3(ii)]. data have important implications for prime-boost vaccination strategies that seek to enhance protective immune responses mediated by Th1 CD4 T cell responses. Introduction CD4 and CD8 T cells play a critical role in the host immune response to intracellular pathogens [1]C[4]. Following the initial exposure to the pathogen, T cells are primed, differentiate into effectors and undergo a phase of rapid growth in numbers. This is followed by a sharp contraction phase in which 90C95% of the effector cells are culled, leaving behind a pool of Ag-experienced T cells that further differentiate into memory populations that can persist for long periods of time. Immunologic memory is usually a hallmark of the adaptive immune response and ensures the host of a swift response that efficiently eliminates the pathogen in the event of re-exposures [1]C[4]. The development of CD8 T cell memory has been examined in great detail in the past few years. For example, there is a general consensus that the initial CD8 T cells that survive the contraction phase express an effector-memory cell (Tem) phenotype, whereas memory CD8 T cell populations found long after clearance of contamination are predominantly composed of central-memory T cells (Tcm) [2], [4], [5]. Tem and Tcm CD8 T cells subsets can be distinguished on the basis of expression of certain surface molecules and the secretion of IL-2. Classically, Tem express low levels of the homing receptors CD62L, CCR7 and produce low amounts of IL-2 while Tcm express higher levels of the CD62L and CCR7 and have a higher fraction of IL-2 producing cells [5]. Following a second exposure to the same pathogen the memory CD8 T cells develop into secondary effectors that eventually differentiate into secondary memory CD8 T cells. Secondary memory CD8 T cells maintain the Tem Ganirelix phenotype for extended time periods, and therefore differ from primary memory CD8 T cells that re-express CD62L more rapidly after priming [6]. This reacquisition of CD62L is also accompanied by improved IL-2 production [6], [7]. In contrast, CD4 T cell memory has not been as extensively studied and is complicated by the existence of multiple Th subsets [8]. Furthermore classification of CD4 T cell memory into Tem and Tcm subsets based primarily on CD62L expression is complicated by the failure of most memory CD4 T cells to re-express this lymph node homing receptor [9]C[11]. In addition, a substantial proportion of CD4 T cells produce IL-2 as early as 1 week after lymphocytic choriomeningitis virus (LCMV) and (Lm) infection and this property is retained as they transition Ganirelix Pten into memory. This differs greatly from the almost complete absence of IL-2 production from effector CD8 T cells [6]. While some reports describe longitudinal analyses of primary and Ganirelix secondary Th1 memory cells [10], [12], [13], little is known about the functional differences induced by secondary immunization. Additionally it is unknown whether the qualities of secondary memory Th1 cells depend on the nature of the boosting agent, and this remains a key question in the development and evaluation of heterologous prime-boost vaccination Ganirelix strategies. In this study we have examined the hypothesis that memory Th1 cells demonstrate phenotypic and functional plasticity and repeat antigenic encounters induce functional maturation of memory Th1 cells. We analyzed both primary and secondary CD4 and CD8 T cell responses occurring simultaneously in the same host after both LCMV and Lm infections. Our data reveal that depending on the nature of the priming agent there are marked differences in the patterns of expression of CD62L, CCR7 and IL-2 production between CD4 and CD8 T cells, and some differences were also noted for a few of the markers between memory CD4 T cell populations generated by either LCMV or Lm. We also examined the impact of repeat antigenic encounters on the ability of memory CD4 T cell subsets to induce LCMV-specific neutralizing antibody (NAb) formation as a read out of helper function and observed a significant improvement in the kinetics of NAb generation in mice harboring secondary memory CD4 T cells. Materials and Methods Mice: C57BL/6 Ganirelix (B6) mice were obtained from the National Cancer Institute, Frederick, MD. B6.PL (Thy1.1+) and B6.SJL (CD45.1+) mice (Jackson Laboratories, Bar Harbor,.