Journal of Experimental Medicine, Vol 141, 1015-1029, Copyright © 1975 by Rockefeller University Press
Separation of antigen-specific lymphocytes. II. Enrichment of hapten- specific antibody-forming cell precursors
W Haas
Normal spleen cells were separated in dishes coated with thin layers of
DNP-gelatin or NIP-gelatin into binding and nonbinding cells and stimulated
in vitro with DNP- and/or NIP-conjugated polymerized flagellin (POL).
Hapten-specific unresponsiveness was induced in the binding cell population
by melting the gel at 37 degrees C or in unfractionated cells by
pretreatment with soluble hapten-gelatin and could be reversed by treatment
with collagenase. A specific enrichment of anti-DNP and anti-NIP
antibody-forming cell precursors (AFCP) could be demonstrated in the
binding cell populations after treatment with collagenase in cultures with
or without "feeder" cells. However, the response of small numbers of
unfractionated and purified hapten- specific spleen cells was suboptimal
even in the presence of mitomycin- treated or irradiated feeder cells.
Optimal numbers of anti-DNP (anti- NIP) antibody-forming cells were
generated by small numbers of normal or purified spleen cells in the
presence of spleen cells depleted of anti-DNP (anti-NIP) AFCP. In this
system the response of only 2 times 10-4 purified hapten-specific cells was
higher than the response of 10- 6 unfractionated cells. Purified
DNP-specific cells responded only to DNP-POL but not to NIP-POL and
purified NIP-specific cells responded only to NIP-POL but not to DNP-POL.
The degree of enrichment of anti- DNP AFCP decreased with increasing
numbers of binding cells. NIP3- gelatin layers bound four to five times
less spleen cells than DNP2- gelatin layers and the enrichment of anti-NIP
AFCP (about 300-fold) was three times greater than the enrichment of
anti-DNP AFCP (about 100- fold). The immunological significance of
hapten-gelatin binding cells which apparently failed to respond to antigen
is discussed.
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