Separation of antigen-specific lymphocytes. II. Enrichment of hapten-specific antibody-forming cell precursors.

Abstract

Clonal selection theories have postulated that individual immunocompetent cells are precommited to the formation of antibodies of a single specificity (1, 2) . There is now experimental evidence that only a distinct subpopulation of lymphocytes can bind and be stimulated by a particular antigen (for review see 3) . Furthermore, it has been shown that individual lymphocytes display only one type of receptor, homogeneous in respect to specificity (4) . This strongly suggests that the precommitment of lymphocytes is expressed by surface receptors for antigen .Depletion of antigen-binding cells by adsorption to antigen-coated beads (5-7) and specific inactivation by radiation (8, 9), provided direct evidence for the specific function of antigen-binding cells in humoral immune responses . However, experiments with lymphocytes depleted in functionally active antigen-specific cells do not prove finally the precommitment hypothesis . Furthermore they do not allow studies on the mechanism of the induction of an immune response . However, isolation and enrichment of lymphocytes bearing surface receptors of a single specificity would be an obvious advantage in studies of the interaction of specific lymphocytes with immunogens, tolerogens, or mitogens . Furthermore, purification of antigen-specific cells would be essential to dissect the various specific and nonspecific interactions of lymphocytes in immune responses .Several attempts have been made to purify antigen-specific lymphocytes and to study their function . Lymphocyte populations from unimmunized mice enriched in sheep red blood cell rosette-forming cells (SRBC-RFC)' contained anti-SRBC antibody-forming cell precursors (AFCP), whose capacity to proliferate and differentiate in vitro (10) or in vivo (11) into antibody-forming cells was dependent on the presence of collaborating cells .Lactoside-specific cells from unimmunized mice were retained in a lactoside-coupled polyacrylamide bead column and could be eluted by an excess of free haptens . The eluted cells were highly enriched in antilactoside AFCP as determined in an adoptive transfer

Sequential use of hapten‐gelatin fractionation and fluorescence‐activated cell sorting in the enrichment of hapten‐specific B lymphocytes

“…Spleen cells were fractionated on FLU-gelatin dishes, using the hapten-gelatin method as previously described for obtaining populations of splenic B lymphocytes enriched for reactivity to the haptens DNP and NIP [1][2][3]6]. FLU-gelatin-binding cells were treated with collagenase ( 100 pg/ml, 15 min at 37 "C) to remove adherent multivalent antigen from their surface prior to culture, or prior to binding of antigen for subsequent sorting on the FACS.…”

“…Therefore, recent experiments from this laboratory [ 1-61 have been aimed at devising techniques for the fractionation of enriched, hapten-specific murine B cells [I 19401 [ 1, 61; for their effective stimulation and assessment in tissue culture [2,3,5,6]; and for the monitoring of early cell surface events that follow the binding of immunogenic or tolerogenic concentrations of antigen [4, 51. The simple haptengelatin method [ 11 can provide populations 100 t o 300-fold enriched for clonable precursors of hapten-specific antibodyforming cells [3].…”

Separation of antigen‐specific lymphocytes. A new general method of releasing cells bound to nylon mesh

“…The second is the mechanical removal of the attached cells which invariably results in at least temporary damage of the cells [ 6-91. The third, which may be called thermal release, consists of binding the cells at 0 "C and releasing them at a higher temperature, either by taking advantage of the capping and shedding mechanism observed with lymphocytes [ 101 or b y melting a spacer substance (gelatine) placed between the solid support and the receptor specific molecule [9,[11][12][13][14]. Still another method uses enzymic degradation of a spacer or of the whole solid support with enzymes [ 15, 161.…”

Antifluorescein affinity columns. Isolation and immunocompetence of lymphocytes that bind fluoresceinated antigens in vivo or in vitro.

“…To test this, we employed BALB/c anti-EL4 (H-2 d anti-H-2 ~) cytotoxic peritoneal exudate lymphocytes, which are a potent population of killer cells (25) and have been shown to bind to EL4 cells or cell monolayers in vitro (9,26). BALB/c peritoneal cells, harvested on day 11 after an intraperitoneal injection of 25 x 106 EIA leukemia cells, were mixed at a 20:1 (BALB:EL4) ratio with FL-EL4 and centrifuged for 5 min at 800 rpm to promote cell contact.…”

“…These include, for example, the use of antigen or antibody bound to nylon fibers (2,3), plastic tubes (4), plastic, polyacrylamide, or agarose beads (5-7), gelatin layers (8,9), as well as "insoluble" antigen in the form of cellular monolayers of tumor (10), fibroblastoid (11), or red blood cell (RBC) (12) origin, resetting (13), and, most recently, the fluorescence-activated cell sorter (FACS) I (14). All of these techniques are dependent on the binding of antigen via specific membrane receptors on normal or sensitized lymphocytes or antibody-forming cells (AFC).…”