Memory cell generation ablated by soluble protein antigen by means of effects on T- and B-lymphocyte compartments.

Abstract

Adult C57BL/6 mice were injected with 100 ,ug of soluble, freshly deaggregated human serum albumin (HSA) to produce partial immunologic tolerance. UnhEjected normal control (N) mice contain only 100 B cells in their spleens with the capacity to (i) be activated in vitro into clonal proliferation by Escherichia coli lipopolysaccharide plus interleukins 2, 4, and 5, (ii) form IgG1 as well as IgM antibody, and (iii) display specificity for HSA when only IgG1 is allowed to score in an enzyme-linked immunosorbent assay (ELISA). Such N mice generate ==50,000 clonable anti-HSA IgG1 antibody-forming cell precursors in their spleens after T-dependent immunization with HSA adsorbed onto alum and given with BordeteUa pertussis adjuvant. Mice prei'jected with soluble HSA (TOL)-generate far fewer anti-HSA IgG1 antibodyforming cell precursors, termed anti-HSA memory cells. Splenocytes were transferred from N or TOL mice into lethally irradiated syngeneic recipients together with syngeneic bone marrow. Whereas N splenocytes generated plentiful memory cells within 2 weeks in antigenically challenged recipients, TOL splenocytes did not. Work with Ly-5 congenic mice ruled out memory cell generation from either the host or the bone marrow inoculum within this limited time. N T cells plus TOL B cells showed consistently lowered memory cell generation. TOL T cells plus N B cells showed an even greater lowering of adoptive memory cell generation. Thus the lowered response capacity of TOL mice resided in the T-and B-cell compartments. Attempts to show a suppressor component within the T-cell population were inconclusive, but a profound defect in capacity to respond to HSA in vitro was exhibited by the CD4' T cells of TOL mice. B lymphocytes were harvested from T-dependently immunized mice 5 days after challenge, incubated with soluble HSA for 18 hr, and then adoptively transferred together with N T cells. The recently activated B cells were not rendered tolerant by this manipulation. The results argue for a major T-cell component in the process whereby soluble protein antigens ablate affinity maturation and memory cell generation.

Soluble antigen profoundly reduces memory B-cell numbers even when given after challenge immunization.

“…In four separate experiments, the numbers were 0, 93, 126, and 240 per spleen. This way of examining the repertoire is made possible by utilizing the capacity of IL-4 to drive the isotype switch and by an ELISA read-out that focuses only on bivalent IgGl antibody (14,15). Through these strategies, the many potentially anti-NP clonotypes of low affinity that would have registered using an IgM read-out are simply ignored.…”

“…To produce two NP epitopes per carrier molecule (NP2-carrier), a ratio of [10][11][12][13][14][15][16][17][18][19][20] ,ug/mg of NP-succinimide to carrier Abbreviations: AFCP, antibody-forming cell precursor; BSA, bovine serum albumin; Cyt-c, cytochrome c; FDC, follicular dendritic cell; HoSA, horse serum albumin; HSA, human serum albumin; IL, interleukin; LPS, lipopolysaccharide; MT-PBS, mouse tonicity phosphate-buffered saline; NP, (4-hydroxy-3-nitrophenyl)acetyl; r, recombinant; V, variable.…”

“…(14,15) and showed that it could prevent the generation of anti-HSA memory B cells in a dose-dependent manner. Adoptive transfer studies showed that the toleragen affected donor T and B cells, the former effect being more profound.…”

“…As tolerance can readily be induced within the primary B-cell repertoire (9-12), we have addressed the question (13) of whether mechanisms exist to prevent fortuitous mutations toward anti-self-reactivity within the secondary B-lymphocyte repertoire-i.e., the memory B-cell compartment. Previously, we used a soluble deaggregated protein antigen, human serum albumin (HSA), as a model toleragen (14,15) and showed that it could prevent the generation of anti-HSA memory B cells in a dose-dependent manner. Adoptive transfer studies showed that the toleragen affected donor T and B cells, the former effect being more profound.…”

Oct-2, although not required for early B-cell development, is critical for later B-cell maturation and for postnatal survival.

“…3T3 feeder cells provide additional support, potentially via IL-6 and steel factor (see Arai et al 1990;Rolink et al 1991b]. At the end of this period, 25 ~1 of each culture supematant was assayed for the presence of IgM or IgG1 by standard ELISA (Karvelas and Nossal 1992 I. Clones with an OD.…”