Identification of prognostic molecular subtypes and model based on CD8+ T cells for lung adenocarcinoma

Background:Cytotoxic T lymphocytes(CD8+T)cells function critically in mediating anti-tumor immune response in cancer patients.Characterizing the specific functions of CD8+T cells in lung adenocarcinoma(LUAD)could help better understand local anti-tumor immune responses and estimate the effect of immunotherapy.Methods:Gens related to CD8+T cells were identified by cluster analysis based on the single-cell sequencing data of three LUAD tissues and their paired normal tissues.Weighted gene co-expression network analysis(WGCNA),consensus clustering,differential expression analysis,least absolute shrinkage and selection operator(LASSO)and Cox regression analysis were conducted to classify molecular subtypes for LUAD and to develop a risk model using prognostic genes related to CD8+T cells.Expression of the genes in the prognostic model,their effects on tumor cell invasion,and interactions with CD8+T cells were verified by cell experiments.Results:This study defined two LUAD clusters(CD8+0 and CD8+1)based on CD8+T cells,with cluster CD8+0 being significantly associated with the prognosis of LUAD.Three heterogeneous subtypes(clusters 1,2,and 3)differing in prognosis,genome mutation events,and immune status were categorized using 42 prognostic genes.A prognostic model created based on 11 significant genes(including CD200R1,CLEC17A,ZC3H12D,GNG7,SNX30,CDCP1,NEIL3,IGF2BP1,RHOV,ABCC2,and KRT81)was able to independently estimate the death risk for patients in different LUAD cohorts.Moreover,the model also showed general applicability in external validation cohorts.Low-risk patients could benefit more from taking immunotherapy and were significantly related to the resistance to anticancer drugs.The results from cell experiments demonstrated that the expression of CD200R1,CLEC17A,ZC3H12D,GNG7,and SNX30 was significantly downregulated,while that of CDCP1,NEIL3,IGF2BP1,RHOV,ABCC2 and KRT81 was upregulated in LUAD cells.Inhibition of CD200R1 greatly increased the invasiveness of the LUAD cells,but inhibiting CDCP1 expression weakened the invasion ability of LUAD cells.Conclusion:This study defined two prognostic CD8+T cell clusters and classified three heterogeneous molecular subtypes for LUAD.A prognostic model predictive of the potential effects of immunotherapy on LUAD patients was developed.

Predicting the Prognosis and Immunotherapeutic Response of Triple-Negative Breast Cancer by Constructing a Prognostic Model Based on CD8+T Cell-Related Immune Genes

Objective Triple-negative breast cancer(TNBC)poses a significant challenge for treatment efficacy.CD8+T cells,which are pivotal immune cells,can be effectively analyzed for differential gene expression across diverse cell populations owing to rapid advancements in sequencing technology.By leveraging these genes,our objective was to develop a prognostic model that accurately predicts the prognosis of patients with TNBC and their responsiveness to immunotherapy.Methods Sample information and clinical data of TNBC were sourced from The Cancer Genome Atlas and METABRIC databases.In the initial stage,we identified 67 differentially expressed genes associated with immune response in CD8+T cells.Subsequently,we narrowed our focus to three key genes,namely CXCL13,GBP2,and GZMB,which were used to construct a prognostic model.The accuracy of the model was assessed using the validation set data and receiver operating characteristic(ROC)curves.Furthermore,we employed various methods,including Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway,immune infiltration,and correlation analyses with CD274(PD-L1)to explore the model's predictive efficacy in immunotherapeutic responses.Additionally,we investigated the potential underlying biological pathways that contribute to divergent treatment responses.Results We successfully developed a model capable of predicting the prognosis of patients with TNBC.The areas under the curve(AUC)values for the 1-,3-,and 5-year survival predictions were 0.618,0.652,and 0.826,respectively.Employing this risk model,we stratified the samples into high-and low-risk groups.Through KEGG enrichment analysis,we observed that the high-risk group predominantly exhibited enrichment in metabolism-related pathways such as drug and chlorophyll metabolism,whereas the low-risk group demonstrated significant enrichment in cytokine pathways.Furthermore,immune landscape analysis revealed noteworthy variations between(PD-L1)expression and risk scores,indicating that our model effectively predicted the response of patients to immune-based treatments.Conclusion Our study demonstrates the potential of CXCL13,GBP2,and GZMB as prognostic indicators of clinical outcomes and immunotherapy responses in patients with TNBC.These findings provide valuable insights and novel avenues for developing immunotherapeutic approaches targeting TNBC.

Tumor-derived DEFB1 induces immune tolerance by inhibiting maturation of dendritic cell and impairing CD8+T cell function in esophageal squamous cell carcinoma

Objective:CD8+T cells are the key effector cells in the anti-tumor immune response.The mechanism underlying the infiltration of CD8+T cells in esophageal squamous cell carcinoma(ESCC)has not been clearly elucidated.Methods:Fresh ESCC tissues were collected and grouped according to the infiltration density of CD8+T cells.After the transcriptome sequencing on these samples and the combined analyses with The Cancer Genome Atlas(TCGA)ESCC data,a secreted protein DEFB1 was selected to explore its potential role in the infiltration of CD8+T cells.Bioinformatics analyses,histological verification and in vitro experiments were then performed.Results:DEFB1 was highly expressed in ESCC,and the high expression of DEFB1 was an independent risk factor for overall survival.Since the up-regulation or down-regulation of DEFB1 did not affect the proliferation,migration and apoptosis of ESCC cells,we speculated that the oncogenic effect of DEFB1 was achieved by regulating microenvironmental characteristics.Bioinformatics analyses suggested that DEFB1 might play a major role in the inflammatory response and anti-tumor immune response,and correlate to the infiltration of immature dendritic cell(imDC)in ESCC.Histological analyses further confirmed that there were less CD8+T cells infiltrated,less CD83+mature DC(mDC)infiltrated and more CD1a+imDC infiltrated in those ESCC samples with high expression of DEFB1.After the treatment with recombinant DEFB1 protein,the maturation of DC was hindered significantly,followed by the impairment of the killing effects of T cells in both 2D and 3D culture in vitro.Conclusions:Tumor-derived DEFB1 can inhibit the maturation of DC and weaken the function of CD8+T cells,accounting for the immune tolerance in ESCC.The role of DEFB1 in ESCC deserves further exploration.

Natural course of chronic hepatitis B is characterized by changing patterns of programmed death type-1 of CD8-positive T cells

AIM:To investigate if and how programmed death type-1(PD-1)expression affects the natural course of hepatitis B virus(HBV)infection. METHODS:Sixty-four patients in different natural stages of chronic HBV infection were enrolled in this study.PD-1 expression in total T cells was detected by flow cytometry.Levels of total CD8+T cell responses and proliferation in relation to PD-1 expression levels were analyzed with intracellular staining and PD-1/ PD-L1 blockage. RESULTS:The PD-1 expression in T cells was dynamically changed during the natural course of chronic HBV infection,did not significantly increase in the immune tolerance phase,and returned to normal in the inactive virus carrier stage.Blockage of the PD-1/PD-L1 pathway could not affect the T-cell response in the immune tolerance and inactive virus carrier stages of chronic HBV infection.However,it could significantly restore the T-cell response in the immune clearance stage of chronic HBV infection.Furthermore,the PD-1 expression level in T cells was associated with the alanine aminotransferase level during the immune clearance stage of chronic HBV infection. CONCLUSION:The PD-l/PD-L1 pathway plays a different role in T-cell response during the natural course of chronic HBV infection.

Analysis of CD4^+CD25^+ Regulatory T Cells and Foxp3 mRNA in the Peripheral Blood of Patients with Asthma

The changes of CD4^＋CD25^＋ regulatory T cells （CD4^＋CD25^＋ Treg） and Foxp3 mRNA in peripheral blood mononuclear cells （PBMCs） from patients with asthma were investigated in order to elucidate the possible roles of CD4^＋CD25^＋ Treg in the development of asthma. The peripheral blood samples were collected from 29 healthy controls （normal control group） and 78 patients with asthma which included 30 patients in exacerbation group, 25 patients in persistent group, and 23 patients in remission group. By using flow cytometry and RT-PCR, the CD4^＋CD25^＋ Treg ratio and Foxp3 mRNA in PBMCs were detected. The CD4^＋CD25^＋ Treg ratio and Foxp3 mRNA in PBMCs of exacerbation and persistent groups were lower than that of remission and normal control groups （P〈0.05）. Although the CD4^＋CD25^＋ Treg ratio and Foxp3 mRNA of remission group were also lower than that of normal control group, there was no significant difference between them （P〉0.05）. As compared with persistent group, exacerbation group had lower CD4^＋CD25^＋ Treg ratio and Foxp3 mRNA （P〈0.05）. It was indicated that the decrease of CD4^＋CD25^＋ Treg ratio and its function in PBMCs may be responsible for pathogenesis of asthma.

Depletion of CD4^+CD25^+ regulatory T cells can promote local immunity to suppress tumor growth in benzo[a]pyrene-induced forestomach carcinoma

AIM： To elucidate the distribution of CD4^＋CD25^＋ regulatory T cells （Tregs） in different lymphoid tissues and its local enhancement on tumor growth before and after depletion of CD4^＋CD25^＋ Tregs. METHODS： Female ICR mice were garaged with benzo[a]pyrene （BaP） to induce forestomach carcinoma. CD4^＋CD25^＋ Tregs were intraperitoneally depleted with monoclonal antibody PC61. These mice were divided into BaP-only, BaP ＋ IgG, BaP ＋ PC61, and control groups. The forestomach of mice was dissected for histological analysis, and tunnel test was performed for apoptosis of tumor cells. CD4^＋CD25^＋ Tregs were sorted from different lymphoid tissues and expression of Foxp3, IL-10, and chemokine receptors was analyzed by flow cytometry, semi-quantitative and veal-time polymerase chain reaction. RESULTS： The mice gavaged with only BaP showed increased forestomach papilloma and carcinoma at wk 16 and 32. The proportion of CD4^＋CD25^＋ Tregs was significantly higher in peri-stomach regional lymph nodes than in other lymphoid tissues. These CD4^＋CD25^＋ Tregs in regional lymph nodes expressed higher levels of Foxp3 and IL-10, enriched in the CD62L-subset, and CCR1 and CCR5 chemokine receptors. In mice gavaged with BaP ＋ PC61, the number of tumor nodules and tumor volume decreased significantly with massive infiltrating cells and apoptosis of tumor cells. In the draining regional lymph nodes, the number of CD4^＋CD25^＋ Tregs also decreased significantly. CONCLUSION： Inducible and activated CD4^＋CD25^＋ Tregs in the draining regional lymph nodes suppress host local immunity during tumor growth. Depletion of CD4^＋CD25^＋ Tregs can promote host local immunity to suppress tumor growth.

Super-resolution immunohistochemistry study on CD4 and CD8 cells and the relation to macrophages in human cochlea

Recently, the human cochlea has been shown to contain numerous resident macrophages under steady-state. The macrophages accumulate in the stria vascularis, among the auditory nerves, and are also spotted in the human organ of Corti. These macrophages may process antigens reaching the cochlea by invasion of pathogens and insertion of CI electrode. Thus, macrophages execute an innate, and possibly an adaptive immunity. Here, we describe the molecular markers CD4 and CD8 of T cells, macrophage markers MHCⅡ and CD11b, as well as the microglial markers TEME119 and P2Y12, in the human cochlea. Immunohistochemistry and the advantageous super-resolution structured illumination microscopy(SR-SIM) were used in the study. CD4^+ and CD8^+ cells were found in the human cochleae. They were seen in the modiolus in a substantial number adjacent to the vessels, in the peripheral region of the Rosenthal's canal, and occasionally in the spiral ligament. While there are a surprisingly large number of macrophages in the stria vascularis as well as between the auditory neurons,CD4^+ and CD8^+ cells are hardly seen in these areas, and neither are seen in the organ of Corti. In the modiolus,macrophages, CD4^+ and CD8^+ cells appeared often in clusters. Interaction between these different cells was easily observed with SR-SIM, showing closely placed cell bodies, and the processes from macrophages reaching out and touching the lymphocytes. Otherwise the CD4^+ and CD8^+ cells in human cochlear tissue are discretely scattered. The possible roles of these immune cells are speculated.

Influence of Danshen Injection on airway inflammation and CD4^+ CD25^+ regulatory T cells of asthmatic rats

Objective： To investigate the influence of Danshen Injection on airway inflammation and CD4^＋CD25^＋ regulatory T cells（CD4^＋CD25^＋ Tr） of asthmatic rats, and elucidate the possible mechanism of Danshen Injection in treatment of asthma. Methods： 30 Wister rats were randomly divided into control group, asthma group and Danshen Injection treated group. Bronchoalveolar lavage fluids （BALF） were collected, and cytology studies were conducted. Lung tissues were obtained and pathologic analyses were done with hematoxylin and eosin stain （HE）. Flow cytometry was used to detect the CD4^＋CD25^＋ Tr ratio in peripheral blood mononuclear cells （PBMCs）. Results： Total cell, the percentage of lymphocytes, neutrophils and eosinophils （Eos） in BALF of Danshen Injection-treated group were lower than that in asthma group （P〈0.05, P〈0.01）. Compared with asthma group, less infiltration of inflammatory cells in lung tissues was observed in Danshen Injection-treated group. CD4^＋CD25^＋ Tr of asthma group was lower than that of control and Danshen Injection treated group （P〈0.05）. Conclusion： Danshen Injection can suppress airway inflammation of asthmatic rats, probably by increasing the number of CD4^＋CD25^＋ Tr.

Glutamine deprivation impairs function of infiltrating CD8^(+)T cells in hepatocellular carcinoma by inducing mitochondrial damage and apoptosis

BACKGROUND The functions of infiltrating CD8^(+)T cells are often impaired due to tumor cells causing nutrient deprivation in the tumor microenvironment.Thus,the mechanisms of CD8^(+)T cell dysfunction have become a hot research topic,and there is increased interest on how changes in metabolomics correlate with CD8^(+)T cell dysfunction.AIM To investigate whether and how glutamine metabolism affects the function of infiltrating CD8^(+)T cells in hepatocellular carcinoma.METHODS Immunohistochemical staining and immunofluorescence were performed on surgically resected liver tissues from patients.Differentially expressed genes in infiltrating CD8^(+)T cells in hepatocellular carcinoma were detected using RNA sequencing.Activated CD8^(+)T cells were co-cultured with Huh-7 cells for 3 d.The function and mitochondrial status of CD8^(+)T cells were analyzed by flow cytometry,quantitative real-time polymerase chain reaction,and transmission electron microscopy.Next,CD8^(+)T cells were treated with the mitochondrial protective and damaging agents.Functional alterations in CD8^(+)T cells were detected by flow cytometry.Then,complete medium without glutamine was used to culture cells and their functional changes and mitochondrial status were detected.RESULTS There were a large number of infiltrating PD-1+CD8^(+)T cells in liver cancer tissues.Next,we cocultured CD8^(+)T cells and Huh-7 cells to explore the regulatory effect of hepatoma cells on CD8^(+)T cells.Flow cytometry results revealed increased PD-1 expression and decreased secretion of perforin(PRF1)and granzyme B(GZMB)by CD8^(+)T cells in the co-culture group.Meanwhile,JC-1 staining was decreased and the levels of reactive oxygen species and apoptosis were increased in CD8^(+)T cells of the co-culture group;additionally,the mitochondria of these cells were swollen.When CD8^(+)T cells were treated with the mitochondrial protective and damaging agents,their function was restored and inhibited,respectively,through the mitochondrial damage and apoptotic pathways.Subsequently,complete medium without glutamine was used to culture cells.As expected,CD8^(+)T cells showed functional downregulation,mitochondrial damage,and apoptosis.CONCLUSION Glutamine deprivation impairs the function of infiltrating CD8^(+)T cells in hepatocellular carcinoma through the mitochondrial damage and apoptotic pathways.

Neutrophils inhibit CD8^(+)T cells immune response by arginase-1 signaling in patients with sepsis

BACKGROUND:Patients with sepsis often exhibit an acute inflammatory response,followed by an immunosuppressive phase with a poor immune response.However,the underlying mechanisms have not been fully elucidated.METHODS:We sought to comprehensively characterize the transcriptional changes in neutrophils of patients with sepsis by transcriptome sequencing.Additionally,we conducted a series of experiments,including real-time quantitative polymerase chain reaction(RT-qPCR)and flow cytometry to investigate the role of arginase-1 signaling in sepsis.RESULTS:Through the analysis of gene expression profiles,we identified that the negative regulation of T cell activation signaling was enriched,and the expression of arginase-1 was high in neutrophils from patients with sepsis.Furthermore,we conducted flow cytometry and found that the function of CD8^(+)T cells in septic patients was impaired.Moreover,neutrophils from septic patients inhibited the percentage of polyfunctional effector CD8^(+)T cells through arginase-1.Additionally,the proportions of granzyme B^(+)IFN^(-)γ^(+)CD8^(+)T and TNF^(-)α^(+)IFN^(-)γ^(+)CD8^(+)T cells increased after inhibition of arginase-1 signaling.CONCLUSION:The impaired effector function of CD8^(+)T cells could be restored by blocking arginase-1 signaling in patients with sepsis.

Isolation and identification of CD4^+CD25^+ regulatory T cells in rat

Objective： To establish a stable and high efficient method for collection of CD4^＋CD25^＋ regulatory T cells from rats in vitro. Methods： CD4^＋CD25^＋ regulatory T cells were isolated from the rat splenic cells through two steps by magic cell sorting （MACS） system. The first step was negative selection of CD4^＋T cells by cocktail antibodies and anti-IgG magic microbeads, and the second step was positive selection of CD25^＋T cells by anti-CD25 PE and anti-PE magic microbeads. The purity and viability of separated cells were measured by flow cytometry （FACS） and Trypan blue staining. The suppressive ability of seperated cells on the proliferation of CD4^＋CD25^- T cells was assessed by cell proliferation assay. Results： The purity of negatively enriched CD4^＋ T cells was 79%-87% （83.6%±2.5% ） , and the purity of positively enriched CD4^＋CD25^＋ T cells was 86%- 93% （ 90.2±1.8% ） with the viability of 92%～95% （92.8% ± 3.4% ）. The enriched cells significantly suppressed the proliferation of CD4^＋CD25^- T cells in mixed lymphocyte culture （P 〈 0.05）. Conclusion： An effective method can be established for enrichment of CD4^＋CD25^＋ regulatory T cells in two steps by MACS, with satisfied cell purity, viability and function.

The Mechanisms of CD8+ T Cells Exhaustion in the Tumor Microenvironment and Immune Therapy

In the tumor immune microenvironment, CD8+ T cells differentiate towards functional failure. The exhaustion of CD8+ T cells (Tex) showed varying degrees of effect dysfunction, loss of proliferation ability, and sustained high expression of a variety of inhibitory receptors, with metabolic and epigenetic changes. Tex cells are heterogeneous, including several subsets with different characteristics at different stages of differentiation. Immune checkpoint inhibitors (ICIs) can restore the effect or function of Tex cells, indicating that this T cell subset plays a key role in tumor immunotherapy. The understanding of the mechanism of CD8+ T cell exhaustion will be helpful to the implementation of tumor immunotherapy. This article reviews the production, differentiation and functional characteristics of Tex cells and their relationship with tumor immunotherapy.

CD4^+CD25^(high) Regulatory Cells in Peripheral Blood of NSCLC Patients

The proportion and changes of CD4^＋CD25^high regulatory T cells （Trs） in peripheral blood of non-small cell lung cancer （NSCLC） patients were analyzed and their clinical significance explored. The peripheral blood was collected from 61 patients with NSCLC and 15 healthy controls. By using monoclonal antibodies, the blood samples were evaluated with the flow cytometry for lymphocyte subsets （CD3^＋, CD4^＋ and CD8^＋） and CD4^＋CD25^high Tr cells. The results showed that the proportion of CD4^＋CD25^high Tr cells in NSCLC group was significantly higher than in control group [（4.36 ±2.07） % vs （2.04±1.03） %, P〈0.01]. The proportion of CD4^＋CD25^ high Tr cells in late stage was higher than that in early stage [stages Ⅰ ＋Ⅱ （2.264±0.6） %; stage Ⅲ（3.284± 1.38） %; stage IV （6.06 4±4.08） %] （P〈0.05）. Kaplan-Meier survival analysis revealed that the prognosis of the patients who had higher proportion of CD4^＋CD25^high Tr cells in peripheral blood was worse （P=0.0026）. In conclusion, the relative increase in CD4^＋CD25^high Tr cells in peripheral blood may be related to im- munosuppression and tumor progression in patients with NSCLC. This finding suggests that CD4^＋CD25^high Tr cells in peripheral blood of NSCLC may be positive for prognosis analysis. The use of depletion of the CD4^＋CD25^high Tr cell therapy to treat NSCLC patients may be an effective strategy.

Effect of CD4^+CD25^+ regulatory T cells in the development of anterior chamber-associated immune deviation

AIM: To investigate whether CD4(+)CD25(+) regulatory T (Treg) cells play a role in the development of anterior chamber-associated immune deviation (ACAID). METHODS: The dynamic changes in the frequency of CD4(+)D25(+) T cells, CD4(+)D25(+) FoxP3(+) T cells and CD4(+)CD25(+) PD-1(+) T cells from spleens of mice with ACAID were analyzed by flow cytometry. Foxp3 mRNA expression in purified CD4(+)CD25(+) T cells was analyzed using real-time PCR. The suppressive effect of purified CD4(+)CD25(+) T cells on the proliferation of CD4(+)CD25(-) T cells was evaluated by [H-3] thymidine incorporation. A blocking experiment was performed to further address the role of CD4(+)CD25(+) T cells in ACAID. The expression of IL-10 in purified CD4(+)CD25(+) T cells was evaluated by ELISA. RESULTS: Increased frequencies of CD4(+)CD25(+) T cells, CD4(+)CD25(+) Foxp3(+) T cells and CD4(+)CD25(+) PD-1(+) T cells were observed in ACAID. The CD4(+)CD25(+) T cells from mice with ACAID showed enhanced suppressive effect on the proliferation of CD4(+)CD25(-) T cells. Treatment of BALB/c mice with anti-CD25 antibody after injection of OVA into the anterior chamber significantly inhibited the induction of ACAID. Furthermore, purified CD4(+)CD25(+) T cells from ACAID mice secreted IL-10. CONCLUSION: Our results demonstrate that Treg cells are induced in the mice undergoing ACAID. These Treg cells may play a role in the development of ACAID.

Peripheral CD4^(+)CD8^(+) double positive T cells:A potential marker to evaluate renal impairment susceptibility during systemic lupus erythematosus

Lupus nephritis(LN) has a high incidence in systemic lupus erythematosus(SLE) patients, but there is a lack of sensitive predictive markers. The purpose of the study was to investigate the association between the CD4^(+)CD8^(+)double positive T(DPT) lymphocytes and LN. The study included patients with SLE without renal impairment(SLE-NRI), LN, nephritic syndrome(NS), or nephritis. Peripheral blood lymphocyte subsets were analyzed by flow cytometry. Biochemical measurements were performed with peripheral blood in accordance with the recommendations proposed by the National Center for Clinical Laboratories. The proportions of DPT cells in the LN group were significantly higher than that in the SLE-NRI group(t=4.012, P<0.001), NS group(t=3.240,P=0.001), and nephritis group(t=2.57, P=0.011). In the LN group, the risk of renal impairment increased significantly in a DPT cells proportion-dependent manner. The risk of LN was 5.136 times(95% confidence interval, 2.115–12.473) higher in cases with a high proportion of DPT cells than those whose proportion of DPT cells within the normal range. These findings indicated that the proportion of DPT cells could be a potential marker to evaluate LN susceptibility, and the interference of NS and nephritis could be effectively excluded when assessing the risk of renal impairment during SLE with DPT cell proportion.

TARGETED ACTIVATION OF CD_(8)^(+) CELLS AND INFECTION OF β_2-MICROGLOBULIN-DEFICIENT MICE FAIL TO CONFIRM A PRIMARY PROTECTIVE ROLE FOR CD_(8)￣(+) CELLS IN EXPERIMENTAL LEISHMANIASIS

CD8+ T cells play an important role in the immunologic control of intracellular pathogens,particularly viruses,Leishmania are obligate intracellular parasites of macrophages in the mammalian host,and previous studies using deletion of CD8+ cells by administration of mAb to infected animals have suggested a protective role for these cells.Two complementary approaches were used to define more carefully the role of CD8+ cells in leishmaniasis.In BALB/c mice susceptible to Leishmania major(L. major)infection, targeted activation of CD8+ cells was attempted by immunization with nonapeptides derived from the conserved major outer surface protein of the organism,8p63, that contained the consensus binding motif for MHC class 1 H-2kd molecules.Two of the nonapeptides induced CTL activity in subsequently infected BALB/c mice that could be elicited against p815 cells pulsed either with peptide or lysates of L.major.purified CD8+ T cells from immunized mice had elevated levels of IFN-γ mRNA transcripts as compared to unimmunized mice.Despite evidence for activation of CD8+ cells,none of the mice immunized with nine different peptides alone or in combination were protected from progressive disease. In a second series of experiments, β2-microglobulin deficient mice that lack CD8+ cells were infected with L. major and the course of infection monitored.These mice cured disease as'rapidly as β2-m +/- and +/+ littermates,and cure was associated with comparable levels of IFN-Y mRNA in the draining lymph node population. Neither of these approaches was able to confirm a substantive role for CD8+ T cells in the primary protective response to L.major.

Stromal CD4+ and CD8+ T Cells in Human Breast Carcinomas. Its Correlation with Chemokine MIG/CXCL9

The interaction between some chemokines with tumoral and stromal cells can influence tumor progression. CXCL9, a monokine induced by interferon gamma (MIG), targets lymphocytes.The aim of our study was to identify and quantify CD4+ and CD8+ T cells in the stroma of human breast cancer and correlate them with the presence of MIG/CXCL9. In 58 specimens of human breast carcinoma and 10 normal breast tissue from mammoplasty surgery, immunohistochemistry and ELISA methods were performed. The number of CD4+ and CD8+ T cells in breast cancer tissue was significantly increased compared with normal breast tissue with a clear predominance of CD8+ T cells. MIG/CXCL9 levels were significantly elevated respect normal breast tissue. This chemokine correlated with the number of CD8+ T cells only in non-metastatic tumors. These data suggest that MIG targets cytotoxic T cells. Their recruitment into breast carcinoma can play a critical role in malignant progression, inhibiting the production of metastasis.

Effect of Mg^(2+)level on the functions of CD8^(+)T lymphocytes and NK cells in patients with COVID-19

Objective:To investigate the changes of Mg^(2+) levels in serum and peripheral blood mononuclear cells(PBMCs)of patients with COVID-19 and its effects on the functions of CD8^(+)T lymphocytes and NK cells.Methods:A total of 165 COVID-19 patients hospitalized in Ezhou Central Hospital from January 20 to February 20,2020 were divided into mild/common group(98 cases)and severe/critical group(67 cases).At the same time,34 healthy persons were selected as the control group.Peripheral blood was collected and PBMCs were isolated,the level of Mg^(2+) in serum and PBMCs was detected.The subsets of CD8^(+)T lymphocytes and NK cell and the expression levels of their surface inhibitory molecular PD-1 and activator molecular NKG2D were detected by flow cytometry.The correlation between Mg^(2+) concentration and the expression levels of PD-1 and NKG2D was also analyzed.Results:Compared with the control group,the concentration of Mg^(2+) in serum and PBMCs,the counts of CD8^(+)T lymphocytes and NK cell in patients with mild/common and severe/critical groups were significantly reduced(P<0.05),while the expression level of surface inhibitory molecular PD-1 were significantly increased(P<0.05),while the expression level of the activation molecule NKG2D were significantly decreased(P<0.05).However,the changes of the above indicators in patients with severe/critical group were greater than those in the mild/common group(P<0.05).In addition,the Mg^(2+) concentration in COVID-19 patients was negatively correlated with the expression level of PD-1 on CD8^(+)T lymphocytes and NK cells(P<0.05),and positively correlated with the expression levels of NKG2D(P<0.05).Conclusion:The concentration of Mg^(2+) in the serum and PBMCs of COVID-19 patients is significantly reduced,which may cause the function of CD8^(+)T lymphocytes and NK cells to be inhibited.

Regulatory T cells suppress autoreactive CD4^+ T cell response to bladder epithelial antigen

AIM： To investigate the role of regulatory T （Treg） cells in CD4^＋ T cell-mediated bladder autoimmune infammation. METHODS： Urothelium-ovalbumin （URO-OVA）/OT-II mice, a double transgenic line that expresses the membrane form of the model antigen （Ag） OVA as a self-Ag on the urothelium and the OVA-specific CD4^＋ T cell receptor specifc for the I-Ab/OVA323-339 epitope in the periphery, were developed to provide an autoimmune environment for investigation of the role of Treg cells in bladder autoimmune infammation. To facilitate Treg cell analysis, we further developed URO-OVA^GFP-Foxp3/OT-II mice, a derived line of URO-OVA/OT-II mice that express the green fuorescent protein （GFP）-forkhead box protein P3 （Foxp3） fusion protein. RESULTS： URO-OVA/OT-II mice failed to develop bladder infammation despite the presence of autoreactive CD4^＋ T cells. By monitoring GFP-positive cells, bladder infltration of CD4^＋ Treg cells was observed in URO-OVA^GFP-Foxp3/OT-II mice. The infiltrating Treg cells were functionally active and expressed Treg cell effector molecule as well as marker mRNAs including transforming growth factor-β, interleukin （IL）-10, fibrinogen-like protein 2, and glucocorticoid-induced tumor necrosis factor receptor （GITR）. Studies further revealed that Treg cells from URO-OVA^GFP-Foxp3/OT-II mice were suppressive and inhibited autoreactive CD4^＋ T cell proliferation and interferon （IFN）-g production in response to OVA Ag stimulation. Depletion of GITR-positive cells led to spontaneous development of bladder infammation and expression of inflammatory factor mRNAs for IFN-γ, IL-6, tumor necrosis factor-α and nerve growth factor in URO-OVA^GFP-Foxp3/OT-II mice. CONCLUSION： Treg cells specifc for bladder epithelial Ag play an important role in immunological homeostasis and the control of CD4^＋ T cell-mediated bladder autoimmune infammation.

CD8 T cell response in a phase I study of therapeutic vaccination of advanced NSCLC with allogeneic tumor cells secreting endoplasmic reticulum-chaperone gp96-Ig-peptide complexes

Antigen containing, allogeneic cells secreting the genetically modified protein and peptide-chaperone gp96-Ig cross, prime and expand antigen specific CD8 T cells with therapeutic antitumor activity in mice. In a first in man phase I study, we now report the results of therapeutic vaccination of non-small cell lung cancer (NSCLC) patients with an established, allogeneic non-small cell lung adenocarcinoma cell line secreting gp96-Ig. Advanced NSCLC-patients stage IIIB or IV of any histological subtype were enrolled and treated with up to 36 vaccinations over the course of 18 weeks. Primary endpoint was safety, secondary endpoints tumor response and overall survival. Measurement of tumor antigen specific CD8 CTL responses is precluded by the lack of known NSCLC associated antigens. Therefore, we measured patient CD8 T cell-IFN-γ responses to allo-antigens of the vaccine cells as surrogate for tumor antigen specific CD8 CTL. In 7 of 18 treated patients tumor growth was stabilized, however none of the 18 patients had an objective tumor response by RECIST criteria. Of 15 patients evaluable for immune response, 11 responded to vaccination with more than twofold increase in CD8-IFN-γ frequency above baseline. These patients had a median survival time of 16.5 months. Four patients who had no CD8 response above base line had survival times from 2.1 to 6.7 months. Our data are consistent with the concept that generation of CD8 CTL by therapeutic vaccination may delay tumor growth and progression and mediate prolonged survival even in the absence of objective tumor responses. Further studies will be required to test this concept and promising result.