title: Limiting dilution analysis of CD4 T-cell cytokine production in mice administered native versus polymerized ovalbumin: directed induction of T-helper type-1-like activation.
abstract: Polarized expression of T-helper type-1 (Th1)- or Th2-like patterns of cytokine production frequently correlates with disease outcome. Previously, we have described the long-lived reciprocal regulation of ovalbumin (OVA)-specific IgE (> 95% inhibition) and IgG2a (300-800-fold increased) production following administration of high MW OVA polymers (OVA-POL), in both de novo and ongoing OVA (alum)-induced responses. Here, limiting dilution analysis (LDA) was used to compare precursor frequencies of CD4 T cells producing interferon-gamma (IFN-gamma), interleukin-4 (IL-4) or IL-10 following OVA versus OVA-POL exposure in vivo. Adjuvants were not used, so as to circumvent their impact on measurement of precursor frequencies. We found that the two forms of antigen elicited T-cell activation of comparable intensity, as indicated by equivalent precursor frequencies of clonogenic antigen-specific CD4 T cells. However, they elicited qualitatively different cytokine responses. OVA-POL treatment led to 10-fold higher (mean of six independent LDA experiments) frequencies of IFN-gamma-producing cells, and a mean fivefold lower frequency of IL-10-producing cells, than was observed following in vivo administration of unmodified OVA. Thus, the high MW polymerized form of antigen acted to steer commitment of naive (for this antigen) CD4 T-cell activation from a situation in which IL-10 producers outnumbered IFN-gamma-producing cells by a factor of 4:1 (found in mice administered OVA), to one where IFN-gamma producers dominated by a factor of 11:1 (in mice given OVA-POL), i.e. a qualitative shift in the nature of the OVA-specific response induced from Th2-like to Th1-like. In vivo co-administration of anti-IFN-gamma monoclonal antibody (mAb) abolished the capacity of OVA-POL to preferentially elicit Th1-like dominance. Interestingly, although the ratios of IFN-gamma:IL-4 and IFN-gamma:IL-10 OVA-specific precursor frequencies were strongly increased following OVA-POL exposure (mean 18- and 47-fold higher), the frequency of IL-4-producing CD4 T cells did not differ significantly. The data suggest that this modified antigen promotes in vivo commitment of naive T cells towards a Th1-like response, with consequent inhibition of IgE and enhancement of IgG2a responses, not through direct effects on IL-4 production, but via decreased frequencies of IL-10 and increased frequencies of IFN-gamma-producing OVA-specific CD4 cells. Collectively, the data (1) demonstrate the ability to manipulate commitment of antigen-driven CD4 T-cell populations in naive mice to specific patterns of cytokine gene expression, and (2) provide in vivo evidence of the regulatory role played by IFN-gamma in limiting induction and/or expansion of IL-4- and IL-10-producing CD4 cells to protein allergens.
authors.affiliation: Department of Immunology, University of Manitoba, Winnipeg, Canada.
authors.forename: R S
authors.initials: RS
authors.lastname: Gieni
authors.forename: X
authors.initials: X
authors.lastname: Yang
authors.forename: A
authors.initials: A
authors.lastname: Kelso
authors.forename: K T
authors.initials: KT
authors.lastname: Hayglass
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D002455:Cell Division / Q000276:immunology; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D007371:Interferon-gamma / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology*; D046911:Macromolecular Substances; D051379:Mice; D008810:Mice, Inbred C57BL; D010047:Ovalbumin / Q000737:chemistry / Q000276:immunology*; D011489:Protein Denaturation; D051381:Rats; D017207:Rats, Sprague-Dawley; D018417:Th1 Cells / Q000276:immunology
chemical_list: D016207:Cytokines; D046911:Macromolecular Substances; D007371:Interferon-gamma; D010047:Ovalbumin