Prognostic impact of enhanced CD96 expression on NK cells by TGF-ß1 in AML.

Acute myeloid leukemia (AML) is one of the most common types of blood cancer in adults and is associated with a poor survival rate. NK cells play a crucial role in combating AML, and alterations in immune checkpoint expression can impair NK cell function against AML. Targeting certain checkpoints may restore this function. CD96, an inhibitory immune checkpoint, has unclear expression and roles on NK cells in AML patients. In this study, we initially evaluated CD96 expression and compared CD96+ NK with the inhibitory receptor and stimulatory receptors on NK cells from AML patients at initial diagnosis. We observed increased CD96 expression on NK cells with dysfunctional phenotype. Further analysis revealed that CD96+ NK cells had lower IFN-γ production than CD96- NK cells. Blocking CD96 enhanced the cytotoxicity of primary NK and cord blood-derived NK (CB-NK) cells against leukemia cells. Notably, patients with a high frequency of CD96+ NK cells at initial diagnosis exhibited poorer clinical outcomes. Additionally, TGF-ß1 was found to enhance CD96 expression on NK cells via SMAD3 signaling. These findings suggest that CD96 is invovled in NK dysfunction against AML blast, and might be a potential target for restoring NK cell function in the fight against AML.

Optimizing sheep B-cell epitopes in Echinococcus granulosus recombinant antigen P29 for vaccine development.

Background: Echinococcus granulosus is a widespread zoonotic parasitic disease, significantly impacting human health and livestock development; however, no vaccine is currently available for humans. Our preliminary studies indicate that recombinant antigen P29 (rEg.P29) is a promising candidate for vaccine. Methods: Sheep were immunized with rEg.P29, and venous blood was collected at various time points. Serum was isolated, and the presence of specific antibodies was detected using ELISA. We designed and synthesized a total of 45 B cell monopeptides covering rEg.P29 using the overlap method. ELISA was employed to assess the serum antibodies of the immunized sheep for recognition of these overlapping peptides, leading to the preliminary identification of B cell epitopes. Utilizing these identified epitopes, new single peptides were designed, synthesized, and used to optimize and confirm B-cell epitopes. Results: rEg.P29 effectively induces a sustained antibody response in sheep, particularly characterized by high and stable levels of IgG. Eight B-cell epitopes of were identified, which were mainly distributed in three regions of rEg.P29. Finally, three B cell epitopes were identified and optimized: rEg.P2971-90, rEg.P29151-175, and rEg.P29211-235. These optimized epitopes were well recognized by antibodies in sheep and mice, and the efficacy of these three epitopes significantly increased when they were linked in tandem. Conclusion: Three B-cell epitopes were identified and optimized, and the efficacy of these epitopes was significantly enhanced by tandem connection, which indicated the feasibility of tandem peptide vaccine research. This laid a solid foundation for the development of epitope peptide vaccine for Echinococcus granulosus.

CD4+T and CD8+T Cells in Uterus Exhibit Both Selective Dysfunction and Residency Signatures.

Unlike T cells in other tissues, uterine T cells must balance strong immune defense against pathogens with tolerance to semiallogeneic fetus. Our previous study fully elucidated the characteristics of γδT cells in nonpregnant uterus and the mechanism modulated by estrogen. However, comprehensive knowledge of the immunological properties of αßT (including CD4+T cells and CD8+T) cells in nonpregnancy uterus has not been acquired. In this study, we fully compared the immunological properties of αßT cells between uterus and blood using mouse and human sample. It showed that most of CD4+T cells and CD8+T cells in murine uterus and human endometrium were tissue resident memory T cells which highly expressed tissue residence markers CD69 and/or CD103. In addition, both CD4+T cells and CD8+T cells in uterus highly expressed inhibitory molecular PD-1 and cytokine IFN-γ. Uterine CD4+T cells highly expressed IL-17 and modulated by transcription factor pSTAT3. Moreover, we compared the similarities and differences between human and murine uterine T cell phenotype. Together, uterine CD4+T cells and CD8+ cells exhibited a unique mixed signature of T cell dysfunction, activation, and effector function which enabled them to balance strong immune defense against pathogens with tolerance to fetus. Our study fully elucidated the unique immunologic properties of uterine CD4+T and CD8+T cells and provided a base for further investigation of functions.

Tissue-resident memory T cells exhibit phenotypically and functionally heterogeneous in human physiological and pathological nasal mucosa.

Pathogens commonly enter mucosal barrier tissues and tissue-resident memory T cells (TRM) are essential for preventing mucosal lesions. However, the immunological properties of TRM cells in nasal mucosa are poorly known. In comparison with control tissues, decreasing CD103+ TRM cells were observed in Chronic rhinosinusitis with nasal polyps (CRSwNPs) and sinonasal inverted papilloma (SNIP), which presented high capability to produce effector cytokines. In CRSwNPs, we found that CD103+ TRM cells with higher cytokine and Granzyme B coexpressed high PD-1, CD103- TRM cells expressed higher IL-10. Homogenates isolated from CRSwNPs induced CD103 expression on peripheral T cells which could be inhibited by blocking TGF-ß. The frequencies of CD103+ TRM cells in CRSwNPs were extremely negatively correlated with neutrophil infiltration. CD103+ TRM cells from Staphylococcus aureus positive CRSwNPs had a stronger response to SEB. Taken together, two phenotypically and functionally distinct subsets of TRM cells exist in nasal tissues and play critical roles in the progress of CRSwNPs and SNIPs.

The NLRP3 molecule influences the therapeutic effects of mesenchymal stem cells through Glut1-mediated energy metabolic reprogramming.

INTRODUCTION: Numerous studies demonstrated that NLRP3 has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Mesenchymal stem cells (MSCs) regulated the NLRP3 inflammasome, which has emerged as a novel therapeutic approach for treating IBD. OBJECTIVES: The exact role of NLRP3 in regulating MSCs' function is unclear. Our study aimed to explore how NLRP3 affects the therapeutic effects of MSCs in colitis. METHODS: We extracted MSCs from the bone marrow of C57BL/6 mice and Nlrp3 KO mice, and identified them using differentiation assays and flow cytometry. In vitro, Both WT MSCs and Nlrp3 KO MSCs were stimulated with inflammatory factor Lipopolysaccharide (LPS), and only WT MSCs were stimulated with varying concentrations of the NLRP3 inhibitor MCC950, then, quantified IL-10 levels in the supernatant. RNA-seq was performed to examine gene expression patterns and Seahorse was used to assess oxidative phosphorylation (OXPHOS) and glycolysis levels. Western blot was used to evaluate protein expression. In vivo, we treated DSS-induced colitis with either WT or Nlrp3 KO MSCs, monitoring weight, measuring colon length, and further evaluation. We also treated DSS-induced colitis with pretreated MSCs (BAY876, oe-Glut1, or oe-NLRP3), following the same experimental procedures as described above. RESULTS: Our results demonstrate that Nlrp3 deletion did not affect MSC phenotypes, but rather promoted osteogenic differentiation. However, the absence of Nlrp3 reduced IL-10 production in MSCs in the presence of LPS, leading to impaired protection on DSS-induced colitis. Conversely, overexpression of NLRP3 promotes the production of IL-10, enhancing therapeutic effects. Further investigation revealed that Nlrp3 deficiency downregulated Glut1 expression and glycolysis activation in MSCs, resulting in decreased IL-10 production. Notably, overexpressing Glut1 in Nlrp3 KO MSCs restored their therapeutic effect that was previously dampened due to Nlrp3 deletion. CONCLUSION: Our findings demonstrate that NLRP3 heightens the therapeutic effects of MSC treatment on DSS-induced colitis.

Tissue resident memory T cells are enriched and dysfunctional in effusion of patients with malignant tumor.

Purpose Most malignant effusion is secondary to metastases to the pleura or peritoneum and portend poor oncological outcomes. Malignant effusion has different tumor microenvironment from primary tumor, containing a variety of cytokines and immune cells and directly contacting with tumor cells. However, the characteristic of CD4+ T cells and CD8+ T cells in malignant effusion remains unclear. Methods Malignant effusion including peritoneal ascites and pleural fluid from thirty-five patients with malignant tumor were collected and compared with matched blood. A detailed characterization of CD4+ T cells and CD8+ T cells in malignant effusion were conducted using flow cytometry and multiple cytokines assay. Results The concentration of IL-6 in malignant effusion was significantly higher than in blood. A substantial portion of T cells in malignant effusion were CD69+ and/ or CD103+ Trm cells. Most CD4+T and CD8+T cells in malignant effusion were exhausted T cells which expressed lower levels of cytokines, cytotoxic molecules and markedly higher levels of inhibitory receptor PD-1 compared with in blood. Conclusion Our study is the first to identify the presence of Trm cells in malignant effusion and will lay the foundation for future research on anti-tumor immunity of Trm cells in malignant effusion.

Immunogenicity of peptide-based vaccine composed of epitopes from Echinococcus granulosus rEg.P29.

Echinococcus granulosus is one of the main causes of economic loss in the livestock industry because of its food-borne transmission. Cutting off the transmission route is a valid prevention method, and vaccines are the most effective means of controlling and eliminating infectious diseases. However, no human-related vaccine has been yet marketed. As a genetic engineering vaccine, recombinant protein P29 of E. granulosus (rEg.P29) could provide protection against deadly challenges. In this study, we generated peptide vaccines (rEg.P29T , rEg.P29B , and rEg.P29T+B ) based on rEg.P29 and an immunized model was established by subcutaneous immunization. Further evaluation showed that peptide vaccine immunization in mice induced T helper type 1 (Th1)-mediated cellular immune responses, leading to high levels of rEg.P29 or rEg.P29B -specific antibodies. In addition, rEg.P29T+B immunization can induce a higher antibody and cytokine production level than single-epitope vaccines, and immune memory is also longer. Collectively, these results suggest that rEg.P29T+B has the potential to be developed as an efficient subunit vaccine for use in areas where E. granulosus is endemic.

Recombinant antigen P29 of Echinococcus granulosus induces Th1, Tc1, and Th17 cell immune responses in sheep.

Echinococcosis is a common human and animal parasitic disease that seriously endangers human health and animal husbandry. Although studies have been conducted on vaccines for echinococcosis, to date, there is no human vaccine available for use. One of the main reasons for this is the lack of in-depth research on basic immunization with vaccines. Our previous results confirmed that recombinant antigen P29 (rEg.P29) induced more than 90% immune protection in both mice and sheep, but data on its induction of sheep-associated cellular immune responses are lacking. In this study, we investigated the changes in CD4+ T cells, CD8+ T cells, and antigen-specific cytokines IFN-γ, IL-4, and IL-17A after rEg.P29 immunization using enzyme-linked immunospot assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA), and flow cytometry to investigate the cellular immune response induced by rEg.P29 in sheep. It was found that rEg.P29 immunization did not affect the percentage of CD4+ and CD8+ T cells in peripheral blood mononuclear cells (PBMCs), and was able to stimulate the proliferation of CD4+ and CD8+ T cells after immunization in vitro. Importantly, the results of both ELISPOT and ELISA showed that rEg.P29 can induce the production of the specific cytokines IFN-γ and IL-17A, and flow cytometry verified that rEg.P29 can induce the expression of IFN-γ in CD4+ and CD8+ T cells and IL-17A in CD4+ T cells; however, no IL-4 expression was observed. These results indicate that rEg.P29 can induce Th1, Th17, and Tc1 cellular immune responses in sheep against echinococcosis infection, providing theoretical support for the translation of rEg.P29 vaccine applications.

Clinical characteristics and antibodies against Echinococcus granulosus recombinant antigen P29 in patients with cystic echinococcosis in China.

OBJECTIVES: Cystic echinococcosis (CE) is a neglected parasitic zoonotic disease caused by the larval stage of the tapeworm Echinococcus granulosus (E. granulosus). This study aimed to understand the clinical characteristics of human CE in Ningxia Hui Autonomous Region (NHAR) located in northwest China and to investigate the antibody profiles against the recombinant E. granulosus antigen P29 (rEg.P29) in plasma of CE patients. METHODS: A total of 37 human CE patients, along with 37 healthy donors enrolled in this study and demographic and clinical data were analyzed, including age, gender, laboratory data, symptoms, and cysts description. Plasma levels of cytokines, total IgG, and total IgE were determined by sandwich ELISA kits. Specific antibodies against rEg.P29 and hydatid cyst fluid (HCF) were assessed by indirect ELISA. RESULTS: The results revealed that females have a higher percentage of CE patients than males. The incidence of CE reached a peak in the 41-50 years-old group. The liver was the most frequent location, accounting for 91.9%. Based on the CT images, cysts of 34 patients who had liver involvement, were classified as 1 (2.9%) CE1, 12 (35.3%) CE2, 5 (14.7%) CE3a, 1 (2.9%) CE3b, and 15 (44.2%) CE5. Twenty-nine (78.4%) patients had a single cyst and 8 (21.6%) had at least two cysts. The most frequently reported symptom was upper abdominal pain. The plasma level of IL-6 and total IgE were significantly increased in CE patients compared with healthy donors. Additionally, IgG response to rEg.P29 in CE patients was significantly higher than in healthy donors, and the dominant IgG subclass was IgG4. Further analysis of different patient groups revealed that rEg.P29-specific IgG and IgG4 were only elevated in CE patients with CE2 type cysts. CONCLUSIONS: This study systematically investigated the clinical characteristics of patients with CE and may provide a reference basis for the diagnosis and treatment of CE in NHAR. Furthermore, tests of specific IgG and IgG4 against rEg.P29 can be used as an assisted method for imaging techniques to identify cystic activity and determine the best therapeutic approach for CE.

Identification of a dominant murine T-cell epitope in recombinant protein P29 from Echinococcus granulosus

Echinococcus granulosus causes echinococcosis, an important zoonotic disease worldwide and a major public health issue. Vaccination is an economical and practical approach for controlling E. granulosus. We have previously revealed that a recombinant protein P29 (rEg.P29) is a good vaccine candidate against E. granulosus. However, T cell immunogenic epitopes have not been identified. In the present study, we use rEg.P29-immunized mice as models to screen immunogenic epitopes for the construction of a novel multi-epitope vaccine. We search for immunodominant epitopes from an overlapping peptide library to screen the peptides of rEg.P29. Our results confirm that rEg.P29 immunization in mice elicits the activation of T cells and induces cellular immune responses. Further analyses show that a T cell epitope within amino acids 86­100 of rEg.P29 elicits significant antigen-specific IFN-γ production in CD4+ and CD8+ T cells and promotes specific T-cell activation and proliferation. Collectively, these results provide a reference for the construction of a novel vaccine against broad E. granulosus genotypes based on epitopes of rEg.P29.

Baseline T-lymphocyte and cytokine indices in sheep peripheral blood.

BACKGROUND: Sheep are an important livestock species worldwide and an essential large-animal model for animal husbandry and veterinary research. Understanding fundamental immune indicators, especially T-lymphocyte parameters, is necessary for research on sheep diseases and vaccines, to better understand the immune response to bacteria and viruses for reducing the use of antibiotics and improving the welfare of sheep. We randomly selected 36 sheep of similar ages to analyze cell-related immune indicators in peripheral blood mononuclear cells (PBMCs). The proportions of CD4+ and CD8+ T cells in PBMCs were detected by flow cytometry. We used Concanavalin A (Con A) and Phorbol-12-myristate-13-acetate (PMA)/Ionomycin to stimulate PBMCs, and measured the expression of IFN-γ, IL-4, and IL-17A using enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISpot). Simultaneously, PMA/Ionomycin/brefeldin A (BFA) was added to PBMCs, then the expression of IFN-γ, IL-4, and IL-17A was detected by flow cytometry after 4 h of culturing. In addition, we observed the proliferation of PBMCs stimulated with Con A for 3, 4, and 5 days. RESULTS: The proportions of CD4+ T lymphocytes (18.70 ± 4.21%) and CD8+ T lymphocytes (8.70 ± 3.65%) were generally consistent among individuals, with a CD4/CD8 ratio of 2.40 ± 0.79. PBMCs produced high levels of IFN-γ, IL-4, and IL-17A after stimulation with PMA/Ionomycin and Con A. Furthermore, PMA/Ionomycin stimulation of PBMC yielded significantly higher cytokine levels than Con A stimulation. Flow cytometry showed that the level of IFN-γ (51.49 ± 11.54%) in CD8+ T lymphocytes was significantly (p < 0.001) higher than that in CD4+ T lymphocytes (14.29 ± 3.26%); IL-4 (16.13 ± 6.81%) in CD4+ T lymphocytes was significantly (p < 0.001) higher than that in CD8+ T lymphocytes (1.84 ± 1.33%), There was no difference in IL-17A between CD4+ (2.83 ± 0.98%) and CD8+ T lymphocytes (1.34 ± 0.67%). The proliferation of total lymphocytes, CD4+ T lymphocytes, and CD8+ T lymphocytes continued to increase between days 3 and 5; however, there were no significant differences in proliferation between the cell types during the stimulation period. CONCLUSIONS: Evaluating primary sheep immune indicators, especially T lymphocytes, is significant for studying cellular immunity. This study provided valuable data and theoretical support for assessing the immune response of sheep to pathogens and improving sheep welfare.

Identification of B-cell dominant epitopes in the recombinant protein P29 from Echinococcus granulosus.

INTRODUCTION: Echinococcus granulosus (E. granulosus) causes a hazardous zoonotic parasitic disease. This parasite can occupy the liver and several areas of the body, causing incurable damage. Our previous studies have provided evidence that the recombinant protein P29 (rEg.P29) exhibit immune protection in sheep and mice against pathological damage induced by E. granulosus, showing its potential as candidate for vaccine development. However, information on the B-cell epitopes of rEg.P29 has not yet been reported. METHODS: Immunological model was established in mice with rEg.P29. SDS-PAGE and Western blot were used to identify protein. Screening for B-cell dominant epitope peptides of rEg.P29 by enzyme-linked immunosorbent assay (ELISA) and immune serum. Dominant epitopes were validated using ELISA and flow cytometry. Multiple sequence alignment analysis was performed using BLAST and UniProt. RESULTS: Immunization with rEg.P29 induced intense and persistent antibody responses, and the epitope of the dominant antigen of B cells are identified as rEg.P29166-185 (LKNAKTAEQKAKWEAEVRKD). Anti-rEg.P29166-185 -specific antibodies lack epitopes against IgA, IgE, and IgG3, compared to anti-rEg.P29-specific antibodies. However, anti-rEg.P29166-185 IgG showed comparatively higher titers, as determined among those peptides by endpoint titration. In addition, rEg.P29 and rEg.P29166-185 promote B-cell activation and proliferation in vitro. The dominant epitopes are relatively conserved in different subtypes of the rEg.P29 sequence. CONCLUSION: rEg.P29166-185 can act as a dominant B-cell epitope for rEg.P29 and promote cell activation and proliferation in the same way as rEg.P29.

Estrogen enhanced the expression of IL-17 by tissue-resident memory γδT cells from uterus via interferon regulatory factor 4.

Tissue-resident memory γδT cells at mucosal and epithelial sites play an important role for pathogen clearance, immunosurveillance, and participating in physiological processes. Different from other barrier sites, the immune cells in uterus face the protection against infections and tolerate an allogeneic fetus during a successful pregnancy. In the previous study, we found that tissue-resident memory γδT cells were enriched both in human and murine uterus and highly expressed IL-17 that promoted the invasion of trophocytes in vitro. In the current study, we found that γδT cells in uterus but not in blood or spleens expressed higher levels of estrogen receptors. The injection of estrogen into mice increased the proportion of γδT cells in uterus but not in spleens in vivo via CXCR3-CXCL10 chemokine axis. In addition, we found that estrogen enhanced the production of IL-17 but not IFN-γ in vivo and in vitro via interferon regulatory factor 4 but not RORγt and pSTAT3 at mRNA and protein levels. The analysis of cell transcriptome sequence further identified multiple differentially expressed genes between estrogen and control γδT cells. Our study demonstrated that estrogen directly act on γδT cells in uterus to enhance the production of IL-17 that might promote the invasion of trophocytes. Furthermore, our study might provide a new idea that estrogen increased the prevalence of autoimmune diseases in women by enhancing γδT cell-derived IL-17 production in uterus and uncover the critical pathological roles for estrogen in the development of autoimmune diseases.

Innate Resistance to Leishmania amazonensis Infection in Rat Is Dependent on NOS2.

Leishmania infection causes diverse clinical manifestations in humans. The disease outcome is complicated by the combination of many host and parasite factors. Inbred mouse strains vary in resistance to Leishmania major but are highly susceptible to Leishmania amazonensis infection. However, rats are highly resistant to L. amazonensis infection due to unknown mechanisms. We use the inducible nitric oxide synthase (Nos2) gene knockout rat model (Nos2 -/- rat) to investigate the role of NOS2 against leishmania infection in rats. Our results demonstrated that diversion toward the NOS2 pathway is the key factor explaining the resistance of rats against L. amazonensis infection. Rats deficient in NOS2 are susceptible to L. amazonensis infection even though their immune response to infection is still strong. Moreover, adoptive transfer of NOS2 competent macrophages into Nos2 -/- rats significantly reduced disease development and parasite load. Thus, we conclude that the distinct L-arginine metabolism, observed in rat macrophages, is the basis of the strong innate resistance to Leishmania. These data highlight that macrophages from different hosts possess distinctive properties and produce different outcomes in innate immunity to Leishmania infections.

PD-1+CXCR5-CD4+ Th-CXCL13 cell subset drives B cells into tertiary lymphoid structures of nasopharyngeal carcinoma.

BACKGROUND: A major current challenge is to exploit tertiary lymphoid structures (TLSs) to promote the lymphocyte infiltration, activation and differentiation by tumor antigens to increase antitumor immune responses. The mechanisms that underlie the role of TLS formation in the adaptive immune responses against nasopharyngeal carcinoma (NPC) remain largely unknown. METHODS: Cell populations and the corresponding markers were identified by single-cell RNA sequencing and fluorescence-activated cell sorting analysis. In vitro differentiation experiments were used to simulate the generation, regulation and function of the Th-CXCL13 cell subset in the tumor microenvironment of NPC. These were followed by histological evaluation of the colocalization of tumor-associated B cells (TABs) and Th-CXCL13 cells within TLSs, and statistical analysis of the relationship between the cells in TLSs and overall survival. RESULTS: A PD-1+CXCR5-CD4+ Th-CXCL13 cell subset was identified in NPC. This subset was a major source of CXCL13, representing the majority of the CD4+ T cells at levels comparable with Th1 and Tfh cells present in the TLSs. Monocytes activated by toll-like receptor 4 agonists served as the antigen-presenting cells that most efficiently triggered the expansion of Th-CXCL13 cells. Transforming growth factor beta 1 (TGF-ß1) stimulation and activation of Sox4 were critical for the induction and polarization of Th-CXCL13 cells in this process. The potential functional contributions of TABs recruited by Th-CXCL13 cells which induced plasma cell differentiation and immunoglobulin production via interleukin-21 and CD84 interactions in the TLSs demonstrated improved survival. CONCLUSIONS: Induction of Th-CXCL13 cells links innate inflammation to immune privilege in tumor-associated TLSs and might predict better survival.

Wogonin Strengthens the Therapeutic Effects of Mesenchymal Stem Cells in DSS-Induced Colitis via Promoting IL-10 Production.

Inflammatory bowel diseases (IBD) are prevalent and debilitating diseases; their clinical remedy is desperately unmet. Mesenchymal stem cells (MSCs) are pluripotent stem cells with multiple immunomodulatory effects, which are attributed to their efficacy in the IBD rodent model. Optimization of MSC regimes in IBD is a crucial step for their further clinical application. Wogonin is a flavonoid-like compound, which showed extensive immunomodulatory and adjuvant effects. This research is aimed at investigating whether and how Wogonin boosted the therapeutic efficiency of MSCs on DSS-induced colitis. Our results showed that the MSC treatment with Wogonin significantly alleviated the intestinal inflammation in IBD mice by increased IL-10 expression. In vitro experiments, Wogonin obviously raised the IL-10 production and ROS levels of MSCs in a dose-dependent manner. Meanwhile, western blot data suggested Wogonin improves the IL-10 production by inducing transcript factor HIF-1α expression via AKT/GSK3ß signal pathway. Finally, the favorable effects of Wogonin on MSCs were confirmed by IL-10 blockade experiment in vivo. Together, our results suggested that Wogonin significantly increased the IL-10 production and enhanced the therapeutic effects of MSCs in DSS-induced colitis. This work suggested Wogonin as a novel optimal strategy for MSC clinical application.

Tissue-Resident Memory-Like CD8+ T Cells Exhibit Heterogeneous Characteristics in Tuberculous Pleural Effusion.

Tissue-resident memory T (TRM) cells are well known to play critical roles in peripheral tissues during virus infection and tumor immunology. Our previous studies indicated that CD69+CD4+ and CD69+CD8+ T cells in tuberculous pleural effusion (TPE) were antigen-specific memory T cells. However, the phenotypical and functional characteristics of CD8+ TRM cells in tuberculosis remain unknown. We found that CD103+CD8+ T cells were the predominant subset of CD103+ lymphocytes in TPE; both CD103 and CD69 expressed on memory CD8+ T cells from TPE were significantly increased compared with those from paired peripheral blood. Phenotypically, CD103+CD69+ and CD103+CD69-CD8+ T cells expressed higher levels of CD45RO than CD103-CD69+CD8+ T cells did; CD103+CD69-CD8+ T cells highly expressed CD27, CD127, and CD62L and some chemokine receptors. We further compared the functional differences among the four distinct CD45RO+CD8+ T subsets identified by CD103 and CD69 expression. In consist with our published results, CD69+CD8+ T cells, but not CD103+CD8+, produced high levels of IFN-γ after treatment with BCG in the presence of BFA. Nevertheless, CD103-CD69+ and CD103+CD69+ memory CD8+ T cells expressed higher levels of Granzyme B, while CD103+CD69- memory CD8+ T cells were characterized as a possibly immunosuppressive subset by highly expressing CTLA-4, CD25, and FoxP3. Furthermore, TGF-ß extremely increased CD103 expression but not CD69 in vitro. Together, CD103+CD8+ T cells form the predominant subset of CD103+ lymphocytes in TPE; CD103 and CD69 expression defines distinct CD8+ TRM-like subsets exhibiting phenotypical and functional heterogeneity. Our findings provide an important theoretical basis to optimize and evaluate new tuberculosis vaccines.

Antigen-Specific Tissue-Resident Memory T Cells in the Respiratory System Were Generated following Intranasal Vaccination of Mice with BCG.

Tissue-resident memory T cells (TRM) are different from effector memory T cells (TEM) and central memory T cells (TCM) and contribute to the protective immunity against local challenges. Currently, we found that CD4+ and CD8+ TRM cells in the nasal mucosa, trachea, lungs, and lavage fluids were heterogeneous on the expression of CD69 and CD103 as well as the production of cytokines including IFN-γ, IL-2, and TNF-α. After intranasal vaccination of mice with BCG, respiratory tissues expressed higher levels of the chemokine CXCL16 and TRM cells expressed CXCR6 to CXCL16. In addition, antigen-specific CD4+ and CD8+ TRM cells expressed cytokines following the stimulation with BCG and persisted in the nasal mucosa, trachea, and lungs for more than a hundred days. At the same time, mice were infected intranasally with live BCG and the results showed that vaccinated mice cleared up live BCG faster than nonvaccinated mice in the respiratory system. Taken together, our data demonstrated that intranasal vaccination of mice with BCG could induce antigen-specific CD4+ and CD8+ TRM cells in the respiratory system and have the ability to provide protection against pulmonary reinfection.

The phenotypic and functional study of tissue B cells in respiratory system provided important information for diseases and development of vaccines.

The field of tissue-resident B cells has received increasing attention, yet the feature of tissue B cells in respiratory system is unclear. Here, we first show that non-circulating B cells obtained from nasal, trachea and lung tissues are numerically and phenotypically distinct from their circulating counterparts. Analysis of single cell transcriptome sequence identified multiple differentially expressed genes between non-circulating B cells and circulating B cells, which illustrated their heterogeneity. Furthermore, we found high expression of CXCR3 on non-circulating B cells, and the chemokine CXCL11 was also up-regulated in the respiratory tissues, suggesting that CXCR3-CXCL11 axis might accelerate the local resident of non-circulating B cells in respiratory tract. Interestingly, intranasal immunization with BCG in mice elicited a sustained humoral immune response via induction of IgA and IgG Abs, which revealed the role of B cells. Meanwhile, tissue-resident B cells, IgA+ and IgG+ memory B cells (MBCs) in respiratory tissues, as well as plasma cells in bone marrow, were expanded and maintained, and these subsets probably developed into antibody-producing cells to participate in the local humoral immunity. Our data illustrate the phenotype and function of tissue B cells in the upper and lower airways, provide references for the prospective development of vaccines.

Local IL-17 positive T cells are functionally associated with neutrophil infiltration and their development is regulated by mucosal microenvironment in nasal polyps.

OBJECTIVE AND DESIGN: IL-17 plays essential roles in neutrophilic inflammation in the lower respiratory tract, however, the characteristics of local IL-17+ T cells in nasal inflammatory mucosa are not fully understood. We investigated the roles of IL-17+ T cells in regulating neutrophil infiltration and the effect of the mucosal microenvironment in modulating IL-17+ T cell differentiation in CRSwNP tissues. SUBJECTS: 47 polyp tissues from chronic rhinosinusitis with nasal polyps (CRSwNP) patients without corticosteroid therapy and 26 tissues from healthy mucosa were obtained. METHODS: Immunohistochemistry and flow cytometry were used to analyze the neutrophil infiltration, local IL-17+ T cell subsets, as well as cytokine producing profiles of IL-17+ T cell; tissue homogenates were used to study neutrophil migration and IL-17+ T cell differentiation. RESULTS: Increase of IL-17+ cells and IL-17+ T cell subsets was significant in polyp tissues versus controls, IL-17+ cell number was positively correlated with neutrophil infiltration; while homogenates from polyp tissues with high IL-17 promoted neutrophil migration in vitro. IL-17 response was found in polyp-derived T cells upon Staphylococcus aureus infection. IL-17+ T cells were also down-regulated in polyps from patients treated with glucocorticoid steroids, and exhibited poly-functionality patterns in polyp tissues. Finally, IL-17+ T cell differentiation could be induced by IL-23, and homogenates from polyps could enhance IL-17+ T cell development. CONCLUSIONS: This study determined a functional association of IL-17+ T cells with neutrophils in CRSwNP, and revealed that polyp microenvironment could promote IL-17+ T cell differentiation, suggesting a potential feedback role for IL-17+ T cell development and local neutrophilic inflammation.