Innate CD4+CD25+ regulatory T cells are required for oral tolerance and inhibition of CD8+ T cells mediating skin inflammation

Blood ◽  
2003 ◽  
Vol 102 (9) ◽  
pp. 3295-3301 ◽  
Author(s):  
Bertrand Dubois ◽  
Ludivine Chapat ◽  
Anne Goubier ◽  
Martine Papiernik ◽  
Jean-François Nicolas ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Oral Tolerance ◽  
Skin Inflammation ◽  
Cd8 T Cell ◽  
Contact Hypersensitivity ◽  
Naturally Occurring

AbstractTo elucidate the role of CD4+CD25+ regulatory T cells in oral tolerance, we used the model of contact hypersensitivity (CHS) to 2,4-dinitrofluorobenzene (DNFB), which is mediated by CD8+ Tc1 effector cells independently of CD4+ T-cell help. Conversely to normal mice, invariant chain knock-out (KO) (Ii°/°) mice, which are deficient in CD4+ T cells, cannot be orally tolerized and develop a chronic hapten-specific CHS response. Transfer of naive CD4+ T cells before hapten gavage into Ii°/° mice restores oral tolerance by a mechanism independent of interleukin-10 (IL-10) production by CD4+ T cells. That naturally occurring CD4+CD25+ T cells are critical for oral tolerance induction is demonstrated by the finding that (1) transfer of CD4+CD25+ but not CD4+CD25– T cells into Ii°/° recipients completely prevents the CHS response and skin infiltration by CD8+ T cells, by blocking development of hapten-specific CD8+ T cells; (2) in vivo depletion of CD4+CD25+ cells by antibody treatment in normal mice impairs oral tolerance; and (3) CD4+CD25+ T cells inhibit hapten-specific CD8+ T-cell proliferation and interferon γ (IFNγ) production, in vitro. These data show that naturally occurring CD4+CD25+ T cells are instrumental for orally induced tolerance and are key actors for the control of antigen-specific CD8+ T-cell effectors mediating skin inflammation.

BK Polyomavirus–Specific CD8 T-Cell Expansion In Vitro Using 27mer Peptide Antigens for Developing Adoptive T-Cell Transfer and Vaccination

2020 ◽  
Author(s):  
Maud Wilhelm ◽  
Amandeep Kaur ◽  
Marion Wernli ◽  
Hans H Hirsch
Keyword(s):  
T Cells ◽  
T Cell ◽  
Kidney Transplant ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Cell Responses ◽  
Bk Polyomavirus

Abstract Background BK polyomavirus (BKPyV) remains a significant cause of premature kidney transplant failure. In the absence of effective antivirals, current treatments rely on reducing immunosuppression to regain immune control over BKPyV replication. Increasing BKPyV-specific CD8 T cells correlate with clearance of BKPyV DNAemia in kidney transplant patients. We characterized a novel approach for expanding BKPyV-specific CD8 T cells in vitro using 27mer-long synthetic BKPyV peptides, different types of antigen-presenting cells, and CD4 T cells. Methods Langerhans cells and immature or mature monocyte-derived dendritic cells (Mo-DCs) were generated from peripheral blood mononuclear cells of healthy blood donors, pulsed with synthetic peptide pools consisting of 36 overlapping 27mers (27mP) or 180 15mers (15mP). BKPyV-specific CD8 T-cell responses were assessed by cytokine release assays using 15mP or immunodominant 9mers. Results BKPyV-specific CD8 T cells expanded using 27mP and required mature Mo-DCs (P = .0312) and CD4 T cells (P = .0156) for highest responses. The resulting BKPyV-specific CD8 T cells proliferated, secreted multiple cytokines including interferon γ and tumor necrosis factor α, and were functional (CD107a+/PD1–) and cytotoxic. Conclusions Synthetic 27mP permit expanding BKPyV-specific CD8 T-cell responses when pulsing mature Mo-DCs in presence of CD4 T cells, suggesting novel and safe approaches to vaccination and adoptive T-cell therapies for patients before and after kidney transplantation.

Universal CD137 Expression upon Activation Allows Efficient Isolation of a Broad Repertoire of Virus-Specific CD8+ and CD4+ T Cells for Adoptive Immunotherapy.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2222-2222
Author(s):  
Maarten L. Zandvliet ◽  
J.H. Frederik Falkenburg ◽  
Inge Jedema ◽  
Roelof Willemze ◽  
Henk-Jan Guchelaar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cd8 T Cell ◽  
Cell Populations ◽  
T Cell Lines ◽  
Kinetics Of ◽  
Ifng Production

Abstract Reactivation of adenovirus (ADV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) can cause serious morbidity and mortality during the prolonged period of immune deficiency following allogeneic stem cell transplantation. It has been shown that adoptive transfer of donor-derived virus-specific T cells can be a successful strategy to control viral reactivation. To provide safe and effective anti-viral immunotherapy, we aimed to generate combined CD8+ and CD4+ T cell lines with high specificity for a broad range of viral epitopes. Isolation by the IFNg capture assay of virus-specific T cells that produce IFNg upon activation allows the generation of highly specific T cell lines without the need for extensive culture. However, it has been recently shown that specific upregulation of the co-stimulatory molecule CD137 upon antigen-specific activation of CD8+ and CD4+ T cells can also be used for isolation. We therefore analyzed IFNg production and CD137 expression by CD8+ and CD4+ T cells upon incubation of peripheral blood mononuclear cells (PBMC) from seropositive donors with peptides corresponding to 17 defined MHC class I restricted minimal epitopes from 10 different ADV, CMV, EBV and influenza (FLU) proteins, and 15-mer or 30-mer peptides containing MHC class II restricted epitopes from CMV pp65 or ADV hexon. Using tetramer and intracellular IFNg staining we first determined the fraction of CD8+ T cells that produced IFNg upon activation with the minimal epitopes. Specific IFNg production was observed for 58–100% of tetramer+ CD8+ T cells specific for CMV pp65 (n=6), and 83% for FLU (n=1), but only 18–58% for CMV pp50 (n=3) or IE-1 (n=3), 4–91% for EBV latent (n=3) and lytic (n=3) epitopes, and 41–63% for ADV hexon (n=2). In contrast to the variation in the fraction of IFNg-producing cells, we observed homogeneous upregulation of CD137 by the virus-specific tetramer+ T cell populations upon activation. In 2 cases where no CD137 expression by tetramer+ T cells could be detected, no IFNg production was observed either. These data suggest that the majority of CD8+ T cells specific for CMV pp65 or FLU can be isolated on basis of IFNg production, but only part of CD8+ T cell populations specific for other viral proteins, while complete virus-specific CD8+ T cell populations may be isolated on basis of CD137 expression. Activation of CD4+ T cells specific for CMV pp65 or ADV hexon with 15-mer or 30-mer peptides induced both specific IFNg production and CD137 expression. To investigate whether multiple virus-specific T cell populations could be isolated simultaneously, we next determined the kinetics of IFNg production after activation with defined MHC class I epitopes or peptides containing MHC class II epitopes. CMV- and EBV-specific CD8+ T cells and CMV-specific CD4+ T cells showed a rapid induction of IFNg production, which peaked after 4 hours and decreased thereafter. In contrast, ADV- and FLU-specific CD8+ T cells and ADV-specific CD4+ T cells, predominantly having a more early differentiation phenotype (CD27+CD28+) compared to CMV- and EBV-specific T cells, showed peak IFNg production after 8 hours that continued for more than 48 hours. This difference in phenotype and IFNg kinetics may suggest that the persistent and frequent presentation of CMV and EBV epitopes in vivo, in contrast to an intermittent exposure to ADV and FLU epitopes, drives differentiation and shapes the kinetics of the IFNg response of specific T cells. Kinetic analysis of CD137 expression showed uniform upregulation by virus-specific CD8+ T cell populations from day 1 to day 4 after activation, which peaked at day 2, suggesting that this may be the optimal time point for CD137-based isolation. In a limited number of experiments, virus-specific CD8+ and CD4+ T cells could be isolated based on CD137 expression within the same timeframe. These data indicate that virus-specific T cell populations can be more efficiently isolated at one time point on basis of CD137 expression than on basis of IFNg production, due to differences in IFNg kinetics. In conclusion, this study shows that T cell lines generated by CD137 isolation may comprise a significant number of virus-specific T cells which do not produce IFNg, but may have other effector functions. Furthermore, CD137-based enrichment may be more robust and allows the efficient simultaneous isolation of multiple virus-specific T cell populations due to uniform kinetics of CD137 expression.

Perforin Is Important For Both CD4+ and CD8+ T Cell-Mediated Graft-Versus-Tumor Effect But Plays Differential Roles In CD4+ and CD8+ T Cell Expansion After Allogeneic Transplantation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3255-3255
Author(s):  
Nicholas Leigh ◽  
Guanglin Bian ◽  
Wei Du ◽  
George L. Chen ◽  
Hong Liu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cell Expansion ◽  
Cd8 T Cell ◽  
Cd4 T Cell ◽  
T Cell Proliferation ◽  
T Cell Expansion ◽  
Time Point

Abstract Graft versus tumor (GVT) effect is the desired and integral outcome for successful allogeneic bone marrow transplantation (allo-BMT) for cancer patients. This effect is dependent on T cell mediated recognition and elimination of residual host tumor cells present after allo-BMT. T cell killing is mediated primarily via three pathways: perforin/granzymes, Fas/FasL, and cytotoxic cytokines. Recent work from our lab has revealed a detrimental role for granzyme B (GzmB) in GVT effect due to its role in activation induced cell death (AICD) of CD8+ T cells. As a result, GzmB-/- CD8+ T cells exhibited higher expansion after allo-BMT and subsequently provided better tumor control. Our current study sought to determine the role of perforin (Prf1) in GVT effect mediated by both CD4+ and CD8+ T cells. Using the MHC-mismatched C57BL/6 (H-2b) to BALB/c (H-2d) allo-BMT model, we first confirmed previous findings that when transplanting CD8+ T cells along with T cell depleted (TCD) BM cells, donor CD8+ T cells require Prf1 to mediate GVT effect against allogeneic A20 lymphoma (Fig 1A, Prf1-/- (n=4) vs WT (n=4), *P<0.05). In addition, our data suggest that Prf1 is also required for CD4+ T cells to effectively mediate GVT effect against A20, as transplant with Prf1-/- CD4+CD25- T cells does not control tumor growth as well as WT controls (Fig 1B). Our previous work showed that GzmB deficiency allows for less AICD and subsequently more CD8+ T cell expansion. New data now show a similar effect for Prf1 in CD8+ T cell accumulation, as Prf1-/- CD8+ T cells outcompete WT CD8+ T cells (CD45.1+) when these two genotypes are mixed in equal numbers and transplanted into tumor bearing BALB/c mice (n=5/time point, *P=0.02 day 9)(Fig 1C). This competitive advantage was due to less AICD in the Prf1-/- CD8+ T cells. However, Prf1 appears to be required for efficient GVT activity, because the higher number of Prf1-/- CD8+ T cells are still less capable than WT counterparts in controlling tumor growth. We next tested the effect of Prf1 in AICD in CD4+CD25- T cells, and again co-transplanted WT CD45.1+ and Prf1-/- CD4+CD25- T cells into tumor bearing mice for a competition assay. Unexpectedly, WT CD4+CD25- T cells accumulate to significantly higher numbers when in direct competition with Prf1-/- CD4+CD25- T cells (n=4/time point, **,P<0.01)(Fig 1D). When we measured apoptotic cells with Annexin V staining, we found that WT CD4+CD25- T cells still had significantly more AICD (Prf1-/- 38.3 ± 4.2% vs. WT 48.1 ± 5.1%, P<0.01 on day 7 post-BMT; Prf1-/- 12.7 ± 1.0% vs. WT 18.1 ± 3.4%, P<0.03 on day 9 post-BMT). This result suggests that while Prf1 has an important role in AICD, it may also play a role in another feature of CD4+ T cell biology. We then explored the hypothesis that may Prf1 promote CD4+ T cell proliferation by evaluating Hoescht staining on day 9 post-BMT. Preliminary results suggest that Prf1 may enhance T cell proliferation, as Prf1-/- CD4+ T cells have less actively dividing cells at this time point. Therefore, Prf1 appears to have a surprising role after allo-BMT in sustaining T cell expansion for CD4+ T cells, but not for CD8+ T cells. Another factor influencing GVT effect may be T cell phenotype. Our previous work with CD8+ T cells suggests that more effector memory (CD62LLOWCD44HIGH) T cells accumulate in the absence of GzmB, and that GzmB-/- CD8+ T cells exhibited higher GVT activity than WT controls. We now found that while Prf1-/- CD4+ T cells also skewed towards the effector memory phenotype (CD62LLOWCD44HIGH), loss of Prf1 still reduced the ability of CD4+ T cells to control tumor growth in this model of allo-BMT. In summary, our results suggest that Prf1 plays an important role in GVT responses mediated not only by CD8+ T cells but also by CD4+ T cells, which were shown in previous literature to mainly utilize Fas ligand and cytokine systems to mediate GVT activity. In addition, Prf1 can cause AICD to both CD4+ and CD8+ T cells after allo-BMT. While Prf1-induced AICD reduces CD8+ T cell expansion, Prf1 appears to play a previously unrecognized role enhancing CD4+ T cell proliferation via an unidentified mechanism. Disclosures: No relevant conflicts of interest to declare.

CD4 T lymphocytes are primed to express Fas ligand by CD4 cross-linking and to contribute to CD8 T-cell apoptosis via Fas/FasL death signaling pathway

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 195-202 ◽  
Author(s):  
Masaki Tateyama ◽  
Naoki Oyaizu ◽  
Thomas W. McCloskey ◽  
Soe Than ◽  
Savita Pahwa
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Interleukin 2 ◽  
Cd8 T Cell ◽  
T Cell Subsets ◽  
Cross Linking ◽  
Induced Apoptosis ◽  
Hiv 1

CD4 molecules serve as coreceptors for the T-cell receptor (TCR)/CD3 complex that are engaged coordinately with TCR and facilitate antigen-specific T-cell activation leading to interleukin 2 (IL-2) production and proliferation. However, cross-ligation of CD4 molecules prior to TCR stimulation has been shown to prime CD4 T cells to undergo apoptosis. Although in vivo and in vitro experiments have implicated the involvement of Fas/FasL interaction in this CD4 cross-linking (CD4XL)-induced apoptosis, detailed mechanisms to account for cell death induction have not been elucidated. In the present study, we demonstrate that CD4XL in purified T cells not only led to Fas up-regulation but also primed CD4 T cells to express FasL upon CD3 stimulation and rendered the T cells susceptible to Fas-mediated apoptosis. Notably, in addition to CD4+ T cells, CD4XL-induced sensitization for apoptosis was observed in CD8+ T cells as well and was associated with Bcl-x down-modulation. Both CD4 and CD8 T-cell subsets underwent apoptosis following cell–cell contact with FasL+ CD4 T cells. CD28 costimulation abrogated CD4XL/CD3-induced apoptosis with restoration of IL-2 production and prevented Bcl-x down-modulation. As CD4 molecules are the primary receptors for human immunodeficiency virus 1 (HIV-1), we conclude that HIV-1 envelope mediated CD4XL can lead to the generation of FasL-expressing CD4+ T cells that can lead to apoptosis of CD4 as well as CD8 T cells. These findings implicate a novel mechanism for CD8 T-cell depletion in HIV disease.

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

Blood ◽  
2008 ◽  
Vol 112 (6) ◽  
pp. 2400-2410 ◽  
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.

scholarly journals c-Jun NH2-Terminal Kinase (JNK)1 and JNK2 Have Distinct Roles in CD8+ T Cell Activation

2002 ◽  
Vol 195 (7) ◽  
pp. 811-823 ◽  
Author(s):  
Dietrich Conze ◽  
Troy Krahl ◽  
Norman Kennedy ◽  
Linda Weiss ◽  
Joanne Lumsden ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Cd8 T Cell ◽  
Cell Surface Expression ◽  
Chain Gene ◽  
Α Chain

The c-Jun NH2-terminal kinase (JNK) signaling pathway is induced by cytokines and stress stimuli and is implicated in cell death and differentiation, but the specific function of this pathway depends on the cell type. Here we examined the role of JNK1 and JNK2 in CD8+ T cells. Unlike CD4+ T cells, the absence of JNK2 causes increased interleukin (IL)-2 production and proliferation of CD8+ T cells. In contrast, JNK1-deficient CD8+ T cells are unable to undergo antigen-stimulated expansion in vitro, even in the presence of exogenous IL-2. The hypoproliferation of these cells is associated with impaired IL-2 receptor α chain (CD25) gene and cell surface expression. The reduced level of nuclear activating protein 1 (AP-1) complexes in activated JNK1-deficient CD8+ T cells can account for the impaired IL-2 receptor α chain gene expression. Thus, JNK1 and JNK2 play different roles during CD8+ T cell activation and these roles differ from those in CD4+ T cells.

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

2006 ◽  
Vol 74 (10) ◽  
pp. 5790-5801 ◽  
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.

Peptide Vaccination Induces Dynamic Changes in CD4+ and CD8+ T Cell Subsets: Report on the First Peptide Vaccination Trial in Patients with Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3159-3159 ◽  
Author(s):  
Krzysztof Giannopoulos ◽  
Malgorzata Kowal ◽  
Anna Dmoszynska ◽  
Jacek Rolinski ◽  
Kamila Mazurek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Immune Responses ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
T Cell Subsets ◽  
Peptide Vaccination ◽  
Tetramer Staining ◽  
Induced Changes

Abstract There is an accumulation of in vivo (graft-versus-leukemia effect) and in vitro (spontaneous remissions after infections) data providing evidence that CLL might be effectively targeted by T-cell based immunotherapy. Earlier, we characterized the receptor for hyaluronic acid mediated motility (RHAMM) as antigen associated with proliferation and negative prognosis in CLL. We also demonstrated that RHAMM-derived epitope(R3)- primed T cells were able to lyse RHAMM+ target CLL cells. Therefore, we initiated a small phase I/II clinical trial with R3 peptide vaccination for patients with CLL. Six CLL patients in Binet stage 0 of the disease were vaccinated four times at a biweekly interval with HLA-A2 restricted RHAMM-derived epitope R3 (ILSLELMKL, 300μg/dose on day 3) emulsified in incomplete Freund’s adjuvant (IFA) with concomitant administration of GM-CSF (100μg/dose, days 1–5). R3-specific T-cell responses were assessed by tetramer staining and ELISPOT assays. T-cell subsets which play a role in regulation of immune responses including CD3+CD4+CD25hiCD127loFOXP3+ T regs, Th17, CD8+CD137+, CD8+CD103+ and IL-17 producing CD8+ T cells (CD8+IL-17+) were evaluated by flow cytometry. No severe adverse events greater than CTC Io skin toxicity could be observed. Four of six patients showed a reduction of WBC during vaccination. Although these WBC changes did not meet the NCI response criteria, we described these favorable hematological changes achieved in short period of immunotherapy as hematological improvement (defined as at least 20% reduction of WBC during vaccination). The immune responses were found in 5/6 patients as assessed by tetramer-staining (positive response defined as an increase of R3-specific CD8+ T cell frequency by more than 100% after vaccination) and confirmed in 4/5 as assessed by ELISPOT assay. Patients included in this study showed median Tregs frequency of 4.2%, range: 2.5–8%. There was no significant difference of Tregs percentages between patients who improved clinically when compared with non-responders (median 6.1% vs. 3.7%). Vaccination induced Tregs in 4 patients (2 non-responders and 2 responders). Two other patients who improved hematologically did not significantly change frequency of Tregs or even reduced it during vaccination (Figure 1). Median expression of CD103 on CD8+ T cells was 1.84%, range: 0.41–5.63%. In one non-responder, we observed an increase in frequency of CD103+CD8+ T-cells during vaccination from 1.46% to 2.56%. During vaccination, changes in CD8+CD103+ T cell subset did not correlate with the frequency of Tregs, nonetheless we could find an inverse correlation with inflammatory Th17 T cells (r2=−0.5, p&lt;0.05). We could find a correlation between the frequency of Tregs and activated CD8+CD69+ T cells (r2=0.51, p&lt;0.05). Interestingly, CD8+CD137+ cells correlated with CD8+IL-17+ T cells (r2=0.54, p&lt;0.05). In conclusion, peptide vaccination in CLL patients is safe and feasible to mount immune responses against the tumor antigen RHAMM. Most of patients benefited hematologically from vaccination. Although in some patients we observed an induction of tumor-specific T cells without induction of Tregs there is a rationale to add novel active agents against Tregs in future vaccination trials. Figure 1. Peptide vaccination induced changes in WBC, percentages of regulatory T cells (Tregs) as well as R3 specific tetramer ‘CD’ T cells (tetra) of A CLL patients. Patients B, C, E and F improved hematologically during vaccination. Figure 1. Peptide vaccination induced changes in WBC, percentages of regulatory T cells (Tregs) as well as R3 specific tetramer ‘CD’ T cells (tetra) of A CLL patients. Patients B, C, E and F improved hematologically during vaccination.

Differential Effects of TCR Stimulation and IL-2 On Apoptotic Pathways in CD4 Regulatory T Cells Compared to Conventional CD4 T Cells and CD8 T Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3280-3280
Author(s):  
Kazuyuki Murase ◽  
Yutaka Kawano ◽  
Jeremy Ryan ◽  
Ken-ichi Matsuoka ◽  
Gregory Bascug ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Mitochondrial Membrane ◽  
Death Receptor ◽  
T Cell Subsets ◽  
Mitochondrial Membrane Depolarization ◽  
Apoptotic Pathways

Abstract Abstract 3280 CD4+CD25+Foxp3+ regulatory T cells (Treg) play an important role in the maintenance of self-tolerance and immune homeostasis and Treg deficiency contributes to the development of autoimmune diseases. CD4Treg, conventional CD4 T cells (Tcon) and CD8 T cells are derived from lymphocyte progenitor cells that differentiate into distinct functional subsets in the thymus before export to the peripheral circulation. As T cells differentiate and expand in the periphery, each T cell subset is differentially regulated and subjected to distinct homeostatic signals. For example, interleukin-2 (IL-2) is a critical regulator of Treg development, expansion and survival and lack of IL-2 results in selective Treg deficiency. In regulating Treg homeostasis, IL-2 has multiple and distinct effects on Treg differentiation, proliferation and susceptibility to apoptosis. To determine the mechanism whereby IL-2 affects susceptibility of Treg to apoptosis, we used a new flow cytometry-based assay (BH3 profiling) to measure the mitochondrial membrane depolarization in response to a panel of pro-apoptotic BH3 peptides (BIM, BID, BAD, NOXA, PUMA, BMF, HRK). This assay allowed us to compare “priming” which we define as susceptibility to BH3 peptide-induced mitochondrial membrane depolarization in different T cell subsets, including CD4 Treg, CD4 Tcon and CD8 T cells. We also examined cell surface expression of CD95 death receptor (Fas) and cytoplasmic expression of Bcl-2 and Ki67 as additional measures of susceptibility to apoptosis and proliferation in each subset. In resting blood obtained from healthy donors (n=10), CD4 Treg were more “primed” than either CD4 Tcon or CD8 T cells when exposed to several BH3 peptides (PUMA, BMF and the combination of BAD+NOXA). CD4 Treg were also found to have decreased expression of Bcl-2 and increased expression of CD95 and Ki67 compared to CD4 Tcon or CD8 T cells. Thus, Treg in healthy individuals have higher proliferative activity and are more susceptible to apoptosis than other major T cell subsets through both mitochondrial and death receptor pathways. To establish the functional effects of TCR stimulation and IL-2, CD4 Treg, CD4 Tcon and CD8 T cells were purified by cell sorting and cultured for 5–6 days with or without TCR stimulation (1μg/ml anti-CD3 + 1μg/ml anti-CD28) and IL-2 (100 IU/ml). Results were compared to cells cultured in media alone. Results are summarized in the table below. CD4 Tcon and CD8 T cells responded in a similar fashion to either TCR stimulation alone or TCR plus IL-2. This response included increased BH3 priming, reduced expression of Bcl-2, increased expression of CD95 and increased proliferation (Ki-67). IL-2 alone had no effect on CD4 Tcon or CD8 T cells. In contrast, TCR stimulation alone had no effect on CD4 Treg but IL-2 alone reduced BH3 priming and increased expression of Bcl-2. Combined TCR stimulation plus IL-2 in Treg increased BH3 priming, reduced expression of Bcl-2, increased expression of CD95 and increased proliferation. Thus, TCR stimulation reversed the anti-apoptotic effects of IL-2 alone and markedly increased susceptibility of Treg to apoptosis. When compared with CD4 Tcon and CD8 T cells, these studies demonstrate distinct effects of TCR stimulation and IL-2 on both mitochondrial and death receptor pathways of apoptosis in CD4 Treg and define mechanisms whereby TCR stimulation and IL-2 interact to regulate Treg homeostasis. Table 1. Effects of in vitro TCR stimulation and IL-2 on apoptotic pathways of T cell subsets TCR Stimulation IL-2 TCR + IL2 BH3 priming Bcl-2 CD95 Ki67 BH3 priming Bcl-2 CD95 Ki67 BH3 priming Bcl-2 CD95 Ki67 CD4 Treg – – – – ↓ ↑ – – ↑ ↓ ↑ ↑ CD4 Tcon ↑ ↓ ↑ ↑ – – – – ↑ ↓ ↑ ↑ CD8 ↑ ↓ ↑ ↑ – – – – ↑ ↓ ↑ ↑ Disclosures: No relevant conflicts of interest to declare.

Transient NKG2D Blockade Attenuates Graft-Versus-Host Disease While Preserving Graft-Versus-Leukemia Effects

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3242-3242 ◽  
Author(s):  
Mobin Karimi ◽  
Theresa M Leichner ◽  
Atsushi Satake ◽  
David Raulet ◽  
Taku Kambayashi
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mouse Model ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cd8 T Cell ◽  
Allogeneic Hsct ◽  
Isotype Control ◽  
Isotype Control Antibody

Abstract In allogeneic hematopoietic stem cell transplantation (HSCT), identification of mechanisms to control GVHD yet maintain GVL responses is of critical importance. One key effector cell that mediates both GVHD and GVL is the CD8+ T cell, which expands in response to T cell receptor (TCR) stimulation by allogeneic MHC class I molecules during allogeneic HSCT. In addition, co-stimulatory molecules facilitate the TCR-mediated activation process and the effector function of CD8+ T cells. Recent data suggest that NKG2D may play a co-stimulatory role in activation and in augmenting anti-tumor cytotoxic responses of CD8+ T cells. NKG2D is an NK cell-associated receptor that is also expressed on all human CD8+ T cells and on activated/memory mouse CD8+ T cells. NKG2D recognizes a diverse array of MHC-related ligands that are expressed by many tumors and induced on cells under stress such as myeloablative conditioning during HSCT. As the role of NKG2D in allogeneic HSCT is unknown, we hereby investigated the role of NKG2D on CD8+ T cells in a mouse model of GVHD and GVL. Our results show that a large fraction (40-50%) of mouse CD8+ T cells inducibly express NKG2D upon activation by allogeneic MHC in vitro and in vivo. To test the role of NKG2D in GVHD pathogenesis, we employed a major MHC-mismatched mouse model of GVHD involving the transplantation of C57BL/6-derived CD8+ T cells and bone marrow (BM) into lethally irradiated Balb/c mice (B6→Balb/c). Using 3 different approaches to block NKG2D on CD8+ T cells (shRNA-mediated silencing, germline NKG2D deficiency, and antibody blockade), we found that weight loss, clinical score, and survival were significantly improved in transplanted mice with NKG2D blockade. The attenuation in GVHD correlated with a significant reduction in TNFα and IFNγ production, cytotoxicity, and proliferation (BrdU incorporation) by CD8+ T cells. Although CD4+ T cells did not express NKG2D, a protective effect of NKG2D blockade was still observed in GVHD induced by a mixture of CD8+ and CD4+ T cells, albeit to a lesser extent. We next tested the effects of NKG2D on CD8+ T cell-mediated GVL. To this end, irradiated Balb/c mice were transplanted with C57BL/6-derived CD8+ T cells and BM, challenged intravenously with luciferase-positive A20 leukemia cells, and followed by total body imaging of luciferase-expressing cells. Given that NKG2D ligands are constitutively expressed on many tumor cells and plays an important role in their eradication, we predicted that continuous NKG2D blockade would inhibit GVL effects. However, as NKGD ligands are upregulated only transiently on stressed normal tissue, we reasoned that transient NKG2D blockade might be sufficient to attenuate GVHD and allow CD8+ T cells to regain their GVL function. To test this hypothesis, we compared the effect of anti-NKG2D antibody as continuous treatment or as 5-day transient treatment to mice receiving isotype control antibody. As expected, mice that received isotype control antibody cleared the A20 cells but developed severe GVHD. Continuous anti-NKG2D antibody-mediated blockade improved GVHD but also blunted the GVL response leading to increased A20 growth. In contrast, a large proportion of mice transiently treated with anti-NKG2D antibody cleared the A20 cells, while maintaining the attenuated GVHD state. Together, these data support a positive role of NKG2D on CD8+ T cells in mediating GVHD and GVL. Given the transient nature of NKG2D ligand upregulation on stressed tissues, a window of opportunity may exist where transient NKG2D blockade could provide a novel therapeutic strategy for treatment of acute GVHD while preserving the GVL function of CD8+ T cells after allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document