A THREE-CELL INTERACTION REQUIRED FOR THE INDUCTION OF THE PRIMARY IMMUNE RESPONSE in vitro

Abstract

The primary immune response involves the interaction of several cell types. [1][2][3][4] In the present experiments, it was possible to evaluate the number of interacting cells by using a combination of recently developed techniques. Mishell and Dutton5 described a method for the induction of the primary immune response to 'sheep erythrocytes in vitro. The numbers of antibody-forming cells in the in vitro response were enumerated by the hemolytic plaque assay described by Jerne and Nordin6 and, independently, by Ingraham and Bussard.7 In a recent paper, one of us8 has shown that cells which adhere to plastic Petri dishes and cells which do not adhere are both required for the induction of the immune response with the methods of Mishell and Dutton. Coppleson and Michie9 have shown that the number of cells interacting in a graft versus host reaction can be estimated from the slope of the log cell dose-log response line. This method of analysis was applied to the adherent and nonadherent cell populations in such a way that the number of interactions contributed by each population could be assayed separately. From these estimates, it appears that at least two, and possibly three, cell types are involved in at least two separate cell interactions during the primary immune response in vitro.Materials and Methods.-Tissue culture: Cell cultures were prepared from the spleens of 8-12-week-old DBA/2 mice. Spleen cell suspensions were prepared by teasing apart with forceps the spleens in cold Hanks' balanced salt solution (HBSS). Cell aggregates were allowed to sediment by gravity, and the resulting suspension of single cells was centrifuged for 10 min at 600 X g at 40C. The pooled sedimented cells were resuspended to a concentration of 1 X 107 cells per ml in Eagle's minimum essential medium (Grand Island Biological) supplemented with nonessential amino acids, pyruvate, and 10% fetal bovine serum (Colorado Serum). Cultures were maintained in 35-mm plastic Petri dishes (Falcon 1008) on a slowly rocking platform (Bellco) in an atmosphere of 7% 02, 10%/ C02, and 83% N2. Each culture dish received daily 50 ul of nutritional mixture8 and 50 ,ul of fetal bovine serum. These conditions closely approximate those originally described by Mishell and Dutton.5 Preparation of adherent and nonadherent cell populations: Adherent and nonadherent cells were prepared by culturing 1 X 107 spleen cells in a plastic Petri dish for 30 min on the rocking platform. Those cells remaining free in the culture medium were aspirated and designated the nonadherent population. The culture dish was then washed three times with cold HBSS, and the washings were discarded. The cells remaining firmly attached to the plastic were designated the adherent population. In each experiment all cell populations were prepared from the same pool of spleen cells.In the main series of experiments, the numbers of either adherent or nonadherent cell populations were varied, and one population was cultured in the presence of a constant excess of the other population. The...

Cellular Differentiation of the Immune System of Mice

“…Limiting dilution experiments for restricted P-PFC and unrestricted ARC, to be reported in the next papers of this series, 1 have confirmed these interpretations. The possibility that two or more different marrow-derived cells cooperate with ARC and SRBC in forming functional splenic ASU has been raised by our own results 1 and by the reconstitution of ASU by mixing spleen cell fractions separated by physical means (22,23).…”

The Role of Nonlymphoid Accessory Cells in the Immune Response to Different Antigens

“…A related aspect is the possible interaction between different lymphoid cells. Evidence for such an interaction in the response to SRC has been provided by in vivo studies (e.g., 19,28) and to some extent from in vitro work (23,30). Again this aspect will be investigated using the density separation technique, but care will be needed to experimentally separate lymphocyte-lymphocyte interaction from the lymphocyte-accessory cell interaction considered in the present report.…”

Antibody and Immunoglobulin Production at the Cellular Level in Bursectomized-Irradiated Chickens

“…An alternative explanation is that the immune deficiency is due to a block in the inductive or early proliferative phase of the immune response. This could be caused, for example, by a functional deficiency of the macrophage processing of SRBC antigen (25,26) or in the failure of the two or possibly three cell types involved in the immune response (27)(28)(29)(30)(31)(32) to interact properly. Studies are presently in progress to evaluate these possibilities.…”