Distinct IFN-Gamma Dependent Effect of the TLR5 Agonist, Flagellin On NK and CD4+ T Cell Immunity Against CMV

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 255-255
Author(s):  
Mohammad Sohrab Hossain ◽  
Andrew T Gewirtz ◽  
Edmund K. Waller
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Cd4 T Cells ◽  
Lethal Dose ◽  
Hematopoietic Cells ◽  
Cmv Infection ◽  
Mcmv Infection ◽  
Radiation Chimeras ◽  
Viral Loads ◽  
Ifn Γ

Abstract Abstract 255 Background: Opportunistic CMV infection remains a major clinical complication in allogeneic BMT (allo-BMT) recipients. We previously published that peri-transplant treatment of highly purified flagellin, a TLR5 agonist extracted from S. typhimurium reduced GvHD in lethally irradiated allo-BMT recipients by reducing early activation and proliferation of donor T cells. Flagellin-treated recipients had enhanced lymphoid immune reconstitution and were protected from lethal MCMV infection. CMV initiates innate immunity through the activation of Toll-like receptors (TLR) 9, 3 and 2 but direct enhancement of anti-CMV immunity through TLR5 has not been studied. Methods: To further explore the effects of a TLR5 agonist on anti-CMV immunity, we infected flagellin-treated C57BL/6 (B6), TLR5KO and Myd88KO mice (B6 background) with sub lethal dose (1×105pfu/mouse i.p) of MCMV. Anti-CMV immunity of NK and T-cells were determined by measuring surface activation markers on the NK and T-cells. Flagellin-treated and MCMV infected IL-12KO, IFN-γRKO and IDO KO mice were used to study the role of IL-12, IFN-γ and IDO on anti-CMV immunity. The relationship between the MCMV lethality and flagellin signaling in hematopoietic or epithelial tissues expressing TLR5 was further investigated by generating radiation chimeras, using WT mice engrafted with TLR5 KO bone marrow (BM) and TLR5 KO mice engrafted with WT B6 BM. Chimeric recipients with WT or TLR5KO hematopoietic cells (or the reverse) were then treated with flagellin or PBS and infected with a sub lethal dose of MCMV infection. Results: Without MCMV infection, flagellin treatment significantly increased numbers of NK cells (p= 0.001) and KLRG1+ NK cells (p=0.009) in WT B6 mice compared with the PBS-treated control mice. Following MCMV infection (day 3), there were significantly increased numbers of splenic NK cells (p= 0.01) and KLRG1+ NK cells (p=0.0008) in flagellin-treated mice compared with the control mice. Flagellin-treatment also enhanced viral clearance, tending to decrease liver viral loads in WT B6 mice. In contrast, MCMV-infected and flagellin-treated TLR5 KO or Myd88KO mice had significantly increased liver viral loads (p=00001) compared with the flagellin- and PBS-treated B6 mice on day 3 post MCMV infection. While flagellin-treatment increased the numbers and activation status of NK cells, it decreased the numbers of activated (CD69+, ICOS+ and KLRG1+) CD4+ T cells on day 3 post MCMV infection. On day 10 post MCMV infection, enhanced numbers of splenic CD8+ T cells and CMV-specific tetramer+ CD8+ T cells (p<0.05) were detected in flagellin-treated mice compared with controls. The numbers of splenic CD4+ T cells increased >2 fold (p= 0.008) associated with significantly increased numbers of splenic CD4+CD25+ Foxp3+ regulatory T cells (p<0.05) on day 10 post MCMV infection in flagellin-treated mice compared with the control mice. Flagellin-induced anti-CMV immunity required IFN-γ signaling as flagellin-treated IFN-γRKO mice had markedly increased susceptibility to MCMV infection whereas flagellin-treated IL-12 KO and IDO KO mice had significantly reduced liver viral loads compared to the PBS-treated IL-12 KO and IDO KO mice on day 10 post MCMV infection. To explore interaction of flagellin with TLR5+ host hematopoietic cells and TLR5+host gut epithelium on CMV immunity, we next infected radiation chimeras expressing either TLR5 on hematopoietic cells or on gut epithelium with sub lethal i.p dose of MCMV. Flagellin-treated TLR5 KO BM → B6 radiation chimeras (TLR5+ host epithelium) had increased mortality compared with other groups following MCMV infection indicating that TLR5+ hematopoietic cells are required for protective immunity against CMV infection. In summary, flagellin caused selective early increased activation of NK cells and reduced activation of CD4+ T cells in presence of MCMV infection. The protective effect of flagellin on anti-CMV immune responses are IFN-γ dependent but independent of IL-12 and IDO. The TLR5+ hematopoietic cells are required for the protective immunity against CMV infection. These data suggest the clinical importance of flagellin in modulating both innate and adaptive immunity against CMV infection and also as an alternative GvHD prophylaxis in allo-BMT recipients. Disclosures: No relevant conflicts of interest to declare.

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

2020 ◽  
Author(s):  
Hasi Chaolu ◽  
Xinri Zhang ◽  
Xin Li ◽  
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.

TLR5 Agonist Recombinant Flagellin, CBLB502 Increases Anti-CMV Immunity By Increasing T-Bet and Eomes Expressing T Cells after MCMV Infection

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3082-3082
Author(s):  
Mohammad Sohrab Hossain ◽  
Zaid Al-Kadhimi ◽  
Edmund K Waller
Keyword(s):  
T Cells ◽  
Transcription Factors ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Conflicts Of Interest ◽  
Infectious Agent ◽  
Cmv Infection ◽  
Mcmv Infection ◽  
Allogenic Bone

Abstract We have previously shown that highly purified cGMP grade recombinant flagellin, CBLB502, a TLR5 agonist, protected allo-HSCT recipients from GvHD without compromising anti-CMV immunity of donor T cells in vivo. A single prophylactic dose (25mg/mouse i.p) of CBLB502 in wild type C57BL/6 (B6) mice also enhanced anti-CMV immunity of NK cells against CMV. CMV is a potential opportunistic infectious agent and most often causes severe life-threatening clinical complications in immunocompromized patients, particularly in cancer patients treated with allogenic bone marrow transplantation (allo-BMT). A mechanism by which CBLB502 protected host against CMV infection has not been well investigated. Transcription factors ROR gammaT, T-bet and EOMES are known to control immune responses of innate and adaptive immunity in vivo. In this study, we have explored the transcription factors ROR gammaT, T-bet and EOMES expressing NK and T cells in CBLB502-treated and MCMV-infected B6 mice. Total numbers of spleen cells increased by 1.7-fold within 48 hours of CBLB502 treatment (day 0 MCMV infection) and by 2.3-fold on day 3 after MCMV infection compared with PBS-treated MCMV-infected mice. T-bet expressing CD8+ T cells (not CD4+ T cells) increased significantly on day 0 of mCMV infection, 48hrs after CBLB502 treatment (CD4, p=0.1; CD8, p<0.001) and both CD4+ and CD8+ T cells increased significantly on day 3 (CD4, p<0.001; CD8, p<0.01) after MCMV infection. CBLB502 treatment had no effect on the ROR gammaT expression on T cells. Interestingly, although EOMES expressing CD4+ and CD8+ T cells increased significantly on day 0 (48 hrs after CBLB502 treatment) (CD4, p<0.01; CD8, p<0.01) and day 3 (CD4, p<0.001; CD8, p<0.001) in the spleens of CBLB502-treated mice compared with the PBS-treated mice, EOMES expression on CMV-specific tetramer+ CD8+ T cells rapidly decreased by day 10 after infection in both CBLB502 and PBS treated mice. However, >90% of CMV-specific CD8+ T cells had persistent expression of T-bet even after 30 days post CMV infection. In contrast, CBLB502 treatment did not have a significant effect on the numbers of ROR gammaT, T-bet and EOMES expressing NK cells compared with PBS-treated mice. These data suggest that 1) CBLB502-induced anti-CMV immunity of T cells is persistently maintained through increased T-bet expression but not by EOMES expression; and 2) CBLB502-induced anti-viral immune regulation is not maintained through transcription factor ROR gammaT. 3) CBLB502-induced increased anti-CMV immunity of NK cells is independent of transcription factors ROR gammaT, T-bet and EOMES. Taken together, these data support the early peri-transplant use of TLR5 agonists as a novel method to enhance antigen-specific anti-viral immunity in recipients of allo-BMT. Disclosures No relevant conflicts of interest to declare.

scholarly journals Circulating Cytokines and Lymphocyte Subsets in Patients Who Have Recovered from COVID-19

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Hasichaolu ◽  
Xinri Zhang ◽  
Xin Li ◽  
Xin Li ◽  
Dongyan Li
Keyword(s):  
T Cells ◽  
B Cells ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Lymphocyte Subsets ◽  
Healthy Controls ◽  
Cell Counts ◽  
Tnf Α ◽  
Ifn Γ

To investigate the immune status of people who previously had COVID-19 infections, we recruited two-week postrecovery patients and analyzed circulating cytokine and lymphocyte subsets. We measured levels of total lymphocytes, CD3+ T cells, CD4+ T cells, CD8+ T cells, CD19+ B cells, and 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 postrecovery 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, two-week postrecovery 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 postrecovery patients, T cells, particularly CD4+ T cells, were positively correlated with CD19+ B cell counts. Additionally, CD8+ T cells were 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, postrecovery decreases in lymphocyte subsets and increases in cytokines were identified as independent predictors of rehabilitation efficacy. These findings indicate that the immune system gradually recovers following COVID-19 infection; however, the sustained hyperinflammatory 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.

scholarly journals Elevated Gamma Interferon-Producing NK Cells, CD45RO Memory-Like T Cells, and CD4 T Cells Are Associated with Protection against Malaria Infection in Pregnancy

2008 ◽  
Vol 76 (4) ◽  
pp. 1678-1685 ◽  
Author(s):  
Caroline Othoro ◽  
Julie M. Moore ◽  
Kathleen A. Wannemuehler ◽  
Sichangi Moses ◽  
Altaf Lal ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Cd4 T Cells ◽  
Malaria Infection ◽  
Immune Cell ◽  
Placental Malaria ◽  
Gamma Interferon ◽  
Ifn Γ ◽  
Cell Subpopulations ◽  
In Pregnancy

ABSTRACT Previous studies have shown that gamma interferon (IFN-γ) production in the placenta is associated with protection against placental malaria. However, it remains unknown which IFN-γ-producing cell subpopulations are involved in this protection and whether the cellular immune components of protection are the same in the peripheral and the placental blood compartments. We investigated cell subpopulations for CD4, CD8, and CD45RO memory-like T cells and CD56+/CD3− natural killer (NK) cells and for IFN-γ production by these cells in maternal peripheral and placental intervillous blood in relation to the status of malaria infection in pregnancy. Of 52 human immunodeficiency virus-negative enrolled pregnant women residing in Western Kenya, 20 had placental parasitemia. We found that the percentages of CD45RO memory-like and CD4 T cells were significantly higher in the periphery than in the placenta, while the CD56/CD3− NK-cell percentage was higher in the placenta than in the periphery, suggesting differences in immune cell profiles between the two blood compartments. Furthermore, the percentages of peripheral CD45RO memory-like and CD4 T cells were significantly elevated in aparasitemic women compared to levels in the parasitemic group, with aparasitemic multigravid women having the highest percentages of CD45RO memory-like and CD4 T cells. In contrast, at the placental level, IFN-γ production by innate NK cells was significantly increased in aparasitemic women compared to parasitemic women, regardless of gravidity. These results suggest that the elevated IFN-γ-producing NK cells in the placenta and CD45RO memory-like and CD4 T cells in peripheral blood may be involved in protection against malaria infection in pregnancy.

scholarly journals High Levels of CD38+ NK Cells Contribute to Immune Imbalance of Th1/Th2 and Th17/Treg in Rheumatoid Arthritis

2020 ◽  
Author(s):  
Hongxing Wang ◽  
Kehua Fang ◽  
Xiaotian Chang
Keyword(s):  
Rheumatoid Arthritis ◽  
Flow Cytometry ◽  
T Cells ◽  
Synovial Fluid ◽  
Nk Cells ◽  
Treg Cell ◽  
Cd4 T Cells ◽  
Th17 Cell ◽  
Ifn Γ ◽  
Cell Proportion

Abstract Background Increased CD38 expression and CD38+ cell proportion as well as their importance had been reported in rheumatoid arthritis (RA).Methods The proportion of lymphocyte subtypes in RA patients and rats with collagen-induced arthritis (CIA) was examined using flow cytometry. CD38+ NK cells, CD38+ NKT cells and CD4+ T cells as well as mononuclear cells (MNCs) depleted of CD38+ cells were isolated from RA synovial fluid using flow cytometry and cocultured in transwell apparatus.Results This study detected a significantly increased CD38+ NK cell proportion and a decreased CD38+ NKT cell proportion in RA peripheral blood and synovial fluid. The CD38+ NK/CD38+ NKT ratio was positively correlated with the disease activity. A similar result was observed in CIA rats. When CD38+ NK cells were cocultured with MNCs, the Treg cell proportion in MNCs and IL-10 level significantly decreased, and Th17 cell proportion and IFN-γ level increased. When the CD38+ NK cells were pretreated with monoclonal anti-CD38 antibody, Treg cell proportion and IL-10 level significantly increased, and the Th17 cell proportion and IFN-γ and IL-6 level decreased. When CD38+ NK cells were cocultured with CD4+ T cells, the Th1/Th2 and Th17/Treg ratios significantly increased, and mTOR signaling was activated in the cells. When the CD38+ NK cells were pretreated with the anti-CD38 antibody, the opposite result was obtained. Coculturing CD38+ NKT cells with MNCs or CD4+T cells showed opposite results. The anti-CD38 antibody also significantly increased TGF-β expression in the CD38+ NK cells.Conclusions Our results suggest that a high CD38+ NK and low CD38+ NKT proportion in RA elevates Th1/Th2 and Th17/Treg ratio to contribute to the pathogenesis.

Host and Donor Immune Responses Contribute to Anti-Viral Effects of Amotosalen-Treated Donor Lymphocytes Following Early Post-Transplant CMV Infection.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3217-3217
Author(s):  
Mohammad S. Hossain ◽  
John D. Roback ◽  
Ned Waller
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Opportunistic Infections ◽  
Lethal Dose ◽  
Mcmv Infection ◽  
Post Transplant ◽  
Allogeneic Bmt ◽  
Tnf Α ◽  
Donor T Cells ◽  
Ifn Γ

Abstract Background: GvHD and opportunistic infections are the major causes of morbidity and mortality in cancer patients treated with allogeneic BMT. In allogeneic BMT patients, donor derived T-cells help eradicate residual cancer and fight against opportunistic infections but they also cause the major deleterious effects, including GvHD which is the result of host allo-antigens recognition by the donor T-cells. Moreover, donor T-cells also play a critical role in promoting stem cell engraftment, encouraging rapid recovery of cellular immunity, and decreasing the probability of disease relapse. Thus, to establish a therapeutically useful adoptive immunotherapy using donor T-cells, separation of the beneficial anti-opportunistic infection and anti-tumor effects of donor T-cells from the deleterious GvHD effect are highly desirable. We previously showed that amotosalen-treated splenocytes rescued recipients from a lethal dose of MCMV administered on day 0 in experimental parent to F1 allogeneic bone marrow transplant (BMT). To model early post-transplant CMV reaction, in this study, we investigated the anti-viral immune responses and GvHD activity of treated donor T-cells after infecting allogeneic BMT recipients with a lethal dose of MCMV on 7 days post transplant. Methods: Using a parent to F1 mouse BMT model, splenocytes (3×106 untreated or 10×106 amotosalen-treated) harvested from the MCMV immunized C57BL/6 donors were transplanted along with 5×106 T-cell depleted bone marrow (TCD BM) from naïve congeneic mice into lethally irradiated (11Gy) CB6F1 (C57BL/6 × Balb/C) recipients. Recipient mice were infected i.p. with a sublethal dose (5×104 pfu per mouse) of MCMV 7 days after transplant. Flow cytometry was used to quantitate T cell chimerism (in recipient spleen and thymus) and MCMV-peptide specific tetramer+ CD8+ T-cells. Serum IFN-γ and TNF-α were determined by ELISA. Liver viral load was determined by counting PFU in tissue homogenates plated onto 3T3 confluent monolayers. Results: MCMV infection in recipients of amotosalen-treated splenocytes did not cause any mortality whereas recipients of untreated splenocytes had 40% early mortality due to acute GvHD. Like the recipients of untreated splenocytes, recipients of amotosalen-treated splenocytes effectively cleared MCMV from their liver within 10 days of infection. In contrast to full donor chimerism in recipients of untreated splenocytes, recipients of amotosalen-treated splenocytes showed mixed chimerism with donor spleen- and host-derived MCMV peptide specific tetramer+ CD8+ T cells that proliferated following day 7 post MCMV infection. Significantly higher numbers of host derived CD4−CD8− (DN) TCRαβT-cells appeared in the spleen with peak on day 3 post MCMV infection among recipients of amotosalen-treated splenocytes compared with the recipients of untreated splenocytes. Lower levels of serum IFN-γ and TNF-α and preservation of thymic function were also noted in the recipients of amotosalen-treated splenocytes compared with the recipients of untreated splenocytes following MCMV infection. Conclusion: Adoptive immunotherapy with amotosalen-treated T cells is an ideal therapeutic approach that facilitates early hematopoietic engraftment, anti-viral donor immune reconstitution and preserves early post-transplant host immunity leading to protection from lethal viral infection without causing aGvHD.

scholarly journals Murine Cytomegalovirus Inhibits Interferon γ–induced Antigen Presentation to CD4 T Cells by Macrophages Via Regulation of Expression of Major Histocompatibility Complex Class II–associated Genes

1998 ◽  
Vol 187 (7) ◽  
pp. 1037-1046 ◽  
Author(s):  
Mark T. Heise ◽  
Megan Connick ◽  
Herbert W. Virgin
Keyword(s):  
T Cells ◽  
Antigen Presentation ◽  
Mhc Class Ii ◽  
Cd4 T Cells ◽  
Class Ii ◽  
Murine Cytomegalovirus ◽  
Cell Surface Expression ◽  
Mrna Levels ◽  
Mcmv Infection ◽  
Ifn Γ

CD4 T cells and interferon γ (IFN-γ) are required for clearance of murine cytomegalovirus (MCMV) infection from the salivary gland in a process taking weeks to months. To explain the inefficiency of salivary gland clearance we hypothesized that MCMV interferes with IFN-γ induced antigen presentation to CD4 T cells. MCMV infection inhibited IFN-γ–induced presentation of major histocompatibility complex (MHC) class II associated peptide antigen by differentiated bone marrow macrophages (BMMφs) to a T cell hybridoma via impairment of MHC class II cell surface expression. This effect was independent of IFN-α/β induction by MCMV infection, and required direct infection of the BMMφs with live virus. Inhibition of MHC class II cell surface expression was associated with a six- to eightfold reduction in IFN-γ induced IAb mRNA levels, and comparable decreases in IFN-γ induced expression of invariant chain (Ii), H-2Ma, and H-2Mb mRNAs. Steady state levels of several constitutive host mRNAs, including β-actin, cyclophilin, and CD45 were not significantly decreased by MCMV infection, ruling out a general effect of MCMV infection on mRNA levels. MCMV effects were specific to certain MHC genes since IFN-γ–induced transporter associated with antigen presentation (TAP)2 mRNA levels were minimally altered in infected cells. Analysis of early upstream events in the IFN-γ signaling pathway revealed that MCMV did not affect activation and nuclear translocation of STAT1α, and had minor effects on the early induction of IRF-1 mRNA and protein. We conclude that MCMV infection interferes with IFN-γ–mediated induction of specific MHC genes and the Ii at a stage subsequent to STAT1α activation and nuclear translocation. This impairs antigen presentation to CD4 T cells, and may contribute to the capacity of MCMV to spread and persist within the infected host.

scholarly journals First Evidence of Restoration of T and NK Cell Compartment after Venetoclax Treatment

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1860-1860 ◽  
Author(s):  
Iris de Weerdt ◽  
Tom Hofland ◽  
Johan Dobber ◽  
Julie Dubois ◽  
Eric Eldering ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Cytokine Production ◽  
Nk Cell ◽  
Advisory Committees ◽  
Ifn Γ

Abstract Introduction Chronic lymphocytic leukemia (CLL) is characterized by a profound immune suppression. In addition, CLL cells evade immune destruction by interacting with cells of the adaptive immune system, resulting in dysfunctional T cells. CD4+ T cells are skewed towards a TH2-profile and the number of regulatory T (Treg) cells, that diminish cellular immune responses, is increased in CLL patients. CD8+ T cells resemble exhausted T cells and have reduced cytotoxic, yet increased cytokine production capacity. The cytotoxic function of NK cells is impaired in CLL patients, but in contrast to CD8+ T cells their cytokine production is also compromised, presumably induced by CLL cells. These data are chiefly obtained from studies on peripheral blood (PB). Although the lymph node (LN) compartment has a central role in the pathobiology of CLL, very little is known about the composition of non-malignant lymphocytes in LN tissue. The Bcl-2 inhibitor venetoclax (Ven) is highly effective in CLL and, especially in combination with anti-CD20 monoclonal antibodies such as obinutuzumab (O), results in high rates of minimal residual disease (MRD) undetectable responses. However, the prospective effects of venetoclax on non-malignant lymphocytes in patient samples remain largely unexplored. Methods PB and LN biopsy specimens were collected at baseline from patients enrolled in the 1st-line FCR-unfit HOVON 139 / GIVE trial. Study treatment consisted of O (cycle 1-2), Ven+O (cycle 3-8) and Ven (cycle 9-14). Immune composition was analyzed by 7-color flow cytometry. Baseline PB samples were compared to paired LN samples. Moreover, PB samples of the first patients that completed 6 cycles of Ven monotherapy (cycle 14) were compared to baseline. Cytokine production and degranulation of T and NK cells was studied after stimulation of PBMCs with PMA/Ionomycin. Results Comparison of LN (n=28) vs PB (n=48) revealed a larger proportion of T cells in LN (13.2% vs 5.1% of the lymphocytes), at the expense of CLL cells, with a skewed CD4:CD8 ratio (5.2 in LN vs 1.8 in PB). Within the CD4+ T cells, significantly higher levels of both follicular T helper cells (15. 7% vs 5.2%) and Tregs (11.5% vs 6.9%) were found in LN (see Table). CD4+ T cells mostly consisted of naïve and memory T cells in both PB and LN. There were fewer CD8+ T cells and especially fewer effector CD8+ T cells in the LN in comparison to PB. CD8+ T cells in LN mostly had a naïve and memory phenotype. An increased percentage of LN-residing CD8+ T cells expressed the exhaustion marker PD-1 as compared to PB CD8+ T cells (30.4% in LN vs 12.4% in PB). We then compared PB baseline samples to PB obtained after cycle 14 (n=11). Ten patients achieved MRD undetectable levels (<10-4, determined by flow cytometry) and 1 patient was MRD intermediate (10-4-10-2). As expected, the treatment regimen led to complete elimination of CD19+ B cells. In contrast, absolute numbers of CD4+ and CD8+ T cells did not change during treatment. Differentiation status of CD4+ and CD8+ T cells remained similar. Interestingly, the proportion and absolute number of Tregs decreased after treatment (6.1% vs 0.9% of CD4+ T cells). After stimulation with PMA/Ionomycin, the percentage of IL-2 producing CD4+ T cells increased after treatment, leading to a higher IL-2:IL-4 ratio, that suggests normalization towards a TH1-profile. Fewer CD8+ T cells expressed PD-1 after treatment. The fraction of CD8+ T cells that produced IFN-γ (69.8% vs 56.2%) and TNF-α (58.4% vs 40.3%) decreased. Degranulation of CD8+ T cells did not change upon treatment. After treatment, the capacity of NK cells to degranulate increased. In addition, a larger proportion of NK cells produced IFN-γ, suggesting recovery of NK cell function after treatment. Conclusion In conclusion, our data strengthen the view that CLL cells reside in an immune suppressive environment in the LN. Moreover, we provide the first evidence that the Ven+O regimen does not harm non-malignant lymphocyte populations other than B cells. Both the improved cytokine production of NK cells and diminished cytokine production of CD8+ T cells may point to normalization of immune function. Collectively, the phenotypical and functional changes observed may reflect the eradication of the immunosuppressive CLL clone by Ven+O and subsequent recovery of the immune microenvironment in CLL patients. Disclosures Eldering: Celgene: Research Funding. Mobasher:F. Hoffmann-La Roche Ltd: Other: Ownership interests non-PLC; Genentech Inc: Employment. Levin:Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Kater:Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2668-2668 ◽  
Author(s):  
Alice Bertaina ◽  
Perla Filippini ◽  
Valentina Bertaina ◽  
Barbarella Lucarelli ◽  
Aurelie Bauquet ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Lymphoblastic Leukemia ◽  
Γδ T Cells ◽  
Cell Phenotype ◽  
Ifn Γ

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.

scholarly journals Osteoclast-expanded super-charged NK-cells preferentially select and expand CD8+ T cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kawaljit Kaur ◽  
Meng-Wei Ko ◽  
Nick Ohanian ◽  
Jessica Cook ◽  
Anahid Jewett
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Nk Cell ◽  
Important Relationship ◽  
Ifn Γ ◽  
Blt Mice ◽  
Tissue Compartments ◽  
Selection Of

AbstractOsteoclasts (OCs) and much less dendritic cells (DCs) induce significant expansion and functional activation of NK cells, and furthermore, the OC-expanded NK cells preferentially increase the expansion and activation of CD8+ T cells by targeting CD4+ T cells. When autologous OCs were used to expand patient NK cells much lower percentages of expanded CD8+ T cells, decreased numbers of expanded NK cells and decreased functions of NK cells could be observed, and the addition of allogeneic healthy OCs increased the patients’ NK function. Mechanistically, OC-expanded NK cells were found to lyse CD4+ T cells but not CD8+ T cells suggesting potential selection of CD8+ T cells before their expansion by OC activated NK cells. In agreement, Increased IFN-γ secretion, and NK cell-mediated cytotoxicity and higher percentages of CD8+ T cells, in various tissue compartments of oral tumor-bearing hu-BLT mice in response to immunotherapy by OC-expanded NK cells were observed. Thus, our results indicate an important relationship between NK and CD8+ T cells.

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