e. the most proximal LN to the site of tumour growth) ex vivo. These cells were markedly enriched in frequency (Fig. 1A) and total numbers (Fig. 1C) in IL-7-driven and not control (Nil)-cultures. It is worth noting that I-Ad/LACK+, CD4+ T cells accumulated in response to IL-7 in spite selleck of a CD4+ T-cell loss during culture time (Fig. 1D). When quantified in independent experiments, the number of LACK (tumour)-specific
cells and in particular of IL-2/IFN-γ-double secreting CD4+ T cells detected in IL-7-driven cultures over control (Nil) cultures was increased by several folds (7.88±0.78 n=8; and 25.3±8.13 n=3, respectively). Memory-like LACK-specific T cells were undetectable in T-dLN of control TS/A tumour-bearing (Fig. 1A and B, lower panels) and tumour-free 16.2β mice (Fig. 2) both ex vivo and after IL-7-driven culture. This indicates that in vivo Ag sensitization is required for the observed in vitro IL-7-driven response. Both in vitro cell division and survival might account for the selective accumulation of LACK-specific lymphocytes in response to IL-7. To analyze proliferation, cells derived from naive (control) and T-dLN, were labeled with CFSE and cultured without (Nil) and with IL-7. In cultures derived from control LN, few CD4+ T cells underwent in vitro cell proliferation in the absence of stimulation (Fig. 2A, Nil), while a fraction of cells with a CFSEdim (i.e.
diminished CFSE content) profile, Selleckchem LBH589 likely undergoing homeostatic-like cell division, was found in IL-7-driven cultures (Fig. 2A, IL-7), as also described previously 26. In the case of cultures derived from T-dLN, a sizeable fraction of CD4+ T cells proliferated in the absence of stimulation (Fig. 2B, Nil, hereafter defined as Interleukin-2 receptor “spontaneous” cell division). LACK-specific IL-2 (not depicted)
and IFN-γ-double secreting cells, identified by intracellular cytokine staining, were mostly found among CFSEdim cells, and selectively enriched after exposure to IL-7 (Fig. 2B, IL-7). Similar results were obtained with highly purified (>97%) CD4+ cell cultures. Although spontaneous cell division was no longer detectable in CD4+ cell cultures (Fig. 2C and D, Nil), suggesting that APC might support the ex vivo expansion of in vivo Ag-sensitized T cells, LACK-specific CFSEdim T cells accumulated in response to IL-7 (Fig. 2D, bottom) to extents comparable to those found in unfractionated T-dLN cultures (compare pie charts in Fig 2B and D). Thus, IL-7-driven in vitro expansion of in vivo Ag-sensitized memory-like T cells accounts, at least in part, for their selective accumulation. We further analyzed IL-7-driven cultures derived from T-dLN of BALB/c mice, which have a physiological polyclonal representation of LACK (tumour)-specific naive CD4+ T cells (∼1/105), and additionally compared IL-7 to other cytokines known to play a pro-survival role in T-cell biology.