The Involvement of the Intestinal Microflora in the Expansion of CD4(+) T Cells with a-Naive Phenotype in the Periphery

It is well known that immune reactivity declines with age. Recently, we demonstrated that the age-related decrease in IL-2 production by CD4(+) T cells was accompanied by an increased production of IL-4 and interferon-γ,(IFN-γ). This age-related shift in the profile of lymphokine production was related to phenotypic changes within the CD4(+) T-cell subset, that is, a decrease in the percentage of CD45RB(++) CD4(+) T cells and an increase in the percentage of Pgp-1(+) CD4(+) T cells. To study whether these age-related changes were due to previous antigenic exposure, we performed a phenotypic and functional analysis on splenic CD4(+) T cells isolated from individual, germ-free (GF), specific pathogen-free (SPF), and clean conventional (CC) mice. Interestingly, the total number of splenic CD4(+) T cells in GF mice was twofold lower as compared to age-matched SPF or CC mice, regardless whether mice were analyzed at young (10 weeks) or at advanced age (13-14 months). Unexpectedly, the phenotypic composition of the CD4(+) T-cell subset was comparable in the GF, SPF, and CC mice as determined by the expression of CD45RB and Pgp-1, indicating that CD4(+) T cells with a naive phenotype (CD45RB(++) Pgp-1 (–)) were not enriched in GF mice. Moreover, at an age of 13–14 months, CD4(+) T cells from GF mice frequently produced more IL-4 and IFN-γ, than their CC counterparts. These lymphokine data showed, therefore, that a relatively high proportion of CD4 T cells with a memory phenotype can also be defined in GF mice on the basis of their function. The contamination of GF mice with a colonization resistant factor (CRF flora) resulted in twofold higher numbers of splenic CD4(+) T cells. Surprisingly, not only CD4(+) T cells with a memory phenotype (CD45RB(–/+) Pgp-1(++)) had expanded, but also CD4(+) T cells with a naive (CD45RB(++) Pgp-1(–)) phenotype. Our results, therefore, strongly suggest that the expansion of naive CD4(+) T cells in the periphery is mediated by the intestinal microflora.