The rationale for the IL-2-independent generation of the self-renewing central memory CD8+ T cells.

Clones of CD8+ T cells that have been selected in the primary response must have a mechanism by which they can continuously or intermittently generate new effector cells. Several years ago, this mechanism was proposed to involve a self-renewing, stem cell-like subset that could avoid the differentiating effects of interleukin-2 (IL-2). The model considered the stem cell subset to be contained within the central memory population of CD8+ T cells (T(CM)). This proposal was inconsistent with subsequent findings suggesting that all antigen-activated CD8+ T cells differentiated to effector cells (T(EFF)) during the primary response and that T(CM) developed during the memory phase by de-differentiating from effector memory cells (T(EM)). However, findings have since been reported that support the stem cell model. First, studies indicate that T(EM) do not serve as the precursors of T(CM). Second, transcriptional repressors of IL-2 signaling do enhance the memory response. Third, memory cells lacking effector functions and with a capacity to replicate in a secondary response develop in the absence of signaling through the IL-2/IL-15 receptor. Taken together, these findings suggest that antigen-activated CD8+ T cells with a stem cell-like capability for maintaining proliferative potential develop by an unknown IL-2-independent process. The challenge is now to identify this unknown pathway of clonal expansion.

Identifying genes important for spermatogonial stem cell self-renewal and survival.

Spermatogonial stem cells (SSCs) are the foundation for spermatogenesis and, thus, preservation of a species. Because of stem cell rarity, studying their self-renewal is greatly facilitated by in vitro culture of enriched biologically active cell populations. A recently developed culture method identified glial cell line-derived neurotrophic factor (GDNF) as the essential growth factor that supports in vitro self-renewal of SSCs and results in an increase in their number. This system is a good model to study mechanisms of stem cell self-renewal because of the well defined culture conditions, enriched cell population, and available transplantation assay. By withdrawing and replacing GDNF in culture medium, we identified regulated expression of many genes by using microarray analysis. The expression levels of six of these genes were dramatically decreased by GDNF withdrawal and increased by GDNF replacement. To demonstrate the biological significance of the identified GDNF-regulated genes, we examined the importance of the most responsive of the six, bcl6b, a transcriptional repressor. By using siRNA to reduce transcript levels, Bcl6b was shown to be crucial for SSC maintenance in vitro. Moreover, evaluation of Bcl6b-null male testes revealed degeneration and/or absence of active spermatogenesis in 24 +/- 7% of seminiferous tubules. These data suggest that Bcl6b is an important molecule in SSC self-renewal and validate the biological relevance of the GDNF-regulated genes identified through microarray analysis. In addition, comparison of data generated in this study to other stem cell types suggests that self-renewal in SSCs is regulated by distinctly different molecular mechanisms.

BCL6b mediates the enhanced magnitude of the secondary response of memory CD8+ T lymphocytes.

A characteristic of the secondary response of CD8(+) T cells that distinguishes it from the primary response is the generation of greater numbers of effector cells. Because effector CD8(+) T cells are derived from a pool of less differentiated, replicating cells in secondary lymphoid organs, and because IL-2 mediates effector differentiation, the enhanced secondary response may reflect the enlargement of this generative pool by the transient repression of IL-2-mediated differentiation. We have examined for this function the transcriptional repressor BCL6b, a homologue of BCL6 that represses IL-2-induced B cell differentiation. BCL6b is expressed in a small subset of antigen-experienced CD8(+) T cells. Ectopic expression of BCL6b in CD8(+) T cells diminishes their growth in response to IL-2 in vitro. Female mice in which the BCL6b gene has been interrupted have normal primary responses of CD8(+) T cells to infection with vaccinia expressing the H-Y epitope, Uty, but Uty-specific, BCL6b(-/-), memory CD8(+) T cells have diminished recall proliferative responses to this epitope in vitro. BCL6b(-/-) mice also have normal primary CD8(+) T cell responses to influenza infection, but nucleoprotein peptide-specific, BCL6b(-/-), memory CD8(+) T cells have a cell autonomous defect in the number of effector cells generated in response to reinfection. Therefore, BCL6b is required for the enhanced magnitude of the secondary response of memory CD8(+) T cells.