Immune Cell Phenotype and Function After Treatment With Blinatumomab For Childhood Relapsed B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL)

Abstract Background Blinatumomab is a bi-specific monoclonal antibody designed to engage and tether cytotoxic T-cells (CTL) to CD19-expressing target B cells. An ongoing phase I multicenter study in pediatric patients with relapsed/refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has shown that blinatumomab induces morphological and molecular remissions, defined as minimal residual disease (MRD) levels <10-4, in 47% of patients [Gore L, et al. J Clin Oncol 31, 2013 (suppl; abstr 10007)]. It is presently unknown whether and to what extent blinatumomab affects T-cell phenotype and function in pediatric patients with BCP-ALL. Patients and Methods Eight children diagnosed with relapsed/refractory BCP-ALL at the Bambino Gesù Children’s Hospital in Rome (median age at diagnosis 5.8 years, range 0.5-14.6) received blinatumomab as continuous intravenous infusion for 28 consecutive days, followed by a 2-week drug-free period. Four out of 8 patients were given repeated treatment courses. Peripheral blood samples were collected before treatment (day 0) and weekly thereafter, for 4 consecutive weeks. Bone marrow (BM) aspirates were available on days 0 and +29 of each drug course. Peripheral blood mononuclear cells (PBMC) were labeled with appropriate combinations of fluorochrome-conjugated monoclonal antibodies to quantitate naïve/memory T cells, αβ/γδ-expressing T cells and other immune effectors with potential anti-leukemia activity, such as CD3+CD56+ natural killer (NK) T cells and CD3-CD56+ NK cells. T-cell production of interferon (IFN)-γ, interleukin (IL)-4 and IL-17 was measured at the single-cell level, after short-term (4-hour) stimulation with phorbol myristate acetate (PMA) and ionomycin. The TCR-Vβ Repertoire Kit® (Beckman Coulter, Milan, Italy) allowed the flow cytometry analysis of 24 different Vβ specificities on T cells, thus covering approximately 70% of the normal human TCR-Vβ repertoire. Results Peripheral blood lymphocytes reached their nadir on day +1 (median 300/µL of blood [inter-quartile range 40-380] compared with 1,080/µL of blood at baseline [inter-quartile range 360-2,310]; p=0.0037 by Mann-Whitney U test for paired data), expanded within 7 days up to 3.5-fold above baseline, and included both CD4+ and CD8+ T cells. By contrast, the frequency of both CD3+CD56+ NK T cells and CD3-CD56+ NK cells remained unchanged compared to baseline. IFN-γ production by patient-derived CD4+ T cells exceeded that observed in CD4+ T cells from healthy controls by 2-fold, indicating robust T helper type 1 (Th1) polarization. The frequency of Th2/Th17 cells, defined as CD4+IL-4+ and CD4+IL-17+ cells, respectively, was not different after treatment compared to baseline. CD31 expression on recovering CD45RA+ naïve T cells, a surrogate phenotypic feature for recent thymic emigrants (RTEs), suggested that thymic output may contribute to T-cell expansion after blinatumomab administration. Non-significant changes in the relative proportion of TCR-αβ and TCR-γδ-expressing CD3+ T cells were detected after treatment (median 79.5% TCR-αβ+ T cells and 19.3% TCR-γδ+ T cells among total CD3+ cells) compared with baseline (median 87.4% TCR-αβ+ T cells and 12.2% TCR-γδ+ T cells among total CD3+ cells). Importantly, both CD3+CD8bright T cells and NK cells expressed lytic granule proteins, such as perforin and granzyme-B, at levels that increased during treatment. The analysis of Vβ TCR repertoire revealed a restricted usage of single Vβ domains by BM-resident CD8+ T cells, but not by CD4+ T cells. Specifically, the sum of Vβ within CD8+ T cells in the BM averaged 56.7±6.2% after blinatumomab, compared with 78±5.1% in healthy controls (p=0.04; Mann-Whitney U test for unpaired data). Conclusions Blinatumomab expands both CD31+CD45RA+ thymic-naïve and memory T cells with heightened IFN-γ production and is highly effective at clearing MRD in children with BCP-ALL. Skewing of the Vβ repertoire within BM-resident CD8+ T cells may be consistent with clonal expansions. Disclosures: Zugmaier: Amgen: Employment.

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Decreased Expression of T-Cell Costimulatory Molecule CD28 on CD4 and CD8 T Cells of Mexican Patients with Pulmonary Tuberculosis

Tuberculosis Research and Treatment ◽

10.1155/2010/517547 ◽

2010 ◽

Vol 2010 ◽

pp. 1-8 ◽

Cited By ~ 3

Author(s):

German Bernal-Fernandez ◽

Patricia Espinosa-Cueto ◽

Rosario Leyva-Meza ◽

Nathalie Mancilla ◽

Raul Mancilla

Keyword(s):

Immune Response ◽

T Cells ◽

T Cell ◽

Pulmonary Tuberculosis ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Costimulatory Molecule ◽

Cell Phenotype ◽

Mexican Patients

Patients with tuberculosis frequently develop anergy, a state of T-cell hyporesponsiveness in which defective T-cell costimulation could be a factor. To know if the expression of T-cell costimulatory molecules was altered in tuberculosis, we analyzed the peripheral blood T-cell phenotype of 23 Mexican patients with pulmonary tuberculosis. There was severe CD4 (P<.001) and CD8 (P<.01) lymphopenia and upregulation of costimulatory molecule CD30 on CD4 and CD8 T cells (P<.05); this increase was higher in relapsing tuberculosis. The main finding was severe downregulation of the major costimulatory molecule CD28 on both CD8 and CD4 T cells (P<.001). Depletion of the CD4/CD28 subset, a hitherto undescribed finding, is relevant because CD4 T cells constitute the main arm of the cell-mediated antimycobacterial immune response.

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Human Bone Marrow as a Source of Multifunctional CMV-Specific CD4+ T Cells for Adoptive Cell Therapy.

Blood ◽

10.1182/blood.v110.11.2973.2973 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2973-2973

Author(s):

Il-Kang Na ◽

Anne Letsch ◽

Ines Noack ◽

Sandra Bauer ◽

Jens Geginat ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cell Therapy ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Human Bone ◽

Ifn Γ

Abstract Introduction Adoptive cell transfer of ex vivo primed and expanded human cytotoxic T lymphocytes (CTLs) has emerged as a promising approach to treat both infectious and malignant diseases in humans. First clinical studies have shown that transfer of Cytomegalovirus (CMV)-specific CD8+ T cells is safe and effective in reconstitution of cellular immunity against CMV disease. Efficacy of adoptive T cell therapy is limited by the numbers of CTLs in vitro and the survival and function after infusion. CD4+ T cells may enhance activity via direct or indirect effector functions (Matloubian 1994 [1]). In this study we have analysed whether bone marrow is superior to peripheral blood for expansion of CMV-specific T cells. Experimental design Paired peripheral blood and bone marrow samples were obtained from patients who underwent total hip arthroplasty. By using two different protocols T cells were expanded in the presence of IL-2 and IL-7 either from bulk culture with exposure of two different peptide pools (IE1 and pp65) or after selection via IFN-γ secretion by stimulation with pp65. CMV specific immune responses were assessed by using multiparameter flow cytometry staining cells for CD3, CD4, CD8, CCR7 and CD45RA and for the cytokines IFN-γ IL-2 and TNF at day 0 and after 10 days of in vitro expansion. Results Similar frequencies of cytokine-producing pp65– and IE1-specific CD4+ and CD8+ T cells were found in unmanipulated paired PB and BM samples. Expansion of CMV-specific T cells from BM resulted in significantly higher frequencies of specific CD4+ T cells than from PB, whereas no difference in frequencies of CMV-specific CD8+ T cells was observed. Interestingly, significantly higher frequencies of BM pp65 and IE1-specific CD4+ T cells were multifunctional, characterized by producing simultaneously IFN-γ, TNF and IL-2 (IE1: BM mean 0.44% ± 0.16; PB mean 0.09% ± 0.05, p=0.031; pp65: BM mean 3.87% ± 2.46; PB mean 1.24% ± 0.90, p=0.031). Expansion of multi-functional CD4+ T cells from BM was observed with both the bulk and selection assay protocol. Both PB and BM CMV-specific CD4+ and CD8+ T cell lines had a predominant CD45RA-CCR7- effector memory phenotype. Conclusions This study implicates the use of human bone marrow as a source for expansion of multifunctional CMV-specific CD4+ T cells. Recent studies in HIV and Leishmania support the crucial role of multifunctional T cells in disease control (Darrah 2007 [2]).

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Redirected CD4+ T Cells Using WT1-Specific T-Cell Receptor Gene Transfer Can Supply Multifactorial Help to Enhance the Anti-Leukemia Reactivity Mediated by Similarly Redirected CD8+ T Cells Using the Identical Gene Transfer

Blood ◽

10.1182/blood.v118.21.645.645 ◽

2011 ◽

Vol 118 (21) ◽

pp. 645-645

Author(s):

Toshiki Ochi ◽

Hiroshi Fujiwara ◽

Sachiko Okamoto ◽

Hiroaki Asai ◽

Yukihiro Miyazaki ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Gene Transfer ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Central Memory ◽

Cell Phenotype ◽

Adoptive Therapy ◽

Ifn Γ ◽

Peptide Ligation

Abstract Abstract 645 [Purpose] Redirected T-cell-based anti-cancer adoptive therapy using cancer antigen-specific T-cell receptor (TCR) gene transfer has clinically shown promise, however there still remain considerable issues of achieving better clinical efficacy. For this purpose, helper function provided by concurrent CD4+ T cells should be evidently considerable. In this study, to achieve the enhanced anti-leukemia functionality mediated by redirected CD8+ T cells using WT1-specific TCR gene transfer, we in detail examined functionalities mediated by similarly redirected CD4+ T cells using the identical TCR gene transfer.[Methods] HLA-A*24:02-restricted and WT1235-243-specific codon-optimized TCR α/β genes were inserted into the novel retroviral vector encoding shRNAs for endogenous TCRs (WT1-siTCR vector). (1) Cognate antigen-responsive cytokine production mediated by WT1-siTCR transduced CD4+ T cells was assessed using cytokine beads array and ELISA assay. (2) Expression of CD40L and OX40 on redirected CD4+ T cells stimulated by WT1 peptide ligation was assessed using flow cytometer. (3) Impact caused by redirected CD4+ T cells on each magnitude of WT1-specific cytotoxicity, target-responsive proliferation and transition to central memory T-cell phenotype of WT1-siTCR transduced CD8+ T cells was measured using 51Cr-release assay, CD107a assay, intracellular IFN-γ assay and CFSE assay. (4) Chemokines produced by redirected CD4+ T cells stimulated using WT1 peptide was comprehensively assessed using real-time PCR. Consequent chemotaxis of redirected CD8+ T cells toward those stimulated redirected CD4+ cells was validated using transwell experiments. (6) Finally, anti-leukemia reactivity against autologous leukemia cells mediated by patients' redirected CD8+ T cells was similarly examined in the presence or absence of such autologous CD4+ T cells. [Results] First, in this study, those redirected CD4+ T cells hardly became positive for intracellular FoxP3, a crucial marker for regulatory T cell phenotype. In response to the WT1 peptide, WT1-siTCR transduced CD4+ T cells produced Th1 cytokines; IL-2, IFN-γ and TNF-α, in the context of HLA-A*24:02, which also needed HLA class II molecules on target cells. Magnitudes of WT1-responsive CD107a expression, IFN-γ production and cytotoxicity mediated by WT1-siTCR transduced CD8+ T cells were efficiently enhanced in the presence of redirected, but not non-redirected CD4+ T cells. Similarly, in the presence of those redirected CD4+ T cells, redirected CD8+ T cells expressing WT1-specific TCR increased in number and the transition to central memory T-cell phenotype (CD45RA−CD62L+) of those CD8+ T cells was stimulated in response to the stimulation with WT1 peptide. WT1 peptide ligation stimulated those redirected CD4+ T cells to express membrane-bound OX40, which is involved in the formation of central memory CD8+ T cells. WT1 peptide ligation also stimulated the redirected CD4+ T cells to produce chemokines; CCL3 and CCL4. Redirected CD8+ T cells expressed the receptor for these chemokines, CCR5; efficient migration of redirected CD8+ T cells toward redirected CD4+ T cells stimulated with WT1 peptide was obviously observed in transwell experiments. Finally, redirected CD4+ T cells isolated from patients with leukemia successfully provided Th1 helper function to autologous redirected CD8+ T cells to enhance the anti-leukemia reactivity; cytotoxicity, proliferation and formation of central memory T cells, in response to autologous leukemia cells, in vitro. [Conclusion] Although further investigations are warranted, concurrently adopted WT1-siTCR introduced CD4+ T cells seems feasible to enhance the efficacy of WT1-targeting redirected T-cell-based adoptive therapy for treatment of human leukemia. Disclosures: No relevant conflicts of interest to declare.

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Adoptive T Cell Therapy of Human CMV Infection in Immunocompromised Hosts: In Vitro Generation of CMV-Specific T Cells from Naïve Precursors

Blood ◽

10.1182/blood.v112.11.206.206 ◽

2008 ◽

Vol 112 (11) ◽

pp. 206-206 ◽

Cited By ~ 1

Author(s):

Sonja Schmucker ◽

Mario Assenmacher ◽

Jurgen Schmitz ◽

Anne Richter

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Peptide Pool ◽

Adoptive T Cell Therapy ◽

T Cell Therapy ◽

A Cell ◽

Ifn Γ

Abstract Adoptive transfer of virus-specific T cells is a promising therapy for the treatment of infections in immunocompromised patients. Virus-specific T cells can readily be obtained from antigen-experienced, but not naïve donors. In this study we describe a cell culture system for the in vitro generation of CMV-specific T cells from naive T cells derived from CMV-seronegative donors. We isolated naïve T cells by magnetic depletion of non-T cells, CD25+ regulatory T cells, and CD45RO+ effector and memory T cells from peripheral blood mononuclear cells (PBMC) of CMV-seronegative donors. These naïve T cells were co-cultured with autologous mature monocyte-derived DC (MoDC) loaded with a pool of overlapping peptides from the CMV protein pp65. CD3-depleted autologous PBMC were used as feeder cells and CD28 antibody, IL-2, IL-7, and IL-15 were added to the culture. Already only 9–13 days after starting the priming culture, frequencies of 0.0024% and 0.009% pp65495–503/A2-tetramer+ cells among CD8+ T cells were found for 2 HLA-A2+ blood donors. In contrast pp65495–503/A2-tetramer+ T cells were not detectable when naive T cells were cultured with unpulsed MoDC. Tetramers are suitable tools for the identification of antigen-specific T cells but are restricted to single epitopes of mainly CD8+ T cells. To analyze primed CD4+ T cells as well as CD8+ T cells having specificities other than for the peptide pp65495–503, we looked for upregulation of the activation marker CD137 after a second stimulation and found increased frequencies of CD137+ CD4+ T cells as well as CD137+ CD8+ T cells in the pp65-primed cell cultures only when restimulated with the peptide pool of pp65. Because IFN-γ is important for the control of CMV infection, we studied the capability of the in vitro primed pp65-specific CD4+ and CD8+ T cells to produce this cytokine. Restimulation of the T cells with pp65 peptide pool induced IFN-γ secretion in up to 3.9% of the CD8+ T cells and up to 3.8% of the CD4+ T cells in each of six donors tested. No specific IFN-γ production was detected after restimulation with an irrelevant IE-1 peptide pool. As expected the frequency of pp65-specific T cells in the priming cultures is low. For generation of T cell lines, we magnetically enrich pp65- specific T cells according to their IFN-γ secretion using the cytokine secretion assay technology. After further cultivation for 2 weeks the antigen-specificity of the expanded T cells was again evaluated. Only if restimulated with the pp65 peptide pool 56.6% of the CD4+ T cells showed upregulated expression of the activation marker CD154 (CD40L). Cytokine analysis of the cells revealed IFN-γ production in 40.2% of the CD4+ T cells, of which 36% co-expressed IL-2, indicating the functionality of the in vitro primed and expanded T cells. In conclusion, we established a cell culture system for in vitro priming of CMV-specific CD4+ and CD8+ T cells derived from peripheral blood of donors not infected by CMV. This should extend the application of adoptive T cell therapy to patients for whom immune donors are not available.

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Cytotoxic CD4+ T cells use granulysin to kill Cryptococcus neoformans, and activation of this pathway is defective in HIV patients

Blood ◽

10.1182/blood-2006-03-009720 ◽

2006 ◽

Vol 109 (5) ◽

pp. 2049-2057 ◽

Cited By ~ 60

Author(s):

Chun Fu Zheng ◽

Ling Ling Ma ◽

Gareth J. Jones ◽

M. John Gill ◽

Alan M. Krensky ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Cryptococcus Neoformans ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Peripheral Blood Lymphocytes ◽

Effector Molecule ◽

Microbicidal Activity ◽

Anticryptococcal Activity

AbstractAn important mechanism of host defense to Cryptococcus neoformans involves the direct microbicidal activity of lymphocytes. The importance of CD4+ T cells is illustrated by the incidence of this infection in the acquired immunodeficiency syndrome (AIDS) patients; however, the relative activity of microbicidal CD4+ T cells compared with CD8+ T cells and natural killer (NK) cells has not been established. Further, although NK cells and CD8+ T cells use perforin or granulysin, respectively, to kill C neoformans, the effector molecule used by CD4+ T cells is not known. Experiments demonstrated that IL-2–activated peripheral blood lymphocytes from healthy adults acquire anticryptococcal activity, and surprisingly, that CD4+ T cells had the most profound effect on this activity. Using SrCl2induced degranulation and siRNA knockdown, granulysin was shown to be the effector molecule. Although activation by anti–CD3 + IL-2 resulted in the additional expression of perforin, this did not improve the anticryptococcal activity. Cryptococcal killing by CD4+ T cells was defective in human immunodeficiency virus (HIV)–infected patients due to dysregulated granulysin and perforin production in response to IL-2 or anti–CD3 + IL-2. In conclusion, CD4+ T cells are the major subset of cells responsible for killing C neoformans in peripheral blood. These cells use granulysin as the effector molecule, and priming is dysregulated in HIV-infected patients, which results in defective microbicidal activity.

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Retrovirally induced CTL degranulation mediated by IL-15 expression and infection of mononuclear phagocytes in patients with HTLV-I–associated neurologic disease

Blood ◽

10.1182/blood-2008-02-138529 ◽

2008 ◽

Vol 112 (6) ◽

pp. 2400-2410 ◽

Cited By ~ 25

Author(s):

Yoshimi Enose-Akahata ◽

Unsong Oh ◽

Christian Grant ◽

Steven Jacobson

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Spastic Paraparesis ◽

Cd8 T Cell ◽

Mononuclear Phagocytes ◽

Type I ◽

Human T Cell ◽

Ifn Γ

AbstractCD8+ T cells contribute to central nervous system inflammation in human T-cell lymphotropic virus type I (HTLV-I)–associated myelopathy/tropical spastic paraparesis (HAM/TSP). We analyzed CD8+ T-cell dysfunction (degranulation and IFN-γ production) and have demonstrated that CD8+ T cells of patients with HAM/TSP (HAM/TSP patients) spontaneously degranulate and express IFN-γ in ex vivo unstimulated culture. CD8+ T cells of HTLV-I asymptomatic carriers and healthy donors did not. Spontaneous degranulation was detected in Tax11-19/HLA-A*201 tetramer+ cells, but not in CMV pp65 tetramer+ cells. Interestingly, degranulation and IFN-γ production in CD8+ T cells was induced by coculture with autologous CD14+ cells, but not CD4+ T cells, of HAM/TSP patients, which correlated with proviral DNA load in CD14+ cells of infected patients. Moreover, the expression of IL-15, which induced degranulation and IFN-γ production in infected patients, was enhanced on surface of CD14+ cells in HAM/TSP patients. Blockade of MHC class I and IL-15 confirmed these results. Thus, CD8+ T-cell dysregulation was mediated by both virus infection and enhanced IL-15 on CD14+ cells in HAM/TSP patients. Despite lower viral expression than in CD4+ T cells, HTLV-I–infected or –activated CD14+ cells may be a heretofore important but under recognized reservoir particularly in HAM/TSP patients.

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Organ- and Disease-Stage-Specific Regulation of Toxoplasma gondii-Specific CD8-T-Cell Responses by CD4 T Cells

Infection and Immunity ◽

10.1128/iai.00098-06 ◽

2006 ◽

Vol 74 (10) ◽

pp. 5790-5801 ◽

Cited By ~ 26

Author(s):

Sonja Lütjen ◽

Sabine Soltek ◽

Simona Virna ◽

Martina Deckert ◽

Dirk Schlüter

Keyword(s):

T Cells ◽

T Cell ◽

Toxoplasma Gondii ◽

Cd8 T Cells ◽

Functional Activity ◽

Cd4 T Cells ◽

Cd8 T Cell ◽

Cd4 T Cell ◽

Ifn Γ

ABSTRACT Toxoplasma gondii induces a persistent central nervous system infection, which may be lethally reactivated in AIDS patients with low CD4 T-cell numbers. To analyze the role of CD4 T cells for the regulation of parasite-specific CD8 T cells, mice were infected with transgenic T. gondii expressing the CD8 T-cell antigen β-galactosidase (β-Gal). Depletion of CD4 T cells prior to infection did not affect frequencies of β-Gal876-884-specific (consisting of residues 876 to 884 of β-Gal) CD8 T cells but resulted in a pronounced reduction of intracerebral β-Gal-specific gamma interferon (IFN-γ)-producing and cytolytic CD8 T cells. After cessation of anti-CD4 treatment a normal T. gondii-specific CD4 T-cell response developed, but IFN-γ production of intracerebral β-Gal-specific CD8 T cells remained impaired. The important supportive role of CD4 T cells for the optimal functional activity of intracerebral CD8 T cells was also observed in mice that had been depleted of CD4 T cells during chronic toxoplasmosis. Reinfection of chronically infected mice that had been depleted of CD4 T cells during either the acute or chronic stage of infection resulted in an enhanced proliferation of β-Gal-specific IFN-γ-producing splenic CD8 T cells. However, reinfection of chronically infected mice that had been depleted of CD4 T cells in the acute stage of infection did not reverse the impaired IFN-γ production of intracerebral CD8 T cells. Collectively, these findings illustrate that CD4 T cells are not required for the induction and maintenance of parasite-specific CD8 T cells but, depending on the stage of infection, the infected organ and parasite challenge infection regulate the functional activity of intracerebral CD8 T cells.

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The Role of β-Catenin in Th1 Immune Response against Tuberculosis and Profiles of Expression in Patients with Pulmonary Tuberculosis

Journal of Immunology Research ◽

10.1155/2021/6625855 ◽

2021 ◽

Vol 2021 ◽

pp. 1-15

Author(s):

Kunlong Xiong ◽

Jinxia Niu ◽

Ruijuan Zheng ◽

Zhonghua Liu ◽

Yanzheng Song ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Mononuclear Cells ◽

Cd4 T Cell ◽

Cell Frequency ◽

Tnf Α ◽

Ifn Γ

β-Catenin is a key molecule of canonical Wnt/β-catenin pathway. Its roles and expression profiles in T cells of tuberculosis (TB) remain unclear. The aim of this study was to explore the role of β-catenin in CD4+ T cells and its expression characteristics in patients with pulmonary tuberculosis (PTB). In this study, CD4+ T cell-specific β-catenin conditional knockout mice (β-CAT-cKO mice) were aerosol infected with Mycobacteria tuberculosis (Mtb) H37RV with wild-type mice as controls. Four weeks after infection, the mRNA expression of IFN-γ, TNF-α, and TCF-7 in the lungs of mice was measured. CD4, CD8, β-catenin, IFN-γ, and TNF-α in mononuclear cells from the lungs and spleens were measured by flow cytometry, and the pathological changes of lungs were also observed. Patients with PTB were enrolled, with blood samples collected and PBMCs isolated. The expressions of β-catenin, IFN-γ, TNF-α, and PD-1 in CD4+ and CD8+ T cells were measured by flow cytometry. Results showed a decreased frequency of and reduced IFN-γ/TNF-α mRNA expression and secretion by CD4+ T cells in the lungs of infected β-CAT-cKO mice compared with infected wild-type controls, and only slightly more inflammatory changes were observed in the lungs. β-catenin expressions in CD4+ and CD8+ T cells were significantly decreased in blood cells of patients with severe PTB compared with those in mild PTB. The stimulation of peripheral blood mononuclear cells (PBMCs) with lithium chloride (LiCl), a stimulant of β-catenin, resulted in the increase in CD4+ T cell frequency, as well as their secretion of IFN-γ and TNF-α. β-Catenin demonstrated a moderately positive correlation with PD-1 in CD4+ T cells. β-Catenin along with PD-1 and IFN-γ in CD4+ T cells had a high correlation with those in CD8+ T cells. In conclusion, β-catenin may be involved in the regulation of Th1 response and CD4+ T cell frequency in TB.

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Circulating cytokines and lymphocyte subsets in patients who have recovered from COVID-19

10.1101/2020.07.22.20160259 ◽

2020 ◽

Author(s):

Hasi Chaolu ◽

Xinri Zhang ◽

Xin Li ◽

Dongyan Li

Keyword(s):

T Cells ◽

B Cells ◽

Nk Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Lymphocyte Subsets ◽

Healthy Controls ◽

Tnf Α ◽

Ifn Γ ◽

Circulating Cytokines

To investigate the immune status of people who previously had COVID-19 infections, we recruited patients 2 weeks post-recovery and analyzed circulating cytokines and lymphocyte subsets. We measured levels of total lymphocytes, CD4+ T cells, CD8+ T cells, CD19+ B cells, CD56+ NK cells, and the serum concentrations of interleukin (IL)-1, IL-4, IL-6, IL-8, IL-10, transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) by flow cytometry. We found that in most post-recovery patients, levels of total lymphocytes (66.67%), CD3+ T cells (54.55%), CD4+ T cells (54.55%), CD8 + T cells (81.82%), CD19+ B cells (69.70%), and CD56+ NK cells(51.52%) remained lower than normal, whereas most patients showed normal levels of IL-2 (100%), IL-4 (80.88%), IL-6 (79.41%), IL-10 (98.53%), TNF-α (89.71%), IFN-γ (100%) and IL-17 (97.06%). Compared to healthy controls, 2-week post-recovery patients had significantly lower absolute numbers of total lymphocytes, CD3+ T cells, CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD56+ NK cells, along with significantly higher levels of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL-17. Among post-recovery patients, T cells, particularly CD4+ T cells, were positively correlated with CD19+ B cell counts. Additionally, CD8+ T cells positively correlated with CD4+ T cells and IL-2 levels, and IL-6 positively correlated with TNF-α and IFN-γ. These correlations were not observed in healthy controls. By ROC curve analysis, post-recovery decreases in lymphocyte subsets and increases in cytokines were identified as independent predictors of rehabilitation efficacy. These findings indicate that the immune system has gradually recovered following COVID-19 infection; however, the sustained hyper-inflammatory response for more than 14 days suggests a need to continue medical observation following discharge from the hospital. Longitudinal studies of a larger cohort of recovered patients are needed to fully understand the consequences of the infection.

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CD8+CD28− T cells: key cytotoxic players impacting disease pathogenesis in chronic HBV infection

Clinical Science ◽

10.1042/cs20190369 ◽

2019 ◽

Vol 133 (17) ◽

pp. 1917-1934

Author(s):

Madhuparna Nandi ◽

Sourina Pal ◽

Sumantra Ghosh ◽

Bidhan Chandra Chakraborty ◽

Debangana Dey ◽

...

Keyword(s):

T Cells ◽

Chronic Hepatitis ◽

T Cell ◽

Hepatitis B ◽

Chronic Hepatitis B ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Hepatitis B E Antigen ◽

Tnf Α ◽

Ifn Γ

Abstract During chronic hepatitis B (CHB), CD8+ T cells down-regulate CD28, the primary co-stimulation molecule for T-cell activation. Diverse functional attributes of CD8+CD28− T cells are suggested in various disease contexts. The present study aimed to characterize CD8+CD28− T cells in different phases of chronic Hepatitis B virus (HBV) infection (CHI)- Immune-tolerance (IT), Hepatitis B e-antigen-positive CHB (EP-CHB), Inactive carriers (IC) and Hepatitis B e-antigen-negative CHB (EN-CHB), to appraise their contribution in HBV-related disease pathophysiology. Flow cytometry analysis of T cells in peripheral blood of study subjects revealed enhanced CD8+CD28− T-cell accumulation in EP-/EN-CHB, compared with IT/IC and they expanded equivalently in HBV-specific and non-specific CD8+ T-cell compartments. Profound increase in CD8+CD28− T cells expressing perforin/granzyme-B/CD57/IFN-γ/TNF-α and markers of terminal differentiation were observed exclusively in EP-/EN-CHB. Further, activation with anti-NKG2D resulted in heightened IFN-γ/TNF-α production selectively from CD8+CD28− T cells, suggesting NKG2D-mediated alternative co-stimulation. CD8+CD28− T cells sorted from CHB patients induced enhanced apoptosis of peripheral blood mononuclear cells (PBMC), including CD4+ T cells. However, NKG2D-ligand (major histocompatibility complex class I chain-related molecule A/B (MICA/B)) was preferentially expressed by HBV-specific CD4+ T cells of CHB patients, making these cells a potential target to NKG2D-dependent CD8+CD28− T-cell killing. Both CD28+ and CD28− T cells in CHB expressed CXCR3 at similar levels and thus capable of homing to the liver. A positive correlation was seen between CD8+CD28− T-cell frequency and serum-alanine transaminase (ALT) levels and CHB-derived CD8+CD28− T cells caused pronounced cell death in HBV-transfected Huh7 cells. Immunofluorescence staining identified greater intrahepatic incidence of CD8+CD28− T cells but decline in CD4+ T cells in CHB than IC. Collectively, CD8+CD28− T cells demonstrated differential distribution and phenotypic/functional skewing in different CHI phases and contribute to disease progression by Perforin-Granzyme- or IFN-γ-TNF-α-mediated cytotoxicity while restraining antiviral immunity through NKG2D-dependent HBV-specific CD4+ T-cell depletion.

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