BACKGROUND: Peripheral helper T (T PH ) cells are uniquely positioned within pathologically inflamed non-lymphoid tissues to stimulate B-cell responses and antibody production. However, the phenotype, function, and clinical relevance of T PH cells in hepatocellular carcinoma (HCC) are currently unknown. METHODS: Blood, tumor, and peritumoral liver tissue samples from 39 HCC patients (Sep 2016-Aug 2017) and 101 HCC patients (Sep 2011-Dec 2012) at the Third Affiliated Hospital of Sun Yat-sen University were used. Flow cytometry was used to quantify the expression, phenotype, and function of T PH cells. Log-rank tests were performed to evaluate disease-free survival and overall survival in samples from 39 patients and 101 patients with HCC. T PH cells, CD19 + B cells, and T follicular helper (T FH ) cells were cultured separately in vitro or isolated from C57/B6L mice in vivo for functional assays. RESULTS: T PH cells highly infiltrated tumor tissues, which was correlated with tumor size, early recurrence, and shorter survival time. The tumor-infiltrated T PH cells showed a unique ICOS hi CXCL13 + IL-21 - MAF + BCL-6 - phenotype and triggered naïve B-cell differentiation into regulatory B cells. Triggering programmed cell death protein 1 (PD-1) induced the production of C-X-C motif chemokine ligand 13 (CXCL13) by T PH cells, which then suppressed tumor-specific immunity and promoted disease progression. CONCLUSION Our study reveals a novel regulatory mechanism of T PH cell-regulatory B-cell-mediated immunosuppression and provides an important perspective for determining the balance between the differentiation of protumorigenic T PH cells and that of antitumorigenic T FH cells in the HCC microenvironment.
Carcinoma, Hepatocellular/metabolism* ; Liver Neoplasms/metabolism* ; Humans ; T-Lymphocytes, Helper-Inducer/metabolism* ; Animals ; Mice ; Male ; Female ; Mice, Inbred C57BL ; Middle Aged ; B-Lymphocytes, Regulatory/metabolism* ; Flow Cytometry ; Interleukin-21 ; Aged ; Chemokine CXCL13/metabolism*

Objective To investigate the differences of type 2 innate lymphocytes (ILC2) and interlukin 9 (IL-9) between chronic lymphocytic leukemia (CLL) patients and healthy controls, and to understand the effects of ILC2 on the function of regulatory T cells (Tregs), CD8⁺ T cells and CLL cells through IL-9. Methods Flow cytometry was used to detect the levels of ILC2 and Tregs in the peripheral blood of 45 newly diagnosed CLL patients and 24 healthy controls, and the expressions of granzyme B and perforin in CD8⁺ T cells in the peripheral blood of 28 patients and 15 healthy controls; ELISA was used to detect the level of IL-9 in the serum. ILC2 of patients and healthy controls was sorted by immunomagnetic beads and cultured separately, and the level of IL-9 in the culture supernatant was measured by ELISA. ILC2 sorted from CLL patients and healthy control-derived peripheral blood mononuclear cells(PBMCs) were co-cultured with the B cell leukemia MEC-1 cells, one group was supplemented with IL-9 antibody and the other group was not. After 72 hours of culture, the ratio of Tregs, programmed death 1 (PD-1), T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), cytotoxic T lymphocyte antigen 4 (CTLA-4) on Tregs, granzyme B and perforin in CD8⁺ T cells were measured by flow cytometry, IL-9 level of the culture supernatant was measured by ELISA, the apoptosis of MEC-1 cells was measured by Annexin V-PI. Results Compared with the healthy control group, the levels of ILC2, Tregs and IL-9 in the CLL group increased significantly. The levels of granzyme B and perforin in CD8⁺ T cells were positively correlated in the peripheral blood of CLL patients. Compared with the healthy control group, IL-9 levels in the supernatant of sorted ILC2 from CLL patients increased. In the anti-IL9 antibody group, the level of PD-1 and TIGIT on Tregs decreased, and the level of granzyme B in CD8⁺ T cells increased significantly. The level of IL-9 in the anti-IL9 antibody group decreased statistically. And MEC-1 cells showed increased early apoptotic rate in the anti-IL9 antibody group statistically. Conclusion In CLL, ILC2 affects CD8⁺ T cells and Tregs through IL-9, which weakens the anti-tumor effect of CD8⁺ T cells, enhances the immunosuppressive effect of Tregs, and plays a role in the occurrence and development of CLL disease.
Humans ; Leukemia, Lymphocytic, Chronic, B-Cell/immunology* ; CD8-Positive T-Lymphocytes/immunology* ; T-Lymphocytes, Regulatory/immunology* ; Middle Aged ; Male ; Female ; Interleukin-9/blood* ; Aged ; Granzymes/metabolism* ; Perforin/metabolism* ; Immunity, Innate ; Adult ; Lymphocytes/immunology*

OBJECTIVE: To investigate the expression of co-inhibitory molecules TIGIT/CD155 and PD-1 on CD4⁺T cells and Treg cells in peripheral blood of patients with chronic lymphocytic leukemia (CLL) and analyze their clinical significance. METHODS: The expression of PD-1 and TIGIT on CD4⁺T cells and Treg cells was detected by flow cytometry in 40 CLL patients and 20 healthy controls. Additionally, the expression of CD155 on peripheral blood B cells and DC cells of the enrolled subjects was detected. RESULTS: The proportions of PD-1⁺TIGIT⁺CD4⁺T cells, PD-1⁺TIGIT⁺Treg cells and CD155⁺DC cells in peripheral blood of CLL patients were significantly higher than those of healthy controls ( P < 0.05). The proportions of PD-1⁺TIGIT⁺CD4⁺T cells and PD-1⁺TIGIT⁺Treg cells in CLL patients were significantly higher than those of PD-1⁺TIGIT^-CD4⁺T cells and PD-1⁺TIGIT^-Treg cells, respectively ( P < 0.05). Both PD-1⁺TIGIT⁺CD4⁺T cells and PD-1⁺TIGIT⁺Treg cells were positively correlated with the level of CD155⁺DC cells (r =0.742, r =0.766). With the progression of Binet stage, the proportions of PD-1⁺TIGIT⁺CD4⁺T cells, PD-1⁺TIGIT⁺Treg cells, and CD155⁺DC cells gradually increased ( P < 0.05), and the aforementioned three types cells were all increased in patients with CD38≥30%, IGVH unmutated, or poor prognosis due to chromosomal abnormalities ( P < 0.05). CONCLUSION Co-inhibitory molecules PD-1 and TIGIT may be involved in immunodepletion in patients with advanced CLL, which has clinical prognostic value. Dual inhibitor molecular targeted therapy provides a new direction for the individualized treatment of CLL.
Humans ; Leukemia, Lymphocytic, Chronic, B-Cell/immunology* ; Receptors, Immunologic/metabolism* ; Programmed Cell Death 1 Receptor/metabolism* ; T-Lymphocytes, Regulatory/metabolism* ; Receptors, Virus/metabolism* ; CD4-Positive T-Lymphocytes/metabolism* ; Male ; Female ; Middle Aged ; Flow Cytometry ; Clinical Relevance

OBJECTIVE: To evaluate the effects of hepatitis B virus (HBV) on helper T lymphocytes 17 (Th17), regulatory T lymphocyte (Treg) and Th17/Treg ratio in chronic hepatitis B patients in different alanine aminetransferase (ALT) stages. METHODS: In the study, 336 chronic hepatitis B patients in the first hospital of Lanzhou University were analyzed. The hepatitis B antigen antibody parameters were measured by chemiluminescence immunoassay analyzer, the liver function parameters were measured by automatic biochemical analyzer, the HBV loads were measured by quantitative PCR, Th17, Treg and Th17/Treg ratios were detected by flow cytometry. Among them, 111 cases (ALT < 40 U/L) of ALT were normal hepatitis B, 108 cases of chronic hepatitis B with ALT above normal upper limit and < 2 times higher (40 U/L≤ALT < 80 U/L), and 117 cases of chronic hepatitis B with ALT above 2 times normal upper limit (80 U/L≤ALT). According to the viral load, they were divided into low replication group with HBV DNA < 4.0 lg copies/mL, medium replication group with 4.0 lg copies/mL≤HBV DNA < 6.0 lg copies/mL and high replication group with HBV DNA ≥ 6.0 lg copies / mL. Dunnett T3 variance analysis were used to analyze the effects of HBV on Th17, Treg and Th17/Treg ratio in the chronic hepatitis B patients in different ALT stages. The changes of virological and immunological indexes before and after treatment were observed for 24 weeks of antiviral therapy in the hepatitis B patients with ALT≥double upper limit of normal group. RESULTS: In the ALT normal group, different virus load HBV had minor effects on Th17, Treg and Th17/Treg ratio. In the ALT≥2 times upper limit of normal group, with the virus load increased, Th17 (3.18%±0.79% in low replication group, 3.78%±0.92% in medium replication group and 4.57%±1.15% in high replication group), Treg cells (5.52%±1.58% in low replication group, 5.89%±1.84% in medium replication group and 6.37%±2.35% in high replication group) and their ratio Th17/Treg (0.57±0.25 in low replication group, 0.65±0.29 in medium replication group and 0.73±0.36 in high replication group) were significantly increased (P < 0.05). After entecavir treatment 24 weeks, the patient' s HBV-DNA decreased significantly, Th17 (3.89%±1.02% vs. 2.06%±0.46%), Treg (6.02%±2.03% vs. 5.06%±1.25%), Th17/Treg ratio (0.65±0.28 vs. 0.41±0.14) decreased significantly (P < 0.05). CONCLUSION Investigation on the effects of HBV on Th17 and Treg cells and their ratios in different ALT states can clarify the effects of HBV on the body from the immunological perspective and can further understand the ALT grouping for antiviral treatment theoretical significance, which is helpful for clinical treatment.
Alanine/therapeutic use* ; Alanine Transaminase/therapeutic use* ; Antiviral Agents/therapeutic use* ; DNA, Viral/therapeutic use* ; Hepatitis B/drug therapy* ; Hepatitis B virus/genetics* ; Hepatitis B, Chronic/drug therapy* ; Humans ; T-Lymphocytes, Regulatory

Langerhans cell histiocytosis (LCH) is a rare proliferative disease dominated by the proliferation of Langerhans cells, which is inflammatory myeloid neoplasms. Its clinical manifestations are variable, occurring at any age and at any site, and it is rarer in adults than in children. The gold standard for diagnosis is histopathological biopsy. Due to the rarity of adult LCH and the heterogeneity of this disease, treatment of adult LCH should be developed according to the extent of the disease and risk stratification. With the discovery of MAPK, PI3K and c-KIT signaling pathway activation, especially BRAF V600E and MAP2K1 mutations, targeted therapy has become a hot spot for therapeutic research. Meanwhile, the discovery of high expression of M2-polarized macrophages and Foxp3⁺ regulatory T cells (Treg) in LCH has provided an important basis for the immunotherapy. In this article, we will focus on reviewing the latest research progress in the treatment of adult LCH in recent years, and provide a reference for clinical research on the treatment of adult LCH patients.
Adult ; Child ; Histiocytosis, Langerhans-Cell/therapy* ; Humans ; Mutation ; Proto-Oncogene Proteins B-raf/metabolism* ; Signal Transduction ; T-Lymphocytes, Regulatory/pathology*

OBJECTIVE: To explore the relationship between Treg cells level in peripheral blood and prognosis of patients with diffuse large B-cell lymphoma (DLBCL). METHODS: The percentage and absolute value of Treg cells in peripheral blood of DLBCL patients were detected by flow lytometry, and their correlation to prognosis was analyzed by survival analysis. The absolute count of Treg cells was detected by using maximally selected Log-rank statistic, and it was used as cutoff point to distinguish difference survival. The new group of Treg based on cutoff point was combined with age, sex, pathological subtype, risk stratification, treatment plan, and other indicators to include in the single factor survival analysis of Kaplan-Meier. Finally, the COX proportional risk model was used to verify the effect of the above indicators on progression-free survival. RESULTS: The absolute count of Treg cells in DLBCL patients was significantly lower in the disease progressed group than those in the remission group. The cutoff point of absolute value of the Treg cell was 19 cells /μl. The absolute count of Treg cells was an independent prognostic factor of the risk stratification. CONCLUSION At the beginning of diagnosis, the reduction of the absolute count of Treg cells in peripheral blood of DLBCL patients show a poor prognosis.
Humans ; Lymphoma, Large B-Cell, Diffuse ; Monocytes ; Prognosis ; Proportional Hazards Models ; Retrospective Studies ; T-Lymphocytes, Regulatory

OBJECTIVE: To explore the role of follicular helper T cell (Tfh)/ follicular regulatory T cell (Tfr) imbalance in B-cell lymphoma (BCL). METHODS: Sixteen BCL patients who were admitted to the Department of Hematology of The First People's Hospital of Yichang and 20 healthy people from December 2019 to November 2020 were enrolled and respectively divided into observation group and control group. The levels of Tfh and Tfr in peripheral blood were detected by flow cytometry. The changes of Tfh, Tfr, and Tfh/Tfr ratio were compared and the relationship between Tfh/Tfr ratio and efficacy, prognosis was analyzed. RESULTS: Compared with the healthy controls, Tfh and Tfh/Tfr ratio in peripheral blood of the BCL patients increased (P<0.05, P<0.01), while levels of Tfr was decreased (P<0.01). After chemotherapy, Tfh and Tfh/Tfr ratio in peripheral blood of the BCL patients decreased significantly than before chemotherapy (P<0.01), but Tfr was no significant difference. Multivariate analysis showed that Tfh and Tfh/Tfr ratio were positively correlated with international prognostic index (IPI) score and Ann Arbor stage (r=0.626, 0.564, 0.573, 0.608, respectively), while Tfr negatively (r=－0.504, －0.542, respectively). According to the average value of Tfh/Tfr ratio at initial diagnosis, BCL patients were divided into Tfh/Tfr high ratio group and low ratio group. It was found that the complete remission (CR) rate, overall response rate (ORR), and survival time in the high ratio group were significantly lower than the low ratio group (P<0.01). CONCLUSION There is an imbalance of Tfh/Tfr ratio in peripheral blood of the BCL patients, and those with a high Tfh/Tfr ratio have lower CR, ORR and shorter survival time.
Flow Cytometry ; Humans ; Lymphoma, B-Cell ; T Follicular Helper Cells ; T-Lymphocytes, Helper-Inducer ; T-Lymphocytes, Regulatory

OBJECTIVE: To study the expression of CD19B cells in immune thrombocytopenia patients, and to analyze the involvement of serum Breg in the pathogenesis of ITP. METHODS: A total of 68 ITP patients admitted in department of hematology in our hospital from August 2015 to August 2018 were selected and enrolled in ITP group. 30 healthy individuals who were admitted to physical examination center at the same time were selected and enrolled in control group. The expression of peripheral blood lymphocyte subsets CD19 and peripheral blood Breg cells were measured by flow cytometry. The difference between the ITP group and the control group was compared. The IL-10 mRNA and TGF-β1 mRNA levels were detected by RT-PCR. Correlations between the ratio of peripheral blood Breg cells and the expression of IL-10 mRNA and TGF-β1 mRNA in patients with immune thrombocytopenia, were analyzed. RESULTS: Expression of CD19 in ITP group was significantly higher than that in control group. The difference between the two groups was statistically significant. Expression level of serum Breg cells in ITP group was lower than that in control group. IL-10 mRNA level in ITP group was significantly lower than that in control group. IL-10 mRNA level after treatment was significantly higher than that before treatment. In addition, TGF-β1 mRNA level in newly diagnosed ITP group was significantly higher than that in control group. After treatment, the TGF-β1 mRNA level was higher than that before treatment. The ratio of peripheral blood Breg cells positively correlated with IL-10 mRNA expression (r=0.968, P＜0.05), however, there was no correlation between peripheral blood Breg cell ratio and TGF-β1 mRNA expression (r=0.502, P＞0.05). CONCLUSION Expression of CD19 cells in ITP patients is increased. Breg cells may participate in the genesis of immune thrombocytopenia. The abnormal expression of Breg cells relates with the pathogenesis of immune thrombocytopenia.
Antigens, CD19 ; B-Lymphocytes, Regulatory ; Cell Count ; Humans ; Purpura, Thrombocytopenic, Idiopathic

OBJECTIVE: To investigate the role of regulatory B cells (Breg) in pathogenesis of immune thrombocytopenia(ITP) and its clinical significance. METHODS: A total of 40 ITP patients and 20 normal controls were enrolled in this study. The content of Breg, Th1, Th2, Th17 and Treg cells were detected by flow cytometry (FCM). The expression level of IL-10,TGF-β, CD40 and CD40L was detected by AimPlex Flow High Throughput Screening Technology. RESULTS: The of Breg cells in ITP patients was significantly lower than that in normal controls (P<0.05)，the expression levels of IL-10,TGF-β and CD40L in ITP patients were also significantly lower than those in normal controls (P<0.05). The contents of Th1 cells in ITP patients were significantly higher than that in normal controls (P<0.05), whereas the contents of Th2, Th17 and Treg cells in ITP patients were significantly lower than those in normal controls (P<0.05). CONCLUSION The Breg cells may play an important role in the pathogenesis of ITP.
B-Lymphocytes, Regulatory ; Humans ; T-Lymphocytes, Regulatory ; Th17 Cells ; Thrombocytopenia

Since primary Sjögren's syndrome (pSS) is an autoummune disease of B cell hyperactivity and pathologic autoantibody response, follicular helper T (Tfh) cells and follicular regulatory T (Tfr) cells are suggested to be key players in pSS. We examined subsets of Tfh and Tfr cells from the blood in pSS patients, and whether these subsets represent disease activity, glandular inflammation, or autoantibody responses in pSS. Circulating Tfh and Tfr cells, along with their specific subsets, were identified from the peripheral blood of 18 pSS patients and 14 age- and sex-matched healthy controls (HCs) using flow cytometry analysis. Blood Tfr and Tfh cell ratios were increased in pSS patients compared with HCs. The CCR7(lo)PD-1(hi) subset of circulating Tfh cells was increased in pSS patients with high degree of focal lymphocytic sialadenitis; whereas circulating Tfh cells did not differ between pSS patients and HCs. The frequency of CCR7(lo)PD-1(hi) Tfh cells was significantly correlated with disease activity scores and differentiated B cells. PD-1 expression on blood Tfh and Tfr cells showed positive correlations with IL-21 in pSS. Increasing trend of blood Tfr cells was observed in pSS patients, and blood Tfr cells (particularly Th1 and Th17 subsets) represented hypergammaglobulinemia in pSS. In summary, circulating CCR7(lo)PD-1(hi) Tfh cells indicated disease activity and glandular inflammation in pSS. Circulating Tfr cells, shifted toward Th1 and Th17 subsets, indicated ongoing IgG production in pSS. Subsets of circulating Tfh or Tfr cells could be biomarkers for disease monitoring and patient stratification in pSS.
Autoantibodies ; B-Lymphocytes ; Biomarkers ; Flow Cytometry ; Humans ; Hypergammaglobulinemia ; Immunoglobulin G ; Inflammation ; Sialadenitis ; T-Lymphocyte Subsets ; T-Lymphocytes ; T-Lymphocytes, Helper-Inducer ; T-Lymphocytes, Regulatory