title: Limiting dilution analysis reveals the precursors of interleukin-4-producing CD4+ cells induced by protein immunization.
abstract: Although cytokine-producing T cells play a key role in the response to vaccination, they are not always revealed by antigen stimulation of primed lymphoid cells in vitro. In this study, mice were immunized subcutaneously with alum-precipitated keyhole limpet haemocyanin (KLH) in adjuvant to activate interleukin-4 (IL-4)-producing CD4+ T cells. IL-4 mRNA was the dominant cytokine mRNA species found in draining lymph nodes (LN) 7 days after immunization and its levels were increased after in vitro stimulation with KLH for 24 hr. IL-4 protein, on the other hand, was not detected in the supernatants of such antigen-stimulated cultures. The presence of T cells primed for IL-4 production was nevertheless suggested by the findings that primed LN cells produced low IL-4 titres in response to anti-CD3 antibody, whereas normal LN cells did not, and primed CD4+ LN cells produced readily detectable IL-4 levels in response to antigen after one or more cycles of in vitro restimulation. Culture at limiting dilution showed that 1-2% of 7 day KLH-primed CD4+ LN cells were clonogenic and specific for KLH without prior expansion in vitro, and that this frequency was markedly increased by repeated stimulation in bulk culture. Most clonogenic cells in primed LN gave rise to IL-4-secreting clones and a smaller number gave rise to interferon-gamma (IFN-gamma)-producing clones. The precursor frequency of IL-4-producing CD4+ cells in primed LN and the average IL-4 titre per cloned cell support the conclusion that these two parameters account for the low levels of IL-4 produced in bulk culture by LN cells from immunized mice.
authors: Walter and Eliza Hall Institute of Medical Research, Parkville, Brisbane, Australia.
authors: P L
authors: PL
authors: Groves
authors: M H
authors: MH
authors: Pech
authors: A B
authors: AB
authors: Troutt
authors: A
authors: A
authors: Kelso
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D002478:Cells, Cultured; D002999:Clone Cells / Q000276:immunology; D005260:Female; D015870:Gene Expression; D006433:Hemocyanins / Q000276:immunology*; D007114:Immunization; D015847:Interleukin-4 / Q000096:biosynthesis* / Q000235:genetics; D008198:Lymph Nodes / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D012333:RNA, Messenger / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D015847:Interleukin-4; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin