Induction of antibody‐forming cell clones in microcultures

Lefkovits, Ivan

doi:10.1002/eji.1830020412

Cited by 138 publications

(66 citation statements)

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“…It is possible that the ability to respond with unusually high antibody levels is associated with or reflected in the number of memory cells circulating in the blood. Estimation of the number of Av-CHO specific memory cells, however, could only be expressed as responding units revealed by the microculture technique (4,(13)(14), because limiting dilution conditions could not be achieved by controlled titration for all cell types (B cells, T cells, and macrophages) involved in the immune reponse in vitro.…”

Section: Clonotype 2 Analysis Of Antibody Produced In Microculturesmentioning

confidence: 99%

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Antibody response of rabbit blood lymphocytes in vitro. Kinetics, clone size, and clonotype analysis in response to streptococcal group polysaccharide antigens.

Braun¹,

Quintáns²,

Luzzati³

et al. 1976

The Journal of Experimental Medicine

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Hyperimmunization of rabbits and mice against streptococcal groups A-variant and A polysaccharides (Av-CHO and A-CHOP leads to the induction of a long-lived immunological memory. The number of clonotypes of specific IgG antibodies remains constant with time and repeated immunizations. This pattern is already stable from the moment when IgG antibodies are first detectable (1).It was previously shown that peripheral blood lymphocytes (PBL) from rabbits primed in vivo with the Av-CHO can be restimulated in vitro. It was observed that the pattern of IgG antibodies overlapped with the clonotype patterns induced in vivo (2). In the present paper the state of immunological memory was investigated by using both conventional and microculture techniques (2-4). Materials and MethodsRabbits. The rabbits used within this study were taken from a colony bred selectively for restricted high responders (5). They had been previously immunized with dead vaccines of Group A-variant (strain A486 variant, M-) streptococci and produced 10-25 mg/ml of antibody at the peak of the response. Rabbit K19-205 was additionally immunized with a Group A (strain J17A4) streptococcal vaccine 6 mo after the irdection course with the Group A-variant vaccine.Cultures. Rabbit blood was collected from the central artery of the ear into heparinized bottles. The erythrocytes were sedimented in 1% pig skin gelatin, as described by Sell and Gel] (6), and the leukocytes were washed and resuspended in RPMI 1640 medium (Microbiological Associates, Bethesda, Md.) containing 10% fetal calf serum (Rehatuin, Rebels Chemical Co., Chicago, Ill., Batch No. H73212), 5 × 10 -5 M 2-mercaptoethanol, and 50 U/ml of both penicillin and streptomycin (Microbiological Associates). Streptococcal A-variant or A vaccines were added as antigen in doses of 2.5-3 ~g rhamnose per ml (2). 1-ml cultures containing 4 × 10 ' or 2 × 10 '; cells were distributed in plastic tubes (Falcon Plastics, Los Angeles, Calif., type 2003

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Section: Clonotype 2 Analysis Of Antibody Produced In Microculturesmentioning

confidence: 99%

“…tures l0 t, 2 × 104, 4 x l0 t, or 8 × l0 t cells were distributed in 10-/~l aliquots in microculture trays (Falcon Plastics, type 3034) as previously described (3)(4). Both 1-ml cultures and microcultures were kept in a 5% CO2 incubator at 37°C without further treatment during the culture period.…”

mentioning

confidence: 99%

Antibody response of rabbit blood lymphocytes in vitro. Kinetics, clone size, and clonotype analysis in response to streptococcal group polysaccharide antigens.

Braun¹,

Quintáns²,

Luzzati³

et al. 1976

The Journal of Experimental Medicine

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“…Much of our recent knowledge about B lymphocyte physiology has come from methods that involve cloning by limiting dilution in liquid tissue culture (1)(2)(3)(4)(5); in such methods the responding B cell is isolated. from most of the effects of the immune network.…”

mentioning

confidence: 99%

Antibody production by single, hapten-specific B lymphocytes: an antigen-driven cloning system free of filler or accessory cells.

Vaux¹,

Pike²,

Nossal³

1981

Proc. Natl. Acad. Sci. U.S.A.

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CBA mouse spleen cells were subjected to hapten affinity fractionation on thin layers of fluorescein (FLU)-gelatin. This procedure yields 97% B cells with varying FLU-binding avidities. One to 30 cells were placed in 10-1.d microcultures without any filler or accessory cells. The T-independent antigen polymerized flagellin coupled to FLU (FLU-POL) was ineffective in stimulating these cells to clonal proliferation or antibody production when used alone. Unpurified preparations rich in interleukins also failed to stimulate the cells. When specific antigen, but not an irrelevant hapten-POL, was combined with the interleukins, clonal proliferation was stimulated and most clones produced anti-FLU antibody-forming cells. The frequency of antibody-forming clones was only slightly lower than that in a system using antigen plus filler cells. In the absence of added interleukins, the mitogens Escherichia coli lipopolysaccharide plus dextran sulfate induced equivalent antibody production. However, a higher frequency of clonal proliferation was noted. Added interleukins did not aid these mitogen-driven responses. Such an antigen-dependent cloning system, free of filler and accessory cells, should permit more precise analysis ofthe respective roles ofantigens and interleukins in the physiology of antibody-forming clone formation.Much of our recent knowledge about B lymphocyte physiology has come from methods that involve cloning by limiting dilution in liquid tissue culture (1-5); in such methods the responding B cell is isolated. from most of the effects of the immune network. However, a major disadvantage of these techniques is their dependence on thymic or x-irradiated splenic cells for support of clonal proliferation. Apart from obscuring visualization, these additional cells may produce interleukins and thereby impede the analysis of the effects of intentionally added stimulatory factors. Conditions have been defined whereby single B lymphocytes can be cloned in agar when physically separated from macrophages, which are necessary for their proliferation (6,7). However, this model allows minimal antibody production (8,9). Given the current interest in the respective roles of antigens or mitogens and macrophage-or T lymphocyte-derived cytokines in lymphocyte proliferation and differentiation (10-16), a tissue culture system in which B lymphocytes can be stimulated as single cells, free of filler cells, is essential. The various stages in the formation ofan antibody-forming cell clone-namely, activation of the small lymphocyte from the Go state, mitosis, and differentiation to large-scale antibody synthesis-could then be sequentially studied.A major step forward in this direction has -been taken by . They showed that individual murine splenic B cells, stimulated by a mixture of the mitogens Escherichia coli lipopolysaccharide (LPS) and dextran sulfate (DXS), proliferated with a cloning efficiency ofup to 80%. Only a minority of the clones produced detectable antibody, and the addition ofsmall numbers ofx-irradiated...

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“…Appropriate numbers of KLH-primed T cells with 1.5 x l0 t m1-1 hapten-primed B cells were cultured in 10-~l vol in wells of Terasaki plates under standard conditions (12) in the presence of 2 btg/ml of TNP-KLH. 30 identical wells were set up for each combination of cells.…”

mentioning

confidence: 99%

Cooperation across the histocompatibility barrier: H2d T cells primed to antigen in an H-2d environment can cooperate with H-2k B cells.

Waldmann¹,

Munro²

1976

The Journal of Experimental Medicine

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H-2d spleen cells derived from either tetraparental or semiallogeneic radiation bone marrow chimeras can be primed to antigen within H-2d recipients to generate helper T cells capable of cooperating in a secondary response with equal efficiency with H-2d or H-2k B cells. Thus it would seem that the cooperative act between T and B cells does not require that the T cell interacts with its target B cells by either cell interaction genes or via an altered self mechanism involving both antigen and the target B-cell I-region products. This does not preclude a requirement for associative recognition or altered self in the interaction of helper T cells with accessory cells.

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Induction of antibody‐forming cell clones in microcultures

Cited by 138 publications

References 6 publications

Antibody response of rabbit blood lymphocytes in vitro. Kinetics, clone size, and clonotype analysis in response to streptococcal group polysaccharide antigens.

Antibody response of rabbit blood lymphocytes in vitro. Kinetics, clone size, and clonotype analysis in response to streptococcal group polysaccharide antigens.

Antibody production by single, hapten-specific B lymphocytes: an antigen-driven cloning system free of filler or accessory cells.

Cooperation across the histocompatibility barrier: H2d T cells primed to antigen in an H-2d environment can cooperate with H-2k B cells.

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