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【Objective】 To understand the memory phenotype and function of SARS-CoV-2 reactive CD4+ T cells in healthy individuals. 【Methods】 In this study, SARS-CoV-2-derived peptides were used to stimulate PBMC from participants.SARS-CoV-2 reactive memory T cells were detected by intracellular staining and flow cytometry, and memory phenotype analysis was performed.CBA was used to detect cytokine secretion after SARS-CoV-2-derived peptides stimulation to evaluate the function of SARS-CoV-2 reactive memory T cells. 【Results】 We found that SARS-CoV-2 reactive CD4+ memory T cells could be detected in 40% (6/15) healthy donors.Phenotypic analysis of memory showed that these T cells were mainly composed of central memory T cells(82.2%), and other memory cells accounted for 17.8%.Compared with negative control, IL-10 was significantly decreased after stimulation of SARS-CoV-2-derived peptides (P<0.05), while the secretion of IFNγ, TNFα, IL-2 and IL-4 showed no significant difference. 【Conclusions】 SARS-CoV-2 reactive CD4+ memory T cells are present in healthy individuals from China.
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ObjectiveTo investigate the disease progression and immunoprotective characteristics in mice re-infected with homogeneous/heterogeneous Plasmodium strains following cure of Plasmodium infections with chloroquine at the peak of parasitemia. MethodsC57BL/6 mice were infected with the non-lethal P. yoelii 17XNL strain, and half of mice were given treatment with chloroquine at the peak of parasitemia (9 days post-infection), while the other mice were self-cured naturally. Then, all cured mice were re-infected with the equivalent lethal P. yoelii 17XL or P. berghei ANKA strain 90 days following primary Plasmodium infections. The parasitemia levels during primary infections and reinfections were measured by microscopic examinations of Giemsa-stained thin blood films, and the levels of the IgG antibody in sera and the percentages of memory T cell subsets in spleen cells were detected in mice using ELISA and flow cytometry before and after parasite reinfections, respectively. Results Following primary infections with the P. yoelii 17XNL strain, the serum IgG antibody levels were (5.047 ± 0.924) pg/mL in the selfcured mice and (4.429 ± 0.624) pg/mL in the chloroquine-treated mice, respectively (t = 0.437, P > 0.05), which were both significantly higher than that in the uninfected mice (1.624 pg/mL ± 0.280 pg/mL) (F = 22.522, P < 0.01). There was no significant difference in the serum IgG antibody level among self-cured and chloroquine-treated mice re-infected with the P. yoelii 17XL strain or the P. berghei ANKA strain (F = 0.542, P > 0.05); however, the serum IgG antibody levels were all significantly higher in selfcured and chloroquine-treated mice re-infected with the P. yoelii 17XLstrain[(15.487±1.173)pg/mLand(15.965±1.150)pg/mL] or the P. berghei ANKA strain [(14.644 ± 1.523) pg/mL and (15.185 ± 1.333) pg/mL] relative to primary infections (F = 67.383, P < 0.01). There was no significant difference in the proportion of CD4+ [(34.208 ± 2.106), (32.820 ± 1.930), (34.023 ± 2.289), (35.608 ± 1.779) pg/mL] or CD8+ T memory cells [(17.935 ± 2.092), (18.918 ± 2.823), (17.103 ± 1.627), (17.873 ± 1.425) pg/mL] in self-cured and chloroquine-treated mice with primary infections with the P. yoelii 17XNL strain followed by re-infections with the P. yoelii 17XL strain or the P. berghei ANKA strain (F = 0.944 and 0.390, both P > 0.05); however, the proportions of the CD4+ or CD8+ T memory cells were significantly greater in self-cured and chloroquine-treated mice with primary infections with the P. yoelii 17XNL strain followed by re-infections with the P. yoelii 17XL strain or the P. berghei ANKA strain than in mice with primary infections (F = 50.532 and 21.751, both P < 0.01). Conclusions The cure of murine Plasmodium infections with chloroquine does not affect the production of effective immune protections in mice during parasite re-infections. Following a primary infection, mice show a protection against re-infections with either homogeneous or heterogeneous Plasmodium strains, and a higher-level resistance to re-infections with homogeneous parasite strains is found than with heterogeneous strains.
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Several studies have demonstrated that memory T cells including stem cell memory (Tscm) T cells and central memory (Tcm) T cells show superior persistence and antitumor immunity compared with effector memory T (Tem) cells and effector T (Teff) cells. Furthermore, the Tcm/Teff ratio has been reported to be a predictive biomarker of immune responses against some tumors. Thus, a system-level understanding of the mechanisms underlying the differentiation of effector and memory T cells is of increasing importance for developing immunological strategies against various tumors. This review focuses on recent advances in efficacy against tumors, the origin, formation mechanisms of memory T cells, and the role of the gut microbiota in memory T cell formation. Furthermore, we summarize strategies to generate memory T cells in (ex) vivo that, might be applicable in clinical practice.
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Several studies have demonstrated that memory T cells including stem cell memory (Tscm) T cells and central memory (Tcm) T cells show superior persistence and antitumor immunity compared with effector memory T (Tem) cells and effector T (Teff) cells. Furthermore, the Tcm/Teff ratio has been reported to be a predictive biomarker of immune responses against some tumors. Thus, a system-level understanding of the mechanisms underlying the differentiation of effector and memory T cells is of increasing importance for developing immunological strategies against various tumors. This review focuses on recent advances in efficacy against tumors, the origin, formation mechanisms of memory T cells, and the role of the gut microbiota in memory T cell formation. Furthermore, we summarize strategies to generate memory T cells in (ex) vivo that, might be applicable in clinical practice.
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ABSTRACT Background: Although chronic lymphocytic leukemia is basically a B cell disease, its pathophysiology and evolution are thought to be significantly influenced by T cells, as these are probably the most important interaction partner of neoplastic B cells, participating in their expansion, differentiation and survival. Chronic lymphocytic leukemia B cells may also drive functional and phenotypic changes of non-malignant T cells. There are few data about the association between memory T cells and prognosis, especially related to ZAP-70, a common reliable surrogate of the gold standard chronic lymphocytic leukemia prognostic markers. Objective: The aim of this study was to investigate whether the expression of ZAP-70 in chronic lymphocytic leukemia patients is associated with abnormal patterns of the distribution of naïve and memory T cells related to crosstalk between these cells. Methods: In this cross-sectional, controlled study, patients with chronic lymphocytic leukemia were compared with healthy blood donors regarding the expression of ZAP-70 and the distribution of naïve and memory T cell subsets in peripheral blood as measured by flow cytometry. Results: ZAP-70 positive patients presented an increased frequency and absolute number of central memory CD4+ T cells, but not CD8+ T cells, compared to ZAP-70 negative patients and age-matched apparently healthy donors. Conclusions: Because central memory CD4+ T cells are located in lymph nodes and express CD40L, we consider that malignant ZAP-70-positive B cells may receive beneficial signals from central memory CD4+ T cells as they accumulate, which could contribute to more aggressive disease.
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Humans , Male , Female , Protein-Tyrosine Kinases , T-Lymphocytes , Leukemia, Lymphocytic, Chronic, B-Cell , ZAP-70 Protein-Tyrosine KinaseABSTRACT
Objective To explore the influence of AKT inhibitors on tumor infiltrating T lymphocytes (TIL)in patients with liver metastasis of colorectal cancer.Methods The tumor tissues from the patients with liv-er metastasis of colorectal cancer in Department of General Surgery,The Affiliated Cancer Hospital of Zhengzhou University from January 2016 to December 2016 were collected.TIL and tumor cells were isolated by percoll densi-ty gradient centrifugation. The profiling of AKT inhibitors on TIL were analyzed by flow cytometry. Results AKT inhibition enhances the expansion of TIL with memory cell without affects its proliferation,also the cells obtained under AKT inhibitor with IL-2 showed higher frequency of IFN-γproducing cells than IL-2. Conclusion Add AKT inhibitors in TIL cultivation system can strengthen the proliferation of central memory T cells,and does not affect the number of CD8+T cells.This might be developed for cell-based immunotherapy of cancer.
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Objective To investigate the relationship of skin-resident memory T cells (Trm cells) with the skin lesions in systemic lupus erythematosus (SLE) so as to deepen our understanding of the pathogenesis of skin lesions in SLE. Methods Peripheral blood and skin samples were collected from SLE patients and matched healthy volunteers. The percentages of effector memory T cells (Tem cells) and effcctor T cells (Teff cells) from peripheral blood were analyzed by flow cytometry. By using direct immunofluoresence, we detected the presence of immune complex,which deposited on the basement membrane of normal appearance skin from SLE patients or healthy individuals. Immunohistochemical staining was performed to detect the two characteristic surface markers of tissue-resident memory T lymphocytes,CD69 and CD103 and to analyze the expression of these T lymphocytes within skins from SLE patients or healthy volunteers. Data analysis was performed using t test. P<0.05 was considered statistically significant. Results As compared with control individuals,the proportions of CD4+Tem (12.6±3.4 vs 8.2±2.5,t=-3.15,P<0.05),CD4+Teff (2.5±1.5 vs 1.3± 0.8,t=-2.79,P<0.05)、CD8+Tem(15.3±3.6 vs 7.0±3.0,t=-6.22,P<0.05)and CD8+Teff cells(13.1±5.4 vs 3.7± 1.3,F=-7.36,P<0.05)in T cell subset were significantly increased. Also,the proportions of CD4+Tem(8.4±2.7 vs 5.8±2.0,t=-2.74,P<0.05),CD4+Teff (1.6±1.0 vs 0.8±0.5,t=-2.84,P<0.05),CD8+Tem (10.4±3.6 vs 5.3±2.4, t=-4.03, P<0.05) and CD8+Teff cells (8.2±4.1 vs 2.6±0.8, t=-6.15, P<0.05) in peripheral blood were increased as well. By direct immunofluorescence,we noticed the deposition of immunoglobulin IgA, IgM and complement C3 on the basement membrane zone of skins from SLE patients but not on heanlhy individuals. The amount of infiltrated lymphocytes in skin samples from SLE patients were significantly iecreased compared to that of healthy individuals,and the quantities of CD4, CD8 and CD103 positive T cells from SLE skin samples were all increased by various degrees than that of controls. Conclusion Skin-resident memory T cells may be involved in the pathogenesis of skin lesions in SLE.
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In recent years,cellular immune therapy represented by antigen-chimeric receptor T cells (CAR-T) has made a great breakthrough in the treatment of hematological malignancies.T memory stem cells (TSCM) and central memory T cells (TCM) can be used as most powerful carrier for T cell immunotherapy,however,how to regulate their differentiation in vitro and in vivo as well as how to construct a strong treatment system while without potential side effect become quite interesting.This article summarized the studies from the 58th America Society of Hematology Annual Meeting regarding to values and associated research progress about TsCM and TCM in hematological malignancies.
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In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43−) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.
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Animals , Male , Mice , B-Lymphocytes/immunology , Heat-Shock Proteins/immunology , Immunomodulation/genetics , /genetics , RNA, Messenger/immunology , T-Lymphocyte Subsets/immunology , B-Lymphocytes/metabolism , Flow Cytometry , Gene Expression/genetics , Heat-Shock Proteins/therapeutic use , Immunologic Memory/physiology , Immunophenotyping/classification , Inflammation Mediators/analysis , Interferon-gamma/analysis , /immunology , /analysis , Mice, Knockout , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , RNA, Messenger/genetics , Spleen/cytology , Spleen/immunology , T-Lymphocyte Subsets/classification , Vaccines, DNA/immunology , Vaccines, DNA/therapeutic useABSTRACT
Objective:To explore the change of initial and memory T cells in peripheral blood and their clinical significance in peripheral T cell lymphoma patients( PTCL) before and after CHOP chemotherapy.Methods:The proportion of CD4+CD45RA+T cells and CD4+CD45RO+T cells,CD8+CD45RA+T cells and CD8+CD45RO+T cells in peripheral blood from 20 PTCL patients before and after chemotherapy was detected by flow cytometry, the relationship between curative effect and T cell subset was further analyzed.Results:Before treatment,the proportion of CD4+and CD4+CD45RO+T cells in PTCL patients was significantly lower than that from the control group,while the proportion of CD4+CD45RA+,CD8+,CD8+CD45RO+and CD8+CD45RA+T cells was significantly higher( P<0.05 );after treatment, proportion of CD4+, CD4+CD45RO+T was significantly increased, CD4+CD45RA+, CD8+, CD8+CD45RO+,CD8+CD45RA+T was slightly decreased( P<0.05).Before and after treatment,higher proportion of CD4+CD45RA+T cells was found in response group compared with no response group.Conclusion: CHOP chemotherapy might influence the thymic output function in PTCL patients,patients with higher thymic output function may have better response to chemotherapy.
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Objective: To establish an optimized method by combining minimally invasive blood sample technique and flow cytometry analysis for monitoring the dynamic changes of memory T cell subsets in mouse peripheral blood. Methods: The blood samples were collected via the saphenous vein, and the four color compensation matrix of flow cytometry was established with fluorescence compensation beads; then the ratios of naive T cells, central memory T cells and effector memory T cells were analyzed using BD Calibur equipped with two laser. The following factors were investigated to optimize the flow cytometry protocol: (1) blood sampling volume; (2) centrifugation of blood or not; (3) the concentration of detecting antibodies; and (4) whether to wash after the lysis of erythrocytes. The optimized protocol was used to investigate the dynamic changes of memory T cells in C57 and apoE knockout (apoE-/-) mice during switching from chow to high fat diet. Results: (1) 10-50 μL blood samples could be collected via the saphenous vein of mice without anesthesia, and the process could be repeated and 10 μL blood sample could meet the requirement for multi-color flow cytometry analysis, which reducing the demand of antibodies. (2) Demand of antibodies could be further reduced by high speed centrifugation and removal of serum or plasma. (3) Optimization of antibody concentration could further reduce the amount of antibodies and potential interference of the background fluorescence without influencing the accuracy and reproducibility. (4) Washing after the lysis of erythrocyte could further decrease the background fluorescence of samples, but it increased the operation time, and it could also be analyzed without washing. (3) The effector memory T cell level of apoE--/- mice was significantly higher than that of C57 mice at the baseline level of chow diet (PC<.05); the level increased to a plateau after 3-week high fat diet in apoK-/- mice and after 2-week high fat diet in C37 mice, indicating immunity dysfunction during early stages of atherosclerosis in apoE -/- mice. Conclusion: Combination of blood sampling via saphenous vein and optimal flow cytometry analysis protocol can help to monitor the dynamic change of memory T cell subsets in vivo in mice.
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Objective The presence of alloreactive memory T cells in recipient is a critical handicap to achieve transplantation tolerance.To make a mouse model which mimics the present transplant patient is important for research at this subject.Thus,we developed a novel re-transplant model and compared the alloresponse in this model with that in the conventional memory T cellstransfer model (transfer control).Methods The re-transplant model was established via microsurgery and vessel cannula techniques,and the experiment was composed of three groups:the re- transplant group,memory T cell-transfer group (transfer control) and the conventional blank group (blank control).The research indexes included survival time of donor heart,rejection score of allograft,and detection of proliferation and differentiation of the alloreactive memory/effector T cells by by flow cytometry (FCM) and in vitro mixed lymphocyte reaction (MLR).Results The median survival time of allograft in re-transplant recipients was significantly shortened compared to that of transfer control,but there was no significant difference in rejection score of graft between them (the score in retransplant group was the most intense of the three groups). Moreover, proliferation and differentiation of the alloreactive effector T cells were more intensive in re- transplant recipients than in the transfer control,which was confirmed by in vitro MLR and by FCM of the splenocytes for detecting CD44highCD62L-memory/effector phenotype cells.Conclusion The recall alloresponse in retransplantation is more intensive than that in memory-transfer setting and this re-transplant model is more close to the clinic situation than the memory-transfer model in rodents.
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Germinal center (GC) is a critical site where the humoral immune responses take place. Especially memory cells are known to be generated from the GC. In this experiment, T cells observed in the GC were studied in the aspect of memory T cells. T cells responding to myelin basic protein (MBP) antigen usually have their own specific T cell receptor complex (TCR) consisting of V 2 and V 8. Therefore, MBP antigen enables to trace specific T cells reacting to MBP antigen. This experiment, in which balb/c mice were injected with MBP into footpad and the popliteal lymph nodes were removed, showed that most of V 2 +/- cells were L3T4 +/- cells, and that initially they were located in the deep cortex near the B cell follicles and later they were observed in the GC. In case of the primary injection of MBP, IL -4 +/- cells were observed for the first time, followed by appearance of CD69 and CD2R. In case of the secondary injection, all of IL -4, CD25, CD69, CD2R and CTLA -4 were observed from the 1st day after injection. However IL -4, CD25 and CD69 among them were not observed any more since 2 wks after the secondary injection. These results strongly suggest that T cells observed in the GC during the immune responses for MBP might be memory T helper cells.
Subject(s)
Animals , Mice , Germinal Center , Immunity, Humoral , Lymph Nodes , Memory , Myelin Basic Protein , Receptors, Antigen, T-Cell , T-Lymphocytes , T-Lymphocytes, Helper-InducerABSTRACT
A study was focused on the analysis of the change of the number and distribution of immunocytes during the tumorigenesis by N-buty1-N-(4-hydroxybutyl)-nitrosamine (BBN) administration to C3H/He inbred mice and intravesical BCG therapy to starch for the subclass of the T cells which could be the major elements in antitumor mechanism of BCG. The number and distribution of memory T(CD44) cells were studied immunohistochemically in the spleen and urinary bladder and those of memory T cells in peripheral lymph nodes and those of lymphocyte homing receptor(Mel-14) positive cells in thymus were studied microflowcytometrically following 4 weekly intravesical BCG instillations. In BBN administered group, changes of immunocytes were not induced by BBN only but as gross tumor developed, increase of memory T cells was observed but was not statistically significant comparing with those of control group. In BCG treated group, memory T cells in bladder and spleen, lymphocyte homing receptor positive cells in thymus and memory T cells in lymph nodes were all increased markedly and de creased with time sequence. In BBN administered and BCG treated group, as the immune cells decreased, gross tumor developed and infiltration of memory T cells to tumor tissues was observed in this group. In conclusion, memory T cells which were activated by BCG can be major element involved in antitumor activity of BCG and the change of the number and the pattern of distribution of immune cells showed us that local reaction as well as systemic cellular immunity seemed to be important in antitumor activity. Also the effect was time limited.