Abstract

Autoreactive T and B cells can be detected in healthy individuals but are normally kept in check by regulatory mechanisms. Among those is an active suppression of naïve T cells by endogenous T regulatory (Tr) cells. Several types of Tr cells exist, including CD4+ T cells which constitutively express the IL-2 receptor α chain (CD25), do not secrete IL-10, and suppress immune responses via direct cell-to-cell interactions. CD4+CD25+ T regulatory cells represent 5%–10% of the endogenous CD4+ T cells subset and are able to suppress CD4+ and CD8+ T cell responses in vitro and in vivo upon TCR ligation. Our recent observation that human platelet factor 4 (PF4; CXCL4) inhibits the proliferative response of human CD4+CD25 T cells, while inducing expansion of CD4+CD25+ Tr cells, and that PF4-induced CD4+CD25+ Tr cells lose their potent suppressor function in vitro, suggests a previously unrecognized role of PF4 in the regulation of immune responsiveness (

Liu, et al.
J Immunol
174
:
2680
–86,
2005
). A large body of evidence suggests that human CD4+CD25+ Tr cells share many of the characteristics of murine CD4+CD25+ Tr cells. McHugh et al. (
Immunity
16
:
311
–23,
2002
), have successfully used the microarray approach to identify genes differentially expressed in resting CD4+CD25+ and CD4+CD25 mouse T cells, but with the only exception of a small preliminary report (
Pati et al.
Ann N Y Acad Sci
.
1005
:
279
–83,
2003
), little information is available on the gene expression profile of human CD4+CD25+ and CD4+CD25 T cells. We performed global gene expression analysis using oligo-DNA microarrays (CodeLink, Amersham Biosciences) that monitor the expression of whole human genome, to define the gene expression profiles in CD4+CD25+ Tr cells stimulated by anti-CD3 mAb and exposed to PF4. CD4+ T cells were isolated from normal donor’s peripheral blood mononuclear cells by positive selection on magnetic beads (Miltenyi Biotec, Auburn, CA), then labeled with PE-conjugated anti-CD4 and FITC-conjugated anti-CD25 and sorted on a FACStar (BD Biosciences, San Jose, CA) to obtain a homogeneous population of T cells consisting of CD4+CD25+ Tr cells expressing CD25 at high levels (CD4+CD25high) and CD4+CD25 T cells (non-regulatory). Total RNA was extracted from the freshly isolated CD4+CD25high and CD4+CD25 T cells subsets, stimulated with anti-CD3 mAb in the presence or the absence of PF4 for 24 hours. Using this approach, we have identified a little over 100 genes that are differentially expressed, in the presence of PF4, in CD4+CD25+ Tr cells following activation with anti-CD3 mAb. We have focused our attention on about 40 target genes whose increased expression has been validated using real time PCR and, were appropriate, at the protein levels, by flow cytometry or Luminex 100 multiplex cytokine quantification (Table 1). Our data suggest that PF4 modulates proliferation and function of CD4+CD25+ Tr cells by the coordinate increasing expression of a relatively large number of genes, coupled with a further enhanced expression of a limited number of growth promoting genes and the specific silencing of a small subset of negative growth regulatory genes.

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