Cytokine Production Profile of CD4+ T Cells From Patients with Immune Thrombocytopenia.

Abstract Abstract 4460 Introduction To balance self-tolerance and immunity, the immune system depends on both activation mechanisms and down regulatory mechanisms. CD4+ T cells are important for the regulation of immune responses and they exert their effects through the secretion of cytokines. Immune thrombocytopenia(ITP) is a common hematologic disorder manifested by immune-mediated platelet destruction. ITP in adults often has a persistent course and frequently requires treatment to prevent bleeding. The mechanism of ITP has historically been attributed to platelet autoantibody production and the resultant platelet destruction. However, more recent evidence suggests that cell immunity pathogenesis plays an important role in ITP. Methods Real-time PCR assays was used for the quantification of mRNA of cytokines IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, IL-23, IL-17, IFN-γ, TNF-α and TGF-β in CD4+ T cells from adult ITP patients and healthy subjects. The mRNA of T-bet, GATA-3, RORγt, and FOXP3 were also analyzed using Real-time PCR. The plasma concentrations of IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, IL-23, IFN-γ, TNF and IL-17 were determined by enzyme-linked immunosorbent assay at the same time. Results The plasma concentrations of IL-2, IL-17 and IFN-γ in CD4+T cells were increased, while levels of IL-4, IL-6, IL-10 and TGF-β were decreased, comparing adult patients with ITP with healthy subjects. Patients with ITP presented significant increased mRNA expression levels on IL-2,IL-17,T-bet and RORγt. Conclusions In adult ITP patients, the expression levels of IL-2 and IL-17 were up-regulated, expression levels of TGF-β and IL-10 were down-regulated, suggesting that ITP is a Th1 and Th17 predominant disease although the precise mechanisms await further study. Disclosures: No relevant conflicts of interest to declare.

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The Immune Regulatory Functions Of Circulating CD5+ B Cells Are Impaired In Adult Patients With Primary Immune Thrombocytopenia

Blood ◽

10.1182/blood.v122.21.3526.3526 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3526-3526

Author(s):

Fanli Hua ◽

Feng Li ◽

Shanhua Zou ◽

Weiguang Wang ◽

Yunfeng Cheng

Keyword(s):

Cell Culture ◽

T Cells ◽

T Cell ◽

B Cells ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Healthy Subjects ◽

Immune Thrombocytopenia ◽

Ifn Γ ◽

Regulatory Functions

Abstract Background and Objective Immune thrombocytopenia (ITP) is a typical autoimmune-mediated bleeding disorder in which isolated thrombocytopenia is resulted from increased platelet clearance and/or impaired thrombocytogenesis. In addition to producing antibodies and acting as antigen presenting cells, B cells have been proven to play an important role in regulating immune response mainly via production of interleukin 10 (IL-10). CD5+ B cells are able to produce IL-10, and CD5 is deemed as a hallmark of regulatory B cells. We have previously demonstrated insufficient IL-10 secretion by CD5+ B cells in patients with ITP. The objective of the current study was to investigate the alteration in regulatory functions of peripheral blood CD5+B cells in ITP patients. Methods Peripheral blood CD5+ B cells, CD5- B cells and CD4+ T cells were magnetically isolated from 7 adult patients with newly diagnosed ITP and 10 healthy controls. CD4+ T cells were labeled with CFSE (5(6)-Carboxyfluorescein N-hydroxysuccinimidyl ester) before cultured in RPMI1640 in the presence of CD3mAb/CD3mAb/IL-2 for 72 hours with or without CD5+/CD5- B cells at gradient B/T ratios. The proliferation of CD4+ T cells was assessed by the dilution of CFSE expression on a flow cytometry. After 72-hour culture, cells were stained with APC-CD4/FITC-Annexin-V/PI to evaluate the apoptosis of CD4+T cells. The concentrations of IL-4 and IFN-γ in cell culture supernatants were determined by ELISA. Results Purified CD5+ B cells from healthy subjects were capable of suppressing the proliferation of CD4+ T cells in a dose-dependent manner when co-cultured for 72 hours. The percentage of CD4+ T cells that underwent proliferation showed a statistically significant reduction (72.8 ± 9.4% vs 61.6 ± 12.8%; p = 0.039) in the presence of CD5+ B cells at 1:3 B/T ratio. With the B/T ratio increased to 1:1, the proportion of proliferating CD4+ T cells decreased to 43.7 ± 16.4% (p < 0.001). The apoptosis of CD4+ T cells was promoted by CD5+ B cells as the percentage of apoptotic CD4+ T cells was increased when co-cultured with CD5+ B cells at 1:1 B/T ratio (3.75 ± 0.82% vs 6.03 ± 2.51%; p = 0.014). In contrast, CD5- B cells did not suppress the proliferation of CD4+ T cells, quite contrary, the proportion of proliferating CD4+ T cells was slightly increased, though not statistically significant, when co-cultured at 1:1 with CD5- B cells for 72 hours as compared with CD4+ T cell cultured alone (72.8 ± 9.4% vs 78.9 ± 8.5%; p = 0.145). No effect of CD5- B cells on the apoptosis of CD4+T cells was observed. In ITP patients, CD5+ B cells showed compromised regulatory functions as the proliferation of CD4+ T cells was not altered by CD5+ B cells until the B/T ratio increased to 3:1 (1:1 B/T: 74.6 ± 12.5% vs 72.1 ± 16.2%, p = 0.752; 3:1 B/T: 74.6 ± 12.5% vs 60.7 ± 10.3%, p = 0.042, respectively). The ability of CD5+ B cells to promote the apoptosis of CD4+ T cells was also impaired in ITP patients: no difference in the percentage of apoptotic CD4+ T cells was found when CD5+ B cells (1:1 ratio of B to T) were added to the T cell culture system (2.97 ± 1.07% vs 3.24 ± 1.45%; p = 0.699). We also evaluated the ability of CD5+ B cells to suppress the secretion of IFN-γ, one of the Th1-type cytokines. In healthy subjects, the IFN-γ secretion by CD4+ T cells was decreased by CD5+ B cells at a minimal B/T ratio of 1:5 (2427.99 ± 632.62 pg/mL vs 1734.41 ± 507.16 pg/mL; p = 0.014) and dramatically dropped to 395.22 ± 136.54 pg/mL when the ratio of B/T increased to 1:1 (p < 0.001). The secretion of IL-4, a representative cytokine of Th2 subset, was not influenced by CD5+ B cells. CD5+ B cells from ITP patients showed no capacity for suppressing the IFN-γ secretion until the ratio of B/T increased as high as to 1:1 (3060.02 ± 493.98 pg/mL vs 2394.57 ± 417.42 pg/mL; p = 0.019) and, even under the condition of 5:1 B/T ratio, CD4+ T cells from ITP patients retained about 30% of the ability of IFN-γ secretion (1067.37 ± 499.70 pg/mL; p < 0.001 ). Conclusions CD5+ B cells from normal controls are competent for inhibiting the proliferation of CD4+ T cells and suppressing the secretion of IFN-γ by CD4+ T cells, suggesting potent immune regulatory functions of CD5+ B cells. While in patients with ITP, CD5+ B cells are severely functionally impaired, indicating that the immune regulatory dysfunctions in CD5+ B cells may participate in the pathogenesis of ITP. Disclosures: No relevant conflicts of interest to declare.

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Effects of RNA Interference on CD70 in Dendritic Cells of Patients with Immune Thrombocytopenia

Blood ◽

10.1182/blood.v128.22.1373.1373 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1373-1373

Author(s):

Zeping Zhou ◽

Yunlong Wang ◽

Renchi Yang ◽

Huiyuan Li

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Dendritic Cells ◽

Real Time ◽

Cd4 T Cells ◽

Immune Thrombocytopenia ◽

Negative Control ◽

Rt Pcr ◽

Ifn Γ ◽

Normal Controls

Abstract Objective: This study aims to assess the biological properties and functions of CD70 costimulatory molecule on dendritic cells (DCs) from patients with chronic immune thrombocytopenia (ITP). Methods: DCs were generated from monocytes isolated from peripheral blood, the immunophenotype markers were detected by flow cytometry. Chemically synthesized siRNA was then transferred into the cells by Lipofectamine 2000 to choose the most effective siRNA to block CD70 expression in three candidates. The mRNA expression and protein synthesis were analyzed by real-time RT-PCR and flow cytometry. The CD4+ T cells were co-cultured with DCs to assess the suppression capacity of DCs in the proliferation of CD4+ T cells and the production of IL-10 and IFN-γ by CD4+ T cells. The CD4+ T cells were co-cultured with DCs to assess the ability of DCs in the induction of Regulatory T cells (Tregs). Results: Dendritic cells were transfected with FITC-labelled scrambled siRNA for 4-6h. The transfection efficiency was average 60%. After transfection (48 h), the results of real-time RT-PCR analysis showed strong and specific downregulation of CD70 mRNA levels in transfected cells (p=0.046, siRNA1\2\3 Inhibition rates (74.5±6.1) %, (78.1±8.0) %, (90.1±2.5) % respectively, but not in negative control or in the absence of siRNA(p=0.157). The CD70-3 siRNA was extraordinarily effective in repressing the expression of CD70. The expression of CD70 on the DCs with siRNA3 was also significantly lower(p=0.043), but showed no difference in negative control (p=0.157). DCs from transfected group could inhibit the proliferation of PHA-activated CD4+ T cells than the negative control in ITP patients(p=0.008), but showed no difference in group of normal control (p=0.08). In chronic ITP patients and normal controls, the DCs from transfected group could inhibit the expression of IFN-γ compared with the group of negative control (p=0.018, p=0.043) and increase the expression of IL-10 (p=0.012, p=0.043). In chronic ITP patients and normal controls, the DCs from transfected group were defective in inducing the CD4+ T cell differentiating to CD4+CD25+CD127low Tregs compared to the negative control (p=0.012, p=0.043). Conclusions: In our study, siRNA is capable of inducing RNAi in DCs, it can knock down the CD70 gene expression specifically and effectively. The DCs from transfected group could inhibit the CD4+ T cells and induce the CD4+ T cells differentiating to Tregs. This approach is a useful tool by which costimulatory molecules of DC can be studied as well as a potential therapeutic option for autoimmune disease. Disclosures No relevant conflicts of interest to declare.

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Efficacy of Immunomodulatory Therapy with All-Trans Retinoid Acid for Adults with Chronic Immune Thrombocytopenia

Blood ◽

10.1182/blood.v120.21.3335.3335 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3335-3335

Author(s):

Lan Dai ◽

Zhaoyue Wang ◽

Ri Zhang ◽

Xia Bai ◽

Mingqing Zhu ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Peripheral Blood ◽

Immune Thrombocytopenia ◽

Pcr Analysis ◽

Therapeutic Effects ◽

Rt Pcr ◽

Platelet Destruction ◽

Prednisone Treatment ◽

Ifn Γ

Abstract Abstract 3335 Objective: Immune thrombocytopenia (ITP) is an immune-mediated disorder characterized by antibody- and cell-mediated platelet destruction and impaired platelet production. In addition of antibody-mediated platelet clearance, T-cell is also involved in platelet destruction or marrow suppression. During immune responses CD4+ T cells can differentiate into a range of cell types, including T helper type 1 (Th1), Th2, Th17 and regulatory T cells (Treg). The goal of the present study is to investigate the therapeutic effects of all-trans-retinoic acid (ATRA) plus prednisone treatment on adult refractory idiopathic thrombocytopenic purpura (RITP) and to further explore the underlying mechanisms. Methods: All 35 patients were treated with ATRA at fixed dose of 10 mg/tid plus prednisone and were started after discontinuation of other previous ITP treatment. Peripheral blood samples were collected from 35 RITP patients and 20 healthy subjects. The concentrations of the peripheral blood CD 4+ T cells (Th1, Th2, TH17, TREG) were analyzed by flow cytometry, the levels of cytokines were confirmed by enzyme-linked immunoassay (ELISA), and the expression of T-bet, GATA-3, RORγt, FOXP3 were detected by semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) in 35 RITP patients before and after treatments, with 20 normal individuals as respective controls. Results: The overall response rate of ATRA plus prednisone treatment in RITP patients was 54.3%, 28.6%(n=10) of patients with a complete response and 25.7%(n=9) of patients with a partial response. Among the 19 responders, the mean platelet count was 34+13×106/ml before ATRA therapy and 106+29×106/ml after treatment. No severe adverse effects happened. The levels of regulory T cells were significantly increased in patients after ATRA plus prednisone treatment (P < 0.05); the levels of Th1, Th2, Th17 were not significantly improved (P >0.05) in effective patients after treatments in flow cytometry analysis. The concentrations of IL-10, TGF-β was increased (P<0.05), whereas IFN-γ, IL-4, IL-17 factor showed no obvious change in the effective groups after treatment (P > 0.05). The expression levels of Foxp3 enhanced dramatically after treatment in real-time RT-PCR analysis (P < 0.05). However, the concentrations of T-bet, GATA-3 and ROR-γ showed no obvious change after the therapy in real-time RT-PCR analysis(P > 0.05). Conclusions: 54.3% of RITP patients recovered after ATRA plus prednisone treatments. The therapeutic effects of ATRA plus prednisone may be involved in the increased levels of regulory T cells in peripheral blood through increased expression of TGF-β, IL-10, and Foxp3. In comparison, the therapeutic effects may not be attributed to the expression of TH1, TH2, TH17, IFN-γ, IL-4, IL-17, T-bet, GATA-3, RORγt. Disclosures: No relevant conflicts of interest to declare.

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Cytokine production by CD4+ T cells specific for coagulation factor VIII in healthy subjects and haemophilia A patients

Thrombosis and Haemostasis ◽

10.1160/th06-09-0519 ◽

2007 ◽

Vol 97 (05) ◽

pp. 788-794 ◽

Cited By ~ 36

Author(s):

Delan Guo ◽

Nigel Key ◽

Bianca Conti-Fine ◽

Genlin Hu

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

Healthy Subjects ◽

Mononuclear Cells ◽

Coagulation Factor ◽

Th2 Cells ◽

Haemophilia A ◽

Strong Inhibitor ◽

Peripheral Blood Mononuclear ◽

Ifn Γ

SummaryHaemophiliaA patients treated with human factorVIII (fVIII) may develop antibody (Ab) inhibitors to fVIII. FVIII-specific CD4+ T cells are common in haemophilia A patients, but also in healthy subjects who do not have a sustained anti-fVIII Ab response. Here, we examined the fVIII-induced IFNγ -, IL-4- and TGF- β 1-producing CD4+ T blasts by culturing peripheral blood mononuclear cells (PBMC) from controls and patients with recombinant fVIII. FVIII exposure significantly increased IFNγ - and IL-4-, but not TGF-β 1-producing CD4+ T blasts in patients with inhibitors. Patients without inhibitors had fVIII-induced IFNγ - andTGF-β 1-,but not IL-4-producing CD4+ T blasts.Controls did not have IL-4-producing CD4+ T blasts. However, controls whose PMBC proliferated in response to fVIII had fVIII-induced CD4+ T blasts that produced IFN-γ, the number of which correlated with the intensity of the proliferative response to fVIII of their PMBC, whereas controls whose PMBC did not proliferate to fVIII had predominantly fVIII-induced CD4+ T blasts that producedTGF- β 1.The presence in controls and patients without inhibitors of fVIII-induced IFN-γ -producing CD4 + T cells, but not IL-4-producing CD4+ T cells, which are abundant in inhibitor patients, suggests a role of Th1 cells in initiating the immune response to fVIII, and of Th2 cells in the development of strong inhibitor production. The polarized high ratios of Th3/Th1 and Th3/Th2 in controls and patients without inhibitors suggest that a preponderance ofTh3 cells in the response to fVIII may help to maintain tolerance to fVIII.

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Induction of Thymic Stromal Lymphopoietin in Mesenchymal Stem Cells by Interaction with Myeloma Cells

Blood ◽

10.1182/blood.v120.21.1825.1825 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1825-1825

Author(s):

Shinji Nakajima ◽

Hiroto Ohguchi ◽

Yasushi Onishi ◽

Mayumi Kamata ◽

Tohru Fujiwara ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Mesenchymal Stem Cells ◽

Real Time ◽

Adhesion Molecule ◽

Real Time Pcr ◽

Cd4 T Cells ◽

Cdna Array ◽

Thymic Stromal Lymphopoietin ◽

Array Analysis

Abstract Abstract 1825 Multiple myeloma (MM) is a B-cell neoplasm characterized by clonal proliferation of malignant plasma cells in the bone marrow (BM). The development of new drugs has increased survival time, but the disease is still incurable. Recently, the BM microenvironment has been reported to play an important role in proliferation and survival of MM cells, suggesting that clarification of the interaction between BM stromal cells and MM cells may provide insights to allow the development of novel treatment strategies for MM. As the signal between MM cells and the BM microenvironment should be bidirectional, it is possible that MM cells produce signals that alter the BM microenvironment to one appropriate for their survival. To validate this hypothesis, we examined the changes in gene expression profile in human BM-derived mesenchymal stem cells (MSC) by co-culture with MM cell line OPM-2. cDNA array analysis indicated that 2 days of co-culture with OPM-2 induced multiple factors in MSC, which are known to have positive effects on survival of MM cells, such as interleukin-6, insulin-like growth factor-1, stromal cell-derived factor 1, vascular cell adhesion molecule 1 (VCAM1), and intercellular adhesion molecule 1 (ICAM1). Other than known factors, thymic stromal lymphopoietin (TSLP), which is a cytokine involved in progression of pancreatic and breast cancer through induction of T helper 2 cell responses, was found to be upregulated by co-culture (ratio 2.22). To confirm the results of cDNA array analysis, MSC co-cultured with OPM-2 or KMS-34 were tested for expression and secretion of TSLP by real-time PCR and ELISA. Real-time PCR revealed that the expression of TSLP in co-cultured MSC was two to three times higher than that in MSC monocultures. Significantly elevated levels of TSLP secretion from MSC after stimulation with PMA/ionomycin were observed by co-culture with OPM-2 or KMS-34 compared with monoculture (201.3 ± 5.0 pg/ml, 176.1 ± 13.3 pg/ml, and 96.6 ± 5.5 pg/ml, respectively; P < 0.01) as measured by ELISA. TSLP did not exhibit any effect on proliferation or drug resistance of MM cells independent of the level of TSLP receptor expression, therefore we evaluated the immunomodulatory effects of TSLP secreted by MSC. Monocyte-derived dendritic cells (MDDC) were first treated with the supernatant of either MSC or MSC co-cultured with KMS-34. Then, allogeneic naïve CD4+ T cells were co-cultured with pretreated MDDC. MDDC treated with the supernatant of co-cultured MSCs expanded IL-13+ CD4+ T cells to a greater extent than MDDC treated with the supernatant of MSC monocultures (10.3 ± 3.7 %, 5.9 ± 4.2 %, respectively; P = 0.02), and addition of anti-TSLP neutralizing antibodies to the supernatant decreased the expansion of IL-13+ CD4+ T cells (10.3 ± 3.7 %, 7.1 ± 2.6 %, respectively; P = 0.04). These results suggested that MM cells act to form more advantageous BM microenvironment for their survival through induction of pleiotropic molecules in MSC involved in not only proliferation and survival of MM cells, but also modifying immunological status. Disclosures: No relevant conflicts of interest to declare.

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HCA587 protein vaccine induces specific antitumor immunity mediated by CD4+ T-cells expressing granzyme B in a mouse model of melanoma

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200728131951 ◽

2020 ◽

Vol 20 ◽

Author(s):

Weiming Yang ◽

Weiheng Zhang ◽

Xiaozhong Wang ◽

Liming Tan ◽

Hua Li ◽

...

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

Antitumor Effect ◽

Granzyme B ◽

Cell Subset ◽

Cell Mediated Immunity ◽

Protein Vaccine ◽

Tumor Tissues ◽

Wide Range ◽

Ifn Γ

Background: The antigen HCA587 (also known as MAGE-C2), which is considered a cancer-testis antigen, exhibits upregulated expression in a wide range of malignant tumors with unique immunological properties, and may thus serve as a promising target for tumor immunotherapy. Objective: To explore the antitumor effect of the HCA587 protein vaccine and the response of humoral and cell-mediated immunity. Methods: The HCA587 protein vaccine was formulated with adjuvants CpG and and ISCOM. B16 melanoma cells were subcutaneously inoculated to C57BL/6 mice, followed by treatment with HCA587 protein vaccine subcutaneously. Mouse survival was monitored daily, and tumor volume was measured every 2 to 3 days. The tumor sizes, survival time and immune cells in tumor tissues were detected. And the vital immune cell subset and effector molecules were explored. Results: After treatment with HCA587 protein vaccine, the vaccination generated elicited significant immune responses, which delayed tumor growth and improved animal survival. The vaccination increased the proportion of CD4+ T cells expressing IFN-γ and granzyme B in tumor tissues. Depletion of CD4+T cells resulted in an almost complete abrogation of the antitumor effect of the vaccination, suggesting that the antitumor efficacy was mediated by CD4+ T cells. In addition, knockout of IFN-γ resulted in a decrease in granzyme B levels which were secreted by CD4+ T cells, and the antitumor effect was also significantly attenuated. Conclusion: The HCA587 protein vaccine may increase the levels of granzyme B expressed by CD4+ T cells, and this increase is dependent on IFN-γ, and the vaccine resulted in a specific tumor immune response and subsequent eradication of the tumor.

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The Impact of Exercise Serum on Selected Parameters of CD4+ T Cell Metabolism

Immuno ◽

10.3390/immuno1030008 ◽

2021 ◽

Vol 1 (3) ◽

pp. 119-131

Author(s):

Jana Palmowski ◽

Kristina Gebhardt ◽

Thomas Reichel ◽

Torsten Frech ◽

Robert Ringseis ◽

...

Keyword(s):

T Cells ◽

Oxygen Consumption ◽

T Cell ◽

Real Time ◽

Cd4 T Cells ◽

Cell Metabolism ◽

Cd4 T Cell ◽

Autologous Serum ◽

Cell Phenotype ◽

The Impact

CD4+ T cells are sensitive to peripheral changes of cytokine levels and metabolic substrates such as glucose and lactate. This study aimed to analyze whether factors released after exercise alter parameters of human T cell metabolism, specifically glycolysis and oxidative phosphorylation. We used primary human CD4+ T cells activated in the presence of autologous serum, which was collected before (CO) and after a 30-min exercise intervention (EX). In the course of activation, cells and supernatants were analyzed for cell viability and diameter, real-time oxygen consumption by using PreSens Technology, mRNA expression of glycolytic enzymes and complexes of the electron transport chain by real-time PCR, glucose, and lactate levels in supernatants, and in vitro differentiation by flow cytometry. EX did not alter T cell phenotype, viability, or on-blast formation. Similarly, no difference between CO and EX were found for CD4+ T cell activation and cellular oxygen consumption. In contrast, higher levels of glucose were found after 48 h activation in EX conditions. T cells activated in autologous exercise serum expressed lower HK1 mRNA and higher IFN-γ receptor 1. We suggest that the exercise protocol used was not sufficient to destabilize the immune metabolism of T cells. Therefore, more intense and prolonged exercise should be used in future studies.

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Circulating CD4 T cells elicited by endemic coronaviruses display vast disparities in abundance and functional potential linked to both antigen specificity and age

The Journal of Infectious Diseases ◽

10.1093/infdis/jiab076 ◽

2021 ◽

Author(s):

Katherine A Richards ◽

Maryah Glover ◽

Jeremy C Crawford ◽

Paul Thomas ◽

Chantelle White ◽

...

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

Protective Immunity ◽

Granzyme B ◽

Antigen Specificity ◽

Functional Potential ◽

Membrane Envelope ◽

Ifn Γ ◽

Human Coronaviruses ◽

Repeated Infections

Abstract Repeated infections with endemic human coronaviruses are thought to reflect lack of long-lasting protective immunity. Here, we evaluate circulating human CD4 T cells collected prior to 2020 for reactivity towards hCoV spike proteins, probing for the ability to produce IFN-γ, IL-2 or granzyme B. We find robust reactivity to spike-derived epitopes, comparable to influenza, but highly variable abundance and functional potential across subjects, depending on age and viral antigen specificity. To explore the potential of these memory cells to be recruited in SARS-CoV-2 infection, we examined the same subjects for cross-reactive recognition of epitopes from SARS-CoV-2 nucleocapsid, membrane/envelope, and spike. The functional potential of these cross-reactive CD4 T cells was highly variable, with nucleocapsid-specific CD4 T cells, but not spike-reactive cells showing exceptionally high levels of granzyme production upon stimulation. These results are considered in light of recruitment of hCoV-reactive cells into responses of humans to SARS-CoV infections or vaccinations.

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