(A) RIP-GP mice were infected with LCMV Arm. Blood glucose levels were measured at multiple time points and used as an indication of islet β cell autoimmune destruction. Blood glucose levels of 1 mouse representative of at least 6 mice are shown for each genotype. (B) P14/RIP-GP animals were infected with LCMV Arm, and blood glucose was monitored as in A. Blood glucose levels of 1 mouse representative of at least 5 mice are shown for each genotype. (C) CFSE-labeled P14 WT or TNF-α^–/– CD8⁺ T cells were adoptively transferred into WT or TNF-α^–/– mice infected 3 days previously with LCMV Arm. The extent of T cell proliferation was assessed by CFSE dilution in spleens 3 days later (solid lines). Histograms are gated on CFSE⁺CD8⁺Vα2⁺ cells and are representative of 3 independent animals per condition. Dashed lines indicate the CFSE profile of undivided cells. (D) Expression of CD69, CD44, and CD62L on CFSE⁺CD8⁺Vα2⁺ cells from C.

CFSE-labeled naive P14 WT or P14/TNF-α^–/– CD8⁺ T cells were adoptively transferred into the indicated RIP(GP × Tag2) hosts. Proliferation of CFSE-labeled cells was assessed 3 days later in PDLN. Histograms are gated on CFSE⁺CD8⁺Vα2⁺ cells. The percentage in each histogram indicates the proportion of dividing cells among the CFSE-labeled population. A summary and mean of the proportion of proliferating cells observed in individual animals is presented. Error bars indicate SEM The indicated differences are statistically significant. Number of mice analyzed: WT→WT: n = 6; WT→TNF-α^+/–: n = 3; WT→TNF-α^–/–: n = 6; TNF-α^–/–→WT: n = 4; TNF-α^–/–→TNF-α^+/–: n = 3; and TNF-α^–/–→TNF-α^–/–: n = 5.

(A) TNFR1 and TNFR2 expression on naive P14 T cells was assessed by gating on CD8⁺Vα2⁺ cells in inguinal LN (iLN) of P14/RIP(GP × Tag2) animals. (B) Proliferating P14 cells display high levels of TNFR2 expression. CFSE-labeled naive P14 WT CD8⁺ T cells were adoptively transferred into WT RIP(GP × Tag2) hosts. TNFR1 and TNFR2 modulation during proliferation was assessed 3 days later in PDLN. Dot plots are gated on CFSE⁺CD8⁺Vα2⁺ cells. The data shown are representative of 3 independent experiments. (C) CFSE-labeled naive P14 WT, P14/TNFR1^–/–, or P14/TNFR2^–/– CD8⁺ T cells were adoptively transferred into WT RIP(GP × Tag2) hosts. Proliferation of CFSE-labeled cells was assessed 3 days later in PDLN. Histograms are gated on CFSE⁺CD8⁺Vα2⁺ cells. The percentage in each histogram indicates the proportion of dividing cells among the CFSE-labeled population. A summary and mean of the proportion of proliferating cells observed in individual animals is presented. Error bars indicate SEM. Number of mice analyzed: P14 WT: n = 7; P14/TNFR1^–/–: n = 6; P14/TNFR2^–/–: n = 7. (D) CFSE-labeled naive P14 WT CD8⁺ T cells were adoptively transferred into WT, TNFR1^–/–, or TNFR2^–/– RIP(GP × Tag2) recipients of the indicated genotype. Proliferation of CFSE-labeled cells was assessed 3 days later in PDLN. Histograms were gated on CFSE⁺CD8⁺Vα2⁺ cells. A summary and mean of the proportion of proliferating cells observed in individual animals is presented. Error bars indicate SEM. A significant reduction in proliferation was observed in TNFR1^–/– RIP(GP × Tag2) mice (P = 0.037). Number of mice analyzed: WT RIP(GP × Tag2): n = 5; RIP(GP × Tag2)/TNFR1^–/–: n = 8; RIP(GP × Tag2)/TNFR2^–/–: n = 3.

(A) P14 WT, P14/TNFR1^–/–, P14/TNFR2^–/–, or P14/TNF-α^–/– CD8⁺ T cells were cocultured with GP33-pulsed thioglycollate-induced macrophages for 4 hours in the presence of an inhibitor of intracellular protein transport. Following incubation, P14 cells were harvested, and the levels of intracellular TNF-α, IL-2, and IFN-γ were evaluated by intracellular flow cytometry. Representative contour plots for IL-2 and TNF-α are shown. The proportion of cells in each quadrant is also indicated. (B) Kinetics of TNF-α, IL-2, and IFN-γ production by P14 WT, P14/TNFR1^–/–, P14/TNFR2^–/–, or P14/TNF-α^–/– CD8⁺ T cells stimulated as described in A. Mean values ± SEM from 4 independent experiments are shown. Statistically significant differences at 4 hours are indicated. For TNF-α, *P = 0.0249, **P = 0.0001. For IL-2, *P = 0.0048, **P = 0.0273. For IFN-γ, *P = 0.0122, **P < 0.0001, ***P = 0.0003.

Bone marrow–derived macrophages were generated from mice of the indicated genotypes and treated for 20 hours with IFN-γ, TNF-α, or LPS. The levels of CD40 and CD86 expression were then evaluated by flow cytometry. Data are representative of 2 independent experiments.

(A) Impaired immune surveillance in TNF-α^–/– P14/RIP(GP × Tag2) animals. The age at onset of hypoglycemia was determined by weekly measurement of blood glucose levels in mice of the indicated genotype. This was used as a surrogate marker of tumor development and immune surveillance, since it is a direct measure of β islet cell mass. Results obtained for individual mice are shown. Differences between WT and TNF-α^–/– P14/RIP(GP × Tag2) animals were statistically significant (P < 0.0001). (B) The number of CD8⁺Vα2⁺ P14 cells in PDLN of P14/RIP(GP × Tag2) animals of the indicated genotypes was calculated after flow cytometry analysis of PDLN. Averages and data obtained from individual mice are shown. Differences between WT and TNF-α^–/– animals were statistically significant (P = 0.0002). (C and D) The proportion of GP-specific P14 CD8⁺ T cells expressing high levels of the activation markers CD69 (C) and CD44 (D) was assessed in PDLN after gating on CD8⁺Vα2⁺ cells. Averages and data obtained from individual mice are shown. iLN data are also shown as a control. Differences between WT and TNF-α^–/– animals were statistically significant for CD44 (P < 0.0001). (E) Pancreas-infiltrating leukocytes (PIL) were isolated from P14/RIP(GP × Tag2) WT, TNF-α^+/–, or TNF-α^–/– pancreas, and the number of infiltrating CD8⁺, CD4⁺, and B220⁺ cells was determined by flow cytometry and analysis of the leukocytic infiltrates. Error bars indicate SEM. The number of CD8⁺ cells in PILs from P14/RIP(GP × Tag2)/TNF-α^–/– mice was significantly lower than in those from P14 WT/RIP(GP × Tag2) animals (P < 0.0001) (WT mice: n = 5; TNF-α^–/– mice: n = 10).

(A) Histological sections of pancreas from mice of the indicated genotypes were stained for VCAM-1 7 hours following i.p. injection of recombinant mouse TNF-α. (B) T cell activation in PDLN of P14/RIP(GP × Tag2)/TNFR1^–/– animals. Comparison of the expression of CD69 and CD44 on CD8⁺Vα2⁺ cells from PDLN or iLN of WT, TNF-α^–/–, or TNFR1^–/– P14/RIP(GP × Tag2) animals. Representative results from at least 4 independent experiments on different mice are shown. (C) Impaired CD8⁺ T cell recruitment to pancreas of P14/RIP(GP × Tag2)/TNFR1^–/– animals. Proportion of CD8⁺ T cells in PILs from WT or TNFR1^–/– animals. A representative result from at least 4 independent mice is shown.

CFSE-labeled naive P14 WT or P14/TNF-α^–/– CD8⁺ T cells were adoptively transferred into the indicated RIP(GP × Tag2) hosts in combination with CD40 agonistic Ab or CD40 Ab and the TLR2 ligand Pam3. Proliferation of CFSE-labeled cells was assessed 3 days later in PDLN. Zebra plots were gated on CFSE⁺CD8⁺Vα2⁺ cells. The percentage in each plot indicates the proportion of dividing cells among the CFSE-labeled population. The data presented are representative of 3 independent experiments.

In conditions when T cells are activated in the presence of optimal stimulation (optimal APC maturation by danger signals, costimulation, cytokines, etc.), TNF-α is totally or partially dispensable for T cell activation (e.g., systemic viral infection), while when such factors become limiting (e.g., spontaneous tumor growth), TNF-α’s roles become relevant and critical at multiple levels of the T cell immune response.