Improvements in vaccine development rely on advances in our knowledge of the cell types and cytokines that contribute to protective immunity. For intracellular pathogens, CD4+ and CD8+ T cells are considered the primary mediators of long-lived protective memory. We show, for the first time, that another T cell type contributes considerably to in vivo control of infections with two intracellular pathogens, M. tb. and F. tularensis LVS. These DN T cells, derived from LVS and M. tb.–immune mice, are pathogen-specific, Th1-like T cells expressing TCR αβ and CD3; importantly, these cells acquire a memory phenotype after infection. DN T cells potently control the intracellular growth of M. tb. and LVS in vitro, expand in vivo, and contribute to control of in vivo infection by several mechanisms, including cytokine production and possibly cytotoxicity.
In both mice and humans, protective immune responses to M. tb.
and to Francisella
depend on cell-mediated immunity provided by T cells and appear to rely heavily on appropriate CD4+
T cell activity (1
). This is clearly illustrated by the impact of HIV infection and subsequent reduction in CD4+
T cell numbers on susceptibility to tuberculosis (1
cells also contribute notably to both infections (1
). The effector mechanisms used by CD4+
T cells remain the subject of intense study, but clearly include production of IFN-γ and TNF-α for the activation of macrophages, leading to production of toxic reactive oxygen and nitrogen species, as well as classical CD8+
T cell cytotoxic activities (1
). We have taken advantage of an in vitro culture system to further understand the various effector mechanisms used by DN T cells. We find that TNF, iNOS, and especially IFN-γ contribute to M. tb.
and LVS growth control by DN T cells (), but other mechanisms that remain to be defined are also important. However, stimulation of macrophage P2X7R by DN T cell products is not a likely alternative mechanism. Others have presented evidence that control of latent M. tb.
infection in mice is dependent on IFN-γ but independent of iNOS (28
). One iNOS-independent candidate is LRG-47, a 47-kD guanosine triphosphatase family member (29
), and another is the recently identified product of the Ipr1
gene, a putative IFN-γ–regulated transcriptional cofactor (30
). The role of these molecules in the control of LVS and M. tb.
intracellular growth by CD4+
, and DN T cells is currently under investigation. Preliminary studies indicate LVS-immune DN T cells derived from perforin KO mice retain anti-LVS activity, and abrogation of DN T cell FasL function has no impact on anti-LVS activity (unpublished data). Interestingly, however, ~20% and 5% of LVS-immune DN T cells express intracellular granzyme B on stimulation through CD3 or via LVS-infected BMMØs, respectively (unpublished data), indicating that these cells possess unconventional cytotoxic activity that merits further detailed study.
Current studies are also seeking to determine the developmental origin and MHC restriction profiles of these DN T cells, which appear to be complex. The lack of expression of the NK1.1 and DX5 markers on these cells indicate that they are very unlikely to be NKT cells. To date, we have obtained functional DN T cells from LVS-immune class II KO mice, β2
microglobulin KO mice, and CD8 KO mice (unpublished data), indicating that DN T cell development and function does not rely exclusively on any one of these elements. For several reasons, we find it unlikely that these DN cells arise from normal CD4+
T cells that have lost expression of their respective costimulatory molecules after activation, such as been observed after HIV infection (31
). First, DN T cells were readily detected in and obtained from normal uninfected mice (see , , and ). Second, in this study, DN cells expanded in mice that were depleted of all CD4 and CD8 cells before infection. Third, retroviruses, in particular, commonly down-regulate their own receptors, whereas LVS and M. tb.
are not known to interact directly with either CD4 or CD8. However, whether these cells naturally lost expression of CD4 and CD8 because of high-avidity TCR signals during thymic development (32
) remains to be determined.
Other studies have described DN T cells in a variety of genetically deficient mice, including TCR transgenic mice (34
), CD4 KO mice (15
), and autoimmune syndromes including lpr
and FasL-deficient mice (14
). These animals may have expanded or abnormal accumulations of DN T cells as a result of disregulated immune systems; cells lacking expression of CD4 and/or CD8 in these mouse models arise as a consequence of the mutation, and do not reflect normal physiology. There have only been sporadic descriptions of DN T cells in normal mice. DN T cells that possess regulatory T cell activities in tissue allograft rejection models have been described (37
); in one case, these cells acquired and presented alloantigen to syngeneic CD8+
T cells, suppressing their function (38
). In addition to these artificial models, isolated reports previously suggested that DN T cells in normal mice might contribute to immune responses. A population of DN T cells proliferates and produces IFN-γ and TNF-α in response to L. monocytogenes
) and mouse CMV (40
) infections. DN T cells are 70–90% of lymphocytes in the genital tract of Chlamydia trachomatis
–infected mice (41
). However, the few murine descriptions of the presence of DN T cells have not addressed their biological importance or determined whether they contribute to memory immune responses. In humans, DN T cell numbers increase during staphylococcal toxic shock syndrome (42
) and HIV infection (43
) and respond to lipid M. tb.
). Thus, it is possible that humans have a similar population with an important role in immune defense.
We demonstrate that a small DN T cell population is found in the spleens of normal WT mice before, during, and after intracellular bacterial infections, and that these cells clearly participate in control of infection. These experiments unambiguously assign a normal physiological function to cells that were previously of uncertain utility and consequence. Most importantly, we show that these cells acquire a memory T cell phenotype and, thus, may be a new target for vaccination.