Abnormalities in CD4+CD25+Foxp3+ regulatory T (T reg) cells have been implicated in susceptibility to allergic, autoimmune, and immunoinflammatory conditions. However, phenotypic and functional assessment of human T reg cells has been hampered by difficulty in distinguishing between CD25-expressing activated and regulatory T cells. Here, we show that expression of CD127, the α chain of the interleukin-7 receptor, allows an unambiguous flow cytometry–based distinction to be made between CD127lo T reg cells and CD127hi conventional T cells within the CD25+CD45RO+RA− effector/memory and CD45RA+RO− naive compartments in peripheral blood and lymph node. In healthy volunteers, peripheral blood CD25+CD127lo cells comprised 6.35 ± 0.26% of CD4+ T cells, of which 2.05 ± 0.14% expressed the naive subset marker CD45RA. Expression of FoxP3 protein and the CD127lo phenotype were highly correlated within the CD4+CD25+ population. Moreover, both effector/memory and naive CD25+CD127lo cells manifested suppressive activity in vitro, whereas CD25+CD127hi cells did not. Cell surface expression of CD127 therefore allows accurate estimation of T reg cell numbers and isolation of pure populations for in vitro studies and should contribute to our understanding of regulatory abnormalities in immunopathic diseases.

The cytotoxic potential of CD8+ T cells and NK cells plays a crucial role in the immune response to pathogens. Although in vitro studies have reported that CD4+ T cells are also able to mediate perforin-mediated killing, the in vivo existence and relevance of cytotoxic CD4+ T cells have been the subject of debate. Here we show that a population of CD4+ perforin+ T cells is present in the circulation at low numbers in healthy donors and is markedly expanded in donors with chronic viral infections, in particular HIV infection, at all stages of the disease, including early primary infection. Ex vivo analysis shows that these cells have cytotoxic potential mediated through the release of perforin. In comparison with more classical CD4+ T cells, this subset displays a distinct surface phenotype and functional profile most consistent with end-stage differentiated T cells and include Ag experienced CD4+ T cells. The existence of CD4+ cytotoxic T cells in vivo at relatively high levels in chronic viral infection suggests a role in the immune response.

Effects of a human immunodeficiency virus (HIV) type 1 protease inhibitor, ritonavir, were evaluated in 21 patients enrolled in a phase I/II study. The magnitude and rates of CD4 and CD8 lymphocyte increase, changes in subsets of CD4 and CD8 lymphocytes, and proliferative responses to mitogen and antigens were analyzed. Significant increases were noted in CD4 and CD8 lymphocyte counts; numbers of CD4CD45RO lymphocytes increased significantly by week 1 of therapy. Increases in the CD4CD45RA subset were observed at week 4. Reductions in the percentage of CD4 and CD8 lymphocytes expressing CD38 were noted. Increases in proliferative responses to phytohemagglutinin were noted in 6 of 7 patients and correlated with duration of virus load suppression. Increased responses to recall antigens and to HIV-specific proteins were observed. Treatment with ritonavir produced alterations in the immune system that included changes in T cell subset distribution and increases in CD4 and CD8 lymphocyte numbers and of lymphocyte function.

CD4 T cells with cytotoxic function were once thought to be an artefact due to long term in vitro cultures but have in more recent years become accepted and reported in the literature in response to a number of viral infections. In this review we focus on cytotoxic CD4 T cells in the context of human viral infections and in some infections that affect mice and non-human primates. We examine the effector mechanisms used by cytotoxic CD4 cells, the phenotypes that describe this population, and the transcription factors and pathways that lead to their induction following infection. We further consider the cells that are the predominant targets of this effector subset, and describe the viral infections in which CD4 cytotoxic T lymphocytes have been shown to play a protective or pathologic role. Cytotoxic CD4 T cells are detected in the circulation at much higher levels than previously realised and are now recognised to have an important role in the immune response to viral infections.

Ag-specific human CD4⁺ memory T lymphocytes have mostly been studied using assays of proliferation in vitro. Intracellular cytokine and ELISPOT assays quantify effector cell populations but barely detect responses to certain recall Ags that elicit strong proliferative responses, e.g., tetanus toxoid, that comprise non-Th1 CD4⁺ cells. We have found that culturing whole blood with Ag for 40–48 h induces specific CD4⁺ T cells to simultaneously express CD25 and CD134. This new technique readily detects responses to well-described CD4⁺ T cell recall Ags, including preparations of mycobacteria, CMV, HSV-1, influenza, tetanus toxoid, Candida albicans, and streptokinase, as well as HIV-1 peptides, with high specificity. The assay detects much higher levels of Ag-specific cells than intracellular cytokine assays, plus the cells retain viability and can be sorted for in vitro expansion. Furthermore, current in vitro assays for human CD4⁺ memory T lymphocytes are too labor-intensive and difficult to standardize for routine diagnostic laboratories, whereas the whole-blood CD25⁺CD134⁺ assay combines simplicity of setup with a straightforward cell surface flow cytometry readout. In addition to revealing the true extent of Ag-specific human CD4⁺ memory T lymphocytes, its greatest use will be as a simple in vitro monitor of CD4⁺ T cell responses to Ags such as tuberculosis infection or vaccines.