Kinetic study of T lymphocytes after sensitization against soluble antigen. I. Separation on density gradients.

Sensitized T cells in inguinal lymph nodes (LN) of rats immunized with ovalbumin and adjuvant can be separated on BSA density gradients from T cells responding to Con A and PHA. Further, these OA-sensitized T cells are separable from B memory cells responsive to OA. On days 9 and 17 after sensitization, OA-reactive cells peak and are found in enriched numbers in the lighter bands A + B (B-band), and C, respectively whereas Con A and PHA reactive cells, on both days, are concentrated in bands C and D, respectively. On day 9 at least 20 to 30% of B-band cells can reinitiate DNA synthesis and produce high concentrations of lymphotoxin in response to OA in culture, but show only a marginal blastogenic response to Con A or PHA and cannot produce lymphotoxin in response to PHA. These cells are defined as OA-sensitized T cells because a) treatment with rat isoantiserum specific for T lymphocytes plus complement abolished their ability to incorporate 3H-thymidine in response to OA and Con A or PHA; b) membrane-bound immunoglobulin could not detected with indirect immunofluorescence staining; c) histologic studies demonstrate massive hypertrophy of thymus-dependent paracortical areas and absence of germinal centers in inguinal LN at this time, and d) they produce lymphotoxin after challenge with OA, but not with PHA, whereas cells of the other bands produce lymphotoxin in response to PHA, but not to OA. Addition of purified macrophages to the various cultures did not affect the levels of response observed. Day 17 C-band cells are regarded as B-memory cells because treatment with T-specific antiserum plus complement enhanced their response to OA, many germinal centers now are present in inguinal LN, and a significant proportion of cells of bands B and C, negative on day 9, show positive immunofluorescence staining at this time.