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CD4(+)CD25(+) regulatory T (T(reg)) cells have a crucial role in maintaining immune tolerance. Mice and humans born lacking T(reg) cells develop severe autoimmune disease, and depletion of T(reg) cells in lymphopenic mice induces autoimmunity. Interleukin (IL)-2 signaling is required for thymic development, peripheral expansion and suppressive activity of T(reg) cells. Animals lacking IL-2 die of autoimmunity, which is prevented by administration of IL-2-responsive T(reg) cells. In light of the emerging evidence that one of the primary physiologic roles of IL-2 is to generate and maintain T(reg) cells, the question arises as to the effects of IL-2 therapy on them. We monitored T(reg) cells during immune reconstitution in individuals with cancer who did or did not receive IL-2 therapy. CD4(+)CD25(hi) cells underwent homeostatic peripheral expansion during immune reconstitution, and in lymphopenic individuals receiving IL-2, the T(reg) cell compartment was markedly increased. Mouse studies showed that IL-2 therapy induced expansion of existent T(reg) cells in normal hosts, and IL-2-induced T(reg) cell expansion was further augmented by lymphopenia. On a per-cell basis, T(reg) cells generated by IL-2 therapy expressed similar levels of FOXP3 and had similar potency for suppression compared to T(reg) cells present in normal hosts. These studies suggest that IL-2 and lymphopenia are primary modulators of CD4(+)CD25(+) T(reg) cell homeostasis.

Identification of differentially expressed genes between normal and diseased states is an area of intense current medical research that can lead to the discovery of new therapeutic targets. However, isolation of differentially expressed genes by subtraction often suffers from unreported contamination of the resulting subtraction library with clones containing DNA sequences not from the original RNA samples. Subtraction using cDNA representational difference analysis (RDA) was performed on human B cells from normal or common variable immunodeficiency patients. The material remaining after the subtraction was cloned and individual clones were sequenced. The sequence of one clone with similarity to integrases (ILG1, integrase-like gene-1) was used to obtain the full length cDNA sequence and as a probe for the presence of this sequence in RNA or genomic DNA samples. After five rounds of cDNA RDA, 23.3% of the clones from the resulting subtraction library contained Escherichia coli DNA. In addition, three clones contained the sequence of a new integrase, ILG1. The full length cDNA sequence of ILG1 exhibits prokaryotic, but not eukaryotic, features. At the DNA level, ILG1 is not similar to any known gene. At the protein level, ILG1 has 58% similarity to integrases from the cryptic P4 bacteriophage family (S clade). The catalytic domain of ILG1 contains the conserved features found in site-specific recombinases. The critical residues that form the catalytic active site pocket are conserved, including the highly conserved R-H-R-Y hallmark of these recombinases. Interestingly, ILG1 was not present in the original B cell populations. By probing genomic DNA, ILG1 could only be detected in the E. coli TOP10F′ strain used in our laboratory for molecular cloning, but not in any of its precursor strains, including TOP10. Furthermore, bacteria cultured from the mouth of the laboratory worker who performed cDNA RDA were also positive for ILG1. In the course of our studies using cDNA RDA, we have isolated and identified ILG1, a likely active site-specific recombinase and new member of the bacteriophage P4 family of integrases. This family of integrases is implicated in the horizontal DNA transfer of pathogenic genes between bacterial species, such as those found in pathogenic strains of E. coli, Shigella, Yersinia, and Vibrio cholera. Using ILG1 as a marker of our laboratory E. coli strain TOP10F′, our evidence suggests that contaminating bacterial DNA in our subtraction experiment is due to this laboratory bacterial strain, which colonized exposed surfaces of the laboratory worker. Thus, identification of differentially expressed genes between normal and diseased states could be dramatically improved by using extra precaution to prevent bacterial contamination of samples.