Andrew J. Carmichael

Identification of Naive or Antigen-Experienced Human CD8 + T Cells by Expression of Costimulation and Chemokine Receptors: Analysis of the Human Cytomegalovirus-Specific CD8 + T Cell Response

Human CMV (HCMV) infection provides an informative model of how long term human CD8+ T cell memory is maintained in the presence of Ag. To clarify the phenotypic identity of Ag-experienced human CD8+ T cells in vivo, we determined the expression of costimulation and chemokine receptors on Ag-specific CD8+ T cells by quantifying individual virus-specific clones in different cell populations using TCR clonotypic probing. In healthy HCMV carriers, expanded CD8+ clones specific for either HCMV tegument protein pp65 or immediate-early protein IE72 are found in both CD45ROhigh cells and the subpopulation of CD45RAhigh cells that lack the costimulatory molecule CD28. In contrast to previous suggested models of CD8+ T cell memory, we found that in healthy virus carriers highly purified CD28−CD45RAhighCCR7− cells are not terminally differentiated, because following stimulation in vitro with specific HCMV peptide these cells underwent sustained clonal proliferation, up-regulated CD45RO and CCR5, and showed strong peptide-specific cytotoxic activity. In an individual with acute primary HCMV infection, HCMV pp65-specific CD8+ T cells are predominantly CD28−CD45ROhighCCR7−. During convalescence, an increasing proportion of pp65-specific CD8+ T cells were CD28−CD45RAhighCCR7−. We conclude that naive human CD8+ T cells are CD28+CD45RAhigh, express CCR7 but not CCR6, and are predominantly CD27+ and L-selectin CD62 ligand-positive. The phenotype CD27+CD45RAhigh should not be used to identify naive human CD8+ T cells, because CD27+CD45RAhigh cells also contain a significant subpopulation of CD28−CD27+ Ag-experienced expanded clones. Thus CD8+ T cell memory to HCMV is maintained by cells of expanded HCMV-specific clones that show heterogeneity of activation state and costimulation molecular expression within both CD45ROhigh and CD28−CD45RAhigh T cell pools.

Large clonal expansions of human virus‐specific memory cytotoxic T lymphocytes within the CD57+ CD28– CD8+ T‐cell population

The proportion of human peripheral blood CD8+ T cells that are CD57+ CD28– is low at birth but increases with age and in individuals infected with human cytomegalovirus (HCMV) or human immunodeficiency virus (HIV). These CD57+ CD28– CD8+ T cells contain large oligoclonal T‐cell expansions whose antigen specificity is unknown. We identified clonal expansions of virus‐specific memory cytotoxic T‐lymphocyte precursors (CTLp) in both healthy carriers of HCMV and in asymptomatic HIV‐infected subjects. In each subject, from the T‐cell receptor (TCR) β‐chain hypervariable sequence of each immunodominant CTL clone, we designed complementary oligonucleotide probes to quantify the size and phenotypic segregation of individual virus‐specific CTL clones in highly purified populations of peripheral blood CD8+ T cells. We found large clonal expansions of virus‐specific CTL clonotypes in CD57+ CD28– CD8+ T cells. Using limiting dilution analysis, we found functional peptide‐specific CTLp at high frequency in CD57+ CD28– cells. Thus, memory CTL specific for persistent viruses account for many oligoclonal expansions within CD57+ CD28– CD8+ T cells.

Human cytomegalovirus and immunopathology

Human cytomegalovirus (HCMV) is a ubiquitous human persistent virus, which causes disease particularly in the setting of immunosuppression. This review deals with aspects of the pathology and pathogenesis of HCMV, which may be immunologically mediated. It deals both with the effects of HCMV on the immune response, given much recent evidence that CMV possesses specific gene functions that interfere with the immune response, and with pathology possibly produced by the immune response against CMV. The CMVs are species specific; thus, although other mammalian species have their own CMV, HCMV cannot be directly studied in animal models. However, there are informative animal models of CMV, particularly in the mouse but also in the rat and the guinea pig. This review deals specifically with HCMV, although in selected areas it refers to evidence from studies on murine CMV (MCMV) that bear on HCMV disease. It begins with a review of the biology and immunology of HCMV in the normal host as a pre-requisite to understanding any putative immunopathogenic mechanisms associated with the virus.

Long-Term Stable Expanded Human CD4+ T Cell Clones Specific for Human Cytomegalovirus Are Distributed in Both CD45RAhigh and CD45ROhigh Populations

T cells play an important role in the control of human CMV (HCMV) infection. Peripheral blood CD4+ T cell proliferative responses to the HCMV lower tegument protein pp65 have been detected in most healthy HCMV carriers. To analyze the clonal composition of the CD4+ T cell response against HCMV pp65, we characterized three MHC class II-restricted peptide epitopes within pp65 in virus carriers. In limiting dilution analysis, we observed high frequencies of pp65 peptide-specific CD4+ T cells, many of which expressed peptide-specific cytotoxicity in addition to IFN-γ secretion. We analyzed the clonal composition of CD4+ T cells specific for defined HCMV peptides by generating multiple independent peptide-specific CD4+ clones and sequencing the TCR β-chain. In a given carrier, most of the CD4+ clones specific for a defined pp65 peptide had identical TCR nucleotide sequences. We used clonotype oligonucleotide probing to quantify the size of individual peptide-specific CD4+ clones in whole PBMC and in purified subpopulations of CD45RAhighCD45ROlow and CD45RAlowCD45ROhigh cells. Individual CD4+ T cell clones could be large (0.3–1.5% of all CD4+ T cells in PBMC) and were stable over time. Cells of a single clone were distributed in both the CD45RAhigh and CD45ROhigh subpopulations. In one carrier, the virus-specific clone was especially abundant in the small CD28−CD45RAhigh CD4+ T cell subpopulation. Our study demonstrates marked clonal expansion and phenotypic heterogeneity within daughter cells of a single virus-specific CD4+ T cell clone, which resembles that seen in the CD8+ T cell response against HCMV pp65.

Effects of Chlamydia trachomatis infection on the expression of natural killer (NK) cell ligands and susceptibility to NK cell lysis

Natural killer (NK) cells are an important component of the immediate immune response to infections, including infection by intracellular bacteria. We have investigated recognition of Chlamydia trachomatis (CT) by NK cells and show that these cells are activated to produce interferon (IFN)‐γ when peripheral blood mononuclear cells (PBMC) are stimulated with CT organisms. Furthermore, infection of epithelial cell lines with CT renders them susceptible to lysis by human NK cells. Susceptibility was observed 18–24 h following infection and required protein synthesis by the infecting chlamydiae, but not by the host cell; heat or UV inactivated chlamydiae did not induce susceptibility to NK cell lysis. CT infection was also shown to decrease the expression of classical and non‐classical major histocompatibility complex (MHC) molecules on infected cells, thus allowing recognition by NK cells when combined with an activating signal. A candidate activating signal is MICA/B, which was shown to be expressed constitutively on epithelial cells.

Human cytomegalovirus-specific immunity following haemopoietic stem cell transplantation

The herpesvirus Human Cytomegalovirus (HCMV) is an important opportunistic infection in recipients of allogeneic haemopoietic stem cell transplants, in whom HCMV-specific CD8+ and CD4+ T-cell responses are impaired. The nature of the HCMV-specific T-cell response in healthy virus carriers has been characterised in detail. High frequencies of circulating CD8+ T-cells that recognise defined viral peptides are maintained for years, and include individual CD8+ clones that have undergone extensive clonal expansion and phenotypic diversification in vivo. Following stem cell transplantation, the kinetics of HCMV-specific CD8+ T-cell reconstitution in the recipient are related to the presence or absence of antigen-experienced CD8+ T-cells transferred via the allograft, and to the presence of the virus in the recipient. We discuss recent progress in our understanding of HCMV-specific immunity in healthy virus carriers and in recipients after alloSCT.

Progressive loss of IL-2-expandable HIV-1-specific cytotoxic T lymphocytes during asymptomatic HIV infection.

In HIV‐1 infection, circulating HIV‐1‐specific cytotoxic T lymphocytes (CTL) exist in different states of activation, including activated cytotoxic cells and memory cells. We report that a subpopulation of HIV‐1‐specific CTL is capable of clonal expansion upon culture with IL‐2 without exogenous antigen. The IL‐2‐expandable HIV‐1‐specific CTL precursor frequency was reduced in patients with advancing infection, although HIV‐1‐specific memory CTL could still be detected by stimulation in vitro with allele‐specific HIV‐1 peptide. Longitudinal analysis during advancing infection showed a progressive decline in the IL‐2‐expandable HIV‐1‐specific CTL precursor (CTLp) frequency without a decline in Epstein‐Barr virus (EBV)‐specific or allo‐specific CTLp frequencies. To address mechanisms that may contribute to the decline in the IL‐2‐expandable HIV‐specific CTL response, the requirements for in vitro generation of HIV‐1‐specific and EBV‐specific effector CTL were examined. In the absence of exogenous IL‐2 in limiting dilution, generation of EBV‐specific CD8+ effector CTL was dependent upon help from CD4+ cells. CD4+ help for EBV‐specific CD8+ CTL was observed in asymptomatic HIV infection but not in advanced infection. In the presence of exogenous IL‐2, CD4+ cells could also provide help for the optimal generation of HIV‐1 peptide‐specific CD8+ CTL, because in vitro depletion of CD4+ cells prior to culture using stimulation with an MHC class I‐restricted HIV‐1 peptide reduced the peptide‐specific CD8+ CTL response. We conclude that there is a decline in the IL‐2‐expandable HIV‐1‐specific CTL response during advancing infection. There are a number of possible mechanisms for this decline, including a reduction in CD4+ T cell help for in vivo antigen‐activated CD8+ T cells.

Large HIV-specific CD8+ cytotoxic T-lymphocyte (CTL) clones reduce their overall size but maintain high frequencies of memory CTL following highly active antiretroviral therapy

Cytotoxic T‐lymphocytes (CTL) play an important role in the control of human immunodeficiency virus (HIV) and of human cytomegalovirus (HCMV) infection. Following highly active antiretroviral therapy (HAART), most studies have demonstrated a decline in the frequency of HIV‐specific CTL. We analysed the effect of HAART on the size, phenotype and function of individual HIV‐ and HCMV‐specific CTL clones, using clonotypic oligonucleotide probing specific for the T‐cell receptor (TCR) β‐chain hypervariable sequence of defined immunodominant CTL clones specific for peptides of HIV or HCMV, and quantified the limiting dilution analysis frequencies of CTL precursors (CTLp) specific for the same viral peptides. We found that the clonal composition of CD8+ T cells specific for HIV gag and env epitopes was highly focused and did not change after HAART. Following HAART, there was progressive contraction of HIV‐specific CD8+ clones, especially in the CD28– CD27– subpopulation – the remaining cells of contracting HIV‐specific clones were predominantly CD28– CD27+ CD45ROhi. We observed maintenance of strong functional HIV‐specific CD8+ T‐cell responses in limiting dilution analysis following HAART, indicating preferential loss of HIV‐specific cells that have reduced cloning efficiency in vitro. Following HAART, we also observed selective expansion of HCMV‐specific CD8+ clones. Most HCMV‐specific CD8+ clones were predominantly CD28– CD27+/– CD45RAhi following HAART. In one subject, a Vβ6.4+ clone specific for HCMV pp65 selectively expanded following HAART, without expansion of two other Vβ6.4+ clones, indicating that individual clonotypes specific for the same peptide can show different kinetics and phenotypes in response to antiretroviral therapy.