The development of robust in vivo memory cytotoxic and Th1/Th2 responses upon exposure of neonates to NIMA-like alloantigens. (A) Neonates (n = 22) and adults (n = 17) were injected with allogeneic spleen cells, as described for Figure 2. Five to 15 weeks later, the percentage of allospecific cytotoxicity in individual spleens was determined as described in “In vivo cytotoxicity assays.” A minimum of 1 × 106 total cells were examined for each sample. Data from individual mice from 3 independent experiments are depicted (variations in responses were observed within each experiment). The average percentage of allospecific cytotoxicity was not significantly different (2-tailed t test) between neonates and adults. (B) Neonates were injected with allogeneic spleen cells (SP), bone marrow (BM), or Lin− BM cells, as described in “Preparation and injection of donor cells.” Five to 6 weeks later, the percentage of allospecific cytotoxicity in individual spleens was determined. Data are pooled from 2 independent experiments; n = 9 for SP, n = 10 for BM, and n = 11 for Lin− BM. The average percentage of allospecific cytotoxicity was not significantly different (2-tailed t test) between groups. (C-H) Neonates and adults were injected with allogeneic cells, as described for Figure 2. Memory CD4+ cytokine responses were then assayed by ELISA (C-E) and the frequency of cytokine-producing memory cells was determined by ELISPOT (F-H), as described in “Culture conditions for cytokine ELISA and ELISPOT.” The in vitro responses of age-matched, naive controls, determined in parallel, were as follows: for neonates: IFNγ, 155 (± 44) ng/mL and 1728 (± 826) secretors/106 cells; IL-4, 1.0 (± 0.7) ng/mL and 2113 (± 636) secretors/106 cells; for adults: IFNγ, 187 (± 41) ng/mL and 2146 (± 2112) secretors/106 cells; IL-4, 0.28 (± 0.1) ng/mL and 2221 (± 1056) secretors/106 cells. Error bars represent the mean (± SD) of data from 4 independent experiments. Statistical significance was determined using a 2-tailed t test; **P < .005
