[show abstract][hide abstract] ABSTRACT: CD4(+) memory is critical for successful protection against pathogenic challenge. As such, understanding the heterogeneity of cells that arise and survive after initial stimulation of naïve CD4(+) T cells will aid in the design of more successful vaccines. In previous studies, in vivo experimental systems have been extensively used to generate functional memory responses by lymphocytes. Here, we have attempted to develop an in vitro experimental system to generate memory CD4(+) T lymphocytes. CD4(+) T cells stimulated through the antigen receptor complex were examined for their memory-like characteristics after 3 weeks of cell culture. A subset of surviving cells expressed high levels of CD44 and low levels of CD45RB (CD44(hi)CD45(lo)), a phenotype that is similar to bonafide memory CD4(+) T cells. In vitro generated memory-like CD4(+) T cells secreted higher levels of IFN-γ, with rapid kinetics, upon re-stimulation than their naïve counterparts. In addition, these memory-like CD4(+) T cells did not produce either IL-2 or IL-4 but readily proliferated when cultured in the presence of IL-7 and IL-4. These observations suggest that CD4(+) cells surviving the expansion phase of immune response produce a Th1-signature cytokine and retain responsiveness to IL-4, a Th-2 cytokine, as well as to a well described survival factor, interleukin-7.