Regulatory T cells in the antibody response to Haemophilus influenzae type b polysaccharide

Abstract

An in vitro culture system for the induction of an antipolysaccharide response was used to study the cellular interactions which determine the magnitude and nature of this B-lymphocyte response. Healthy adult volunteers were vaccinated with the Haemophilus influenzae type b polysaccharide (PRP)-tetanus toxoid (TT) conjugate vaccine. Optimal in vitro anti-PRP and anti-TT antibody responses were obtained when B cells were cultured with equal amounts of T cells. The in vitro response is antigen dependent and antigen specific. Culturing with PRP mixed with TT in the presence of T cells induces the highest number of anti-PRP antibody-secreting cells (ASC) (128.4 x// 15.9 [geometric mean x// standard deviation] immunoglobulin M [IgM] anti-PRP ASC/10(6) cells; 9.3 x// 7.6 IgG anti-PRP ASC/10(6) cells). Culturing without T cells induced no anti-PRP ASC; culturing with only PRP, in the presence of T cells, yielded low numbers of anti-PRP ASC (3.7 x// 5.2 IgM anti-PRP ASC/10(6) cells and 1.2 x// 2.2 IgG anti-PRP ASC/10(6) cells). Transwell studies showed that the requirements for the antibody response against the polysaccharide are different from those of an antiprotein response. Cytokines formed as a consequence of contact between protein-specific B and T cells were on their own not sufficient to activate TT-specific B cells (8.4 x// 1.4 anti-TT ASC/10(6) cells); direct contact between T and B cells appeared to be an absolute requirement. However, physical contact between B and T cells in one compartment of the Transwell system resulted in the release of soluble factors able to stimulate B cells in the other compartment to secrete antipolysaccharide antibodies (164 x// 1.6 anti-PRP ASC/10(6) cells).

FIG. 1

In vitro anti-PRP response of blood B cells obtained 3 to 4 weeks after vaccination with PRPTT. B cells (0.5 × 10⁶/ml) were cultured with an equal number of irradiated T cells and 5% monocytes in medium containing 10% heat-inactivated pooled human AB serum and various antigens as indicated. After 6 days of culture, IgG and IgM anti-PRP ASC were determined with a spot-forming cell assay and are expressed per 10⁶ lymphocytes. Final concentrations: PRP, 5 ng/ml; TT, 1.5 μg/ml; PRPTT, 0.5 μg of PRP and 1.2 μg of TT/ml. The data are from 21 independent experiments with different donors. Not all antigens were included in all experiments.

FIG. 2

In vitro IgG anti-TT response of blood B cells obtained 3 to 4 weeks after vaccination with PRPTT. See the legend to Fig. 1 for details.

FIG. 3

In vitro anti-PRP (○) and anti-TT (•) responses of blood B cells under various culture conditions in the Transwell system. Lymphocytes were obtained 3 to 4 weeks after vaccination with PRPTT. In culture a, B cells (0.7 × 10⁶) were grown in medium containing 10% heat-inactivated pooled human AB serum and PRP plus TT. In the control experiment (culture b), B cells (0.7 × 10⁶) were grown with an equal number of irradiated T cells and 5% monocytes in medium containing 10% heat-inactivated pooled human AB serum and PRP plus TT. By means of a Transwell system, B cells (0.7 × 10⁶) were also grown separately from T cells (0.7 × 10⁶) with 5% monocytes (culture c). d and e, results of culturing B cells (0.7 × 10⁶) separated by the Transwell system from the lower compartment containing B cells (0.7 × 10⁶) and an equal number of irradiated T cells and 5% monocytes. After 6 days of culture, anti-PRP as well as anti-TT ASC were determined with a spot-forming cell assay and are expressed per 10⁶ lymphocytes. All cultures were stimulated with PRP (5 ng/ml) plus TT (1.5 μg/ml). *, compartment from which the ASC are depicted. The data represent six experiments conducted separately with six different donors. Note that not all combinations were included in every experiment.