(a and b) Reconstitution of TNF-α in rBCG-infected TNF^–/– mice reduced the level of type 1 immune activation. TNF^–/– mice were injected intramuscularly and intranasally with an adenoviral TNF-α gene transfer vector (AdTNF) or a control vector (Addl) at the time of mycobacterial infection. At day 14, isolated splenocytes were subjected to ELISPOT assay to determine the relative frequency of mycobacterial antigen-specific T cells (a) or an antigen stimulation assay to determine the level of IFN-γ release (b). The difference between TNF^–/–/Addl and TNF^–/–/AdTNF under M.tb CF stimulation conditions is statistically very significant (P ≤ 0.05). (c) Improved survival of rBCG-infected TNF^–/– mice depleted of CD4 or CD8 or both T cells. CD4 and CD8 T cells were depleted by 4 repeated injections of anti-CD4 and/or anti-CD8 monoclonal antibodies starting at 3 weeks after infection. Ten, seven, and seven TNF^–/– mice received anti-CD4/CD8, anti-CD4 and anti-CD8 treatment, respectively. Eight TNF^–/– mice were treated with normal rat IgG as control. (d) Reduced IFN-γ levels in TNF^–/– mice depleted of T cells. At day 37, several CD4/CD8 T cell–depleted TNF^–/– (TNF^–/– T cell^–/–) and control mice were sacrificed. The level of IFN-γ in blood and BAL fluids was evaluated. (e and f) Lung histopathogy in control TNF^–/– and CD4/CD8 T cell-depleted TNF^–/– mice on day 37 after infection. The control TNF^–/– lung underwent massive necrosis of both granuloma and lung structure (e). Note that part of the bronchus was also necrotic and filled with necrotic tissue and nucleic debris. The lung of TNF^–/– mice depleted of CD4/CD8 T cells contained largely intact granuloma with typical epithelioid cells (f).