Clones of antibody‐forming cells in pokeweed mitogen stimulated microcultures. II. Estimation of the frequency of precursor cells and the average clone size

Quintáns, José; Lefkovits, Ivan

doi:10.1002/eji.1830040908

Cited by 28 publications

(12 citation statements)

References 9 publications

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“…The microculture system allows one to estimate both the number of precursor B cells which are involved in a response and the size of that part of clonal progeny which can be expressed as PFC (13)(14).…”

Section: Antibody Response In Microculturesmentioning

confidence: 99%

“…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: Antibody Response In Microculturesmentioning

confidence: 99%

Section: Clonotype 2 Analysis Of Antibody Produced In Microculturesmentioning

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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“…1 c), or upon addition of allogeneic factor ( Fig. 1 d) which had been shown t o enhance an anti-SRC response [35]. The straight line observed under the various experimental conditions tested seems to indicate that cell types other than B cells are either not required for the anti-PC response or they are not limiting at any tested cell input.…”

Section: Estimation Of the Frequency Of Precursor Cellsmentioning

confidence: 79%

“…One obvious difference is that SRC are T-dependent antigens and we know of effective ways of helping a larger number of B cells t o respond t o these antigens in microcultures [35]. On …”

Section: Epitope Exclusivenessmentioning

confidence: 99%

Antibody response to phosphorylcholine in vitro. I. Studies on the frequency of precursor cells, average clone size and cellular cooperation

Cosenza¹,

Quintáns²,

Lefkovits³

1975

Eur J Immunol

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The anti-phosphorylcholine (PC) antibodies synthesized by BALB/c spleen cells in microcultures upon immunization with heat-killed vaccine of Pneumococci R36A (Pn) are directed exclusively to the PC epitope. These antibodies are of very restricted avidity and 88% of the responding clones express the idiotype characteristic of the TEPC-15 PC-binding myeloma. This idiotypic restriction appears to be due to the absence of clones capable of expressing other idiotypes, rather than to "clonal dominance". The estimated frequency of precursor cells for the PC epitope is 1 X 10(-5) to 2.5 X 10(-5). These precursors give rise to clones with an average size of 9 plaque-forming cells. When the logarithm of the number of negative wells was plotted against the number of spleen cells/well, the fraction of nonresponding cultures decreased exponentially as the number of spleen cells was increased. This indicated that only one cell type was limiting in our assay, presumably a B cell. Furthermore, treatment of spleen cells with AKR anti serum completely abolished the response to sheep red cells without affecting the response to PC. It is concluded that PC is a T cell-independent antigen. Of interest was the finding that PC requires adherent (A) cells and this is a particular characteristic of PC, since most T cell-independent antigens have been found not to require A cells. Reasons for the possible homogeneity of the response to PC are also discussed.

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“…Each microculture well was assayed separately for PFC [22]. The trays were spun for 5 min at 500 x g, and an excess of cold BSS was added to wash out the culture fluid.…”

Section: Hemolytic Plaque Assaymentioning

confidence: 99%

Antibody response to phosphorylcholine in vitro. II. Analysis of T‐dependent and T‐independent responses

Quintáns¹,

Cosenza²

1976

Eur J Immunol

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Spleen cells from BALB/c mice primed with keyhole limpet hemocyanin (KLH), were stimulated with heat-killed vaccine of rough Pneumococcus pneumoniae R36A (Pn) and/or phosphorylcholine (PC)-coupled KLH to induce an anti-PC response in vitro. The response to PC-KLH was found to be T-dependent while it is T-independent to Pn. The antibodies induced with either antigen had similar avidity and expressed the TEPC 15 idiotype exclusively; thus T cell involvement in the response to PC-KLH failed to alter these parameters of the anti-PC response. At the precursor cell level, Pn induced small clones with an average size of 10 plaque-forming cells (PFC), whereas PC-KLH gave rise to larger clones of 40-50 PFC. This difference in the proliferative potential of PC precursor B cells hinted at the possibility that Pn and PC-KLH were stimulating different precursors. This was corroborated by the observation that a) when Pn and PC-KLH were added to the same cultures a synergistic effect was seen, i.e. the number of plaques was greater than the sum of the responses induced by each antigen, and b) in microcultures, under conditions limiting B cells only, Pn plus PC-KLH induced a higher fraction of responding wells than either antigen on its own. We postulate that Pn and PC-KLH stimulate subpopulations of PC precursor cells which are T-independent and T-dependent, respectively.

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Clones of antibody‐forming cells in pokeweed mitogen stimulated microcultures. II. Estimation of the frequency of precursor cells and the average clone size

Cited by 28 publications

References 9 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 response to phosphorylcholine in vitro. I. Studies on the frequency of precursor cells, average clone size and cellular cooperation

Antibody response to phosphorylcholine in vitro. II. Analysis of T‐dependent and T‐independent responses

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