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Cited by 287T-cell and B-cell ImmunologyRheumatoid Arthritis Research and TherapiesCytokine Signaling Pathways and Interactions

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Chronic Tumor Necrosis Factor Alters T Cell Responses by Attenuating T Cell Receptor Signaling

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Cope, Andrew P., Liblau, Roland S., Yang, Xiao-Dong, Congia, Mauro, Laudanna, Carlo, Schreiber, Robert D., Probert, Lesley, Kollias, George, McDevitt, Hugh O.·1997·The Journal of Experimental Medicine
DOI 10.1084/jem.185.9.1573PMC PMC2196294
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Abstract

Repeated injections of adult mice with recombinant murine TNF prolong the survival of NZB/W F1 mice, and suppress type I insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice. To determine whether repeated TNF injections suppress T cell function in adult mice, we studied the responses of influenza hemagglutinin-specific T cells derived from T cell receptor (HNT-TCR) transgenic mice. Treatment of adult mice with murine TNF for 3 wk suppressed a broad range of T cell responses, including proliferation and cytokine production. Furthermore, T cell responses of HNT-TCR transgenic mice also expressing the human TNF-globin transgene were markedly reduced compared to HNT-TCR single transgenic littermates, indicating that sustained p55 TNF-R signaling is sufficient to suppress T cell function in vivo. Using a model of chronic TNF exposure in vitro, we demonstrate that (a) chronic TNF effects are dose and time dependent, (b) TNF suppresses the responses of both Th1 and Th2 T helper subsets, (c) the suppressive effects of endogenous TNF produced in T cell cultures could be reversed with neutralizing monoclonal antibodies to TNF, and (d) prolonged TNF exposure attenuates T cell receptor signaling. The finding that anti-TNF treatment in vivo enhances T cell proliferative responses and cytokine production provides evidence for a novel regulatory effect of TNF on T cells in healthy laboratory mice. These effects are more pronounced in chronic inflammatory disease. In addition, our data provide a mechanism through which prolonged TNF exposure suppresses disease in animal models of autoimmunity.

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Figure 5 In vitro model for studying chronic TNF effects. HNTTCR transgenic T cells derived from lymph node and/or spleen were stimulated with splenic APC, 2 μg/ml HA 126-138 and 20 U/ml IL-2 in the presence or absence of repeated additions of murine TNF or antiTNF for up to 14 d, as described in Materials and Methods. All rechallenge assays were performed after extensive washing using equal numbers of T cells, fresh, irradiated, non-transgenic splenic APC and HA peptide, in the absence of exogenous TNF or antibody.
Figure 5 In vitro model for studying chronic TNF effects. HNTTCR transgenic T cells derived from lymph node and/or spleen were stimulated with splenic APC, 2 μg/ml HA 126-138 and 20 U/ml IL-2 in the presence or absence of repeated additions of murine TNF or antiTNF for up to 14 d, as described in Materials and Methods. All rechallenge assays were performed after extensive washing using equal numbers of T cells, fresh, irradiated, non-transgenic splenic APC and HA peptide, in the absence of exogenous TNF or antibody.CC BY-NC-SA · PubMed Central
Figure 1 The effects of chronic TNF treatment on peptide-specific responses of transgenic T cells. 8–12-wk-old male BALB/c HNT-TCR transgenic mice were injected i.p. with PBS or 3 μg murine TNF on alternate days for 3 wk before study. (a) The proliferative responses of lymph node (LN), splenic (Spl) or LN + Spl pools of T cells from pairs of transgenic littermates treated with PBS (shaded bars) or TNF (hatched bars) were compared after stimulation with 0.5 μg/ml HA peptide (left), or 20 U/ml recombinant IL-2 (right), as described in Materials and Methods. Data are expressed as mean cpm ± SEM from 9 independent experiments. Background cpm ± SD in the absence of peptide, which ranged from 889– 2537 cpm, were as follows: PBS, 2167 ± 575; TNF, 1992 ± 568 (P >0.1). Significant differences between treatments were observed for responses to peptide (P <0.008, paired T test), but not to IL-2 (P >0.2). (b) 7 × 106 lymph node T cells were stimulated with 0.5 μg/ml HA peptide and culture supernatants harvested at the indicated times. Each data point represents IL-2 production of transgenic lymph node T cells from one mouse, as determined by immunoassay. Data are pooled from three independent experiments. Differences between PBS and TNF treatments were statistically significant at 36 and 72 h (P <0.0005, independent T test).
Figure 1 The effects of chronic TNF treatment on peptide-specific responses of transgenic T cells. 8–12-wk-old male BALB/c HNT-TCR transgenic mice were injected i.p. with PBS or 3 μg murine TNF on alternate days for 3 wk before study. (a) The proliferative responses of lymph node (LN), splenic (Spl) or LN + Spl pools of T cells from pairs of transgenic littermates treated with PBS (shaded bars) or TNF (hatched bars) were compared after stimulation with 0.5 μg/ml HA peptide (left), or 20 U/ml recombinant IL-2 (right), as described in Materials and Methods. Data are expressed as mean cpm ± SEM from 9 independent experiments. Background cpm ± SD in the absence of peptide, which ranged from 889– 2537 cpm, were as follows: PBS, 2167 ± 575; TNF, 1992 ± 568 (P >0.1). Significant differences between treatments were observed for responses to peptide (P <0.008, paired T test), but not to IL-2 (P >0.2). (b) 7 × 106 lymph node T cells were stimulated with 0.5 μg/ml HA peptide and culture supernatants harvested at the indicated times. Each data point represents IL-2 production of transgenic lymph node T cells from one mouse, as determined by immunoassay. Data are pooled from three independent experiments. Differences between PBS and TNF treatments were statistically significant at 36 and 72 h (P <0.0005, independent T test).CC BY-NC-SA · PubMed Central
Figure 3 Kinetics of the proliferative responses of transgenic T cells after anti-TNF injections. (a) Pairs of B10.D2 HNT-TCR transgenic littermates were treated i.p. on alternate days with 6–18 injections of antiTNF or control Ig (100 μg/injection); mice injected for shorter periods (2–4 injections) received 200 μg antibody per injection. After the indicated number of injections, mice were killed and transgenic T cells stimulated in vitro with 0.5 μg/ml HA peptide, as described for Fig. 1. *Differences between treatments were significant (P <0.02, independent T test). (b) Mice were injected with anti-TNF or control Ig for 4 wk. At the end of the treatment period, 105 purified lymph node/splenic CD4+ T cells were stimulated in vitro with increasing concentrations of HA peptide and 5 × 105 fresh irradiated splenic APC from non-transgenic, untreated B10.D2 mice. Data represent mean cpm ± SEM in 72-h proliferation assays. Con A responses in a representative experiment were as follows: control Ig, 38483 ± 1784, anti-TNF, 36457 ± 5288 cpm ± SEM.
Figure 3 Kinetics of the proliferative responses of transgenic T cells after anti-TNF injections. (a) Pairs of B10.D2 HNT-TCR transgenic littermates were treated i.p. on alternate days with 6–18 injections of antiTNF or control Ig (100 μg/injection); mice injected for shorter periods (2–4 injections) received 200 μg antibody per injection. After the indicated number of injections, mice were killed and transgenic T cells stimulated in vitro with 0.5 μg/ml HA peptide, as described for Fig. 1. *Differences between treatments were significant (P <0.02, independent T test). (b) Mice were injected with anti-TNF or control Ig for 4 wk. At the end of the treatment period, 105 purified lymph node/splenic CD4+ T cells were stimulated in vitro with increasing concentrations of HA peptide and 5 × 105 fresh irradiated splenic APC from non-transgenic, untreated B10.D2 mice. Data represent mean cpm ± SEM in 72-h proliferation assays. Con A responses in a representative experiment were as follows: control Ig, 38483 ± 1784, anti-TNF, 36457 ± 5288 cpm ± SEM.CC BY-NC-SA · PubMed Central
Figure 2 The effects of antiTNF treatment on cytokine production by transgenic T cells. B10.D2 (a) or BALB/c (b) HNT-TCR transgenic mice were injected i.p. on alternate days with 100 μg anti-TNF (hatched bars) or control Ig (shaded bars) for 3 wk (6 mice per treatment group). Peptide-specific cytokine production by transgenic T cells was determined as described for Table 2. Differences between treatments were significant only for B10.D2 HNT-TCR transgenic mice (P <0.0015 for IL-2, and P <0.05 for IFN-γ, independent T test).
Figure 2 The effects of antiTNF treatment on cytokine production by transgenic T cells. B10.D2 (a) or BALB/c (b) HNT-TCR transgenic mice were injected i.p. on alternate days with 100 μg anti-TNF (hatched bars) or control Ig (shaded bars) for 3 wk (6 mice per treatment group). Peptide-specific cytokine production by transgenic T cells was determined as described for Table 2. Differences between treatments were significant only for B10.D2 HNT-TCR transgenic mice (P <0.0015 for IL-2, and P <0.05 for IFN-γ, independent T test).CC BY-NC-SA · PubMed Central
Figure 4 Responses of HNT-TCR transgenic T cells from mice expressing human TNF globin transgenes. (a) Human TNF-globin transgenic mice (line Tg197) were crossed to HNT-TCR transgenic mice and backcrossed 3 generations to BALB/c or B10.D2. At 6–8 wk of age, the responses to 0.5 μg/ml HA 126-138 of purified CD4+ T cells from HNT-TCR single and HNT-TCR × TNF-globin double transgenic littermates were compared in 72-h proliferation assays; (b) 5 × 105 lymph node T cells from 8-wk-old BALB/c HNT-TCR single or HNT-TCR × Tg197 double transgenic littermates were stimulated with increasing concentrations of HA 126-138 and proliferative responses determined as for a; (c and d) human TNF-globin transgenic line Tg3647 was crossed to B10.D2 HNT-TCR transgenic mice. F1 mice derived from this cross were monitored for arthritis development. From these litters, pairs of single HNT-TCR transgenic mice were studied for T cell proliferative responses (c) as described for (b) above, and for IFN-γ production in response to 0.5 μg/ml HA peptide (d), and the responses compared with those observed for arthritic or non-arthritic HNT-TCR × TNF-globin double transgenic mice. In representative experiments, Con A responses (mean cpm ± SD) were as follows: HNT-TCR single transgenic mice, 48513 ± 7728; HNT-TCR × human TNF-globin double transgenic mice, 51677 ± 10806 (n = 2 per group).
Figure 4 Responses of HNT-TCR transgenic T cells from mice expressing human TNF globin transgenes. (a) Human TNF-globin transgenic mice (line Tg197) were crossed to HNT-TCR transgenic mice and backcrossed 3 generations to BALB/c or B10.D2. At 6–8 wk of age, the responses to 0.5 μg/ml HA 126-138 of purified CD4+ T cells from HNT-TCR single and HNT-TCR × TNF-globin double transgenic littermates were compared in 72-h proliferation assays; (b) 5 × 105 lymph node T cells from 8-wk-old BALB/c HNT-TCR single or HNT-TCR × Tg197 double transgenic littermates were stimulated with increasing concentrations of HA 126-138 and proliferative responses determined as for a; (c and d) human TNF-globin transgenic line Tg3647 was crossed to B10.D2 HNT-TCR transgenic mice. F1 mice derived from this cross were monitored for arthritis development. From these litters, pairs of single HNT-TCR transgenic mice were studied for T cell proliferative responses (c) as described for (b) above, and for IFN-γ production in response to 0.5 μg/ml HA peptide (d), and the responses compared with those observed for arthritic or non-arthritic HNT-TCR × TNF-globin double transgenic mice. In representative experiments, Con A responses (mean cpm ± SD) were as follows: HNT-TCR single transgenic mice, 48513 ± 7728; HNT-TCR × human TNF-globin double transgenic mice, 51677 ± 10806 (n = 2 per group).CC BY-NC-SA · PubMed Central
Figure 6 The effects of TNF on T cell responses in vitro are dependent on the exposure period. (a) Acute TNF exposure. Fresh BALB/c splenocytes (5 × 105/well) were stimulated for 72 h with the indicated concentrations of TNF, in the presence or absence of suboptimal concentrations of Con A (50 ng/ml). Proliferative responses (mean cpm ± SEM) were determined as described in Materials and Methods. (b) Chronic TNF exposure. BALB/c HNT-TCR transgenic lymph node/splenic T cells were stimulated with HA 126-138 and IL-2 in the presence or absence of repeated additions of the indicated concentrations of TNF, as described in Fig. 5. After 12 d of culture, cells were washed before restimulation with 0.5 μg/ml HA peptide and fresh splenic APC. Data represent proliferative responses (mean cpm ± SEM) obtained in 72 h rechallenge assays (left), or IL-2 production 36 h after restimulation with peptide (4 independent experiments shown; right panel). (c) Chronic TNF blockade. Transgenic T cells were stimulated as for b above in the presence of repeated additions of 10 μg/ml anti-TNF, control Ig or medium alone. Proliferative responses and IL-2 production were determined as described for b. Data from four independent experiments are shown.
Figure 6 The effects of TNF on T cell responses in vitro are dependent on the exposure period. (a) Acute TNF exposure. Fresh BALB/c splenocytes (5 × 105/well) were stimulated for 72 h with the indicated concentrations of TNF, in the presence or absence of suboptimal concentrations of Con A (50 ng/ml). Proliferative responses (mean cpm ± SEM) were determined as described in Materials and Methods. (b) Chronic TNF exposure. BALB/c HNT-TCR transgenic lymph node/splenic T cells were stimulated with HA 126-138 and IL-2 in the presence or absence of repeated additions of the indicated concentrations of TNF, as described in Fig. 5. After 12 d of culture, cells were washed before restimulation with 0.5 μg/ml HA peptide and fresh splenic APC. Data represent proliferative responses (mean cpm ± SEM) obtained in 72 h rechallenge assays (left), or IL-2 production 36 h after restimulation with peptide (4 independent experiments shown; right panel). (c) Chronic TNF blockade. Transgenic T cells were stimulated as for b above in the presence of repeated additions of 10 μg/ml anti-TNF, control Ig or medium alone. Proliferative responses and IL-2 production were determined as described for b. Data from four independent experiments are shown.CC BY-NC-SA · PubMed Central
Figure 7 TNF suppresses the responses of Th1 and Th2 transgenic T cells. Transgenic T cells derived from BALB/c (closed symbols) and B10.D2 (open symbols) HNT-TCR transgenic mice were stimulated as described for Fig. 6 in the presence or absence of repeated additions of medium alone or 10 ng/ml TNF (a), or 10 μg/ml anti-TNF or control Ig (b). Cytokine production (mean pg/ml) was determined by immunoassay at 36 h for IFN-γ (circles), and 72 h for IL-4 (triangles) and IL-10 (diamonds) after restimulation with 0.5 μg/ml HA peptide. Data from four independent comparisons are shown. Statistically significant differences between treatments are shown (paired T test).
Figure 7 TNF suppresses the responses of Th1 and Th2 transgenic T cells. Transgenic T cells derived from BALB/c (closed symbols) and B10.D2 (open symbols) HNT-TCR transgenic mice were stimulated as described for Fig. 6 in the presence or absence of repeated additions of medium alone or 10 ng/ml TNF (a), or 10 μg/ml anti-TNF or control Ig (b). Cytokine production (mean pg/ml) was determined by immunoassay at 36 h for IFN-γ (circles), and 72 h for IL-4 (triangles) and IL-10 (diamonds) after restimulation with 0.5 μg/ml HA peptide. Data from four independent comparisons are shown. Statistically significant differences between treatments are shown (paired T test).CC BY-NC-SA · PubMed Central
Figure 8 The effects of chronic TNF exposure on intracellular Ca2+ mobilization. BALB/c HNT-TCR transgenic T cells were stimulated in vitro with HA 126-138 peptide in the presence or absence of repeated additions of 10 ng/ml TNF (a), or 10 μg/ml anti-TNF or control Ig (b). Calcium fluxing was determined after 11 d in culture by spectrofluorometric (a), or by flow cytometric analyses (b), after restimulation of T cells with fresh splenic APC loaded with 10 μg/ml peptide. Data are shown for T cell/splenic APC cells in the presence or absence of 10 μg/ml HA peptide, and expressed as \xc6 Ca2+ flux (335/380 nm) ratio (a: medium, solid line, TNF, dotted line) as a function of time (min), or the % indo-1–positive, FITC-negative cells (b) fluxing during the first 60 s of analysis after peptide stimulation.
Figure 8 The effects of chronic TNF exposure on intracellular Ca2+ mobilization. BALB/c HNT-TCR transgenic T cells were stimulated in vitro with HA 126-138 peptide in the presence or absence of repeated additions of 10 ng/ml TNF (a), or 10 μg/ml anti-TNF or control Ig (b). Calcium fluxing was determined after 11 d in culture by spectrofluorometric (a), or by flow cytometric analyses (b), after restimulation of T cells with fresh splenic APC loaded with 10 μg/ml peptide. Data are shown for T cell/splenic APC cells in the presence or absence of 10 μg/ml HA peptide, and expressed as \xc6 Ca2+ flux (335/380 nm) ratio (a: medium, solid line, TNF, dotted line) as a function of time (min), or the % indo-1–positive, FITC-negative cells (b) fluxing during the first 60 s of analysis after peptide stimulation.CC BY-NC-SA · PubMed Central

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Metadata

Authors
Cope, A. P., Liblau, R. S., Yang, X.-D., Congia, M., Laudanna, C., Schreiber, R. D., Probert, L., Kollias, G., & McDevitt, H. O.
Affiliations
  • Washington University in St. Louis (US)
  • From the *Department of Microbiology and Immunology, ‡Department of Pathology, and §Department of Medicine, Stanford University School of Medicine, Stanford, California 94305; ‖Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110; and ¶Department of Molecular Genetics, Hellenic Pasteur Institute, Athens, Greece
ORCID
Venue
The Journal of Experimental Medicine
Publisher
Rockefeller University Press
Published
1997-05-05
Volume
185
Issue
9
Pages
1573-1584
ISSN
  • 0022-1007 (ISSN-L)
  • 1540-9538
DOI
10.1084/jem.185.9.1573
PMC
PMC2196294
OpenAlex
W2153191296
Cited by
287
References
38
Citations by year
2026: 3 · 2025: 4 · 2023: 4 · 2022: 1 · 2021: 8 · 2020: 4 · 2019: 6 · 2018: 3 · 2017: 4 · 2016: 11 · 2015: 6 · 2014: 11 · 2013: 8 · 2012: 13
FWCI
8.32
Type
journal article
Language
en
Open access
Locations
  • The Journal of Experimental Medicine (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
Funders
  • Wellcome Trust
  • Institut National de la Santé et de la Recherche Médicale
  • National Institutes of Health
  • School of Medicine, Stanford University
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
  • Health Sciences › Medicine › Rheumatology › Rheumatoid Arthritis Research and Therapies
  • Life Sciences › Immunology and Microbiology › Immunology › Cytokine Signaling Pathways and Interactions
Keywords
TNF-α, T cell receptor signaling, T cell suppression, TNF receptor p55, T cell proliferation, cytokine production, anti-TNF therapy, autoimmunity, type 1 diabetes, chronic inflammation
MeSH terms
Animals, Calcium, Immunosuppressive Agents, Interferon-gamma, Interleukin-2, Lymph Nodes, Lymphocyte Activation, Male, Mice, Inbred BALB C, Mice, Transgenic, Receptors, Antigen, T-Cell, Recombinant Proteins, T-Lymphocytes, Tumor Necrosis Factor-alpha, Signal Transduction, CD4-Positive T-Lymphocytes, Interleukin-4, Interleukin-10, Receptors, Tumor Necrosis Factor, Th1 Cells, Th2 Cells, Mice

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