Cellular Requirements for the Primary in Vitro Antibody Response to DNP-Ficoll

Abstract

Antibody formation to most antigens requires collaboration between T lymphocytes and B lymphocytes, but certain antigens (e.g., polymerized bacterial flagellin, bacterial lipopolysaccharides, polyvinylpyrrolidone, levan [1][2][3][4][5]) induce antibody synthesis in the absence of detectable numbers of T lymphocytes, and hence are termed "T independent". We have examined the in vitro response of mouse spleen cells to e-2,4 dinitrophenyl (DNP)-lysyl-subsfituted Ficoll (a synthetic polymer of sucrose and epichlorhydrin) and find that nanogram quantities of DNP-Ficoll stimulate a primary anti-DNP response of considerable magnitude which appears to require neither T lymphocytes nor splenic adherent cells for its generation. The wide availability of Ficoll and ease of preparation of hapten derivatives makes this powerful T-independent antigen of particular value in the study of direct activation of precursors of antibody-forming cells. Materials and MethodsMice.--8-to 12-wk old female BALB/c mice obtained from the Animal Production Section of the NIH were used in most experiments. Outbred congenitally athymie nu/nu mice or heterozygous nu/-J-(or %/+) mice were obtained from the same source. C57BL/10 Sn, B10.A, B10.Br, B10.D2, DBA/2, C3H/J, AKR, and SJL mice were obtained from Jackson Laboratories, Bar Harbor, Maine.Cell Culture.--Mouse spleen cells were cultured by the method of Mishell and Dutton (6) as previously modified (7). Briefly, single cell suspensions at a concentration of 10 X 106 cells per ml were cultured in modified Eagle's minimal essential medium in 3S-ram plastic culture dishes on a rocking platform at 37°C in an atmosphere of 7% O~, 10% COs, and 83% N2. 50 #1 of nutritional cocktail and fetal calf serum were added to cultures daily. Altering these conditions by adding 2-mercaptoethanol (10-3M) to the medium, omitting the daily nutritional cocktail and fetal calf serum, and leaving the cultures stationary did not significantly change the response to DNP-Ficoll but did reduce parallel control responses to sheep red blood cells (SRBC) or trinitrophenyl-substituted horse red blood cells (TNP-HRBC).Antigens.--e-dinitrophenyl-substituted Ficoll was prepared as described elsewhere, z Briefly, the substitution procedure involved reaction of Ficoll with cyanuric chloride at 40C, followed by the addition of e-DNP-lysine and subsequent reaction at room temperature. The commonly used preparation had a molar ratio of DNP-Ficoll of 32:1 (DNP32Ficoll). Other substitution ratios (DNPz.2Ficoll, DNPssFicoll) were used in some experiments. A copolymer of D-glutamic acid and D-lysine was dlnitrophenylated (DNP44D-GL) as previz McMaster, P. R. B., manuscript in preparation.

Ontogeny of mouse lymphocyte function. II. Development of the ability to produce antibody is modulated by T lymphocytes.

“…The in vitro PFC response to DNP-Ficoll has been shown to be independent of T lymphocytes in adult (9) and neonatal (Mosier, unpublished experiments) mice. The response to SRBC or TNP-SRBC is quite dependent upon the presence of T lymphocytes (15) .…”

“…Preparation of DNP-Ficoll has been described recently (9). DNP,,Ficoll (32 mol DNP/400,000 average mol wt of Ficoll) was used for these experiments .…”

“…Spleen cell suspensions were prepared from pooled spleens of several mice of the same age and cultured according to a modification of the Mishell-Dutton technique (10) as previously described (9). Plaque-forming cells (PFC) were assayed after 3, 4, or 5 days of culture by a slide modification of the Jerne hemolytic plaque assay (11) .…”

Ribavirin treatment in murine autoimmune disease

“…The effect of ribavirin on the primary in vitro PFC response to SRBC was determined by the method of Mosier et a1 (28). Primary in vitro PFC were generated in the presence of varying dilutions of ribavirin (moles/liter) and the number of PFC/culture well was determined on day 5.…”

IR gene regulation of the response to trinitrophenyl-polysaccharides. Two independent genes are required for antibody production.

“…TNP-Ficoll, a prototypic TI-2 antigen, requires Ia + macrophages (14), a specific subset of B cells (Lyb-5 +) (15), and elicits a response in congenitally athymic mice (16). The response of nu/nu mice to TI-2 antigens suggested that T cells were not required for specific antibody production.…”