Our data show that the HIV+ and aged conditions foster a change in the CD8+ T cell repertoire characterized by expansion of a CD8+ CD28- phenotype capable of enhanced IFN-γ and TNF-α production. These results contrast with other studies concluding that the production of these cytokines was downregulated or unchanged in T cells from HIV+ and aged individuals [21, 28–32, 37, 38]. Reliability in our findings derives from use of flow cytometric analysis which measures de novo synthesis per cell and thus obviates numerous artifacts possibly associated with ELISA or procedures that measure mRNA synthesis. Secondly, similar results were obtained with two different inhibitors of Golgi transport. Thirdly, of the cytokines examined, including 11–4, 11–6 and 11–10, only IFN-γ and TNF-α were enhanced in the HIV+ or aged states. Fourthly, CD4+ T cells did not show a significant change in IFN-γ production, but did show a small but significant increase in TNF-α production in aged and HIV+-derived cells. Fifthly, the mRNA and ELISA studies support the conclusions of the flow cytometric data.
The question arises whether or not our results have physiological relevance since PMA and anti-CD3 was used to elicit the cytokine responses we observed. The question resolves into two parts: are TNF-α and IFN-γ levels increased in the HIV+ and aged states, and do the CD8+ T cells show increased production of these cytokines. There is considerable evidence supporting both parts of this question. Regarding part one, IFN-γ levels are elevated in the sera of HIV+ patients . Serum levels of both TNF-α and IFN-γ were markedly higher in HIV+ patients over controls, but varied depending on the skin disease exhibited by the patient . AIDS patients also showed elevated TNF-α levels in the plasma . Studies of lymphocytes from aged donors showed increase serum levels of TNF-α and IFN-γ that correlate with upregulation of HLA-DR on CD3+-lymphocytes .
Regarding part two, there are many studies showing that physiological activation of CD8+ T cells from HIV+ or aged donors prompts increased IFN-γ and/or TNF-α production. HIV+ lymph nodes exhibit a large (6 fold) increase in CD8+ T cells, most of which are involved in IFN-γ production . Amison et al  concluded that IFN-γ is secreted by the CD8+ T cell in HIV+ infections. In CD8+ T cells from African HIV+ patients, stimulated with env peptide, both IFN-γ and TNF-α production were increased over controls . PBMC from the elderly (almost healthy donors) showed upregulation of TNF-α in unstimulated cultures . HLA-DR activation of CD8+ CD57+ cells from elderly subjects strongly produced TNF-α . Lipopolysaccharide-activation of T cells from aged donors induced higher levels of TNF-α than controls in two separate studies [53, 31]. Finally, in HIV+ patients receiving HIV type 1 antigen, both INF-γ and TNF-α were significantly increased in memory T cells activated by antigen .
Thus there appears to be considerable support for the enhanced presence of TNF-α and INF-γ in the HIV+ and the aged states, and for a physiologic role of the CD8+ T cell subset in their production. Our data shows that it is the CD8+ CD28- cell that is primarily responsible. Why the CD8+ CD28- subset should dominated in such apparently diverse conditions as the HIV+ and the aged states is not known. However, the notion that similarities exist between the HIV+ and the aged states with respect to the CD8+ CD28- T cell has been presented previously . It has also been proposed that the CD8+ CD28- T cell in the HIV+ state is derived from the CD8+ CD28+ cells by clonal expansion induced by massive antigenic stimulus (HIV proteins) since both showed the same epitope repertoire . Dolad et al  have shown that the CD8+ CD28- cell, not the CD8+ CD28+ cell, comprises the major CTL activity in the HIV+ state, a finding confirmed by Vingerhoets et al , who associated cytotoxic T cell activity with the CD8+ CD28- T cell in HIV-infected subjects. Thus, the CD8+ CD28- T cell assumes a crucial role in HIV infection. Likewise in the aged state, we can speculate that chronic viral and bacterial exposure over a lifetime may induce the clonal expansion of the CD8+ CD28- T cell.
There is evidence that the cytotoxic activity of the CD28- cell can lyse and suppress HIV-infected cells [58, 59] and thus play a protective role in HIV infection. Perhaps in the aged cell as well, the CD28- memory cell serves a similar protective role against a variety of viruses. The production of IFN-γ and TNF-α can result in strong anti-viral consequences as shown with hepatitis B virus , but the cytokines have a dual role since they are associated with inflammatory activity. IFN-γ, in its defensive role against infection, is a foremost factor in converting resting macrophages into the active state  where they produce reactive oxidants , nitric oxide , TNF-α , and neopterin. Not only can these factors damage DNA and host tissues but can modulate inflammatory reactions  and stimulate apoptosis , a major mechanism leading to loss of CD4+ T cells . TNF-α also has the capacity to perturb the clinical condition in AIDS by strongly upregulating the transcription of the HIV genome . In fact, it has been proposed  that AIDS is in many ways a TNF-α disease since this cytokine it is a potent activator of NF-kB, and an inducer of HIV genome transcription and apoptosis in HIV-infected T cells. Moreover, the wasting, weight loss, and cachexia associated with AIDS has also been related to TNF-α activity . Thus both IFN-γ and TNF-α have the potential for a double role in the pathogenesis associated with AIDS i.e. antiviral and inflammatory responses.
Based on the AIDS scenario, it is reasonable to propose that chronic viral and bacterial infections suffered by an individual over a lifetime may lead to similar consequences as those occurring in acute HIV infection, i.e., clonal expansion of the CD8+ CD28- subset at the expense of the CD8+ CD28- subset. The data argue that the CD28-cell may serve as a natural endpoint in the differentiation of CD8+ cells, as proposed by Effros et al , where aging represents a "normal endpoint" that occurs after many years of immune activity, while AIDS represents an accelerated endpoint due to the increase rate in T cell formation during attempted compensation for CD4+ loss induced by HIV. By virtue of their inflammatory roles, TNF-α and IFN-γ likely contribute to the exacerbation of the inflammatory conditions associated with the aged. For example, autoimmune diseases are more prevalent in the aged, where certain diseases such as arthritis are often associated with chronic debilitation. Interestingly, both IFN-γ and TNF-α are prominent in the pathogenesis of autoimmune diseases in general; the former by activation of local auto antigen presentation to appropriate T cells, and the latter by mediation of inflammatory tissue damage . Apparently TNF-α plays a central role in the chronic release of highly arthritogenic II-1 , and thus serves as a major factor in the pathogenesis of rheumatoid arthritis (RA) . Recent studies show that treatment with anti-TNF-α gives significant clinical improvement in RA . Thus our findings in aged individuals of a prominent CD8+ CD28- T cell subset with enhanced ability to produce TNF-α, may assume clinical relevance. It is logical to infer that the increased susceptibility of aged individuals to rheumatoid arthritis and other autoimmune pathologies, may be due to increased TNF-α (and IFN-γ) production by the CD8+ CD28- T cell. It should be noted that the action of IFN-γ on macrophages induces TNF-α production , thus amplifying the effects of the latter.
Although IFN-γ production in the HIV+ individual by the CD4+ T cells (TH1) has been emphasized [13, 45, 49] there appears to be little doubt that the production of IFN-γ by CD8+ CD28- cells may have greater clinical relevance as discussed above on the increased serum levels [45, 46]. With TNF-α as well, our data suggest it is likely that the increased serum levels in AIDS [46–48] may derive in part from increased numbers of CD8+ CD28-cells. However, TNF-α levels could also originate from monocytes stimulated by the enhanced IFN-γ production. In any case, the HIV+ state is replete with antagonistic complexities of antiviral effects of IFN-γ and TNF-α on one hand, and their inflammatory effects on the other. Overall, our data presents an intriguing parallel between AIDS and the aging state with regard to their cytokine and CD8+ T cell profiles, and the potential for new insight into aging mechanisms. It is noteworthy that our results are compatible with recent aging studies using gene arrays to measure gene expression in the neocortex and cerebellum of aged and young mice. Lee et al  found that in both brain regions, one hallmark of aging was enhanced gene expression of the inflammatory response, including proinflammatory peptides and many interferon-related proteins. Thus the advent of the CD8+ CD28- cell in aging, with its potential for proinflammatory cytokines, could have profound effects not only on autoimmune diseases, but on Alzheimer's disease as well since cytokines may contribute to the neuropathology .