scholarly journals The CD8 Population in CD4-deficient Mice Is Heavily Contaminated with MHC Class II–restricted T Cells

2004 ◽  
Vol 199 (4) ◽  
pp. 559-565 ◽  
Author(s):  
Aaron J. Tyznik ◽  
Joseph C. Sun ◽  
Michael J. Bevan
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Class Ii ◽  
T Cell Response ◽  
Class I ◽  
Cytotoxic T Cell ◽  
Similar Response ◽  
Wild Type ◽  
The Impact

In experiments to study the impact of deficiency in CD4+ T cell help on the magnitude of CD8+ cytotoxic T cell response to pathogens, it was noted that in CD4 gene knockout mice, the CD8 population made significant responses to several nominally major histocompatibility complex (MHC) class II–restricted epitopes in addition to the expected responses to MHC class I–restricted epitopes. A similar response by CD8+ T cells to class II–restricted epitopes was not observed in wild-type mice, or in mice that had been acutely depleted of CD4+ T cells just before the immunization. Coincident with this unexpected response to class II–restricted epitopes, it was also observed that the CD8+ response to the class I–restricted epitopes was consistently lower in CD4−/− mice than in wild-type mice. Further experiments suggested that these two observations are linked and that the CD8 population in CD4−/− mice may contain a majority of T cells that were actually selected by recognition of MHC class II molecules in the thymus. These results have implications for understanding CD4 versus CD8 lineage commitment in the thymus, and for the practical use of CD4−/− mice as models of helper deficiency.

Double Loading of Dendritic Cell MHC Class I and MHC Class II with an AML Antigen Repertoire Enhances Primary and Secondary T-Cell Responses In Vitro.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2529-2529
Author(s):  
William K. Decker ◽  
Dongxia Xing ◽  
Sufang Li ◽  
Simon N. Robinson ◽  
Hong Yang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Cd8 T Cells ◽  
Class Ii ◽  
T Cell Response ◽  
Class I ◽  
Cross Presentation ◽  
Ifn Γ

Abstract Despite improvements in therapy for acute myelogenous leukemia (AML), a significant percentage of patients still relapse and succumb to their disease. Dendritic cell immunotherapy offers the promise of potentially effective supportive therapy for a variety of neoplastic conditions; and the use of DCs loaded with tumor antigens is now recognized as an important investigational therapy. Though a variety of methods have been used to load DC vaccines, the loading of the MHC class II compartment with tumor lysate has predominated. The priming of a class II-mediated (CD4) T-cell response may be crucial to the success of DC immunotherapy as such a response is likely required for the development of memory CD8+ T-cells. DC cross-presentation is credited with the ability of lysate-loaded DCs to prime both CD4 and CD8 T-cell responses, enabling the generation of CD8+ CTLs without the loading of the MHC class I compartment (i.e. the cytoplasm). Recently, however, several reports have raised doubts as to the efficiency of cross-presentation as a mechanism for CTL priming in vivo. To examine this issue, we have loaded human DCs with both AML tumor lysate and mRNA. This technique allows the full repertoire of class I antigens to be presented without dependence upon cross-presentation; and, moreover, provides a full complement of class II antigens necessary for CD4 T-cell priming and the generation of memory responses. Methods: CD14+ precursors were isolated from normal donor PBPCs by magnetic separation. Immature DCs were then generated by culturing precursors for six days in GM-CSF and IL-4. Lysate was produced by three successive freeze/thaw cycles of blasts. mRNA was extracted from blasts using Trizol and oligo-dT separation. Immature DCs were pulsed for three hours with AML lysate and subsequently electroporated with AML mRNA. Loaded DCs were matured for 48 hours with IL-1β, TNF-α, IL-6, and PGE2 and then used to prime autologous T-cells. Short-term responses were assayed on day 5 of the 1st stimulation. Memory responses were assayed on day 10 of a tertiary stimulation. Results: Doubly-loaded DCs can prime a superior T-cell response in vitro in comparison to that of singly-loaded DCs, demonstrating a 30–70% increase in IFN-γ ELISpots over lysate-loaded DCs (p<0.001) and a 3–4 fold increase in ELISpots in comparison to mRNA loaded DCs (p<0.001). These results were verified by flow cytometry which showed 35% of CD8+ T-cells primed by doubly-loaded DCs were CD69+/IFN-γ+ vs. 14% of CD8+ T-cells primed by lysate-loaded DCs (p<0.001). This enhancement may be based upon both an upregulation of CD83 surface expression (p<0.0019) of doubly-loaded DCs and/or the upregulation of B7.1/B7.2 that accompanies elevated CD40L signaling. Memory responses were also greatly improved, with a 126% increase in total ELISpots (double loaded DCs versus lysate loaded DCs; p<0.03) and a 187% increase in total IFN-γ secretion (p<0.03). Unloaded (p<0.01) and mRNA (p<0.007) loaded DCs exhibited a virtual inability to generate memory T-cells in vitro, suggesting that the perpetuation of the memory response is reliant upon T-cell help. Conclusion: DCs doubly-loaded with lysate and mRNA are more efficient in the generation of primary and secondary immune responses than are singly-loaded DCs. The clinical administration of such doubly-loaded DCs may offer an important therapeutic option to patients with AML.

scholarly journals Mechanisms of mouse spleen dendritic cell function in the generation of influenza-specific, cytolytic T lymphocytes.

1992 ◽  
Vol 176 (2) ◽  
pp. 519-529 ◽  
Author(s):  
R Nonacs ◽  
C Humborg ◽  
J P Tam ◽  
R M Steinman
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Mhc Class I ◽  
Mhc Class Ii ◽  
Cd4 T Cells ◽  
Cell Function ◽  
Class Ii ◽  
T Cell Response ◽  
Class I

We have evaluated the capacity of dendritic cells to function as antigen-presenting cells (APCs) for influenza and have examined their mechanism of action. Virus-pulsed dendritic cells were 100 times more efficient than bulk spleen cells in stimulating cytotoxic T lymphocyte (CTL) formation. The induction of CTLs required neither exogenous lymphokines nor APCs in the responding T cell population. Infectious virus entered dendritic cells through intracellular acidic vacuoles and directed the synthesis of several viral proteins. If ultraviolet (UV)-inactivated or bromelain-treated viruses were used, viral protein synthesis could not be detected, and there was poor induction of CTLs. This indicated that dendritic cells were not capable of processing noninfectious virus onto major histocompatibility complex (MHC) class I molecules. However, UV-inactivated and bromelain-treated viruses were presented efficiently to class II-restricted CD4+ T cells. The CD4+ T cells crossreacted with different strains of influenza and markedly amplified CTL formation. Cell lines that lacked MHC class II, and consequently the capacity to stimulate CD4+ T cells, failed to induce CTLs unless helper lymphokines were added. Similarly, dendritic cells pulsed with the MHC class I-restricted nucleoprotein 147-155 peptide were poor stimulators in the absence of exogenous helper factors. We conclude that the function of dendritic cells as APCs for the generation of virus-specific CTLs in vitro depends measurably upon: (a) charging class I molecules with peptides derived from endogenously synthesized viral antigens, and (b) stimulating a strong CD4+ helper T cell response.

scholarly journals Peptide vaccination directed against IDO1-expressing immune cells elicits CD8+ and CD4+ T-cell-mediated antitumor immunity and enhanced anti-PD1 responses

2020 ◽  
Vol 8 (2) ◽  
pp. e000605
Author(s):  
Souvik Dey ◽  
Erika Sutanto-Ward ◽  
Katharina L Kopp ◽  
James DuHadaway ◽  
Arpita Mondal ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Mhc Class I ◽  
Adoptive Transfer ◽  
Immune Cells ◽  
Class Ii ◽  
T Cell Response ◽  
Class I ◽  
Antitumor Effects

BackgroundThe tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which subverts T-cell immunity at multiple levels, is itself subject to inherent T-cell reactivity. This intriguing deviation from central tolerance has been interpreted as counterbalancing IDO1-mediated immunosuppression. Based on this hypothesis, clinical studies employing an IDO1 peptide-based vaccine approach for cancer treatment have been initiated, but there remains a pressing need to further investigate the immunological ramifications of stimulating the anti-IDO1 T-cell response in this manner.MethodsCT26 colon carcinoma tumors were evaluated for expression of IDO1 protein by western blot analysis, immunofluorescence microscopy and flow cytometry. Mouse IDO1-derived peptides, predicted to bind either major histocompatibility complex (MHC) class I or II of the H2d BALB/c strain, were emulsified in 50% Montanide for prophylactic or therapeutic vaccine treatment of CT26 tumor-bearing mice initiated either 7 days prior to or following tumor cell injection, respectively. In some therapeutic treatment experiments, administration of programmed cell death protein 1-binding antibody (anti-PD1 antibody) or epacadostat was concurrently initiated. Tumor size was determined by caliper measurements and comparative tumor growth suppression was assessed by longitudinal analyses of tumor growth data. For adoptive transfer, T cells from complete responder animals were isolated using paramagnetic beads and fluorescence-activated cell sorting.ResultsThis study identifies mouse MHC class I-directed and II-directed, IDO1-derived peptides capable of eliciting antitumor responses, despite finding IDO1 expressed exclusively in tumor-infiltrating immune cells. Treatment of established tumors with anti-PD1 antibody and class I-directed but not class II-directed IDO1 peptide vaccines produced an enhanced antitumor response. Likewise, class I-directed and II-directed IDO1 peptides elicited an enhanced combinatorial response, suggesting distinct mechanisms of action. Consistent with this interpretation, adoptive transfer of isolated CD8+ T cells from class I and CD4+ T cells from class II peptide-vaccinated responder mice delayed tumor growth. The class II-directed response was completely IDO1-dependent while the class I-directed response included an IDO1-independent component consistent with antigen spread.ConclusionsThe in vivo antitumor effects demonstrated with IDO1-based vaccines via targeting of the tumor microenvironment highlight the utility of mouse models for further exploration and refinement of this novel vaccine-based approach to IDO1-directed cancer therapy and its potential to improve patient response rates to anti-PD1 therapy.

scholarly journals A Class II-Restricted CD8γ13 T-Cell Clone Protects During Chlamydia muridarum Genital Tract Infection

2020 ◽  
Vol 221 (11) ◽  
pp. 1895-1906
Author(s):  
Raymond M Johnson ◽  
Norma Olivares-Strank ◽  
Gang Peng
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Cd8 T Cells ◽  
Genital Tract ◽  
Cell Clone ◽  
Class Ii ◽  
Class I ◽  
T Cell Clone ◽  
Tract Infections

Abstract Background The T-cell response to chlamydia genital tract infections in humans and mice is unusual because the majority of antigen-specific CD8 T cells are not class I restricted (referred to here as “unrestricted” or “atypical”). We previously reported that a subset of unrestricted murine chlamydia-specific CD8 T cells had a cytokine polarization pattern that included interferon (IFN)-γ and interleukin (IL)-13. Methods In this study, we investigated the transcriptome of CD8γ13 T cells, comparing them to Tc1 clones using microarray analysis. That study revealed that CD8γ13 polarization included IL-5 in addition to IFN-γ and IL-13. Adoptive transfer studies were performed with Tc1 clones and a CD8γ13 T-cell clone to determine whether either influenced bacterial clearance or immunopathology during Chlamydia muridarum genital tract infections. Results To our surprise, an adoptively transferred CD8γ13 T-cell clone was remarkably proficient at preventing chlamydia immunopathology, whereas the multifunctional Tc1 clone did not enhance clearance or significantly alter immunopathology. Mapping studies with major histocompatibility complex (MHC) class I- and class II-deficient splenocytes showed our previously published chlamydia-specific CD8 T-cell clones are MHC class II restricted. Conclusions The MHC class II-restricted CD8 T cells may play an important role in protection from intracellular pathogens that limit class I antigen presentation or diminish CD4 T-cell numbers or impair their function.

Human T Cells with Distinct Specificities Mediate Graft-Versus-Leukemia Reactivity and Xenogeneic Graft-Versus-Host Disease in a NOD/Scid Mouse Model for Human Acute Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1330-1330
Author(s):  
Sanja Stevanovic ◽  
Bart Nijmeijer ◽  
Marianke LJ Van Schie ◽  
Roelof Willemze ◽  
Marieke Griffioen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class I ◽  
Mhc Class Ii ◽  
Class Ii ◽  
Cd4 T Cell ◽  
Class I ◽  
T Cell Clones ◽  
Cell Clones ◽  
Human T Cells

Abstract Abstract 1330 Poster Board I-352 Immunodeficient mice inoculated with human leukemia can be used as a model to investigate Graft-versus-Leukemia (GvL) effects of donor lymphocyte infusions (DLIs). In addition to GvL reactivity, treatment with DLI induces xenogeneic Graft-versus-Host Disease (GvHD) in mice, characterized by pancytopenia and weight loss. In patients treated with DLI for relapsed or residual leukemia after allogeneic stem cell transplantation, immune responses against non-leukemic cells may also cause GvHD. It has been suggested that GvL reactivity and GvHD, which co-develop in vivo, can be separated and that distinct T cells exist with the specific capacity to mediate GvL reactivity or GvHD. Since adoptive T cell transfer models that allow analysis of separation of GvL and GvHD are rare, we aimed to establish whether GvL reactivity and xenogeneic GvHD could be separated using our model of human leukemia-engrafted NOD/scid mouse after treatment with human donor T cells. In this study, non-conditioned NOD/scid mice engrafted with primary human acute lymphoblastic leukemic cells were treated with CD3+ DLI. Established tumors were effectively eliminated by emerging human T cells, but also induced xenogeneic GvHD. Flowcytometric analysis demonstrated that the majority of emerging CD8+ and CD4+ T cells were activated (HLA-DR+) and expressed an effector memory phenotype (CD45RA-CD45RO+CCR7-). To investigate whether GvL reactivity and xenogeneic GvHD were mediated by the same T cells showing reactivity against both human leukemic and murine cells, or displaying distinct reactivity against human leukemic and murine cells, we clonally isolated and characterized the T cells during the GvL response and xenogeneic GvHD. T cell clones were analyzed for reactivity against primary human leukemic cells and primary NOD/scid hematopoietic (BM and spleen cells) and non-hematopoietic (skin fibroblasts) cells in IFN-g ELISA. Isolated CD8+ and CD4+ T cell clones were shown to recognize either human leukemic or murine cells, indicating that GvL response and xenogeneic GvHD were mediated by different human T cells. Flowcytometric analysis demonstrated that all BM and spleen cells expressed MHC class I, whereas only 1-3 % of the cells were MHC class II +. Primary skin fibroblasts displayed low MHC class I and completely lacked MHC class II expression. Xeno-reactive CD8+ T cell clones were shown to recognize all MHC class I + target cells and xeno-reactive CD4+ T cells clones displayed reactivity only against MHC class II + target cells. To determine the MHC restriction of xeno-reactive T cell clones, NOD/scid bone marrow (BM) derived dendritic cells (DC) expressing high levels of murine MHC class I and class II were tested for T cell recognition in the presence or absence of murine MHC class I and class II monoclonal antibodies in IFN-g ELISA. Xeno-reactive CD8+ T cell clones were shown to be MHC class I (H-2Kd or H-2Db) restricted, whereas xeno-reactive CD4+ T cell clones were MHC class II (I-Ag7) restricted, indicating that xeno-reactivity reflects genuine human T cell response directed against allo-antigens present on murine cells. Despite production of high levels of IFN-gamma, xeno-reactive CD8+ and CD4+ T cell clones failed to exert cytolytic activity against murine DC, as determined in a 51Cr-release cytotoxicity assay. Absence of cytolysis by CD8+ T cell clones, which are generally considered as potent effector cells, may be explained by low avidity interaction between human T cells and murine DC, since flowcytometric analysis revealed sub-optimal activation of T cells as measured by CD137 expression and T cell receptor downregulation upon co-culture with murine DC, and therefore these results indicate that xenogeneic GvHD in this model is likely to be mediated by cytokines. In conclusion, in leukemia-engrafted NOD/scid mice treated with CD3+ DLI, we show that GvL reactivity and xenogeneic GvHD are mediated by separate human T cells with distinct specificities. All xeno-reactive T cell clones showed genuine recognition of MHC class I or class II associated allo-antigens on murine cells similar as GvHD-inducing human T cells. These data suggest that our NOD/scid mouse model of human acute leukemia may be valuable for studying the effectiveness and specificity of selectively enriched or depleted T cells for adoptive immunotherapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Efficiency of CD4 Recruitment to Ligand-engaged TCR Controls the Agonist/Partial Agonist Properties of Peptide–MHC Molecule Ligands

1997 ◽  
Vol 185 (2) ◽  
pp. 219-230 ◽  
Author(s):  
Joaquín Madrenas ◽  
Luan A. Chau ◽  
Judy Smith ◽  
Jeffrey A. Bluestone ◽  
Ronald N. Germain
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Partial Agonist ◽  
Interleukin 2 ◽  
Class Ii ◽  
Wild Type ◽  
Functional Responses ◽  
Mhc Molecules ◽  
Partial Agonists

One hypothesis seeking to explain the signaling and biological properties of T cell receptor for antigen (TCR) partial agonists and antagonists is the coreceptor density/kinetic model, which proposes that the pharmacologic behavior of a TCR ligand is largely determined by the relative rates of (a) dissociation of ligand from an engaged TCR and (b) recruitment of lck-linked coreceptors to this ligand-engaged receptor. Using several approaches to prevent or reduce the association of CD4 with occupied TCR, we demonstrate that consistent with this hypothesis, the biological and biochemical consequence of limiting this interaction is to convert typical agonists into partial agonist stimuli. Thus, adding anti-CD4 antibody to T cells recognizing a wild-type peptide–MHC class II ligand leads to disproportionate inhibition of interleukin-2 (IL-2) relative to IL-3 production, the same pattern seen using a TCR partial agonist/antagonist. In addition, T cells exposed to wild-type ligand in the presence of anti-CD4 antibodies show a pattern of TCR signaling resembling that seen using partial agonists, with predominant accumulation of the p21 tyrosine-phosphorylated form of TCR-ζ, reduced tyrosine phosphorylation of CD3ε, and no detectable phosphorylation of ZAP-70. Similar results are obtained when the wild-type ligand is presented by mutant class II MHC molecules unable to bind CD4. Likewise, antibody coligation of CD3 and CD4 results in an agonist-like phosphorylation pattern, whereas bivalent engagement of CD3 alone gives a partial agonist-like pattern. Finally, in accord with data showing that partial agonists often induce T cell anergy, CD4 blockade during antigen exposure renders cloned T cells unable to produce IL-2 upon restimulation. These results demonstrate that the biochemical and functional responses to variant TCR ligands with partial agonist properties can be largely reproduced by inhibiting recruitment of CD4 to a TCR binding a wild-type ligand, consistent with the idea that the relative rates of TCR–ligand disengagement and of association of engaged TCR with CD4 may play a key role in determining the pharmacologic properties of peptide–MHC molecule ligands. Beyond this insight into signaling through the TCR, these results have implications for models of thymocyte selection and the use of anti-coreceptor antibodies in vivo for the establishment of immunological tolerance.

scholarly journals Genetic Dissection of Primary and Secondary Responses to a Widespread Natural Pathogen of the Gut,Eimeria vermiformis

2000 ◽  
Vol 68 (11) ◽  
pp. 6273-6280 ◽  
Author(s):  
Adrian L. Smith ◽  
Adrian C. Hayday
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Class Ii ◽  
Primary Response ◽  
Class I ◽  
Class Ii Mhc ◽  
Ifn Γ ◽  
Αβ T Cells

ABSTRACT Because most pathogens initially challenge the body at epithelial surfaces, it is important to dissect the mechanisms that underlie T-cell responses to infected epithelial cells in vivo. The coccidian parasites of the genus Eimeria are protozoan gut pathogens that elicit a potent, protective immune response in a wide range of host species. CD4+ αβ T cells and gamma interferon (IFN-γ) are centrally implicated in the primary immunoprotective response. To define any additional requirements for the primary response and to develop a comparison between the primary and the secondary response, we have studied Eimeria infections of a broad range of genetically altered mice. We find that a full-strength primary response depends on β2-microglobulin (class I major histocompatibility complex [MHC] and class II MHC and on IFN-γ and interleukin-6 (IL-6) but not on TAP1, perforin, IL-4, Fas ligand, or inducible nitric oxide synthetase. Indeed, MHC class II-deficient and IFN-γ-deficient mice are as susceptible to primary infection as mice deficient in all αβ T cells. Strikingly, the requirements for a highly effective αβ-T-cell-driven memory response are less stringent, requiring neither IFN-γ nor IL-6 nor class I MHC. The class II MHC dependence was also reduced, with adoptively transferable immunity developing in MHC class II−/− mice. Besides the improved depiction of an immune response to a natural gut pathogen, the finding that effective memory can be elicited in the absence of primary effector responses appears to create latitude in the design of vaccine strategies.

scholarly journals Deciphering the Plasmodium falciparum malaria-specific CD4+ T cell response: ex vivo detection of high frequencies of PD-1+TIGIT+ EXP1-specific CD4+ T cells using a novel HLA-DR11-restricted MHC class II tetramer

2021 ◽  
Author(s):  
Sophia Schulte ◽  
Janna Heide ◽  
Christin Ackermann ◽  
Sven Peine ◽  
Michael Ramharter ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Cell Response ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Class Ii ◽  
T Cell Response ◽  
Cd4 T Cell ◽  
Inhibitory Receptors

Abstract Relatively little is known about the ex vivo frequency and phenotype of the P. falciparum-specific CD4+ T cell response in humans. The exported protein 1 (EXP1) is expressed by plasmodia at both, the liver stage and blood stage, of infection making it a potential target for CD4+ and CD8+ effector T cells. Here, a fluorochrome-labelled HLA-DRB1*11:01-restriced MHC class II tetramer derived from the P. falciparum EXP1 (aa62-74) was established for ex vivo tetramer analysis and magnetic bead enrichment in ten patients with acute malaria. EXP1-specific CD4+ T cells were detectable in nine out of ten (90%) malaria patients expressing the HLA-DRB1*11 molecule with an average ex vivo frequency of 0.11% (0-0.22%) of total CD4+ T cells. The phenotype of EXP1-specific CD4+ T cells was further assessed using co-staining with activation (CD38, HLA-DR, CD26), differentiation (CD45RO, CCR7, KLRG1, CD127), senescence (CD57) and co-inhibitory (PD-1, TIGIT, LAG-3, TIM-3) markers as well as the ectonucleotidases CD39 and CD73. EXP1-specific tetramer+ CD4+ T cells had a distinct phenotype compared to bulk CD4+ T cells and displayed a highly activated effector memory phenotype with elevated levels of co-inhibitory receptors and activation markers: EXP1-specific CD4+ T cells universally expressed the co-inhibitory receptors PD-1 and TIGIT as well as the activation marker CD38 and showed elevated frequencies of CD39. These results demonstrate that MHC class II tetramer enrichment is a sensitive approach to investigate ex vivo antigen-specific CD4+ T cells in malaria patients that will aid further analysis of the role of CD4+ T cells during malaria.

Histone Deacetylase Inhibitors Induce Immuno-Dominant Suppression of Dendritic Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 456-456 ◽  
Author(s):  
Pavan Reddy ◽  
Yoshinobu Maeda ◽  
Raimon Duran-Struuck ◽  
Oleg Krijanovski ◽  
Charles Dinarello ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Cd4 T Cells ◽  
Hdac Inhibitors ◽  
Class Ii ◽  
Wild Type ◽  
Tnf Α

Abstract We and others have recently demonstrated that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor with anti-neoplastic properties, reduces experimental acute graft-versus-host disease (GVHD). We have now investigated the mechanisms of action of two HDAC inhibitors, SAHA and ITF 2357, on allogeneic immune responses. Bone marrow derived dendritic cells (DCs) were preincubated with the HDAC inhibitors at nanomolar concentrations for 16–18 hours and stimulated with lipopolysaccharide (LPS). Pretreatment of DCs caused a significant reduction in the secretion of TNF-α, IL-12p70 and IL-6 compared to the untreated controls (P< 0.005). Similar effects were seen using human peripheral blood mononuclear cell derived DCs. Pre-treatment of both murine and human DCs also significantly reduced their in vitro stimulation of allogeneic T cells as measured by proliferation and IFN-γ production (P<0.01). We determined the in vivo relevance of these observations utilizing a mouse model where the responses of allogeneic donor bm12 T cells depended on the function of injected host B6 DCs would stimulate. Recipient Class-II −/− B6 (H-2b) received 11 Gy on day -1 and were injected with 4–5 x 106 wild type B6 DCs treated with SAHA or with media on days -1 and 0 and then transplanted with 2 x 106 T cells and 5 x 106 TCDBM cells from either syngeneic B6 or allogeneic bm12 donors. SAHA treatment of DCs significantly reduced expansion of allogeneic donor CD4+ T cells on day +7 after BMT compared to controls (P<0.05). SAHA treatment induced a similarly significant reduction in the expansion of CD8+ cells in Class I disparate [bm1→β2M−/−] model. In vitro, SAHA treatment significantly suppressed the expression of CD40 and CD80 but did not alter MHC class II expression. Surprisingly, when mixed with normal DCs at 1:1 ratio, SAHA treated DCs dominantly suppressed allogeneic T cell responses. The regulation of T cell proliferation was not reversible by addition of IL-12, TNF-α, IL-18, anti-IL-10 or anti-TGFβ, either alone or in combination. Suppression of allogeneic responses was contact dependent in trans-well experiments. To address whether the regulation of SAHA treated DCs required contact with T cells, we devised a three cell experiment where SAHA treated DCs lacked the capacity to present antigens to T cells. DCs from B6 MHC Class II deficient (H-2b) were treated with SAHA and co-cultured with wild type B6 (H-2b) DCs along with purified allogeneic BALB/c (H-2d) CD4+ T cells in an MLR. Allogeneic CD4+ T cells proliferated well, demonstrating the regulation to be dependent on contact between SAHA treated DCs and T cells. To address the in vivo relevance of this suppression, we utilized a well characterized [BALB/c →B6] mouse model of acute GVHD. Recipient B6 animals received 11Gy on day -1 and were injected with of 5 million host type SAHA treated or control DCs on days −1, 0, and +2. Mice were transplanted on day 0 with 2 x 106 T cells and 5 x 106 BM from either syngeneic B6 or allogeneic BALB/c donors. Injection of SAHA treated DCs resulted in significantly better survival (60% vs. 10%, P < 0.01) and significantly reduced serum levels of TNF-α, donor T cell expansion and histopathology of GVHD on day +7 after BMT compared to the controls. We conclue that HDAC inhibitors are novel immunomodulators that regulate DC function and might represent a novel strategy to prevent GVHD.

Interferon Gamma (IFNγ)/STAT1 Signaling in Host Antigen Present Cells Suppresses MHC Class II-Dependent Presentation of Self-Antigens and Development of Graft Versus Host Disease (GVHD)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2147-2147
Author(s):  
Caisheng Lu ◽  
Huihui Ma ◽  
Liangsong Song ◽  
Shirong Li ◽  
Suzanne Lentzsch ◽  
...  
Keyword(s):  
T Cells ◽  
Mhc Class Ii ◽  
Cd4 T Cells ◽  
Class Ii ◽  
Invariant Chain ◽  
Wild Type ◽  
Mhc Ii ◽  
Exogenous Antigen ◽  
Stat1 Signaling ◽  
The Impact

Abstract IFNγ signaling plays a critical role in the pathogenesis of GVHD. In this study, we observed that LPS-maturated bone marrow-derived dendritic cells (BMDCs) lacking IFNγ receptor (IFNγR, GRKO) or signal transducer and activator of transcription 1 (STAT1KO) had increased expression of major histocompatibility complex class II (MHC II), CD86, CD80, and enhanced allo-stimulatory capacity. This was further confirmed using fully MHC-mismatched bone marrow transplantation (BMT) studies. APC of GRKO or STAT1KO recipients had increased MHC II expression, which was associated with enhanced activation and expansion of donor CD4 and CD8 T cells and subsequently accelerated GVHD mortality compared to wild type (WT) controls. This increased GVHD mortality and increased MHC II expression on host APCs was further observed in the absence of recipient conditioning in the B6→CB6F1 mouse model. This was associated with increased presentation of host derived endogenous Eα52-68 peptide via I-Ab on recipient CD11c+ cells as detected by staining with the YA-e antibody. Furthermore, we could demonstrate that absence of IFNγR in BMDC promotes presentation Eα52-68 peptide and subsequently elicits pronounced activation, expansion and Th1 differentiation of TEa-TCR-tg CD4 T cells which recognize the Eα52-68 peptide presented by I-Ab. Next, we assessed the impact of this pathway on presentation of exogenous antigens. Interestingly, when lysate prepared from BALB/c splenocytes was incubated with BMDCs from B6 mice, Y-Ae expression on STAT1-/- BMDCs was significantly reduced compared to wild type BMDCs allowing us to hypothesize that IFNγ/STAT1 signaling may play an important role in promoting presentation of exogenous antigen while suppressing presentation of endogenous antigen. To further confirm this hypothesis, we used ovalbumin (OVA) as a second model antigen. To assess the impact of IFNγ/STAT1 signaling on presentation of exogenous antigen, WT, GRKO or STAT1KO BMDC were directly pulsed with OVA. To address the role in endogenous antigen presentation we studied act-mOVA-transgenic wildtype, act-mOVA.GRKO or act-mOVA.STAT1KO BMDCs. Transgenic OT-II CD4 T cells express a TCR specific for the OVA peptide 323-33 presented by I-Ab. The proliferation/activation of OT II T cells was monitored by flow cytometer as readout for effective Ag presentation. Our data demonstrated that IFNγR- or STAT1-deficient BMDCs loaded with exogenous intact OVA protein were compromised in promoting OT II proliferation. In contrast, responder OT-II CD4 T cells proliferated much more vigorously when stimulated with IFNγR/STAT1-deficient m-Act-OVA BMDCs compared to controls. We further observed significantly impaired OT-II cell proliferation in IFNγR or STAT1-deficient mice immunized with OVA indicating impaired presentation of exogenous antigens. However, OT-II CD4 T cells injected into lethally irradiated act-mOVA.STAT1KO transgenic mice proliferated more robustly and displayed increased Th1 differentiation compared to control mice when tested 3 days after OT II administration. We next started to assess several key factors (Ii [invariant chain, CD74], Cathepsin S [CTSS], H2-M, CIITA and MARCH1), known to be involved in the process of MHC class II antigen presentation and MHC II expression. We found retention of Invariant chain (CD74) expression as well as reduced CTSS and H2M expression in GRKO or STAT1KO BMDC following LPS-maturation. Furthermore, we observed significantly reduced lysosome formation/function in STAT1KO BMDCs compared to wild type BMDCs after LPS maturation. These data suggest that exogenous protein-derived peptide exchange in the MHCII compartment (MIIC) is impaired in STAT1KO BMDCs. Immature and LPS-maturated STAT1-/-BMDCs had significantly increased autophagy, which could explain enhanced endogenous Ag presentation since autophagy has been demonstrated to be critical in MHC II Ag presentation of cytoplasmic constituents. Finally, we found evidence of enhanced MHC II synthesis as supported by increased CIITA mRNA expression and conversely reduced MHC II degradation as indicated by reduced MARCH1 expression. In summary our data suggest that absence of IFNγR/STAT1 signaling in DC leads to abnormal surface MHC II turnover, promotes presentation of endogenous peptides and concomitantly impairs processing and presentation of exogenous antigens. Disclosures Lentzsch: BMS: Consultancy; Foundation One: Consultancy; Celgene: Consultancy, Honoraria.

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