Detecting 24 Kinds of Cytokines Via Flow Cytometry Aimplex Kit Is an Effective Way to Monitor CRS after CAR-T Cells Infusion

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 50-51
Author(s):  
Dongchu Wang ◽  
Xiang Li ◽  
Liyuan Qiu ◽  
Shizhe Fu ◽  
Lingyun Cui ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Early Prevention ◽  
Tnf Α ◽  
Car T ◽  
Variation Curve ◽  
Ifn Γ ◽  
The Relationship

Introduction: Cytokine release syndrome (CRS) is a serious side effect of chimeric antigen receptor-T cell (CAR-T) therapy. Effective cytokine monitoring can provide support for early prevention and treatment. As more and more promising markers have been identified to be associated with CRS responses, traditional ELISA method is inapplicable because it's sample-consuming, low flexibility, and difficult to detect multiple cytokines simultaneously. Aimplex kit, a new high-throughput technique based on flow cytometry(FCM) and micro beads has been testified a effective way to monitor multiple cytokines in peripheral blood(PB) samples. If a new suitable cytokines panel is established to monitor the variation curve of multiple cytokines in patients' PBs post CAR-T therapy, study the relationship between clinical symptoms and the lab results, even build a cytokines database of CAR-T therapy, it would offer a promising support to prevent and handle CRS. On the other hand, by studying the relationship between cytokines and activated cells, we might investigate mechanisms of CRS and explore more related markers. Methods: A two tubes panel was designed to detect 24 cytokines, including IFN-γ/IL-1β/IL-2/IL-4/IL-5/IL-6/IL-8/IL-10/IL-12p70/IL-17A/IL-17F/IL-22/TNF-α/TNF-β, and sCD25/GM-CSF/IL-15/MCP-1/GranzymeB/Reg3A/ST2/TNFRSF1A/Elafin/MIP-1 alpha. 50 PB samples from complete response(CR) patients without CAR-T therapy were detested as normal controls to establish the normal values of 24 cytokines. 81 patients who infused CAR-T cell in Hebei Yanda Ludaopei Hospital from January to June 2020 were selected. Serum were collected on 0d, 4d, 7d, 11d, 15d, 20d and 30d after infusion, and 24 cytokines were detected by FCM Aimplex. Of which 31 patients were selected for methodological comparison between FCM and ELISA by detecting four routine cytokines, IL-6, IFN-γ, TNF-α and sCD25. 66 Patients were divided into 2 groups according to clinical manifestations, 60 patients without or mild CRS were classified as low grade CRS group, 6 patients with grade 2 or 3 CRS were classified as high grade CRS group. Results: The comparative test showed that a good relationship between the results of ELISA and Aimplex kit, showing similar time-cytokines variation curve of PBs after CAR-T treatment. The concentrations of IL-2, IL-10, IL-12p70, IL-17A, IL-17F, MCP-1, ST-2s, IL-5, IL-6 and IFN-γ were significantly higher in high CRS grade group. By comparing the proportion of CAR-T+, CD8+ and CD4+ T cells in CD3+ cells and the concentration of cytokines in PB specimens, there were obvious positive correlations between percentages of CAR-T+, CD8+ T cells and most cytokines, and negative correlation between proportion of CD4+ T cells and most cytokines. Conclusions: Detecting 24 kinds of cytokines by Aimplex kit is a promising method, which is time and specimens saving, and can cost-effectively reflect the cytokine situation in PBs from patients post-CAR-T treatment. Increasing the sample size and establishing a cytokines database will be helpful for monitoring, early prevention and control of side effects after CAR-T treatment. Disclosures No relevant conflicts of interest to declare.

Abstract 001: Axl Expression by CD4+ T Lymphocytes Promotes Salt-Dependent Hypertension

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Kristine M Wadosky ◽  
Sri N Batchu ◽  
Angie Hughson ◽  
Kathy Donlon ◽  
Craig N Morrell ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Cd4 T Cells ◽  
White Blood Cells ◽  
Interleukin 4 ◽  
Th2 Cells ◽  
Salt Hypertension ◽  
Ifn Γ

Introduction: Our laboratory has shown that Axl, a receptor tyrosine kinase, is important in both vascular and immune functions during deoxycorticosterone acetate (DOCA)-salt hypertension. We hypothesized that Axl activity specifically in T lymphocytes could explain the dependence of hypertension on Axl. Methods and Results: We did adoptive transfers of either Axl+/+ or Axl-/- CD4+ T cells to RAG1-/- mice that lack mature T cells. Once CD4+ T cell repopulations were confirmed, we induced DOCA-salt hypertension for 6 weeks. Systolic blood pressure (BP, mmHg) increased by 20±5 in Axl+/+RAG-/- mice after DOCA-salt, but Axl-/- RAG-/- mice had increases in BP by only 6+3 after 6 weeks of DOCA-salt. We isolated naïve CD4+ T cells from both Axl+/+ and Axl-/- littermates and primed them under either Th1 or Th2 polarizing conditions in culture. Production of interferon gamma (IFN-γ ng/mL) was significantly decreased (-23%, p<0.05) in Axl-/- (396±23) compared to Axl+/+ (512±42) under Th1-priming. However, Axl had no effect on interleukin 4 (IL-4, ng/mL) production under Th2 polarizing conditions. Intracellular staining of the Th1/Th2 cells with IFN-γ and IL-4 antibodies by flow cytometry confirmed expression of cytokines in culture media. Complete blood counts showed that Axl-/- mice had significantly lower white blood cells due to decreased numbers of lymphocytes (4.5±0.7x10 9 ) compared to Axl+/+ mice (7.8±0.7x10 9 ). We found a higher population of AnnexinV (marker of early apoptosis)-positive peripheral leukocytes in Axl-/- mice (10±1%) compared to Axl+/+ (4±1%) by flow cytometry; while the percentages of dead cells (~10%) were similar between Axl+/+ and Axl-/- mice. Conclusions: Altogether we show that expression of Axl by T cells drives salt-induced hypertension. The mechanism of Axl-dependent effects on T cells occurs via T-cell-dependent expression of the pro-inflammatory cytokine IFN-γ. In addition, Axl plays a role in inhibiting lymphocyte apoptosis in the circulation. Future work will focus on how Axl expression in T cells affects T cell-dependent vascular remodeling during hypertension.

Targeting bioenergetics prevents CD4 T cell–mediated activation of synovial fibroblasts in rheumatoid arthritis

Rheumatology ◽  
2020 ◽  
Vol 59 (10) ◽  
pp. 2816-2828 ◽  
Author(s):  
Andreea Petrasca ◽  
James J Phelan ◽  
Sharon Ansboro ◽  
Douglas J Veale ◽  
Ursula Fearon ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adhesion Molecule ◽  
Cd4 T Cells ◽  
Reciprocal Relationship ◽  
Angiogenic Factor ◽  
Flux Analysis ◽  
Intracellular Cytokines ◽  
Tnf Α ◽  
Ifn Γ

Abstract Objectives We investigated the reciprocal relationship linking fibroblast-like synoviocytes (FLS) and T lymphocytes in the inflamed RA synovium and subsequently targeted cellular metabolic pathways in FLS to identify key molecular players in joint inflammation. Methods RA FLS were cultured with CD4 T cells or T cell conditioned medium (CD4CM); proliferation, expression of adhesion molecules and intracellular cytokines were examined by flow cytometry. FLS invasiveness and secreted cytokines were measured by transwell matrigel invasion chambers and ELISA, while metabolic profiles were determined by extracellular Seahorse flux analysis. Gene expression was quantified by real-time quantitative RT-PCR. Results Our results showed mutual activation between CD4 T cells and FLS, which resulted in increased proliferation and expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 by both CD4 T cells and FLS. Furthermore, interaction between CD4 T cells and FLS resulted in an increased frequency of TNF-α+, IFN-γ+ and IL-17A+ CD4 T cells and augmented TNF-α, IFN-γ, IL-17A, IL-6, IL-8 and VEGF secretion. Moreover, CD4CM promoted invasiveness and boosted glycolysis in FLS while downregulating oxidative phosphorylation, effects paralleled by increased glucose transporters GLUT1 and GLUT3; key glycolytic enzymes GSK3A, HK2, LDHA and PFKFB3; angiogenic factor VEGF and MMP-3 and MMP-9. Importantly, these effects were reversed by the glycolytic inhibitor 2-DG and AMP analogue 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Conclusion This study demonstrates that CD4 T cells elicit an aggressive phenotype in FLS, which subsequently upregulate glycolysis to meet the increased metabolic demand. Accordingly, 2-DG and AICAR prevent this activation, suggesting that glycolytic manipulation could have clinical implications for RA treatment.

Transcription Factor BACH2 Inhibits AP1 Proteins JunB and FosL1 in Umbilical Cord Blood (UCB) CD4+ T-Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1743-1743
Author(s):  
Mathew L. Lesniewski ◽  
Laura R. Fanning ◽  
Margeret Kozik ◽  
Richard P. Weitzel ◽  
Yeal Hegerfeldt ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Transcription Factor ◽  
T Cell ◽  
Cd4 T Cells ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Tnf Α ◽  
Ifn Γ ◽  
Sirna Knockdown

Abstract Introduction: Umbilical cord blood (UCB) CD4+ T-cells have been shown to express significant levels of BACH2 transcription factor protein compared to adult blood (AB) CD4+ T-cells. Previously, NFAT1 siRNA knockdown of UCB T-cells exhibited a significantly higher BACH2 mRNA expression, and IFN-γ, TNF-α. and CTLA-4 mRNA levels were significantly suppressed. BACH2, a member of the b-Zip family, has been shown to act as a heterodimer with the bZip protein MafK, as a transcriptional inhibitor via recruitment of a histone deacetylase class II complex (HDAC II) in differentiating B-cells, and neurons. Due to observed inverse expression of BACH2 and NFAT1 in UCB CD4+ T-cells, we hypothesized that BACH2 may regulate transcription factors known to bind with NFAT1 including AP-1 proteins JunB and FosL1. We tested this by siRNA knockdown of BACH2 in primary UCB-derived CD4+ T-cells. Key developmental transcription factors JUNB, FosL1, NFAT1 and downstream IFN-γ, and TNF-α were mRNA analyzed. Methods: UCB T-cells were purified using autoMACs system (Miltenyi). After overnight culture, T-cells were transfected with BACH2 siRNA (Dharmacon) using Amaxa Nucleofector system (Amaxa Inc). Both siRNA treated and control cells were incubated in media for 18 hours, and then stimulated using anti-CD3/anti-CD28 antibodies (BD BioScience). Aliquots of cells were collected at specified time points post-stimulation for protein and total RNA isolation. The relative change in mRNA levels for BACH2, JUNB, FosL1, IFN-γ, NFAT1, and TNF-α were determined by Lightcycler SybrGreen real time RT-PCR system (Roche). siRNA knockdown of BACH2 protein in transfected UCB T-cells was confirmed by western blot. Results: Real-time RT-PCR of BACH2 siRNA treated UCB CD4+ T-cells stimulated with anti-CD3/CD28 antibodies and analyzed after 6 hrs of stimulation showed a 4 log increase in FosL1 and NFAT1 mRNA, a 3 log increase in JunB mRNA, a 5 log increase in IFN-γ as compared to stimulated control UCB T-cells. TNF-α mRNA was decreased by 5 logs in BACH2 siRNA treated UCB T-cells as compared to control. CD3/CD28 stimulated untransfected UCB T-cells were previously shown to have decrease expression of NFAT1, JunB, FosL1, IFN-γ, and TNF-α, and in UCB T-cells compared to stimulated AB T-cells. Conclusions: BACH2 expression correlates with an inhibition of expression of AP1 transcription regulatory proteins in UCB T-cells during primary CD3/CD28 stimulation. The complete activation of the T-cell requires the activation of AP1 by CD28 pathway otherwise the antigen presenting cell signals the T-cell to enter anergy. In UCB CD4+ T-cells express BACH2, which acts as a transcriptional inhibitor of two critical AP1 genes, JUNB and FosL1, which mediate the CD28 co-stimulatory pathway. These results further suggests that expression of BACH2 in UCB T-cells may contribute to lower incidence of alloreactivity observed in leukemia patients receiving UCB stem cells compared to AB bone marrow stem cells and thus leads to low GVHD, and contribute to the weak Th1 response seen in stimulated UCB T-cells by reduced amounts of AP1 protein available for activating the T-cell.

Opposing Effects of PD-1/PD-L1/L2 Engagement and IFN-γ/TNF-α in the Treatment of AML w/ Anti-CD33 Chimeric Antigen Receptor-Modified T Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5891-5891
Author(s):  
Jacob Halum Basham ◽  
Terrence L. Geiger
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Chimeric Antigen Receptor ◽  
Short Term ◽  
Car T Cells ◽  
Tnf Α ◽  
Car T ◽  
Ifn Γ

Abstract Chimeric antigen receptor-modified T lymphocytes (CART cells) have shown benefit as an adjuvant immunotherapy in the treatment of B cell malignancies. This success of re-targeted T cells has not been extended to other hematologic malignancies. We have developed an immunotherapeutic approach to treat acute myeloid leukemia (AML) using CAR T cells re-directed against the myeloid-specific antigen CD33 (CART-33). CART-33 cells are potent and specific in eliminating AML cells in vitro and in vivo. Despite this, CART-33 cells have shown poor in vivo expansion and persistence in NOD-SCID IL2rγ (-/-) (NSG) AML xenograft models. To address the reason for this, we assessed the impact of AML-expressed programmed death ligands 1 & 2 (PD-L1/2) on CART-33 cell activity. PD-L1 inhibits T cell functions upon binding PD-1, which is upregulated with T cell activation. Less is known about PD-L2's effect. Interferon-gamma (IFN-γ), a primary effector cytokine secreted by CD4+ and CD8+ effector T cells, is a known potent inducer of PD-L1 on AML blasts. Using AML cell lines U937, Oci-AML3, CMK, and MV4-11 we show that IFN-γ, TNF-α, and activated CART-33 supernatant can induce up-regulation of PD-L1 and PD-L2 on AML. IFN-γ and TNF-α synergize strongly in up-regulating PD-1 ligands on AML. The kinetics and induction of PD-L2 are distinct from that of PD-L1. Although PD-L1 is well documented to suppress T cell function via ligation of T cell expressed PD-1, induction of PD-L1/L2 had no effect on the cytolytic activity of CART-33 cells against AML in short term (<48 h) cultures. Paradoxically, 24 hr pre-treatment of AML with either IFN-γ or CART-33 supernatant increased AML susceptibility to killing by CART-33 cells despite elevated expression of PD-L1/L2 by AML. Our results highlight the regulatory complexity of AML cytolysis by re-targeted T lymphocytes, and argue that tumor-expressed PD-L1 and PD-L2 impacts the sustainability, but not short-term killing activity, of adoptively transferred CAR T cells in the treatment of AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Protein Energy Malnutrition during Vaccination Has Limited Influence on Vaccine Efficacy but Abolishes Immunity if Administered during Mycobacterium tuberculosis Infection

2015 ◽  
Vol 83 (5) ◽  
pp. 2118-2126 ◽  
Author(s):  
Truc Hoang ◽  
Else Marie Agger ◽  
Joseph P. Cassidy ◽  
Jan P. Christensen ◽  
Peter Andersen
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
T Cell ◽  
Cd4 T Cells ◽  
Protein Energy Malnutrition ◽  
T Cell Subsets ◽  
Content Type ◽  
Protein Energy ◽  
Tnf Α ◽  
Ifn Γ

Protein energy malnutrition (PEM) increases susceptibility to infectious diseases, including tuberculosis (TB), but it is not clear how PEM influences vaccine-promoted immunity to TB. We demonstrate that PEM during low-level steady-state TB infection in a mouse model results in rapid relapse ofMycobacterium tuberculosis, as well as increased pathology, in bothMycobacterium bovisBCG-vaccinated and unvaccinated animals. PEM did not change the overall numbers of CD4 T cells in BCG-vaccinated animals but resulted in an almost complete loss of antigen-specific cytokine production. Furthermore, there was a change in cytokine expression characterized by a gradual loss of multifunctional antigen-specific CD4 T cells and an increased proportion of effector cells expressing gamma interferon and tumor necrosis factor alpha (IFN-γ+TNF-α+and IFN-γ+cells). PEM duringM. tuberculosisinfection completely blocked the protection afforded by the H56-CAF01 subunit vaccine, and this was associated with a very substantial loss of the interleukin-2-positive memory CD4 T cells promoted by this vaccine. Similarly, PEM during the vaccination phase markedly reduced the H56-CAF01 vaccine response, influencing all cytokine-producing CD4 T cell subsets, with the exception of CD4 T cells positive for TNF-α only. Importantly, this impairment was reversible and resupplementation of protein during infection rescued both the vaccine-promoted T cell response and the protective effect of the vaccine againstM. tuberculosisinfection.

scholarly journals TNF-α Regulates Mast Cell Functions by Inhibiting Cell Degranulation

2017 ◽  
Vol 44 (2) ◽  
pp. 751-762 ◽  
Author(s):  
Yuwei Gao ◽  
Baixue Xu ◽  
Peng Zhang ◽  
Yanlong He ◽  
Xin Liang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mast Cells ◽  
Cd4 T Cells ◽  
Cell Function ◽  
Mapk Signaling ◽  
Cell Functions ◽  
Tnf Α ◽  
Ifn Γ ◽  
Stimulation Of

Background/Aims: The aim of this study was to investigate the involvement of inducible co-stimulatory ligand (ICOSL) expression in stimulation of mast cells (MCs) by TNF-α and the ability of TNF-α stimulation of MCs to influence CD4+ T cell differentiation and function. The mechanisms underlying TNF-α stimulation of MCs were also explored. Methods: Mast cells and CD4+ T cells were prepared from C57BL/6 mice (aged 6–8 weeks). ICOSL expression by MCs was measured by real-time PCR and flow cytometry, and levels of IL-4, IL-10 and IFN-γ were measured by ELISA. Results: ICOSL expression by MCs was increased by TNF-α stimulation, and resulted in interaction with CD4+ T cells. The IL-4 and IL-10 levels in the co-culture system increased, while IFN-γ levels decreased. Furthermore, CD4+CD25+Foxp3+ T cell proliferation was induced by co-culture with TNF-α-stimulated MCs. The mechanism by which TNF-α stimulated MCs was dependent on the activation of the MAPK signaling pathway. Conclusion: TNF-α upregulated the expression of ICOSL on mast cells via a mechanism that is dependent on MAPK phosphorylation. TNF-α-treated MCs promoted the differentiation of regulatory T cells and induced a shift in cytokine expression from a Th1 to a Th2 profile by up-regulation ICOSL expression and inhibition of MC degranulation. Our study reveals a novel mechanism by which mast cells regulate T cell function.

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

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.

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

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.

The Role of Pretransplant Conditioning On Graft-Versus-Leukemia Effects: Migration Matters.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3563-3563
Author(s):  
Ji-Young Lim ◽  
Mi-Sun Choi ◽  
Eun Young Choi ◽  
Hyewon Youn ◽  
Chang-Ki Min
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Cd4 T Cells ◽  
Tumor Tissue ◽  
Cell Trafficking ◽  
Tnf Α ◽  
Ifn Γ ◽  
Conditioning Intensity

Abstract Abstract 3563 Poster Board III-500 The therapeutic potential of allogeneic hematopoietic stem cell transplantation (HSCT) relies on the graft-versus-leukemia (GVL) effect to eradicate residual tumor cells by immunologic mechanisms. However, the relationship of conditioning intensity to GVL effect has not been clearly established independent of immunosuppression or the tolerance induced by mixed donor-host chimerism. Using a murine allogeneic HSCT model, we have compared two total body irradiation (TBI) doses (1,300 vs. 900 cGy), both of which provided complete donor engraftment and elimination of host lympho-hematopoetic cells. We used C57BL/6 (H-2b) → B6D2F1 (H-2b/d) model of GVHD, which differ at major and minor histocompatibility loci, to address the role of conditioning intensity on the GVL effect. Lethally irradiated (either 900 or 1300 cGy) recipient mice were transplanted with either C57BL/6 (allogeneic) or B6D2F1 (syngeneic) bone marrow (5 × 106) and spleen T cells (1 × 106) on day 0 and then P815 (H-2d) mastocytoma cells (1 × 106) injected subcutaneously on day 1 to generate a GVL model. As expected, GVHD morbidity after the higher TBI dose was aggravated compared to the lower TBI dose (P<.05). Among the syngeneic recipients, the injection of P815 cells into the recipient skin led to progressive tumor growth and death of about 100% 21 days after transplant regardless of the TBI dose. In contrast, tumor growth was remarkably suppressed and tumor death was not observed in the allogeneic recipients. Surprisingly, tumors in the allogeneic recipients receiving 1300 cGy TBI exhibited markedly delayed growth in vivo compared to those with 900 cGy (tumor volume on day 42, 428 vs. 8735mm3, P<.01), which was associated with an increase in the in vivo cytotoxicity using comparing the clearance of infused allogeneic B cells labeled with CFSE reflecting the enhanced alloimmune reactivity. To ask whether the diminished GVL effect after the lower TBI dose was due to reduced production of inflammatory cytokines, we measured the levels of TNF-α or IFN-γ in recipient sera on days 6, 28 and 42 after transplantation and did not find any significant difference according to the intensity of radiation dose (P>.05). In parallel, the in vitro P815-specific TNF-α or IFN-γ responses of splenocytes were comparable between the two doses. The percentages of donor T cells to undergo proliferation or apoptosis in response to alloantigens in vivo between the two TBI doses also were comparable (P>.05). Collectively, these data indicate that the impaired ability of alloreacive T cells to inhibit tumor growth after the lower TBI dose was not attributed to an intrinsic defect in T-cell expansion and activation. We next analyzed the spleen for the number of donor CD4+ and CD8+ T cells and observed no difference between the two TBI doses. In contrast to spleen, the number of CD8+ but not CD4+ T cells from the recipients that had received 1300 cGy was significantly increased in the skin (P<05). The effector function of donor CD8+ and CD4+ cells in both spleen and tumor tissue was examined by intracellular staining for IFN-γ. In the spleen, the percentages of CD8+ and CD4+ T cells expressing IFN-γ were not different between the two TBI doses. (5.9% vs 4.8%, P>.05, and 7.6% vs. 6.5%, P>.05 respectively) By contrast, 45.5% and 50.3% of CD8+ and CD4+ T cells, respectively, isolated from the tumor tissue of recipients receiving the higher TBI dose were IFN-γ; secreting cells, whereas only 25.5% and 16.3% of those cells from the tumor tissue of recipients treated with the lower dose showed this phenotype (P<.01 and <.05, respectively). After the higher TBI dose, secondary lymphoid organ homing receptors including CD62L and CCR7 were down-regulated on donor CD8+ T cells while CD44 expression was up-regulated compared to the lower TBI dose, which may facilitate migration to the tumor sites. In summary, the higher TBI dose (1300 vs. 900 cGy) resulted in significantly enhanced GVL effect, and the alterations in effector T cell trafficking into tumor tissue are the most likely mechanism. Moreover, T-cell activation and function were largely comparable between these conditioning regimens. This provides the rationale for targeting T cell trafficking by inflammation, possibly in combination with integrin or chemokine receptor agonists as a new therapeutic approach in leukemia relapse after allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.

scholarly journals T-Cell Cytokine Response in Salmonella Typhimurium-Vaccinated versus Infected Pigs

Vaccines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 845
Author(s):  
Selma Schmidt ◽  
Heinrich Kreutzmann ◽  
Maria Stadler ◽  
Kerstin H. Mair ◽  
Melissa R. Stas ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Salmonella Typhimurium ◽  
Cd4 T Cells ◽  
T Cell Subsets ◽  
Local Immunity ◽  
Effective Measure ◽  
Treatment Groups ◽  
Tnf Α ◽  
Ifn Γ

Vaccination with the live attenuated vaccine Salmoporc is an effective measure to control Salmonella Typhimurium (STM) in affected swine populations. However, the cellular immune response evoked by the Salmoporc vaccine including differences in vaccinated pigs versus non-vaccinated pigs upon STM infection have not been characterized yet. To investigate this, tissue-derived porcine lymphocytes from different treatment groups (vaccination-only, vaccination and infection, infection-only, untreated controls) were stimulated in vitro with heat-inactivated STM and abundances of IFN-γ, TNF-α and/or IL-17A-producing T-cell subsets were compared across organs and treatment groups. Overall, our results show the induction of a strong CD4+ T-cell response after STM infection, both locally and systemically. Low-level induction of STM-specific cytokine-producing CD4+ T cells, notably for the IFN-γ/TNF-α co-producing phenotype, was detected after vaccination-only. Numerous significant contrasts in cytokine-producing T-cell phenotypes were observed after infection in vaccinated and infected versus infected-only animals. These results suggest that vaccine-induced STM-specific cytokine-producing CD4+ T cells contribute to local immunity in the gut and may limit the spread of STM to lymph nodes and systemic organs. Hence, our study provides insights into the underlying immune mechanisms that account for the efficacy of the Salmoporc vaccine.

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