RESUMO
Despite the beneficial therapeutic effects of intravenous immunoglobulin (IVIg) in inflammatory diseases, consistent therapeutic efficacy and potency remain major limitations for patients and physicians using IVIg. These limitations have stimulated a desire to generate therapeutic alternatives that could leverage the broad mechanisms of action of IVIg while improving therapeutic consistency and potency. The identification of the important anti-inflammatory role of fragment crystallizable domain (Fc) sialylation has presented an opportunity to develop more potent Ig therapies. However, translating this concept to potent anti-inflammatory therapeutics has been hampered by the difficulty of generating suitable sialylated products for clinical use. Therefore, we set out to develop the first, to our knowledge, robust and scalable process for generating a well-qualified sialylated IVIg drug candidate with maximum Fc sialylation devoid of unwanted alterations to the IVIg mixture. Here, we describe a controlled enzymatic, scalable process to produce a tetra-Fc-sialylated (s4-IVIg) IVIg drug candidate and its qualification across a wide panel of analytic assays, including physicochemical, pharmacokinetic, biodistribution, and in vivo animal models of inflammation. Our in vivo characterization of this drug candidate revealed consistent, enhanced anti-inflammatory activity up to 10-fold higher than IVIg across different animal models. To our knowledge, this candidate represents the first s4-IVIg suitable for clinical use; it is also a valuable therapeutic alternative with more consistent and potent anti-inflammatory activity.
Assuntos
Anti-Inflamatórios/uso terapêutico , Desenho de Fármacos , Imunoglobulinas Intravenosas/uso terapêutico , Ácido N-Acetilneuramínico/metabolismo , Receptores Fc/metabolismo , Animais , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Artrite Experimental/tratamento farmacológico , Artrite Experimental/patologia , Vesícula/complicações , Vesícula/tratamento farmacológico , Vesícula/patologia , Modelos Animais de Doenças , Epidermólise Bolhosa Adquirida/complicações , Epidermólise Bolhosa Adquirida/tratamento farmacológico , Epidermólise Bolhosa Adquirida/patologia , Glicosilação/efeitos dos fármacos , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulinas Intravenosas/farmacocinética , Imunoglobulinas Intravenosas/farmacologia , Camundongos , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Púrpura Trombocitopênica Idiopática/patologia , Distribuição Tecidual/efeitos dos fármacos , Resultado do TratamentoRESUMO
PURPOSE: This global phase I trial investigated the safety, efficacy, pharmacokinetics, and pharmacodynamics of lisaftoclax (APG-2575), a novel, orally active, potent selective B-cell lymphoma 2 (BCL-2) inhibitor, in patients with relapsed or refractory chronic lymphocytic leukemia or small lymphocytic lymphoma (R/R CLL/SLL) and other hematologic malignancies (HMs). PATIENTS AND METHODS: Maximum tolerated dose (MTD) and recommended phase II dose were evaluated. Outcome measures were safety and tolerability (primary) and pharmacokinetic variables and antitumor effects (secondary). Pharmacodynamics in patient tumor cells were explored. RESULTS: Among 52 patients receiving lisaftoclax, MTD was not reached. Treatment-emergent adverse events (TEAEs) included diarrhea (48.1%), fatigue (34.6%), nausea (30.8%), anemia and thrombocytopenia (28.8% each), neutropenia (26.9%), constipation (25.0%), vomiting (23.1%), headache (21.2%), peripheral edema and hypokalemia (17.3% each), and arthralgia (15.4%). Grade ≥ 3 hematologic TEAEs included neutropenia (21.2%), thrombocytopenia (13.5%), and anemia (9.6%), none resulting in treatment discontinuation. Clinical pharmacokinetic and pharmacodynamic results demonstrated that lisaftoclax had a limited plasma residence and systemic exposure and elicited rapid clearance of malignant cells. With a median treatment of 15 (range, 6-43) cycles, 14 of 22 efficacy-evaluable patients with R/R CLL/SLL experienced partial responses, for an objective response rate of 63.6% and median time to response of 2 (range, 2-8) cycles. CONCLUSIONS: Lisaftoclax was well tolerated, with no evidence of tumor lysis syndrome. Dose-limiting toxicity was not reached at the highest dose level. Lisaftoclax has a unique pharmacokinetic profile compatible with a potentially more convenient daily (vs. weekly) dose ramp-up schedule and induced rapid clinical responses in patients with CLL/SLL, warranting continued clinical investigation.
Assuntos
Anemia , Antineoplásicos , Neoplasias Hematológicas , Leucemia Linfocítica Crônica de Células B , Linfoma de Células B , Neutropenia , Trombocitopenia , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Antineoplásicos/efeitos adversos , Linfoma de Células B/patologia , Neoplasias Hematológicas/tratamento farmacológico , Neutropenia/induzido quimicamente , Anemia/induzido quimicamente , Anemia/tratamento farmacológico , Trombocitopenia/induzido quimicamente , Proteínas Proto-Oncogênicas c-bcl-2RESUMO
OBJECTIVE: Rheumatoid arthritis culminates in joint destruction that, in mouse models of disease, is supported by innate immune molecules, including Fcγ receptors (FcγR) and complement. However, these findings may not be predictive of the outcome in humans, given the structural differences between murine and human activating FcγR on neutrophils, a prominent component of joint exudates. The aim of this study was to examine the role of human neutrophil FcγRIIa in the development of arthritis and probe the underlying mechanism by which FcγRIIa initiates disease. METHODS: K/BxN mouse serum transfer-induced arthritis was examined in mice expressing human FcγRIIa on neutrophils but lacking their own activating FcγR (γ-chain-deficient mice). The role of mast cells, complement (C3 and C5a), and CD18 integrins in FcγRIIa-initiated disease was examined using cell reconstitution approaches, inhibitors, and functional blocking antibodies, respectively. Crosstalk between the complement receptor C5aR and FcγRIIa on neutrophils was evaluated in vitro. RESULTS: The expression of human FcγRIIa on neutrophils was sufficient to restore susceptibility to K/BxN serum-induced neutrophil recruitment, synovitis, and bone destruction in γ-chain-deficient mice. Joint inflammation was robust and proceeded even in the absence of mast cells and vascular permeability, features shown to contribute to disease in wild-type mice. Neutrophil recruitment was dependent on the presence of a CD18 integrin, lymphocyte function-associated antigen 1, and C5aR. In addition, C5aR significantly enhanced FcγRIIa-mediated phagocytosis and oxidative burst in vitro. CONCLUSION: Human and murine activating FcγR on neutrophils are not functionally equivalent, and in humans, they may play a primary role in arthritis. Crosstalk between neutrophil C5aR and FcγRIIa is essential for disease progression, thus highlighting a new aspect of complement during the effector phase of inflammatory arthritis.
Assuntos
Artrite Experimental/imunologia , Neutrófilos/imunologia , Receptor da Anafilatoxina C5a/imunologia , Receptores de IgG/imunologia , Transferência Adotiva , Animais , Artrite Experimental/metabolismo , Transplante de Medula Óssea , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Fagocitose/imunologia , Receptor Cross-Talk/imunologia , Receptor da Anafilatoxina C5a/metabolismo , Receptores de IgG/metabolismo , Especificidade da Espécie , Sinovite/imunologia , Sinovite/metabolismoRESUMO
Immunomodulatory therapeutics such as monoclonal antibodies (mAb) carry an inherent risk of undesired immune reactions. One such risk is cytokine release syndrome (CRS), a rapid systemic inflammatory response characterized by the secretion of pro-inflammatory cytokines from immune cells. It is crucial for patient safety to correctly identify potential risk of CRS prior to first-in-human dose administration. For this purpose, a variety of in vitro cytokine release assays (CRA) are routinely used as part of the preclinical safety assessment of novel therapeutic mAbs. One of the challenges for the development and comparison of CRA performance is the lack of availability of standard positive and negative control mAbs for use in assay qualification. To address this issue, the National Institute for Biological Standards and Control (NIBSC) developed a reference panel of lyophilised mAbs known to induce CRS in the clinic: human anti-CD52, mouse anti-CD3 and human superagonistic (SA) anti-CD28 mAb manufactured according to the respective published sequences of Campath-1H® (alemtuzumab, IgG1) , Orthoclone OKT-3® (muromonab, IgG2a) and TGN1412 (theralizumab, IgG4), as well as three isotype matched negative controls (human IgG1, mouse IgG2a and human IgG4, respectively). The relative capacity of these control mAbs to stimulate the release of IFN-γ, IL-2, TNF-α and IL-6 in vitro was evaluated in eleven laboratories in an international collaborative study mediated through the HESI Immuno-safety Technical Committee Cytokine Release Assay Working Group. Participants tested the NIBSC mAbs in a variety of CRA platforms established at each institution. This paper presents the results from the centralised cytokine quantification on all the plasma/supernatants corresponding to the stimulation of immune cells in the different CRA platforms by a single concentration of each mAb. Each positive control mAb induced significant cytokine release in most of the tested CRA platforms. There was a high inter-laboratory variability in the levels of cytokines produced, but similar patterns of response were observed across laboratories that replicated the cytokine release patterns previously published for the respective clinical therapeutic mAbs. Therefore, the positive and negative mAbs are suitable as a reference panel for the qualification and validation of CRAs, comparison of different CRA platforms (e.g. solid vs aqueous phase), and intra- and inter-laboratory comparison of CRA performance. Thus, the use of this panel of positive and negative control mAbs will increase the confidence in the robustness of a CRA platform to identify a potential CRS risk for novel immunomodulatory therapeutic candidates.
RESUMO
The signals linking neutrophil opsonic receptors, FcgammaRs and complement receptor 3 (Mac-1) to cellular cytotoxic responses are poorly understood. Furthermore, because a deficiency in activating FcgammaRs reduces both IgG-mediated neutrophil recruitment and tissue injury, the role of FcgammaRs specifically in mediating neutrophil cytotoxicity in vivo remains unclear. In this study, we demonstrate that neutrophil Vav 1 and 3, guanine exchange factors for Rac GTPases, are required for IgG/FcgammaR-mediated hemorrhage and edema in the reverse passive Arthus in the lung and skin. Rac GTPases are also required for development of the reverse passive Arthus reaction. A deficiency in Vav 1 and 3 does not affect neutrophil accumulation at the site of immune complex deposition, thus uncoupling neutrophil recruitment and tissue injury. Surprisingly, Vav and Rac proteins are dispensable for the development of the local Shwartzman reaction in vivo and phagocytosis of complement-opsonized RBC in vitro, processes strictly dependent on Mac-1 and complement C3. Thus, FcgammaR signaling through the Vav and Rac proteins in neutrophils is critical for stimulating immune complex disease while Vav- and Rac-independent pathways promote Mac-1/complement C3-dependent functions.
Assuntos
Complemento C3/imunologia , Imunoglobulina G/imunologia , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Proteínas Proto-Oncogênicas c-vav/imunologia , Transdução de Sinais/imunologia , Animais , Complexo Antígeno-Anticorpo/imunologia , Reação de Arthus/genética , Reação de Arthus/imunologia , Complemento C3/genética , Edema/genética , Edema/imunologia , Hemorragia/genética , Hemorragia/imunologia , Imunoglobulina G/genética , Pulmão/imunologia , Antígeno de Macrófago 1/genética , Antígeno de Macrófago 1/imunologia , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos/genética , Fagocitose/imunologia , Proteínas Proto-Oncogênicas c-vav/genética , Receptores de IgG/genética , Receptores de IgG/imunologia , Fenômeno de Shwartzman/genética , Fenômeno de Shwartzman/imunologia , Pele/imunologia , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/imunologiaRESUMO
CD18 integrins promote neutrophil recruitment, and their engagement activates tyrosine kinases, leading to neutrophil activation. However, the significance of integrin-dependent leukocyte activation in vivo has been difficult to prove. Here, in a model of thrombohemorrhagic vasculitis, the CD18 integrin Mac-1 on neutrophils recognized complement C3 deposited within vessel walls and triggered a signaling pathway involving the Src-family kinase Hck and the Syk tyrosine kinase. This led to neutrophil elastase release, causing hemorrhage, fibrin deposition, and thrombosis. Mice genetically deficient in any of these components (C3, Mac-1, Hck, Syk, or elastase) were resistant to disease despite normal tissue neutrophil accumulation. Disease was restored in Mac-1-deficient mice infused with wild-type, but not kinase- or elastase-deficient, neutrophils. Elastase release in the inflamed tissue was reduced in Mac-1-deficient mice, and a deficiency of Mac-1 or the kinases blocked neutrophil elastase release in vitro. These data suggest that Mac-1 engagement of complement activates tyrosine kinases to promote elastase-dependent blood vessel injury in vivo.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Antígeno de Macrófago 1/metabolismo , Elastase Pancreática/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fenômeno de Shwartzman/metabolismo , Transdução de Sinais , Trombose/metabolismo , Quinases da Família src/metabolismo , Animais , Adesão Celular , Movimento Celular , Complemento C3/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Ligantes , Antígeno de Macrófago 1/genética , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/metabolismo , Neutrófilos/citologia , Neutrófilos/enzimologia , Peptídeo Hidrolases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fenômeno de Shwartzman/genética , Fenômeno de Shwartzman/patologia , Quinase Syk , Trombose/genética , Trombose/patologiaRESUMO
We developed an approach that increases CD4+CD25+ regulatory T-cell potency by antigen-specifically redirecting them against pathologic T lymphocytes. The regulatory cells are transgenically modified with chimeric receptors that link antigen-major histocompatibility complex (MHC) extracellular and transmembrane domains with the cytoplasmic signaling tail of T-cell receptor zeta (TCR-zeta). The receptors' antigen-MHC recognizes the TCR of cognate T lymphocytes. Receptor engagement stimulates the receptor-modified T cell (RMTC) through the linked zeta chain. CD4+CD25+ RMTCs expressing a myelin basic protein (MBP) 89-101-IAs-zeta receptor, unlike unmodified CD4+CD25+ T cells or CD4+CD25- RMTCs, prevented and treated experimental allergic encephalomyelitis (EAE) induced with MBP89-101. The RMTCs were effective even after the autoreactive T-cell repertoire had diversified to include specificities not directly targeted by the chimeric receptor. Remissions were sustained and mortality was decreased from more than 50% to 0%. These results provide proof of principal for a novel approach to enforce the interaction of regulatory and pathologic T lymphocytes, thereby facilitating the treatment of autoimmune disease.
Assuntos
Transferência Adotiva/métodos , Linfócitos T CD4-Positivos/transplante , Encefalomielite Autoimune Experimental/terapia , Receptores de Interleucina-2 , Animais , Células Cultivadas , Feminino , Proteínas de Membrana/imunologia , Camundongos , Camundongos Transgênicos , Proteína Básica da Mielina/imunologia , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Especificidade do Receptor de Antígeno de Linfócitos TRESUMO
How small numbers of CD4+CD25+ regulatory T cells suppress autoimmune responses in vivo is unclear. In this report we analyze the immunomodulatory activity of CD4+CD25+ T cells that are antigen-specifically redirected against myelin basic protein (MBP)89-101-specific autoreactive T cells by a MBP89-101-IA(s)-zeta chimeric receptor. We have previously shown that these redirected regulatory T cells are highly potent in treating a model autoimmune disease, experimental allergic encephalomyelitis. We show here that they have only limited effect in vivo on autoreactive T cell proliferation and therefore do not act by deleting or suppressing the expansion of pathologic effector cells. Rather, the redirected CD4+CD25+ T cells divert the pathologic T helper 1 self-specific T cell response to one characterized by high IL-10 and lower IL-4 production. Significantly, when isolated from the inducing CD4+CD25+ regulatory T cells, these self-specific T cells can independently suppress the autoreactive T cell response and experimental allergic encephalomyelitis development in an IL-10-dependent manner. These results provide evidence that CD4+CD25+ regulatory T cells can manipulate the adaptive immune response in vivo through the infectious induction of tolerance, specifically by promoting the formation of antigen-specific, IL-10-secreting regulatory T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Encefalomielite Autoimune Experimental/imunologia , Tolerância Imunológica/imunologia , Imunoterapia , Interleucina-10/imunologia , Receptores de Interleucina-2/imunologia , Animais , Proliferação de Células , Citocinas/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Infecções/imunologia , Infecções/patologia , CamundongosRESUMO
We previously showed that transgenically expressed chimeric Ag-MHC-zeta receptors can Ag-specifically redirect T cells against other T cells. When the receptor's extracellular Ag-MHC domain engages cognate TCR on an Ag-specific T cell, its cytoplasmic zeta-chain stimulates the chimeric receptor-modified T cell (RMTC). This induces effector functions such as cytolysis and cytokine release. RMTC expressing a myelin basic protein (MBP) 89-101-IAs-zeta receptor can be used therapeutically, Ag-specifically treating murine experimental allergic encephalomyelitis (EAE) mediated by MBP89-101-specific T cells. In initial studies, isolated CD8+ RMTC were therapeutically effective whereas CD4+ RMTC were not. We re-examine here the therapeutic potential of CD4+ RMTC. We demonstrate that Th2-differentiated, though not Th1-differentiated, CD4+ MBP89-101-IAs-zeta RMTC prevent actively induced or adoptively transferred EAE, and treat EAE even after antigenic diversification of the pathologic T cell response. The Th2 RMTC both Th2-deviate autoreactive T cells and suppress autoantigen-specific T cell proliferation. IL-10 is critical for the suppressive effects. Anti-IL-10R blocks RMTC-mediated modulation of EAE and suppression of autoantigen proliferation, as well as the induction of IL-10 production by autoreactive T cells. In contrast to IL-10, IL-4 is required for IL-4 production by, and hence Th2 deviation of autoreactive T cells, but not the therapeutic activity of the RMTC. These results therefore demonstrate a novel immunotherapeutic approach for the Ag-specific treatment of autoimmune disease with RMTC. They further identify an essential role for IL-10, rather than Th2-deviation itself, in the therapeutic effectiveness of these redirected Th2 T cells.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/prevenção & controle , Interleucina-10/imunologia , Células Th2/imunologia , Animais , Proliferação de Células , Citocinas/biossíntese , Encefalomielite Autoimune Experimental/patologia , Feminino , Tolerância Imunológica , Interleucina-10/biossíntese , Interleucina-4/antagonistas & inibidores , Interleucina-4/biossíntese , Interleucina-4/imunologia , Camundongos , Proteína Básica da Mielina/imunologia , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Interleucina/antagonistas & inibidores , Receptores de Interleucina-10 , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Células Th1/imunologia , Células Th1/patologia , Células Th2/patologiaRESUMO
Prior reports have shown that CD4(+)CD25(+) regulatory T cells suppress naive T cell responses by inhibiting IL-2 production. In this report, using an Ag-specific TCR transgenic system, we show that naive T cells stimulated with cognate Ag in the presence of preactivated CD4(+)CD25(+) T cells also become refractory to the mitogenic effects of IL-2. T cells stimulated in the presence of regulatory T cells up-regulated high affinity IL-2R, but failed to produce IL-2, express cyclins or c-Myc, or exit G(0)-G(1). Exogenous IL-2 failed to break the mitotic block, demonstrating that the IL-2 production failure was not wholly responsible for the proliferation defect. This IL-2 unresponsiveness did not require the continuous presence of CD4(+)CD25(+) regulatory T cells. The majority of responder T cells reisolated after coculture with regulatory cells failed to proliferate in response to IL-2, but were not anergic and proliferated in response to Ag. The mitotic block was also dissociated from the antiapoptotic effects of IL-2, because IL-2 still promoted the survival of T cells that had been cocultured with CD4(+)CD25(+) T cells. IL-2-induced STAT5 phosphorylation in the cocultured responder cells was intact, implying that the effects of the regulatory cells were downstream of receptor activation. Our results therefore show that T cell activation in the presence of CD4(+)CD25(+) regulatory T cells can induce an alternative stimulation program characterized by up-regulation of high affinity IL-2R, but a failure to produce IL-2, and uncoupling of the mitogenic and antiapoptotic effects of IL-2.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Ciclo Celular/fisiologia , Interleucina-2/imunologia , Ativação Linfocitária/imunologia , Receptores de Interleucina-2/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Western Blotting , Células Cultivadas , Técnicas de Cocultura , Proteínas de Ligação a DNA/imunologia , Citometria de Fluxo , Camundongos , Camundongos Transgênicos , Proteínas do Leite/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT5 , Transdução de Sinais/imunologia , Transativadores/imunologiaRESUMO
As CTL adhere to and lyze their targets, they extract cognate Ag-MHC and represent this on their own cell surface. Whether such self-presented cognate Ag stimulate the TCR of a CTL is uncertain. To analyze this, we examined TCR capping in response to self-presented Ag. We found that OVA peptide-specific OT-1 CTL that were pulsed with cognate peptide Ag did not cap their TCR, implying that the autologously presented MHC-Ag complex does not normally stimulate the TCR. However, this functional separation of the TCR and its ligand on the cell surface was not absolute. Treatment of Ag-pulsed OT-1 CTL with agents that alter cell surface charge, including trypsin, papain, tunicamycin, neuraminidase, and polybrene, allowed Ag-specific TCR capping. The TCR capped together with the restricting MHC molecule on the surface of the cell, implying an interaction between the TCR and cell-associated Ag. Further, the treated CTL underwent a time- and dose-dependent suicidal death that was both Fas- and perforin-dependent. Therefore, our results indicate that the association of the TCR with its MHC-peptide ligand on the surface of a CTL is normally proscribed by biophysical properties of the plasma membrane. Overcoming this restriction allows TCR stimulation and induces CTL effector functions and cell suicide.
Assuntos
Apresentação de Antígeno , Antígenos H-2/fisiologia , Agregação de Receptores/imunologia , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Animais , Morte Celular/imunologia , Membrana Celular/imunologia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Células Cultivadas , Espaço Extracelular/imunologia , Glicosilação , Antígenos H-2/metabolismo , Líquido Intracelular/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/imunologia , Ovalbumina/metabolismo , Ovalbumina/farmacologia , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/ultraestrutura , Tripsina/farmacologiaRESUMO
T cells play a central role in the recognition and elimination of foreign pathogens. Signals through the T cell receptor (TCR) control the extent and duration of the T cell response. To ensure that T cells are not inappropriately activated, signaling pathways downstream of the TCR are subject to multiple levels of positive and negative regulation. Herein, we describe two related proteins, Sts-1 and Sts-2, that negatively regulate TCR signaling. T cells from mice lacking Sts-1 and Sts-2 are hyperresponsive to TCR stimulation. The phenotype is accompanied by increased Zap-70 phosphorylation and activation, including its ubiquitinylated forms. Additionally, hyperactivation of signaling proteins downstream of the TCR, a marked increase in cytokine production by Sts1/2(-/-) T cells, and increased susceptibility to autoimmunity in a mouse model of multiple sclerosis is observed. Therefore, Sts-1 and Sts-2 are critical regulators of the signaling pathways that regulate T cell activation.