Regulatory T (T reg) cells are critical regulators of immune tolerance. Most T reg cells are defined based on expression of CD4, CD25, and the transcription factor, FoxP3. However, these markers have proven problematic for uniquely defining this specialized T cell subset in humans. We found that the IL-7 receptor (CD127) is down-regulated on a subset of CD4+ T cells in peripheral blood. We demonstrate that the majority of these cells are FoxP3+, including those that express low levels or no CD25. A combination of CD4, CD25, and CD127 resulted in a highly purified population of T reg cells accounting for significantly more cells that previously identified based on other cell surface markers. These cells were highly suppressive in functional suppressor assays. In fact, cells separated based solely on CD4 and CD127 expression were anergic and, although representing at least three times the number of cells (including both CD25+CD4+ and CD25−CD4+ T cell subsets), were as suppressive as the “classic” CD4+CD25hi T reg cell subset. Finally, we show that CD127 can be used to quantitate T reg cell subsets in individuals with type 1 diabetes supporting the use of CD127 as a biomarker for human T reg cells.

Functional subsets of human T cells were delineated by analyzing patterns of lymphokines produced by clones from individuals with leprosy and by T cell clones of known function. CD4 clones from individuals with strong cell-mediated immunity produced predominantly interferon-gamma, whereas those clones that enhanced antibody formation produced interleukin-4. CD8 cytotoxic T cells secreted interferon-gamma. Interleukin-4 was produced by CD8 T suppressor clones from immunologically unresponsive individuals with leprosy and was found to be necessary for suppression in vitro. Both the classic reciprocal relation between antibody formation and cell-mediated immunity and resistance or susceptibility to certain infections may be explained by T cell subsets differing in patterns of lymphokine production.

Adhesion measurements between CD8 and 48 point mutants of HLA-A2.1 show that the CD8 alpha-chain binds to the alpha 3 domain of HLA-A2.1. Three clusters of alpha 3 residues contribute to the binding, with an exposed, negatively charged loop (residues 223-229) playing a dominant role. CD8 binding correlates with cytotoxic T-cell recognition and sensitivity to inhibition by anti-CD8 antibodies. Impaired alloreactive T-cell recognition of an HLA-A2.1 mutant with reduced affinity for CD8 is not restored by functional CD8 binding sites on an antigenically irrelevant class I molecule. Therefore, complexes of CD8 and the T-cell receptor bound to the same class I major histocompatibility complex molecule seem to be necessary for T-cell activation.

Granulysin, a protein located in the acidic granules of human NK cells and cytotoxic T cells, has antimicrobial activity against a broad spectrum of microbial pathogens. A predicted model generated from the nuclear magnetic resonance structure of a related protein, NK lysin, suggested that granulysin contains a four α helical bundle motif, with the α helices enriched for positively charged amino acids, including arginine and lysine residues. Denaturation of the polypeptide reduced the α helical content from 49 to 18% resulted in complete inhibition of antimicrobial activity. Chemical modification of the arginine, but not the lysine, residues also blocked the antimicrobial activity and interfered with the ability of granulysin to adhere to Escherichia coli and Mycobacterium tuberculosis. Granulysin increased the permeability of bacterial membranes, as judged by its ability to allow access of cytosolic β-galactosidase to its impermeant substrate. By electron microscopy, granulysin triggered fluid accumulation in the periplasm of M. tuberculosis, consistent with osmotic perturbation. These data suggest that the ability of granulysin to kill microbial pathogens is dependent on direct interaction with the microbial cell wall and/or membrane, leading to increased permeability and lysis.

Summary When killer lymphocytes recognize infected cells, perforin delivers cytotoxic proteases (granzymes) into the target cell to trigger apoptosis. What happens to intracellular bacteria during this process is unclear. Human, but not rodent, cytotoxic granules also contain granulysin, an antimicrobial peptide. Here we show that granulysin delivers granzymes into bacteria to kill diverse bacterial strains. In E. coli, granzymes cleave electron transport chain complex I and oxidative stress defense proteins, generating ROS that rapidly kill bacteria. ROS scavengers and bacterial antioxidant protein overexpression inhibit bacterial death. Bacteria overexpressing a GzmB-uncleavable mutant of the complex I subunit nuoF or strains that lack complex I still die, but more slowly, suggesting that granzymes disrupt multiple vital bacterial pathways. Mice expressing transgenic granulysin are better able to clear L. monocytogenes. Thus killer cells play an unexpected role in bacterial defense.