS: Postoperative cognitive function in patients did not show significant alteration when compared with pre-operation levels (p > 0.05). There were notable reduction in the levels of interleukin-18 (IL-18), Caspase-3, Gasdermin-D (GSDMD) and NLR Family Pyrin Domain Containing 3 (NLRP3) (p < 0.001), accompanied by an increase in the proportion of CD4+T cells and an decrease in CD8+T cells after operation (p < 0.01). In aged rats, postoperative exploratory activities increased compared to their preoperative state. Compared with preoperative levels, the levels of interleukin-1ß (IL-1ß), IL-18, Caspase-3, GSDMD, and NLRP3 were significantly decreased (p < 0.001), the proportion of CD4+T cells was increased, and the proportion of CD8+T cells was decreased postoperatively (p < 0.01). CONCLUSIONS: Although there was no significant alteration in postoperative cognitive function in patients, dexmedetomidine may still play a role in mitigating POCD potentially due to its effects on reducing immune inflammation and pyroptosis markers. Further research is needed to fully understand the underlying mechanisms and its clinical implications.

How do adjuvants enhance immune responses?

By altering which peptide antigens are presented to CD4+ T cells, adjuvants affect the specificity of the immune response.

The dynamics and longevity of circulating CD4+ memory T cells depend on cell age and not the chronological age of the host.

Quantifying the kinetics with which memory T cell populations are generated and maintained is essential for identifying the determinants of the duration of immunity. The quality and persistence of circulating CD4 effector memory (TEM) and central memory (TCM) T cells in mice appear to shift with age, but it is unclear whether these changes are driven by the aging host environment, by cell age effects, or both. Here, we address these issues by combining DNA labelling methods, established fate-mapping systems, a novel reporter mouse strain, and mathematical models. Together, these allow us to quantify the dynamics of both young and established circulating memory CD4 T cell subsets, within both young and old mice. We show that that these cells and their descendents become more persistent the longer they reside within the TCM and TEM pools. This behaviour may limit memory CD4 T cell diversity by skewing TCR repertoires towards clones generated early in life, but may also compensate for functional defects in new memory cells generated in old age.

Differentiation and regulation of CD4+ T cell subsets in Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease, and its hallmark pathological features are the loss of dopaminergic (DA) neurons in the midbrain substantia nigra pars compacta (SNpc) and the accumulation of alpha-synuclein (α-syn). It has been shown that the integrity of the blood-brain barrier (BBB) is damaged in PD patients, and a large number of infiltrating T cells and inflammatory cytokines have been detected in the cerebrospinal fluid (CSF) and brain parenchyma of PD patients and PD animal models, including significant change in the number and proportion of different CD4+ T cell subsets. This suggests that the neuroinflammatory response caused by CD4+ T cells is an important risk factor for the development of PD. Here, we systematically review the differentiation of CD4+ T cell subsets, and focus on describing the functions and mechanisms of different CD4+ T cell subsets and their secreted cytokines in PD. We also summarize the current immunotherapy targeting CD4+ T cells with a view to providing assistance in the diagnosis and treatment of PD.

The profile and prognostic significance of bone marrow T-cell differentiation subsets in adult AML at diagnosis.

Background: T lymphocytes in tumor microenvironment play a pivotal role in the anti-tumor immunity, and the memory of T cells contributes to the long-term protection against tumor antigens. Compared to solid tumors, studies focusing on the T-cell differentiation in the acute myeloid leukemia (AML) bone marrow (BM) microenvironment remain limited. Patients and methods: Fresh BM specimens collected from 103 adult AML patients at diagnosis and 12 healthy donors (HDs) were tested T-cell differentiation subsets by multi-parameter flow cytometry. Results: CD4 and CD8 T-cell compartments had different constituted profiles of T-cell differentiated subsets, which was similar between AML patients and HDs. Compared to HDs, AML patients as a whole had a significantly higher proportion of CD8 effector T cells (Teff, P = 0.048). Moreover, the T-cell compartment of AML patients with no DNMT3A mutations skewed toward terminal differentiation at the expense of memory T cells (CD4 Teff: P = 0.034; CD8 Teff: P = 0.030; CD8 memory T: P = 0.017), whereas those with mutated DNMT3A had a decrease in CD8 naïve T (Tn) and CD4 effector memory T cells (Tem) as well as an increase in CD4 central memory T cells (Tcm) (P = 0.037, 0.053 and 0.053). Adverse ELN genetic risk correlated with a lower proportion of CD8 Tn. In addition, the low proportions of CD4 Tem and CD8 Tn independently predicted poorer relapse-free survival (RFS, HR [95%CI]: 5.7 (1.4-22.2), P = 0.017 and 4.8 [1.3-17.4], P = 0.013) and event-free survival (EFS, HR [95% CI]: 3.3 (1.1-9.5), P = 0.029; 4.0 (1.4-11.5), P = 0.010), respectively. Conclusions: AML patients had abnormal profiles of BM T-cell differentiation subsets at diagnosis, which was related to DNMT3A mutations. The low proportions of CD4 Tem and CD8 Tn predicted poor outcomes.