41 Assessment of the spatial distribution of CD4+ T cells subpopulations in the tumor microenvironment by Brightplex®, a sequential chromogenic multiplex assay

BackgroundCancer immunotherapy reinvigorates tumor-specific T cell responses of CD8+ cytotoxic T lymphocytes that detect intracellular antigens that are presented by MHC class I molecules expressed by all tumor cell types. Because most tumors do not express MHC class II, the potential antitumor protective role of CD4 T cells, which bind MHC class II molecules on target cells, has been less studied. However, CD4+ T cells are also required for efficacious antitumor immunity; they are core components of adaptive immunity that differentiate into lineages responsible for effector activities. Both TH1 and TH2 cell types mediate antitumor immunity, although TH1 cells may be more potent due to the production of large amounts of IFN-γ, as well as chemokines that enhance the priming and expansion of CD8 cells. TH1 cells help in recruiting natural killer cells and type I macrophages to tumor sites, which can act in concert toward tumor eradication. The ability of TH2 cells to mobilize innate cells, may represent a general pathway for their impact on the host antitumor response. Tumor infiltrating TFH cells play a key role in immune cell recruitment to the tumor and in the formation of intratumoral follicular structures, which correlate with a positive prognosis. On the contrary, cells from the TH17 subset induces inflammatory responses resulting in a tumor-promoting environment. CD4+ Tregs which are critically important for the maintenance of self-tolerance, impede effective immunity against the tumor when they are present in the tumor microenvironment (TME).Therefore, beyond the detection of total CD4+ T cells within the TME, it is of critical importance to determine to which subpopulation each CD4+ T cell belongs to decipher their roles in tumor rejection.MethodsWe have developed a multiplex 7-plex panel of of antibodies against biomarkers to identify main types of CD4+ T cellsResultsOn a single FFPE tissue section, main types of CD4+ T cells: TH1, TH2, TFH, TH17 and Tregs are identified by a combination of antibodies against transcription factors and membrane proteins. Following images registration, complex cells phenotypes can be detected and quantified. Furthermore, digital pathology tools allow the evaluation of the spatial distribution of CD4+ T cells within the TME.ConclusionsThis new tool unravels the diversity of CD4+ T cells in TME and could help clinical researchers to design more effective immunotherapies in cancer treatment. Integrated into an Immunogram, this new Brightplex® Panel will also be critical to understand the immune contexture of tumors.

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Induction and Break of Immune Tolerance to Human FVIII in a HLA-DRB1*1501 Humanized Hemophilic Mouse Model

Blood ◽

10.1182/blood.v118.21.2280.2280 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2280-2280

Author(s):

Katharina Nora Steinitz ◽

Brigitte Binder ◽

Christian Lubich ◽

Rafi Uddin Ahmad ◽

Markus Weiller ◽

...

Keyword(s):

T Cells ◽

Immune System ◽

T Cell ◽

Mouse Model ◽

Mhc Class Ii ◽

Immune Tolerance ◽

Cd4 T Cells ◽

Hemophilia A ◽

Class Ii ◽

T Cell Epitopes

Abstract Abstract 2280 Development of neutralizing antibodies against FVIII is the major complication in the treatment of patients with hemophilia A. Although several genetic and environmental risk factors have been identified, it remains unclear why some patients develop antibodies while others do not. Understanding the underlying mechanisms that drive the decision of the immune system whether or not to make antibodies against FVIII would help to design novel therapeutics. We used a new humanized hemophilic mouse model that expresses the human MHC-class II molecule HLA-DRB1*1501 on the background of a complete knock out of all murine MHC-class II genes. Initial studies had indicated that only a fraction of these mice developed antibodies when intravenously (i.v.) treated with human FVIII. These findings which resemble the situation in patients with severe hemophilia A, evoked the question if the lack of antibody development in non-responder mice reflects the induction of specific immune tolerance after i.v. application of FVIII or represent non-responsiveness for other reasons. We addressed this question by choosing another application route (subcutaneous, s.c.) and by combining i.v. application with a concomitant activation of the innate immune system applying LPS, a well characterized ligand for toll-like receptor 4, together with FVIII. Both strategies resulted in the development of antibodies in all mice included in the study what suggested that non-responsiveness against i.v. FVIII does not reflect an inability to develop antibodies against FVIII. Next, we asked if i.v. FVIII does induce immune tolerance in non-responder mice. We pretreated mice with i.v. FVIII, selected non-responder mice and challenged them with s.c. FVIII. None of the mice developed antibodies what indicated that i.v. pretreatment had induced immune tolerance in non-responder mice. Currently, we test the hypothesis that immune tolerance after i.v. application is induced and maintained by FVIII-specific regulatory T cells. The differences in responder rates after i.v. and s.c. application of FVIII raised the question if there are differences in FVIII T-cell epitopes involved in the initial activation of FVIII-specific CD4+ T cells. We obtained spleen cells from mice treated with either i.v. or s.c. FVIII and generated CD4+ T-cell hybridoma libraries that were tested for peptide specificities. For this purpose we used a FVIII peptide library containing 15 mers with an offset of 3 amino acids. Our results indicate that the pattern of FVIII-specific T-cell epitopes involved in the activation of FVIII-specific CD4+ T cells after i.v. and s.c. application of FVIII is almost identical and represents a small set of FVIII peptides distributed over the A1, A2, B, A3 and C1 domains. Based on our results we conclude that the new HLA-DRB1*1501 hemophilic mouse model represents an interesting opportunity to uncover the mechanisms that drive the decision of the immune system whether or not to develop antibodies against FVIII. Disclosures: Steinitz: Baxter BioScience: Employment. Binder:Baxter BioScience: Employment. Lubich:Baxter BioScience: Employment. Ahmad:Baxter BioScience: Employment. Weiller:Baxter BioScience: Employment. de la Rosa:Baxter BioScience: Employment. Schwarz:Baxter BioScience: Employment. Scheiflinger:Baxter BioScience: Employment. Reipert:Baxter Innovations GmbH: Employment.

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Endogenous human cytomegalovirus gB is presented efficiently by MHC class II molecules to CD4+ CTL

Journal of Experimental Medicine ◽

10.1084/jem.20050162 ◽

2005 ◽

Vol 202 (8) ◽

pp. 1109-1119 ◽

Cited By ~ 61

Author(s):

Nagendra R. Hegde ◽

Claire Dunn ◽

David M. Lewinsohn ◽

Michael A. Jarvis ◽

Jay A. Nelson ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Glial Cells ◽

Human Cytomegalovirus ◽

Cd4 T Cells ◽

Class Ii ◽

Host Cells ◽

Cd4 T Cell

Human cytomegalovirus (HCMV) infects endothelial, epithelial, and glial cells in vivo. These cells can express MHC class II proteins, but are unlikely to play important roles in priming host immunity. Instead, it seems that class II presentation of endogenous HCMV antigens in these cells allows recognition of virus infection. We characterized class II presentation of HCMV glycoprotein B (gB), a membrane protein that accumulates extensively in endosomes during virus assembly. Human CD4+ T cells specific for gB were both highly abundant in blood and cytolytic in vivo. gB-specific CD4+ T cell clones recognized gB that was expressed in glial, endothelial, and epithelial cells, but not exogenous gB that was fed to these cells. Glial cells efficiently presented extremely low levels of endogenous gB—expressed by adenovirus vectors or after HCMV infection—and stimulated CD4+ T cells better than DCs that were incubated with exogenous gB. Presentation of endogenous gB required sorting of gB to endosomal compartments and processing by acidic proteases. Although presentation of cellular proteins that traffic into endosomes is well known, our observations demonstrate for the first time that a viral protein sorted to endosomes is presented exceptionally well, and can promote CD4+ T cell recognition and killing of biologically important host cells.

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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

Clinical & Experimental Immunology ◽

10.1093/cei/uxab027 ◽

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.

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

Blood ◽

10.1182/blood.v106.11.456.456 ◽

2005 ◽

Vol 106 (11) ◽

pp. 456-456 ◽

Cited By ~ 1

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.

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Treatment of Established B16 Murine Melanoma Tumors with Tyrp-1 Specific CD4+ Lymphocytes.

Blood ◽

10.1182/blood.v108.11.3688.3688 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3688-3688

Author(s):

Pawel Muranski ◽

Andrea Boni ◽

Crystal M. Paulos ◽

Kari R. Irvine ◽

Paul A. Antony ◽

...

Keyword(s):

T Cells ◽

Immune System ◽

T Cell ◽

Solid Tumors ◽

Mhc Class Ii ◽

Cd4 T Cells ◽

Tumor Response ◽

Class Ii ◽

B16 Melanoma ◽

Cd4 Cells

Abstract T-cell mediated response against solid tumors has been mostly associated with CD8+ cytotoxic lymphocytes, which act directly on the MHC class I expressing tumors. In the previously published model, gp-100 melanoma antigen-specific pmel-1 CD8+ T cells required co-administration of IL-2 and vaccine to induce significant regression of poorly immunogenic B16 melanoma in mice. MHC class II restricted CD4+ T-cells (T helpers) may have multiple direct and indirect effects on the immune response, but their role in adoptive cell transfer (ACT) therapy of solid tumors remains mainly undefined and based on highly manipulated models involving foreign antigens. In order to investigate the function of tumor specific CD4+ T-cells we have generated a transgenic mouse expressing a TRP-1 T cell receptor (TCR) directed against class II restricted murine melanocyte differentiation antigen tyrp-1. In vitro expanded TRP1 CD4+ cells secreted Th1-like cytokines upon antigen stimulation and caused direct cytotoxic effect against B16 melanoma. In vivo they mediated a highly effective response against large (>1cm2) B16 melanoma tumors after ACT of as few as 2.5×105 cells/mouse into C57B6 animals, which was associated with a massive tumor infiltration with CD11b+, MAC3+, GR1+ cells. TRP-1 T cells caused partial tumor rejection and prolonged survival in MHC class II−/− hosts implying the ability to directly recognize low level MHC class II on the tumor. This suboptimal effect was significantly enhanced after co-transfer of MHC class II+ APCs into MHC class II−/− hosts allowing for antigen cross- presentation. Interestingly, Rag1−/− hosts, deficient in all T and B lymphocytes, demonstrated excellent initial response to treatment, but were not cured and succumbed to late relapse of the melanoma. Long-term responses were even more impaired in Rag1−/− γc−/− hosts, while complete and durable cure was observed in TCRα−/−, CD4−/− and C57B6 mice, suggesting involvement of other arms of the adaptive immune system. Similarly, co-transfer of 0.1×106 CD4+ TRP-1 cells and 1×106 CD8+ pmel-1 cells resulted in effective tumor regression, while the same numbers of each cells transferred individually were not sufficient to initiate a rejection. Introduction of tumor-specific CD4+ cells therefore eliminates the previously sine qua non need for co-administration of vaccine and IL-2 for effective treatment with CD8+ pmel-1 cells. Overall, we show that antigen-specific CD4+ T cells are highly effective in mediating the anti-tumor response by causing both the direct anti-tumor effect and by activating innate and adaptive arms of the immune system. These findings suggest that CD4+ T helper cells may play a key role in improving efficacy of ACT immunotherapy as central activators of the anti-tumor response.

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Humanized E17 Hemophilic Mice Are a Major Breakthrough in the Design of New Preclinical Models for Developing Factor VIII Products with Reduced Immunogenicity.

Blood ◽

10.1182/blood.v110.11.782.782 ◽

2007 ◽

Vol 110 (11) ◽

pp. 782-782 ◽

Cited By ~ 1

Author(s):

Birgit M. Reipert ◽

Christina Hausl ◽

Maria Sasgary ◽

Maria Schuster ◽

Rafi U. Ahmad ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Immune Responses ◽

Factor Viii ◽

Mhc Class Ii ◽

Cd4 T Cells ◽

Plasma Cells ◽

Hemophilia A ◽

Class Ii ◽

Mhc Class Ii Molecules

Abstract MHC class II molecules are crucial for regulating adaptive immune responses against self and foreign protein antigens. They determine the antigenic peptides that are presented to CD4+ T cells and are essential for shaping the CD4+ T-cell repertoire in the thymus. Thus, the structure of MHC class II molecules is a major determinant for protein antigen immunogenicity. Structural differences between murine and human MHC class II complexes fundamentally limit the use of conventional murine hemophilia A models for dissecting immune responses to human factor VIII and developing new factor VIII products with reduced immunogenicity. To overcome this limitation, we humanized the murine E17 model of hemophilia A by introducing the human MHC class II haplotype HLA-DRB1*1501 on the background of a complete knockout of all murine MHC class II genes. Any anti-FVIII antibody response in this new humanized hemophilia A model is driven by CD4+ T cells that recognize FVIII-derived peptides that are presented by human HLA-DRB1*1501. The MHC class II haplotype HLA-DRB1*1501 is particularly relevant for the situation in hemophilia A patients because it is found in about 25% of Caucasians and 32% of Africans and has been shown to be associated with an increased risk that patients with severe hemophilia A have for developing FVIII inhibitors. We validated the relevance of this new model by asking the question whether HLA-DRB1*1501 hemophilic E17 mice develop FVIII inhibitors that are similar to those observed in patients with hemophilia A. Furthermore, we wanted to show that anti-FVIII antibody responses in these mice depend on the expression of the human DRB1*1501 molecule. Mice were treated with 8 intravenous doses of human FVIII and tested for anti-FVIII antibodies, anti-FVIII antibody-producing plasma cells and FVIII-specific T cells. About 90% of all humanized hemophilic E17 mice tested developed anti-FVIII antibodies that were similar to FVIII inhibitors found in patients. These antibodies were not restricted isotypically and contained mainly IgG1, IgG2a and IgG2b antibodies. Detection of antibodies in the circulation correlated with the presence of anti-FVIII antibody-producing plasma cells in the spleen. Development of anti-FVIII antibodies depended on the activation of FVIII-specific T cells and strictly depended on the expression of the HLA-DRB1*1501 molecule. Mice that did not express any MHC class II molecules did not develop anti-FVIII antibodies. We conclude that this new humanized E17 model for hemophilia A is a major advance towards developing suitable animal models needed to design future immunomodulatory strategies for patients with FVIII inhibitors and develop new FVIII products with reduced immunogenicity. Furthermore, it provides a tool for identifying T-cell epitopes of human FVIII restricted by MHC class II molecules that can be used for monitoring FVIII-specific T cells in patients who receive replacement therapy with FVIII products.

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Antigen presentation by mouse CD4+ T cells involving acquired MHC class II:peptide complexes: another mechanism to limit clonal expansion?

Blood ◽

10.1182/blood-2002-04-1230 ◽

2003 ◽

Vol 101 (7) ◽

pp. 2704-2710 ◽

Cited By ~ 72

Author(s):

Julia Y. S. Tsang ◽

Jian Guo Chai ◽

Robert Lechler

Keyword(s):

T Cells ◽

T Cell ◽

Antigen Presentation ◽

Mhc Class Ii ◽

Cd4 T Cells ◽

Interleukin 2 ◽

Clonal Expansion ◽

Class Ii ◽

Hybridoma Cells ◽

Mhc Class Ii Molecules

Antigen presentation by activated human and rat CD4+ T cells has long been known to induce hyporesponsiveness due to a combination of anergy and apoptosis. It has been assumed that no such phenomenon occurs in mice due to the inability of mouse T cells to synthesize major histocompatibility complex (MHC) class II molecules. There have been several recent descriptions of the transfer of molecules, including MHC molecules, from antigen-presenting cells (APCs) to T cells. Here, we describe the acquisition of MHC class II molecules by T-cell receptor (TCR)–transgenic T cells and T-hybridoma cells following culture with APCs. Acquisition was markedly enhanced by T-cell activation either due to cognate recognition of antigen or anti-CD3 activation. When activation was induced by antigen recognition, preferential acquisition of complexes of class II molecules displaying cognate peptide was observed; in contrast, following activation by anti-CD3 the acquisition of class II molecules was MHC unrestricted. T cells that had acquired MHC class II:peptide complexes were able to act as APCs and induced proliferation and interleukin-2 secretion by resting T cells. However, when activated T cells that had acquired MHC class II:peptide complexes engaged in T:T interactions, this led to an increase in apoptosis and the induction of hyporesponsiveness. These results raise the possibility that the acquisition of MHC class II:peptide complexes by T cells during an immune response may serve to limit clonal expansion, including that induced by alloantigen following tissue or stem cell transplantation.

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Quantitative Analysis of the T Cell Repertoire Selected by a Single Peptide–Major Histocompatibility Complex

Journal of Experimental Medicine ◽

10.1084/jem.187.11.1871 ◽

1998 ◽

Vol 187 (11) ◽

pp. 1871-1883 ◽

Cited By ~ 39

Author(s):

Laurent Gapin ◽

Yoshinori Fukui ◽

Jean Kanellopoulos ◽

Tetsuro Sano ◽

Armanda Casrouge ◽

...

Keyword(s):

T Cells ◽

Major Histocompatibility Complex ◽

T Cell ◽

Mhc Class Ii ◽

Cd4 T Cells ◽

Class Ii ◽

T Cell Repertoire ◽

Cell Repertoire ◽

Histocompatibility Complex ◽

Single Peptide

The positive selection of CD4+ T cells requires the expression of major histocompatibility complex (MHC) class II molecules in the thymus, but the role of self-peptides complexed to class II molecules is still a matter of debate. Recently, it was observed that transgenic mice expressing a single peptide–MHC class II complex positively select significant numbers of diverse CD4+ T cells in the thymus. However, the number of selected T cell specificities has not been evaluated so far. Here, we have sequenced 700 junctional complementarity determining regions 3 (CDR3) from T cell receptors (TCRs) carrying Vβ11-Jβ1.1 or Vβ12-Jβ1.1 rearrangements. We found that a single peptide–MHC class II complex positively selects at least 105 different Vβ rearrangements. Our data yield a first evaluation of the size of the T cell repertoire. In addition, they provide evidence that the single Eα52-68–I-Ab complex skews the amino acid frequency in the TCR CDR3 loop of positively selected T cells. A detailed analysis of CDR3 sequences indicates that a fraction of the β chain repertoire bears the imprint of the selecting self-peptide.

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Aberrant Expression of MHC Class II in Melanoma Attracts Inflammatory Tumor-Specific CD4+ T- Cells, Which Dampen CD8+ T-cell Antitumor Reactivity

Cancer Research ◽

10.1158/0008-5472.can-14-2956 ◽

2015 ◽

Vol 75 (18) ◽

pp. 3747-3759 ◽

Cited By ~ 47

Author(s):

Marco Donia ◽

Rikke Andersen ◽

Julie W. Kjeldsen ◽

Paolo Fagone ◽

Shamaila Munir ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Cd4 T Cells ◽

Cd8 T Cell ◽

Class Ii ◽

Aberrant Expression

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Ex Vivo Blockade of NF-[dappa]B Signaling in Alloreactive T Cells Prevents Murine Graft-Versus-Host Disease (GVHD).

Blood ◽

10.1182/blood.v104.11.3058.3058 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3058-3058

Author(s):

Matthew J. O’Shaughnessy ◽

Christine Vogtenhuber ◽

Jonathon S. Serody ◽

Raquel Sitcheran ◽

Albert S. Baldwin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Cd4 T Cells ◽

Ex Vivo ◽

Tolerance Induction ◽

Class Ii ◽

Maximal Effect ◽

Alloreactive T Cells ◽

Iκb Kinase

Abstract A failure of IL-2 transcription has been associated with tolerance induction. We hypothesized that inhibition of the NF-κB pathway in alloreactive T-cells, which is critical for IL-2 transcription, could lead to alloantigen-specific hyporesponsiveness and prevention of GVHD. PS1145, a potent inhibitor of IκB kinase, and hence NF-κB activation, was added to an MLR culture consisting of CD4+ T-cells and MHC class II-disparate stimulators. Inhibition of NF-κB activity was verified by EMSA and confocal microscopy. Global inhibition of cytokine production and T-cell hyporesponsiveness was observed which persisted after washing T-cells and re-exposure to alloantigen. Responses to non-specific mitogens remained largely intact and alloantigen hyporesponsiveness was reversed by exogenous IL-2. Treatment of T cells and stimulator cells with PS1145 was required for maximal effect. Depletion of CD4+CD25+ cells from the MLR indicated that these cells were not required for tolerance induction in this system. Using an MLR system containing alloreactive and non-alloreactive transgenic T cells indicated that PS1145 treatment increased the rate of T-cell apoptosis selectively in alloreactive cells. Data from each of 4 experiments showed that GVHD in recipients of ex vivo PS1145 treated cells was profoundly inhibited, whereas CD4+ T-cells recovered from a vehicle-treated 7-day MLR were uniformly fatal upon adoptive transfer into sublethally irradiated MHC class II-disparate recipients. Studies addressing non-alloreactive in vivo responses of PS1145 treated T cells will also be presented. Our studies indicate that the NF-κB pathway is a critical regulator of productive alloresponses and provide a novel ex vivo approach to induce alloantigen-specific tolerance as a means of preventing GVHD.

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