This paper describes an in vitro system for the study of the factors that control and regulate the cellular events in the immune response. It was reported earlier (1) that dissociated cell suspensions obtained from the spleens of normal mice could be immunized in vitro to heterologous erythrocytes. The response is primary in the sense that it follows the first experimental exposure to the erythrocyte antigen. The response was measured by the increase in the number of hemolytic plaque-forming cells and by assay of antibody in the culture supernatants. The critical conditions for culture and immunization of the spleen cells included low oxygen tension, gentle agitation of the cultures, the inclusion of fetal bovine serum in the medium, adequate spleen cell density, and daily feeding of the cultures with a nutritional mixture. This paper presents a more detailed account of the experimental system and some comparisons between in vitro and in vivo responses under a variety of experimental conditions. The in vitro response closely parallels that seen in vivo with respect to size, early kinetics, effect of antigen dose, and the inhibitory effect of passive antibody. These findings encourage the belief that observations made with the in vitro system are relevant to our understanding of the in vivo response. The in vitro differs from the in vivo response in that it appears to show a greater capacity to discriminate between different homologous erythrocyte antigens and that it shows no limitation or termination of the increase in 19S antibody-forming cells 4 or 5 days after the initiation of the response. These differences have been characterized in the hope that their further study may uncover underlying regulatory mechanisms involved in the response. * This is publication No. 217 from

Immunological memory can be defined as the faster and stronger response of an animal that follows reexposure to the same antigen. By this definition, it is an operational property of the whole animal or the immune system. Memory cells express a different pattern of cell surface markers, and they respond in several ways that are functionally different from those of naive cells. Murine memory cells are CD44 high and low in the expression of activation markers such as CD25 (IL-2R), whereas human memory cells are CD45RA-, CD45RO+. In contrast to naive cells, memory cells secrete a full range of T cell cytokines and can be polarized to secrete particular restricted patterns of secretion for both CD4 and CD8 T cells. The requirements for the activation of memory cells for proliferation and cytokine production are not quite as strict as those of naive cells, but costimulation in the broad sense is required for optimum responses and for responses to suboptimum antigen concentrations. It would appear that memory cells can persist in the absence of antigenic stimulation and persist as nondividing cells. Reencounter with the same antigen can expand the population to a new, stable, higher level and generate a separate population of CD44 high effectors that may be required for protection, while competition from other antigens can drive it down to a lower stable level. It is unclear how or where memory cells arise, but once generated they have different pathways of recirculation and homing.

The poor correlation between cellular immunity to respiratory virus infections and the numbers of memory CD8+ T cells in the secondary lymphoid organs suggests that there may be additional reservoirs of T cell memory to this class of infection. Here we identify a substantial population of Ag-specific T cells in the lung that persist for several months after recovery from an influenza or Sendai virus infection. These cells are present in high numbers in both the airways and lung parenchyma and can be distinguished from memory cell populations in the spleen and peripheral lymph nodes in terms of the relative frequencies among CD8+ T cells, activation status, and kinetics of persistence. In addition, these cells are functional in terms of their ability to proliferate, express cytolytic activity, and secrete cytokines, although they do not express constitutive cytolytic activity. Adoptive transfer experiments demonstrated that the long-term establishment of activated T cells in the lung did not require infection in the lung by a pathogen carrying the inducing Ag. The kinetics of persistence of Ag-specific CD8+ T cells in the lung suggests that they play a key role in protective cellular immunity to respiratory virus infections.