Loss of CD127 expression defines an expansion of effector CD8+ T cells in HIV-infected individuals

The immunodeficiency that follows HIV infection is related to the virus-mediated killing of infected CD4(+) T cells, the chronic activation of the immune system, and the impairment of T cell production. In this study we show that in HIV-infected individuals the loss of IL-7R (CD127) expression defines the expansion of a subset of CD8(+) T cells, specific for HIV as well as other Ags, that show phenotypic (i.e., loss of CCR7 and CD62 ligand expression with enrichment in activated and/or proliferating cells) as well as functional (i.e., production of IFN-gamma, but not IL-2, decreased ex vivo proliferative potential and increased susceptibility to apoptosis) features of effector T cells. Importantly, in HIV-infected individuals the levels of CD8(+)CD127(-) T cells are directly correlated with the main markers of disease progression (i.e., plasma viremia and CD4(+) T cell depletion) as well as with the indices of overall T cell activation. In all, these results identify the expansion of CD8(+)CD127(-) effector-like T cells as a novel feature of the HIV-associated immune perturbation. Further studies are thus warranted to determine whether measurements of CD127 expression on CD8(+) T cells may be useful in the clinical management of HIV-infected individuals.     

HIV-infected humans, but not chimpanzees, have circulating cytotoxic T lymphocytes that lyse uninfected CD4+ cells

It has been suggested that autoimmune phenomena contribute to the depletion of CD4+ T cells and the development of AIDS in HIV-1 infected humans based, in part, on observations that some HIV-1-infected humans have autoantibodies reactive with Ag expressed on uninfected CD4+ cells. In this study, 11 of 14 asymptomatic HIV-1-infected homosexuals and hemophiliacs, but none of 17 uninfected homosexuals or heterosexuals, were found to have cytotoxic lymphocytes in blood that can lyse uninfected CD4+ T cells from humans and chimpanzees but not human B lymphoblastoid cells or mouse T cells. The cytotoxic PBL were concluded to be CTL rather than NK cells, with the phenotype being CD3+, TCR-1 alpha beta+, CD8+, CD4-, CD16- based on findings that PBL-mediated lysis of uninfected CD4+ cells was 1) blocked by a mAb to CD3, which inhibits CTL but not NK activity; 2) diminished by treatment of PBL with a mAb to CD8 and C, but not by treatment with mAb to CD4 or CD16 and C; and 3) blocked by mAb WT31 directed against the TCR-1 alpha beta. In contrast, PBL from HIV-1-infected chimpanzees, which to date have not developed AIDS, lacked detectable CTL lytic for uninfected CD4+ cells.     

Virus-specific CD4+ and CD8+ cytotoxic T-cell responses and long-term T-cell memory in individuals vaccinated against polio

The presence of poliovirus (PV)-specific CD4(+) T cells in individuals vaccinated against polio has been shown, but CD8(+) T-cell responses have not been described. Here, we functionally characterize the CD4(+) T-cell response and show for the first time that dendritic cells and macrophages can stimulate PV-specific CD8(+) T-cell responses in vitro from vaccinees. Both CD4(+) T and CD8(+) T cells secrete gamma interferon in response to PV antigens and are cytotoxic via the perforin/granzyme B-mediated pathway. Furthermore, the T cells also recognize and kill Sabin 1 vaccine-infected targets. The macrophage-stimulated CD4(+) T and CD8(+) T cells most likely represent memory T cells that persist for long periods in vaccinated individuals. Thus, immunity to PV vaccination involves not only an effective neutralizing antibody titer but also long-term CD4(+) and CD8(+) cytotoxic T-cell responses.     

Circulating cytotoxic CD8+ CD28- T cells in ankylosing spondylitis

Circulating CD8⁺ CD28^- T cells were found to be expanded more in patients with ankylosing spondylitis than in an age-matched healthy population (41.2 ± 17.7% versus 18.6 ± 7.6%). The level of CD8⁺CD28^- T cells was dependent on the disease status, but was independent of age. Most of the CD8⁺ CD28^- T cells produced perforin after stimulation in vitro, in contrast to their CD8⁺CD28⁺ counterparts. From the clinical perspective, the percentage of the cytotoxic CD8⁺ CD28^- T cells reflected a more severe course of disease, as it correlated with distinct movement restrictions, as well as the metrology score summarizing cervical rotation (in sitting position), chin-to-jugulum distance, thoracic Schober, chest expansion, and fingers-to-floor distance (P = 0.032).     

Antigen presentation by Toxoplasma gondii-infected cells to CD4+ proliferative T cells and CD8+ cytotoxic cells

Cytotoxic cells specific for Toxoplasma gondii-infected cells were detected in the peripheral blood leukocytes from a patient with acute toxoplasmosis. The cytotoxicity was mediated by CD5+, CD4-, CD8+ cells. The cytotoxic T cells lysed Toxoplasma-infected target cells with HLA class I restriction. Two types of T cell clones were established from peripheral blood leukocytes of a patient with chronic toxoplasmosis; one was a CD5+, CD4-, CD8+ cytotoxic cell specific for Toxoplasma-infected cells, and the other was a CD5+, CD4+, CD8- proliferative cell that responded to Toxoplasma antigen. Toxoplasma-infected cell-specific cytotoxic cloned T cells recognize the infected target cells in the context of the HLA class I molecules, and the CD8 molecule was involved in the cytotoxicity. Toxoplasma antigen-specific proliferative cloned T cells were stimulated by Toxoplasma antigen-pulsed or Toxoplasma-infected cells in conjunction with HLA-DR molecule on the target cells. Thus, antigen presentation by Toxoplasma-infected cells for activation of both cytotoxic and proliferative T cells has been demonstrated.     

Differential in vitro activation of CD4+CD8- and CD8+CD4- herpes simplex virus-specific human cytotoxic T cells

In order to clarify the differential activation of CD4+ and CD8+ HSV-specific CTL, we compared the characteristics of CTL generated by different methods of in vitro HSV stimulation by treatment of effectors with anti-CD4 and anti-CD8 mAb and C after the elimination of nonspecific cytotoxic effector cells. Cell-free HSV mainly activated CD4+ CTL precursors, whereas HSV-infected fibroblasts were more effective in activating CD8+ CTL precursors than CD4+ CTL precursors. In addition, limiting dilution analyses with enriched T cells from two HSV-seropositive donors revealed that the frequency of HSV-specific CD4+ CTL precursors responsive to stimulation with free HSV was approximately 1/4,000 to 6,000 CD4+ T cells, whereas that of precursors responsive to stimulation with HSV-infected fibroblasts was approximately 1/19,000 to 22,000 CD4+ T cells. Conversely, the frequency of CD8+ CTL precursors in peripheral blood responsive to stimulation with free HSV was approximately 1/28,000 to 30,000 CD8+ T cells, whereas that of precursors responsive to stimulation with HSV-infected fibroblasts was approximately 1/10,000 to 11,000 CD8+ T cells. The present data suggest that generalized viral infection due to cell-free viruses is fought mainly by CD4+ CTL, which have previously been reported to possess both cytotoxicity and helper function, and that localized viral infection on HLA class II-negative fibroblasts is prevented from spreading to adjacent cells mainly by CD8+ CTL. Such differential activation of CD4+ and CD8+ CTL seems probable when considering the protective mechanisms against viral infection.     

Murine CD8+ cytotoxic T lymphocytes lyse Toxoplasma gondii-infected cells

Studies were performed to determine whether CTL against Toxoplasma gondii-infected cells could be induced in a murine model of T. gondii infection in which CD8+ T lymphocytes have been shown to play a major role in resistance against this parasite. In 51Cr release assays, nylon wool nonadherent spleen cells from BALB/c (H-2d) mice immunized with the temperature-sensitive (ts-4) mutant strain of T. gondii were cytotoxic for T. gondii-infected P815 (H-2d) mastocytoma cells but not for uninfected cells. This cytotoxic activity was remarkably increased after in vitro stimulation with T. gondii-infected syngeneic spleen cells. The effector cells were shown to be CD8+ T lymphocytes, because the cytotoxicity was significantly inhibited by depletion of CD8+ T lymphocytes but not by depletion of CD4+ T lymphocytes. This cytotoxic activity was genetically restricted. Spleen cells from T. gondii-immune BALB/c mice were not cytotoxic for T. gondii-infected EL4 (H-2b) thymoma cells, whereas spleen cells from T. gondii-immune C57B1/6 (H-2b) mice were cytotoxic for T. gondii-infected EL4 cells but not for T. gondii-infected P815 cells. The cytolytic activity of CD8+ T lymphocytes against T. gondii-infected cells might be a mechanism whereby these cells confer resistance against T. gondii.     

Cross-primed CD8+ cytotoxic T cells induce severe Helicobacter-associated gastritis in the absence of CD4+ T cells

BACKGROUND: Although previous studies have reported important roles of CD4(+) type 1-helper T cells and regulatory T cells in Helicobacter-associated gastritis, the significance of CD8(+) cytotoxic T cells remains unknown. To study the roles of CD8(+) T cells, we examined the immune response in the gastric mucosa of Helicobacter felis-infected major histocompatibility complex (MHC) class II-deficient (II(-/-)) mice, which lack CD4(+) T cells. MATERIALS AND METHODS: Stomachs from H. felis-infected wild-type and infected MHC II(-/-) mice were examined histologically and immunohistochemically. Gastric acidity and serum levels of anti-H. felis antibodies were measured. The expression of pro-inflammatory and anti-inflammatory cytokine, Fas-ligand, perforin, and Foxp3 genes in the gastric mucosa was investigated. RESULTS: H. felis-infected MHC II(-/-) mice developed severe gastritis, accompanied by marked infiltration of CD8(+) cells. At 1 and 2 months after inoculation, mucosal inflammation and atrophy were more severe in MHC II(-/-) mice, although gastritis had reached similar advanced stages at 3 months after inoculation. There was little infiltration of CD4(+) cells, and no Foxp3-positive cells were detected in the gastric mucosa of the infected MHC II(-/-) mice. The expression of the interleukin-1beta and Fas-ligand genes was up regulated, but that of Foxp3 was down regulated in the infected MHC II(-/-) mice. Serum levels of anti-H. felis antibodies were lower in the infected MHC II(-/-) mice, despite severe gastritis. CONCLUSIONS: The present study suggests that cross-primed CD8(+) cytotoxic T cells can induce severe H.-associated gastritis in the absence of CD4(+) helper T cells and that Foxp3-positive cells may have an important role in the control of gastric inflammation.     

Generation of EBV-specific CD4+ cytotoxic T cells from virus naive individuals.

Adoptive immunotherapy with EBV-specific CTL (EBV-CTL) effectively prevents and treats EBV-driven lymphoproliferation in immunocompromised hosts. EBV-seronegative solid organ transplant recipients are at high risk of EBV-driven lymphoproliferation because they lack EBV-specific memory T cells. For the same reason, standard techniques for generating EBV-CTL in vitro from EBV-naive individuals are unsuccessful. To overcome this problem, we compared several methods of expanding EBV-CTL from seronegative adults and children. First, the standard protocol, using EBV-transformed lymphoblastoid B cell lines (LCL) as the source of APC, was compared with protocols using EBV-Ag-loaded dendritic cells as APC. Surprisingly, the standard protocol effectively generated CTL from all seronegative adults. The additional finding of EBV-DNA in the peripheral blood of three of these four adults suggested that some individuals may develop cellular, but not humoral, immune responses to EBV. By contrast, LCL failed to reactivate EBV-CTL from any of the six EBV-seronegative children. EBV-Ag-loaded dendritic cells could expand EBV-CTL, but only in a minority of children. However, the selective expansion of CD25-expressing T cells, 9-11 days after activation with LCL alone, proved to be a simple and reliable method for generating EBV-CTL from all seronegative children. The majority of these CTL were CD4(+) (71 +/- 26%) and demonstrated HLA class II-restricted, EBV-specific killing. Our results suggest that a negative EBV serology does not accurately identify EBV-negative individuals. In addition, our method for selecting EBV-specific CTL from naive individuals by precursor cell enrichment may be applicable to the immunotherapy of cancer patients with a low frequency of tumor- or virus-specific CTL.     

Oral administration of hapten inhibits in vivo induction of specific cytotoxic CD8+ T cells mediating tissue inflammation: a role for regulatory CD4+ T cells

We investigated whether oral tolerance could block the development of an inflammatory response mediated by CD8+ T cells, using a mouse model of oral tolerance of contact sensitivity (CS) to the hapten 2, 4-dinitrofluorobenzene (DNFB). In this system, the skin inflammatory response is initiated by hapten-specific class I-restricted cytotoxic CD8+ T (CTL) cells, independently of CD4 help. Oral delivery of DNFB before skin sensitization blocked the CS response by impairing the development of DNFB-specific CD8+ effector T cells in secondary lymphoid organs. This was shown by complete inhibition of DNFB-specific CTL and proliferative responses of CD8+ T cells, lack of specific IFN-gamma-producing CD8+ T cells, and inability of CD8+ T cells to transfer CS in RAG20/0 mice. RT-PCR and immunohistochemical analysis confirmed that recruitment of CD8+ effectors of CS in the skin at the site of hapten challenge was impaired in orally tolerized mice. Sequential anti-CD4 Ab treatment showed that only depletion of CD4+ T cells during the afferent phase of CS abrogated oral tolerance induction by restoring high numbers of specific CD8+ effectors in lymphoid organs, whereas CD4 depletion during the efferent phase of CS did not affect oral tolerance. These data demonstrate that a single intragastric administration of hapten can block in vivo induction of DNFB-specific CD8+ CTL responsible for tissue inflammation and that a subset of regulatory CD4+ T cells mediate oral tolerance by inhibiting expansion of specific CD8+ effectors in lymph nodes.     

Suppression of human immunodeficiency virus replication by CD8+ cells from infected and uninfected chimpanzees.

Over a 4-year period, infectious human immunodeficiency virus type 1 (HIV-1) has been recovered from cultured peripheral blood mononuclear cells (PBMC) of virus-infected animals only intermittently and at relatively low titers. In examining the possible mechanism for this observation, CD4+ cells or CD8+ cells were removed by panning from the PMBC before culture. A dramatic increase in frequency of HIV-1 recovery as well as in the level of virus replication was observed in the CD4+ cell-enriched or CD8+ cell-depleted cultures of PBMC from 3/3 infected animals. Moreover, addition of purified CD8+ effector cells from all 6 HIV-infected and 5/10 uninfected animals to an equal number of HIV-1 acutely infected purified CD4+ target cells resulted in 75-100% suppression of virus production. CD8+ cells from 3 additional uninfected animals caused delayed replication kinetics and moderate to low suppression of peak virus production. These findings contrast with the previously recognized absence of this HIV-1-suppressing activity of CD8+ cells from seronegative humans. This CD8+ cell-mediated suppression of viral replication could help explain the natural resistance of chimpanzees to HIV-induced disease.     

CD8+ T lymphocyte control of HIV replication in cultured CD4+ cells varies among infected individuals

Production of the human immunodeficiency virus (HIV) by cultured peripheral blood mononuclear cells (PMC) from many seropositive individuals is inhibited by the presence of CD8+ T lymphocytes. In a study of 10 subjects, high levels of virus replication could be detected in cultures of purified CD4+ cells, but not in unseparated PMC. Addition of highly purified, autologous CD8+ cells to the enriched CD4+ cells resulted in a dose-dependent inhibition of HIV growth and revealed that for some individuals, even low numbers of CD8+ cells can prevent replication of the virus. The data also indicated that culturing enriched CD4+ cells could greatly enhance detection of infectious virus in blood specimens and demonstrated that the CD4+ molecule is expressed on infected T cells isolated directly from the peripheral blood.     

Therapeutic induction of regulatory, cytotoxic CD8+ T cells in multiple sclerosis

In the setting of autoimmunity, one of the goals of successful therapeutic immune modulation is the induction of peripheral tolerance, a large part of which is mediated by regulatory/suppressor T cells. In this report, we demonstrate a novel immunomodulatory mechanism by an FDA-approved, exogenous peptide-based therapy that incites an HLA class I-restricted, cytotoxic suppressor CD8+ T cell response. We have shown previously that treatment of multiple sclerosis (MS) with glatiramer acetate (GA; Copaxone) induces differential up-regulation of GA-reactive CD8+ T cell responses. We now show that these GA-induced CD8+ T cells are regulatory/suppressor in nature. Untreated patients show overall deficit in CD8+ T cell-mediated suppression, compared with healthy subjects. GA therapy significantly enhances this suppressive ability, which is mediated by cell contact-dependent mechanisms. CD8+ T cells from GA-treated patients and healthy subjects, but not those from untreated patients with MS, exhibit potent, HLA class I-restricted, GA-specific cytotoxicity. We further show that these GA-induced cytotoxic CD8+ T cells can directly kill CD4+ T cells in a GA-specific manner. Killing is enhanced by preactivation of target CD4+ T cells and may depend on presentation of GA through HLA-E. Thus, we demonstrate that GA therapy induces a suppressor/cytotoxic CD8+ T cell response, which is capable of modulating in vivo immune responses during ongoing therapy. These studies not only explain several prior observations relating to the mechanism of this drug but also provide important insights into the natural immune interplay underlying this human immune-mediated disease.     

Generation and ex vivo expansion of HTLV-1 specific CD8+ cytotoxic T-lymphocytes for adoptive immunotherapy

CD8(+) cytotoxic T-lymphocytes (CTLs) have been proven, in multiple animal models, to be the most powerful antiviral and antitumor components of the immune system. We have developed a protocol to activate and expand tumor and virus peptide-specific CD8(+) T-lymphocytes from the peripheral blood of healthy, human trophic leukemia virus-1 (HTLV-1) seronegative human leucocyte antigen (HLA)-A*0201 individuals. A combination of density-based separation and culture conditions was employed to isolate dendritic cells (DCs), which are the most potent antigen-presenting cells (APCs), and T-lymphocytes. The DCs were pulsed with HLA-A*0201 binding peptides and cultured with autologous T-lymphocytes to generate peptide-specific CTLs. The CTLs were generated against a nine-amino-acid peptide from the Tax protein of HTLV-1. The CTLs were expanded according to a restimulation schedule employing peptide-pulsed autologous monocytes and low-dose interleukin-2 (IL-2) to numbers in excess of 100 x 10(6) cells following 5 weeks of culture. Expanded cells contained primarily CD3(+) T-cells, of which CD8(+) T-lymphocytes constituted greater than two-thirds of the cell population. Obtained CTLs exhibited potent antigen-specific lysis of peptide-pulsed target cells in a dose-dependent fashion in in vitro (51)Cr release cytotoxicity assay. This antigen-specific killing was shown to be HLA class I restricted and mediated by CD8(+) T-lymphocytes. Since the T-lymphocytes were obtained from HTLV-1 seronegative donors, the generation of peptide-specific CTLs represents reliable and reproducible elicitation of a primary immune response in vitro against naive antigens and subsequent expansion of generated CTLs for adoptive immunotherapy. (c) 1996 John Wiley & Sons, Inc.     

Maintenance of large numbers of virus-specific CD8+ T cells in HIV-infected progressors and long-term nonprogressors

The virus-specific CD8+ T cell responses of 21 HIV-infected patients were studied including a unique cohort of long-term nonprogressors with low levels of plasma viral RNA and strong proliferative responses to HIV Ags. HIV-specific CD8+ T cell responses were studied by a combination of standard cytotoxic T cell (CTL) assays, MHC tetramers, and TCR repertoire analysis. The frequencies of CD8+ T cells specific to the majority of HIV gene products were measured by flow cytometric detection of intracellular IFN-gamma in response to HIV-vaccinia recombinant-infected autologous B cells. Very high frequencies (0.8-18.0%) of circulating CD8+ T cells were found to be HIV specific. High frequencies of HIV-specific CD8+ T cells were not limited to long-term nonprogressors with restriction of plasma virus. No correlation was found between the frequency of HIV-specific CD8+ T cells and levels of plasma viremia. In each case, the vast majority of cells (up to 17.2%) responded to gag-pol. Repertoire analysis showed these large numbers of Ag-specific cells were scattered throughout the repertoire and in the majority of cases not contained within large monoclonal expansions. These data demonstrate that high numbers of HIV-specific CD8+ T cells exist even in patients with high-level viremia and progressive disease. Further, they suggest that other qualitative parameters of the CD8+ T cell response may differentiate some patients with very low levels of plasma virus and nonprogressive disease.     

Elevated ATP via enhanced miRNA-30b, 30c,and 30e downregulates the expression of CD73 in CD8+ T cells of HIV-infected individuals

CD8⁺ T cells play a crucial role against chronic viral infections, however, their effector functions are influenced by the expression of co-stimulatory/inhibitory receptors. For example, CD73 works with CD39 to convert highly inflammatory ATP to adenosine. However, its expression on T cells in the context of viral infections has not been well defined. Here, we analyzed the expression of CD73 on human T cells in a cohort of 102 HIV-infected individuals including those on antiretroviral therapy (ART), ART-naïve, and long-term non-progressors who were not on ART. We found that the frequency of CD73⁺ T cells was markedly lower among T cell subsets (e.g. naïve, effector or memory) in the peripheral blood of all HIV-infected individuals. Notably, CD73 was decreased at the cell surface, intracellular and gene levels. Functionally, CD8⁺CD73⁺ T cells exhibited decreased cytokine expression (TNF-α, IFN-γ and IL-2) upon global or antigen-specific stimulation and impaired expression of cytolytic molecules at the gene and protein levels. In contrast, CD8⁺CD73⁺ T cells expressed elevated levels of homing receptors such as CCR7, α4β7 integrin, which suggests a migratory advantage for these cells as observed in vitro. We also observed significant migration of CD73⁺CD8⁺ T cells into the cerebrospinal fluids of multiple sclerosis (MS) patients at the time of disease relapse. Moreover, we found that elevated levels of ATP in the plasma of HIV-infected individuals upregulates the expression of miRNA30b-e in T cells in vitro. In turn, inhibition of miRNAs (30b, 30c and 30e) resulted in significant upregulation of CD73 mRNA in CD8⁺ T cells. Therefore, we provide a novel mechanism for the downregulation of CD73 via ATP-induced upregulation of miRNA30b, 30c and 30e in HIV infection. Finally, these observations imply that ATP-mediated downregulation of CD73 mainly occurs via its receptor, P2X1/P2RX1. Our results may in part explain why HIV-infected individuals have reduced risk of developing MS considering the role of CD73 for efficient T cell entry into the central nervous system.     

Invariant NKT cells promote CD8+ cytotoxic T cell responses by inducing CD70 expression on dendritic cells

Activation of invariant NK T (iNKT) cells with the glycolipid alpha-galactosylceramide promotes CD8(+) cytotoxic T cell responses, a property that has been used to enhance the efficacy of antitumor vaccines. Using chimeric mice, we now show that the adjuvant properties of iNKT cells require that CD40 triggering and Ag presentation to CD8(+) T cells occur on the same APCs. We demonstrate that injection of alpha-galactosylceramide triggers CD70 expression on splenic T cell zone dendritic cells and that this is dependent on CD40 signaling. Importantly, we show that blocking the interaction between CD70 and CD27, its costimulatory receptor on T cells, abrogates the ability of iNKT cells to promote a CD8(+) T cell response and abolishes the efficacy of alpha-GalCer as an adjuvant for antitumor vaccines. These results define a key role for CD70 in linking the innate response of iNKT cells to the activation of CD8(+) T cells.     

CD4+ T cells in the absence of the CD8+ cytotoxic T cells are critical and sufficient for NKT cell-dependent tumor rejection

NKT cells perform crucial roles in tumor surveillance, functioning as regulators of early host response. In this study, we have assessed the effects of NKT activation at the time of tumor Ag immunization, and have evaluated the contributions of CD4+ and CD8+ T cells in tumor rejection during adaptive immune response against live tumor cells. Our data indicate that CD4+ T cells play critical roles, not only in assisting CTL, but also in the orchestration of host response against the tumor. The CD4+ T cells were found to reject the transplanted tumor cells very efficiently under conditions in which the CTLs were removed either genetically, or via the action of anti-CD8 Ab in mice that had been immunized with tumor extracts and alpha-galactosylceramide. Immunization resulted in an NKT cell-dependent antitumor adaptive immune response, which was associated with both CD4+ T cells and cytokine IFN-gamma.