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1.
J Immunother Cancer ; 12(7)2024 Jul 04.
Article in English | MEDLINE | ID: mdl-38964788

ABSTRACT

BACKGROUND: OX40 has been widely studied as a target for immunotherapy with agonist antibodies taken forward into clinical trials for cancer where they are yet to show substantial efficacy. Here, we investigated potential mechanisms of action of anti-mouse (m) OX40 and anti-human (h) OX40 antibodies, including a clinically relevant monoclonal antibody (mAb) (GSK3174998) and evaluated how isotype can alter those mechanisms with the aim to develop improved antibodies for use in rational combination treatments for cancer. METHODS: Anti-mOX40 and anti-hOX40 mAbs were evaluated in a number of in vivo models, including an OT-I adoptive transfer immunization model in hOX40 knock-in (KI) mice and syngeneic tumor models. The impact of FcγR engagement was evaluated in hOX40 KI mice deficient for Fc gamma receptors (FcγR). Additionally, combination studies using anti-mouse programmed cell death protein-1 (mPD-1) were assessed. In vitro experiments using peripheral blood mononuclear cells (PBMCs) examining possible anti-hOX40 mAb mechanisms of action were also performed. RESULTS: Isotype variants of the clinically relevant mAb GSK3174998 showed immunomodulatory effects that differed in mechanism; mIgG1 mediated direct T-cell agonism while mIgG2a acted indirectly, likely through depletion of regulatory T cells (Tregs) via activating FcγRs. In both the OT-I and EG.7-OVA models, hIgG1 was the most effective human isotype, capable of acting both directly and through Treg depletion. The anti-hOX40 hIgG1 synergized with anti-mPD-1 to improve therapeutic outcomes in the EG.7-OVA model. Finally, in vitro assays with human peripheral blood mononuclear cells (hPBMCs), anti-hOX40 hIgG1 also showed the potential for T-cell stimulation and Treg depletion. CONCLUSIONS: These findings underline the importance of understanding the role of isotype in the mechanism of action of therapeutic mAbs. As an hIgG1, the anti-hOX40 mAb can elicit multiple mechanisms of action that could aid or hinder therapeutic outcomes, dependent on the microenvironment. This should be considered when designing potential combinatorial partners and their FcγR requirements to achieve maximal benefit and improvement of patient outcomes.


Subject(s)
Receptors, OX40 , Animals , Humans , Mice , Receptors, OX40/agonists , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Neoplasms/drug therapy , Neoplasms/immunology , Cell Line, Tumor , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/drug effects , Disease Models, Animal
2.
J Immunother Cancer ; 10(1)2022 01.
Article in English | MEDLINE | ID: mdl-35017153

ABSTRACT

BACKGROUND: Despite extensive clinical use, the mechanisms that lead to therapeutic resistance to anti-programmed cell-death (PD)-1 monoclonal antibodies (mAbs) remain elusive. Here, we sought to determine how interactions between the Fc region of anti-PD-1 mAbs and Fcγ receptors (FcγRs) affect therapeutic activity and how these are impacted by the immune environment. METHODS: Mouse and human anti-PD-1 mAbs with different Fc binding profiles were generated and characterized in vitro. The ability of these mAbs to elicit T-cell responses in vivo was first assessed in a vaccination setting using the model antigen ovalbumin. The antitumor activity of anti-PD-1 mAbs was investigated in the context of immune 'hot' MC38 versus 'cold' neuroblastoma tumor models, and flow cytometry performed to assess immune infiltration. RESULTS: Engagement of activating FcγRs by anti-PD-1 mAbs led to depletion of activated CD8 T cells in vitro and in vivo, abrogating therapeutic activity. Importantly, the extent of this Fc-mediated modulation was determined by the surrounding immune environment. Low FcγR-engaging mouse anti-PD-1 isotypes, which are frequently used as surrogates for human mAbs, were unable to expand ovalbumin-reactive CD8 T cells, in contrast to Fc-null mAbs. These results were recapitulated in mice expressing human FcγRs, in which clinically relevant hIgG4 anti-PD-1 led to reduced endogenous expansion of CD8 T cells compared with its engineered Fc-null counterpart. In the context of an immunologically 'hot' tumor however, both low-engaging and Fc-null mAbs induced long-term antitumor immunity in MC38-bearing mice. Finally, a similar anti-PD-1 isotype hierarchy was demonstrated in the less responsive 'cold' 9464D neuroblastoma model, where the most effective mAbs were able to delay tumor growth but could not induce long-term protection. CONCLUSIONS: Our data collectively support a critical role for Fc:FcγR interactions in inhibiting immune responses to both mouse and human anti-PD-1 mAbs, and highlight the context-dependent effect that anti-PD-1 mAb isotypes can have on T-cell responses. We propose that engineering of Fc-null anti-PD-1 mAbs would prevent FcγR-mediated resistance in vivo and allow maximal T-cell stimulation independent of the immunological environment.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Immune Checkpoint Inhibitors/therapeutic use , Immunotherapy/methods , Neoplasms/drug therapy , Programmed Cell Death 1 Receptor/immunology , Animals , Antibodies, Monoclonal/pharmacology , Disease Models, Animal , Humans , Immune Checkpoint Inhibitors/pharmacology , Mice , Tumor Microenvironment
3.
J Immunother Cancer ; 8(2)2020 12.
Article in English | MEDLINE | ID: mdl-33428585

ABSTRACT

BACKGROUND: Previous data suggests that anti-OX40 mAb can elicit anti-tumor effects in mice through deletion of Tregs. However, OX40 also has powerful costimulatory effects on T cells which could evoke therapeutic responses. Human trials with anti-OX40 antibodies have shown that these entities are well tolerated but to date have delivered disappointing clinical responses, indicating that the rules for the optimal use of anti-human OX40 (hOX40) antibodies is not yet fully understood. Changes to timing and dosages may lead to improved outcomes; however, here we focus on addressing the role of agonism versus depleting activity in determining therapeutic outcomes. We investigated a novel panel of anti-hOX40 mAb to understand how these reagents and mechanisms may be optimized for therapeutic benefit. METHODS: This study examines the binding activity and in vitro activity of a panel of anti-hOX40 antibodies. They were further evaluated in several in vivo models to address how isotype and epitope determine mechanism of action and efficacy of anti-hOX40 mAb. RESULTS: Binding analysis revealed the antibodies to be high affinity, with epitopes spanning all four cysteine-rich domains of the OX40 extracellular domain. In vivo analysis showed that their activities relate directly to two key properties: (1) isotype-with mIgG1 mAb evoking receptor agonism and CD8+ T-cell expansion and mIgG2a mAb evoking deletion of Treg and (2) epitope-with membrane-proximal mAb delivering more powerful agonism. Intriguingly, both isotypes acted therapeutically in tumor models by engaging these different mechanisms. CONCLUSION: These findings highlight the significant impact of isotype and epitope on the modulation of anti-hOX40 mAb therapy, and indicate that CD8+ T-cell expansion or Treg depletion might be preferred according to the composition of different tumors. As many of the current clinical trials using OX40 antibodies are now using combination therapies, this understanding of how to manipulate therapeutic activity will be vital in directing new combinations that are more likely to improve efficacy and clinical outcomes.


Subject(s)
Immunoglobulin Isotypes/immunology , Immunotherapy/methods , Receptors, OX40/immunology , Animals , Female , Humans , Mice
4.
Immunity ; 49(5): 958-970.e7, 2018 11 20.
Article in English | MEDLINE | ID: mdl-30446386

ABSTRACT

The costimulatory receptor 4-1BB is expressed on activated immune cells, including activated T cells. Antibodies targeting 4-1BB enhance the proliferation and survival of antigen-stimulated T cells in vitro and promote CD8 T cell-dependent anti-tumor immunity in pre-clinical cancer models. We found that T regulatory (Treg) cells infiltrating human or murine tumors expressed high amounts of 4-1BB. Intra-tumoral Treg cells were preferentially depleted by anti-4-1BB mAbs in vivo. Anti-4-1BB mAbs also promoted effector T cell agonism to promote tumor rejection. These distinct mechanisms were competitive and dependent on antibody isotype and FcγR availability. Administration of anti-4-1BB IgG2a, which preferentially depletes Treg cells, followed by either agonistic anti-4-1BB IgG1 or anti-PD-1 mAb augmented anti-tumor responses in multiple solid tumor models. An antibody engineered to optimize both FcγR-dependent Treg cell depleting capacity and FcγR-independent agonism delivered enhanced anti-tumor therapy. These insights into the effector mechanisms of anti-4-1BB mAbs lay the groundwork for translation into the clinic.


Subject(s)
Antibodies, Monoclonal/pharmacology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Immunomodulation/drug effects , Neoplasms/immunology , Neoplasms/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Tumor Necrosis Factor Receptor Superfamily, Member 9/antagonists & inhibitors , Animals , Gene Expression , Humans , Immunoglobulin G/pharmacology , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Mice , Mice, Knockout , Neoplasms/genetics , Tumor Necrosis Factor Receptor Superfamily, Member 9/genetics , Tumor Necrosis Factor Receptor Superfamily, Member 9/metabolism
5.
Sci Rep ; 8(1): 2278, 2018 02 02.
Article in English | MEDLINE | ID: mdl-29396470

ABSTRACT

CD134 (OX40) is a member of the tumour necrosis factor receptor superfamily (TNFRSF). It acts as a costimulatory receptor on T cells, but its role on NK cells is poorly understood. CD137, another TNFRSF member has been shown to enhance the anti-tumour activity of NK cells in various malignancies. Here, we examine the expression and function of CD134 on human and mouse NK cells in B-cell lymphoma. CD134 was transiently upregulated upon activation of NK cells in both species. In contrast to CD137, induction of CD134 on human NK cells was dependent on close proximity to, or cell-to-cell contact with, monocytes or T cells. Stimulation with an agonistic anti-CD134 mAb but not CD134 ligand, increased IFNγ production and cytotoxicity of human NK cells, but this was dependent on simultaneous antibody:Fcγ receptor binding. In complementary murine studies, intravenous inoculation with BCL1 lymphoma into immunocompetent syngeneic mice resulted in transient upregulation of CD134 on NK cells. Combination treatment with anti-CD20 and anti-CD134 mAb produced a synergistic effect with durable remissions. This therapeutic benefit was abrogated by NK cell depletion and in Fcγ chain -/- mice. Hence, anti-CD134 agonists may enhance NK-mediated anti-tumour activity in an Fcγ receptor dependent fashion.


Subject(s)
Antibodies/metabolism , Killer Cells, Natural/immunology , Lymphoma, B-Cell/immunology , Receptors, OX40/metabolism , Animals , Antibodies, Monoclonal/administration & dosage , Cell Adhesion , Cells, Cultured , Cytotoxicity, Immunologic , Gene Expression Profiling , Humans , Interferon-gamma/metabolism , Mice , Monocytes/immunology , Neoplasm Transplantation , Receptors, OX40/analysis , T-Lymphocytes/immunology , Treatment Outcome
6.
Proc Natl Acad Sci U S A ; 114(7): E1178-E1187, 2017 02 14.
Article in English | MEDLINE | ID: mdl-28137869

ABSTRACT

Memory CD8+ T cells confer long-term immunity against tumors, and anticancer vaccines therefore should maximize their generation. Multiple memory CD8+ T-cell subsets with distinct functional and homing characteristics exist, but the signaling pathways that regulate their development are ill defined. Here we examined the role of the serine/threonine kinase Akt in the generation of protective immunity by CD8+ T cells. Akt is known to be activated by the T-cell antigen receptor and the cytokine IL-2, but its role in T-cell immunity in vivo has not been explored. Using CD8+ T cells from pdk1K465E/K465E knockin mice, we found that decreased Akt activity inhibited the survival of T cells during the effector-to-memory cell transition and abolished their differentiation into C-X-C chemokine receptor 3 (CXCR3)loCD43lo effector-like memory cells. Consequently, antitumor immunity by CD8+ T cells that display defective Akt signaling was substantially diminished during the memory phase. Reduced memory T-cell survival and altered memory cell differentiation were associated with up-regulation of the proapoptotic protein Bim and the T-box transcription factor eomesodermin, respectively. These findings suggest an important role for effector-like memory CD8+ T cells in tumor immune surveillance and identify Akt as a key signaling node in the development of protective memory CD8+ T-cell responses.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Immunologic Memory/immunology , Immunologic Surveillance/immunology , Neoplasms, Experimental/immunology , Proto-Oncogene Proteins c-akt/immunology , Animals , Binding Sites/genetics , CD8-Positive T-Lymphocytes/metabolism , CX3C Chemokine Receptor 1/immunology , CX3C Chemokine Receptor 1/metabolism , Cell Line, Tumor , Immunologic Surveillance/genetics , Mice, Inbred C57BL , Mice, Transgenic , Mutation , Neoplasms, Experimental/genetics , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/immunology , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Pyruvate Dehydrogenase Acetyl-Transferring Kinase , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , Receptors, CXCR3/immunology , Receptors, CXCR3/metabolism , Signal Transduction/immunology
7.
Mol Immunol ; 83: 13-22, 2017 03.
Article in English | MEDLINE | ID: mdl-28092803

ABSTRACT

OX40 is a type 1 transmembrane glycoprotein, reported nearly 30 years ago as a cell surface antigen expressed on activated T cells. Since its discovery, it has been validated as a bone fide costimulatory molecule for T cells and member of the TNF receptor family. However, many questions still remain relating to its function on different T cell sub-sets and with recent interest in its utility as a target for antibody-mediated immunotherapy, there is a growing need to gain a better understanding of its biology. Here, we review the expression pattern of OX40 and its ligand, discuss the structure of the receptor:ligand interaction, the downstream signalling it can elicit, its function on different T cell subsets and how antibodies might engage with it to provide effective immunotherapy.


Subject(s)
OX40 Ligand/immunology , Receptors, OX40/immunology , T-Lymphocytes/immunology , Animals , Humans , Lymphocyte Activation/immunology , Structure-Activity Relationship
8.
Cancer Cell ; 27(1): 138-48, 2015 Jan 12.
Article in English | MEDLINE | ID: mdl-25500122

ABSTRACT

Monoclonal antibody (mAb) drugs that stimulate antitumor immunity are transforming cancer treatment but require optimization for maximum clinical impact. Here, we show that, unlike other immunoglobulin isotypes, human IgG2 (h2) imparts FcγR-independent agonistic activity to immune-stimulatory mAbs such as anti-CD40, -4-1BB, and -CD28. Activity is provided by a subfraction of h2, h2B, that is structurally constrained due its unique arrangement of hinge region disulfide bonds. Agonistic activity can be transferred from h2 to h1 by swapping their hinge and CH1 domains, and substitution of key hinge and CH1 cysteines generates homogenous h2 variants with distinct agonistic properties. This provides the exciting opportunity to engineer clinical reagents with defined therapeutic activity regardless of FcγR expression levels in the local microenvironment.


Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/therapeutic use , Immunoglobulin G/chemistry , Immunoglobulin G/therapeutic use , Receptors, IgG/immunology , Thymoma/prevention & control , Thymus Neoplasms/prevention & control , Animals , Antibodies, Monoclonal/immunology , CD28 Antigens/immunology , CD40 Antigens/immunology , Cells, Cultured , Humans , Immunoglobulin G/immunology , Mice , Mice, Inbred C57BL , Thymoma/drug therapy , Thymoma/immunology , Thymus Neoplasms/drug therapy , Thymus Neoplasms/immunology , Tumor Necrosis Factor Receptor Superfamily, Member 9/immunology , Vaccination/methods
9.
J Immunol ; 193(1): 244-51, 2014 Jul 01.
Article in English | MEDLINE | ID: mdl-24860188

ABSTRACT

The factors that determine differentiation of naive CD8 T cells into memory cells are not well understood. A greater understanding of how memory cells are generated will inform of ways to improve vaccination strategies. In this study, we analyzed the CD8 T cell response elicited by two experimental vaccines comprising a peptide/protein Ag and an agonist that delivers a costimulatory signal via CD27 or 4-1BB. Both agonists increased expansion of Ag-specific CD8 T cells compared with Ag alone. However, their capacity to stimulate differentiation into effector and memory cells differed. CD27 agonists promoted increased expression of perforin and the generation of short-lived memory cells, whereas stimulation with 4-1BB agonists favored generation of stable memory. The memory-promoting effects of 4-1BB were independent of CD4 T cells and were the result of programing within the first 2 d of priming. Consistent with this conclusion, CD27 and 4-1BB-stimulated CD8 T cells expressed disparate amounts of IL-2, IFN-γ, CD25, CD71, and Gp49b as early as 3 d after in vivo activation. In addition, memory CD8 T cells, generated through priming with CD27 agonists, proliferated more extensively than did 4-1BB-generated memory cells, but these cells failed to persist. These data demonstrate a previously unanticipated link between the rates of homeostatic proliferation and memory cell attrition. Our study highlights a role for these receptors in skewing CD8 T cell differentiation into effector and memory cells and provides an approach to optimize vaccines that elicit CD8 T cell responses.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Immunization , Immunologic Memory , Peptides/pharmacology , Tumor Necrosis Factor Receptor Superfamily, Member 7/immunology , Tumor Necrosis Factor Receptor Superfamily, Member 9/immunology , Animals , Antigens/pharmacology , Antigens, CD/genetics , Antigens, CD/immunology , CD8-Positive T-Lymphocytes/cytology , Interferon-gamma/genetics , Interferon-gamma/immunology , Interleukin-2/genetics , Interleukin-2/immunology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Mice , Mice, Transgenic , Receptors, Immunologic/genetics , Receptors, Immunologic/immunology , Receptors, Transferrin/genetics , Receptors, Transferrin/immunology , Tumor Necrosis Factor Receptor Superfamily, Member 7/genetics , Tumor Necrosis Factor Receptor Superfamily, Member 9/agonists , Tumor Necrosis Factor Receptor Superfamily, Member 9/genetics
10.
Eur J Immunol ; 43(12): 3314-23, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24002868

ABSTRACT

Naive T cells require signals from multiple costimulatory receptors to acquire full effector function and differentiate to long-lived memory cells. The costimulatory receptor, CD27, is essential for optimal T-cell priming and memory differentiation in a variety of settings, although whether CD27 is similarly required during memory CD8(+) T-cell reactivation remains controversial. We have used OVA and anti-CD40 to establish a memory CD8(+) T-cell population and report here that their secondary expansion, driven by peptide and anti-CD40, polyI:C, or LPS, requires CD27. Furthermore, antigenic peptide and a soluble form of the CD27 ligand, CD70 (soluble recombinant CD70 (sCD70)), is sufficient for secondary memory CD8(+) T-cell accumulation at multiple anatomical sites, dependent on CD80/86. Prior to boost, resting effector- and central-memory CD8(+) T cells both expressed CD27 with greater expression on central memory cells. Nonetheless, both populations upregulated CD27 after TCR engagement and accumulated in proportion after boosting with Ag and sCD70. Mechanistically, sCD70 increased the frequency of divided and cytolytic memory T cells, conferred resistance to apoptosis and enabled retardation of tumor growth in vivo. These data demonstrate the central role played by CD27/70 during secondary CD8(+) T-cell activation to a peptide Ag, and identify sCD70 as an immunotherapeutic adjuvant for antitumor immunity.


Subject(s)
Antigens/immunology , CD8-Positive T-Lymphocytes/immunology , Immunologic Memory/physiology , Lymphocyte Activation/immunology , Peptides/immunology , Tumor Necrosis Factor Receptor Superfamily, Member 7/immunology , Adjuvants, Immunologic , Animals , Antibodies/immunology , Antibodies/pharmacology , CD27 Ligand/immunology , CD27 Ligand/pharmacology , CD40 Antigens/immunology , CD8-Positive T-Lymphocytes/cytology , Immunotherapy/methods , Interferon Inducers/pharmacokinetics , Interferon Inducers/pharmacology , Lipopolysaccharides/pharmacology , Lymphocyte Activation/drug effects , Lymphocyte Activation/genetics , Mice , Mice, Knockout , Neoplasms/immunology , Neoplasms/pathology , Neoplasms/therapy , Poly I-C/pharmacology , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Tumor Necrosis Factor Receptor Superfamily, Member 7/genetics
11.
Eur J Immunol ; 41(9): 2606-11, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21688261

ABSTRACT

TNFRSF25 is a member of the TNF receptor superfamily (TNFRSF) that binds to the TNF-like protein TL1A. Although recent studies have demonstrated a role for TNFRSF25 in regulating CD4(+) T-cell responses, it remains to be determined if TNFRSF25 functions as a costimulatory receptor for CD8(+) T cells. Here, we demonstrate that ectopic expression of TL1A on mouse plasmacytomas promotes elimination of tumor cells in a CD8(+) T-cell-dependent manner and renders mice immune to a subsequent challenge with tumor cells. To gain further insight into the role of TNFRSF25 in CD8(+) T-cell responses, we analyzed the effect of TNFRSF25 triggering on OT-I TCR transgenic T cells. We demonstrate that TNFRSF25 triggering in vivo with soluble TL1A promotes the proliferation and accumulation of antigen-specific CD8(+) T cells as well as their differentiation into CTLs. Furthermore, we show that TNFRSF25 also functions as a costimulatory receptor for memory CD8(+) T cells. Thus, TNFRSF25 triggering enhances the secondary expansion of endogenous antigen-specific memory CD8(+) T cells. Our data suggest that TNFRSF25 agonists, such as soluble TL1A, could potentially be used to enhance the immunogenicity of vaccines that aim to elicit human anti-tumor CD8(+) T cells.


Subject(s)
CD8-Positive T-Lymphocytes/metabolism , Cancer Vaccines , Plasmacytoma/metabolism , Receptors, Tumor Necrosis Factor, Member 25/metabolism , Tumor Necrosis Factor Ligand Superfamily Member 15/metabolism , Animals , Antigens, Neoplasm/immunology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Cell Line, Tumor , Cell Proliferation , Cytotoxicity, Immunologic , Humans , Immunologic Memory , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Transgenic , Plasmacytoma/immunology , Plasmacytoma/pathology , Receptors, Tumor Necrosis Factor, Member 25/immunology , Transgenes/genetics , Tumor Necrosis Factor Ligand Superfamily Member 15/genetics
12.
J Immunol ; 185(2): 1082-92, 2010 Jul 15.
Article in English | MEDLINE | ID: mdl-20554967

ABSTRACT

The ternary complex factors (TCFs; SAP-1, Elk-1, and Net) are serum response factor cofactors that share many functional properties and are coexpressed in many tissues. SAP-1, the predominant thymus TCF, is required for thymocyte positive selection. In this study, we assessed whether the different TCFs are functionally equivalent. Elk-1 deletion, but not the hypomorphic Net(delta) mutation, exacerbated the SAP-1 positive selection phenotype, but triply deficient thymocytes were no more defective than SAP-1(-/-) Elk-1(-/-) cells. Inactivation of the other TCFs did not affect SAP-1-independent processes, including beta-selection, regulatory T cell selection, and negative selection, although reduced marginal zone B cells were observed in SAP-1(-/-) Elk-1(-/-) animals. Ectopic expression of Elk-1, but not Net, rescued positive selection of SAP-1(-/-) thymocytes; thus, SAP-1 and Elk-1 are functionally equivalent in this system, and the SAP-1 null selection phenotype reflects only its high expression in the thymus. Array analysis of TCR-stimulated double-positive cells identified SAP-1-dependent inducible genes whose transcription was further impaired in SAP-1(-/-) Elk-1(-/-) cells; thus, these genes, which include Egr-1 and Egr-2, represent candidate mediators of positive selection. Chromatin immunoprecipitation revealed subtly different promoter targeting between the different TCFs. Ectopic expression of Egr-1 restored positive selection in SAP-1 null thymocytes, establishing it (and possibly other Egr family members) as the major effector for ERK-SAP-1 signaling in thymocyte positive selection.


Subject(s)
Proto-Oncogene Proteins c-ets/immunology , Thymus Gland/immunology , ets-Domain Protein Elk-1/immunology , ets-Domain Protein Elk-4/immunology , Animals , Cell Differentiation/genetics , Cell Differentiation/immunology , Cells, Cultured , Chromatin Immunoprecipitation , Female , Flow Cytometry , Gene Expression Profiling , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Proto-Oncogene Proteins c-ets/genetics , Proto-Oncogene Proteins c-ets/metabolism , Retroviridae/genetics , Thymus Gland/cytology , Thymus Gland/metabolism , Transduction, Genetic , ets-Domain Protein Elk-1/genetics , ets-Domain Protein Elk-1/metabolism , ets-Domain Protein Elk-4/genetics , ets-Domain Protein Elk-4/metabolism
13.
J Immunol ; 179(10): 6836-44, 2007 Nov 15.
Article in English | MEDLINE | ID: mdl-17982074

ABSTRACT

Regulatory T cells (T(reg)) play an important role in immune regulation. Their development in the thymus requires TCR activation and recognition of peptide-MHC, although the downstream signals controlling commitment to the lineage are unclear. To compare the requirements for positive selection and T(reg) development, we studied knockout and transgenic mice defective in Raf signaling and the ERK effector SRF accessory protein 1 (SAP-1), a member of the ternary complex factor family of Ets domain transcription factors. Although SAP-1 deficient mice display a severe defect in thymocyte positive selection, T(reg) development was unimpaired as assessed by expression of Foxp3 and the activation markers CD25, GITR, CTLA4, and CD103 in the CD4(+) cell population. In contrast, inhibition of Raf signaling by the interfering dominant negative Raf derivative reduced both Foxp3(+) and Foxp3(-) CD4(+) populations. In SAP-1-deficient CD4(+)CD25(+) T(reg) cells, TCR crosslinking efficiently induced ERK activation, but transcriptional induction of the immediate early gene Egr-1 was impaired. Nevertheless, neither deletion of SAP-1 nor expression of a dominant negative Raf derivative affected the ability of CD4(+)CD25(+) T(reg) cells to suppress CD4(+)CD25(-) cell proliferation in vitro. Finally the suppressive activity of CD4(+)CD25(+) T(reg) cells lacking SAP-1 in an in vivo colitis model was not significantly impaired. The signaling requirements for development of T(reg) cells in the thymus are thus distinct from those required for "conventional" T cell positive selection, and ERK signaling to SAP-1 is not required for the suppressive activity of T(reg) cells.


Subject(s)
Extracellular Signal-Regulated MAP Kinases/immunology , Signal Transduction/immunology , T-Lymphocytes, Regulatory/immunology , Thymus Gland/immunology , ets-Domain Protein Elk-4/immunology , raf Kinases/immunology , Animals , Antigens, Differentiation , Cell Proliferation , Colitis/immunology , Colitis/metabolism , Colitis/pathology , Disease Models, Animal , Early Growth Response Protein 1/biosynthesis , Early Growth Response Protein 1/genetics , Early Growth Response Protein 1/immunology , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation/immunology , Mice , Mice, Transgenic , Signal Transduction/genetics , T-Lymphocytes, Regulatory/metabolism , T-Lymphocytes, Regulatory/pathology , Thymus Gland/metabolism , Thymus Gland/pathology , ets-Domain Protein Elk-4/genetics , ets-Domain Protein Elk-4/metabolism , raf Kinases/genetics , raf Kinases/metabolism
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