CD30 expression by CD8+ T cells producing type 2 helper cytokines. Evidence for large numbers of CD8+CD30+ T cell clones in human immunodeficiency virus infection

A large panel of CD8+ T cell clones generated from peripheral blood lymphocytes (PBL) of healthy donors or human immunodeficiency virus (HIV)-infected individuals were assessed for both cytokine secretion profile and CD30 expression and release. The great majority of CD8+ T cell clones generated from healthy individuals showed the ability to produce interferon gamma (IFN-gamma), but not interleukin 4 (IL-4), and none of them either expressed membrane CD30 or released substantial amounts of soluble CD30 (sCD30) in their supernatant. In contrast, high numbers of CD8+ T cell clones generated from HIV-infected individuals, which produced IL-4 (and IL-5) in addition to IFN-gamma or IL-4 (and IL- 5) alone, expressed membrane CD30 and released detectable amounts of sCD30 in their supernatants. Indeed, CD30 expression appeared to be positively correlated with the ability of CD8+ T cell clones to produce IL-4 and IL-5 and inversely correlated with their ability to produce IFN-gamma, whereas no correlation between CD30 expression and production of IL-10 was observed. These data suggest that CD30 is a marker for CD8+ T cells that have switched to the production of type 2 helper cytokines.     

CDw60: a marker for human CD8+ T helper cells

The existence of helper cells among the CD8+ T cell subset has been recognized for a long time. However, the phenotype of these cells has remained elusive. In this study, we provide evidence that the expression of the CDw60 antigen on human CD8+ T cell allows one to distinguish between CD8+ T helper cells and CD8+ T cells with cytotoxic and suppressor capacity. CDw60 monoclonal antibodies (mAb) recognize the 9-O-acetylated disialosyl group on ganglioside GD3 expressed on 20- 40% of CD8+ cells. By use of the direct and indirect mAb-rosetting technique, we were able to isolate the CDw60+CD8+ and CDw60-CD8+ cells at high purity. The alloantigen-specific cytotoxic activity of CD8+ cells resided entirely in the CDw60- population. Helper and suppressor capacity of both CD8 subsets was assayed by the pokeweed mitogen- induced differentiation of B cells into immunoglobulin-secreting cells. These studies clearly indicate that the CDw60+CD8+ subset provided substantial help to B lymphocytes, whereas the CD8+ cells with the CDw60- phenotype were suppressing B cell differentiation. Both subsets produced similar amounts of interleukin 2 (IL-2) after stimulation with phytohemagglutinin. Activation with phorbol myristate acetate in combination with Ca-ionophore induced IL-4 secretion in both populations, but preferentially in the CDw60+ subset, whereas the vast majority of interferon gamma was produced by the CDw60-CD8+ cells. When used in combination with other markers, CDw60 may prove to be useful in defining CD8+ subsets with reciprocal functional activities.     

FBL-reactive CD8+ cytotoxic and CD4+ helper T lymphocytes recognize distinct Friend murine leukemia virus-encoded antigens

Immunization of C57BL/6 (B6) mice with FBL, a Friend murine leukemia virus (F-MuLV), induces both tumor-specific cytolytic CD8+ (CTL) and lymphokine-producing CD4+ Th that are effective in adoptive therapy of B6 mice bearing disseminated FBL leukemia. The current study evaluated the F-MuLV antigenic determinants expressed on FBL that are recognized by FBL-reactive CD8+ and CD4+ T cells. To identify the specificity of the FBL-reactive CD8+ CTL, Fisher rat embryo fibroblast (FRE) cells transfected with plasmids encoding F-MuLV gag or envelope (env) gene products plus the class I-restricting element Db were utilized. FBL-reactive CTL recognized FRE target cells transfected with the F-MuLV gag-encoded gene products, but failed to recognize targets expressing F-MuLV env. Attempts to generate env-specific CD8+ CTL by immunization with a recombinant vaccinia virus containing an inserted F-MuLV env gene were unsuccessful, despite the generation of a cytolytic response to vaccinia epitopes, implying that B6 mice fail to generate CD8+ CTL to env determinants. By contrast, CD4+ Th clones recognized FRE target cells transfected with env and not gag genes, and immunization with the recombinant vaccinia virus induced an env-specific CD4+ T cell response. These data show that in a Friend retrovirus-induced tumor model in which tumor rejection can be mediated by either CTL or Th, antigens derived from discrete retroviral proteins are predominantly responsible for activation of each T cell subset.     

Th2-like CD8+ T cells showing B cell helper function and reduced cytolytic activity in human immunodeficiency virus type 1 infection

We analyzed at clonal level the functional profile of circulating or skin-infiltrating T lymphocytes from two individuals infected with the human immunodeficiency virus type 1 (HIV-1), suffering from a Job's- like syndrome (eczematous dermatitis, recurrent skin and sinopulmonary infections, and hypergammaglobulinemia E) and showing virtually no circulating CD4+ T cells. Most of the CD3+ T cell clones generated from both patients were CD4- CD8+ TCR alpha beta +. The others were CD4- CD8- TCR alpha beta + which exhibited reduced mRNA expression for the CD8 molecule or no mRNA expression for either CD4 or CD8 molecules. The great majority of both CD4- CD8+ and CD4- CD8- did not produce interferon (IFN) gamma and exhibited reduced cytolytic activity. Rather, most of them produced large amounts of both interleukin (IL) 4 and IL-5 and provided B cell helper function for IgE synthesis. These data suggest that a switch of cytolytic CD8+ T cells showing a Th1-like cytokine secretion profile to cells that make Th2-type cytokines, exhibit reduced cytolytic potential, and provide B cell helper function can occur in the course of HIV-1 infection. These cells may contribute to the reduced defense against viral infections and intracellular parasites and account for the elevated IgE serum levels, eosinophilia, and the allergic-like clinical manifestations seen in a proportion of HIV-1-infected individuals.     

Complementary dendritic cell-activating function of CD8+ and CD4+ T cells: helper role of CD8+ T cells in the development of T helper type 1 responses

Dendritic cells (DCs) activated by CD40L-expressing CD4+ T cells act as mediators of "T helper (Th)" signals for CD8+ T lymphocytes, inducing their cytotoxic function and supporting their long-term activity. Here, we show that the optimal activation of DCs, their ability to produce high levels of bioactive interleukin (IL)-12p70 and to induce Th1-type CD4+ T cells, is supported by the complementary DC-activating signals from both CD4+ and CD8+ T cells. Cord blood- or peripheral blood-isolated naive CD8+ T cells do not express CD40L, but, in contrast to naive CD4+ T cells, they are efficient producers of IFN-gamma at the earliest stages of the interaction with DCs. Naive CD8+ T cells cooperate with CD40L-expressing naive CD4+ T cells in the induction of IL-12p70 in DCs, promoting the development of primary Th1-type CD4+ T cell responses. Moreover, the recognition of major histocompatibility complex class I-presented epitopes by antigen-specific CD8+ T cells results in the TNF-alpha- and IFN-gamma-dependent increase in the activation level of DCs and in the induction of type-1 polarized mature DCs capable of producing high levels of IL-12p70 upon a subsequent CD40 ligation. The ability of class I-restricted CD8+ T cells to coactivate and polarize DCs may support the induction of Th1-type responses against class I-presented epitopes of intracellular pathogens and contact allergens, and may have therapeutical implications in cancer and chronic infections.     

Quantitative changes in T helper inducer (CD4+ CD45RA-), T suppressor inducer (CD4+ CD45RA+), T suppressor (CD8+ CD11b+), and T cytotoxic (CD8+ CD11b-) subsets in human immunodeficiency virus infection

To determine changes in subsets of CD4 and CD8 in relation to HIV infection and progression to AIDS, we quantitated peripheral blood lymphocytes obtained from 17 heterosexual controls, 22 asymptomatic HIV-seronegative and 18 HIV-seropositive intravenous drug users. 50 patients with AIDS-related complex (ARC), and 9 patients with AIDS using an EPICS "C" flow cytometer by two-color analysis. Both T helper inducer (CD4+ CD45RA-) and suppressor inducer (CD4+ CD45RA+) lymphocytes were decreased significantly in patients with ARC or AIDS. In contrast, T cytotoxic (CD8+ CD11b-) cells were significantly increased and accompanied by a significant decrease in the T suppressor (CD8+ CD11b+) subset in patients with ARC or AIDS. These results suggest that both T helper inducer and T suppressor inducer subsets of the CD4+ population and the T suppressor subset of CD8+ are depleted after HIV infection, while the T cytotoxic subset of CD8+ was increased after HIV infection.     

Both CD8+ and CD16+ human T cell clones can provide B cell help for immunoglobulin production

Summary The functional properties of two CD4⁺CD8⁻CD16⁻, five CD4⁻CD8⁺CD16⁻ and three CD4⁻CD8⁻CD16⁺ human T cell clones were compared. All CD4⁻ T cell clones displayed strong cytolytic activity in the lectin-dependent lytic assay against the P815 murine mastocytoma cell line, but only the CD4⁻CD8⁻CD16⁺ T cell clones exhibited lytic activity against the natural killer-sensitive K562 cell line. Upon activation with anti-CD3 monoclonal antibody, all T cell clones were able to support IgM and IgA synthesis in autologous B cells. Both CD4⁺ and CD4⁻ T cell clones required cell-to-cell interaction with the B cells in order to exert their helper activity for immunoglobulin production. However, unlike CD4⁺, CD4⁻CD8⁺CD16⁻ and CD4⁻CD8⁻CD16⁺ T cell clones provided helper function for immunoglobulin synthesis only when low T/B cell ratios were used in culture. At higher T/B cell ratios, there was a decline in the B cell helper activity of CD4⁻ T cell clones that was probably related to the expression of cytolytic capacity against the antigen-presenting B cell. These data support the notion that under certain experimental conditions even cytotoxic T lymphocytes and natural killer cells may provide B cell helper function.     

Antigen-specific targeting of CD8+ T cells with receptor-modified T lymphocytes

Chimeric receptors that link ligand recognition domains, such as antibody Fv fragments, with TCR signaling domains can redirect T lymphocytes against MHC-unrestricted targets. Such receptor-modified T lymphocytes have shown promise in the treatment of infectious diseases and cancer. We hypothesized that receptor-modified T lymphocytes may also be designed to target antigen-specific T cells. We synthesized chimeric receptors consisting of the extracellular and transmembrane domains of the class I MHC H-2K(b) molecule linked to the signaling domains of either TCR-zeta, CD28 and zeta, or CD28, zeta, and lck. T lymphocytes modified to express these receptors and pulsed with antigenic peptide specifically killed precursor CTL. Cytolysis was efficient, even at effector:target ratios of less than one, and specific, selectively killing antigen-specific precursor CTL among a mixed population of T cells. Cytolysis required activation of the receptor-modified T cells, and did not occur with a signaling-deficient chimeric receptor. In contrast to precursor CTL, differentiated CTL proved resistant to lysis by the receptor-modified T cells. These data demonstrate the feasibility of redirecting T lymphocytes against antigen-specific T cells. Receptor-modified T cells expressing chimeric MHC receptors have potential application in autoimmune and alloimmune diseases.     

Activation-induced death by apoptosis in CD4+ T cells from human immunodeficiency virus-infected asymptomatic individuals.

In immature thymocytes, T cell receptor for antigen (TCR) mobilization leads to an active T cell suicide process, apoptosis, which is involved in the selection of the T cell repertoire. We have proposed that inappropriate induction of such a cell death program in the mature CD4+ T cell population could account for both early qualitative and late quantitative CD4+ T lymphocyte defects of human immunodeficiency virus (HIV)-infected individuals (Ameisen, J.C., and A. Capron. 1991. Immunol. Today. 4:102). Here, we report that the selective failure of CD4+ T cells from 59 clinically asymptomatic HIV-infected individuals to proliferate in vitro to TCR mobilization by major histocompatibility complex class II-dependent superantigens and to pokeweed mitogen (PWM) is due to an active CD4+ T cell death process, with the biochemical and ultrastructural features of apoptosis. Activation-induced cell death occurred only in the CD4+ T cell population from HIV-infected asymptomatic individuals and was not observed in T cells from any of 58 HIV-seronegative controls, including nine patients with other acute or chronic infectious diseases. Activation-induced CD4+ T cell death was prevented by cycloheximide, cyclosporin A, and a CD28 monoclonal antibody (mAb). The CD28 mAb not only prevented apoptosis but also restored T cell proliferation to stimuli, including PWM, superantigens, and the tetanus and influenza recall antigens. These findings may have implications for the understanding of the pathogenesis of acquired immune deficiency syndrome and for the design of specific therapeutic strategies.     

Both CD8+ and CD16+ human T cell clones can provide B cell help for immunoglobulin production.

The functional properties of two CD4+CD8-CD16-, five CD4-CD8+CD16- and three CD4-CD8-CD16+ human T cell clones were compared. All CD4- T cell clones displayed strong cytolytic activity in the lectin-dependent lytic assay against the P815 murine mastocytoma cell line, but only the CD4-CD8-CD16+ T cell clones exhibited lytic activity against the natural killer-sensitive K562 cell line. Upon activation with anti-CD3 monoclonal antibody, all T cell clones were able to support IgM and IgA synthesis in autologous B cells. Both CD4+ and CD4- T cell clones required cell-to-cell interaction with the B cells in order to exert their helper activity for immunoglobulin production. However, unlike CD4+, CD4-CD8+CD16- and CD4-CD8-CD16+ T cell clones provided helper function for immunoglobulin synthesis only when low T/B cell ratios were used in culture. At higher T/B cell ratios, there was a decline in the B cell helper activity of CD4- T cell clones that was probably related to the expression of cytolytic capacity against the antigen-presenting B cell. These data support the notion that under certain experimental conditions even cytotoxic T lymphocytes and natural killer cells may provide B cell helper function.     

Selection of CD8+PD-1+ lymphocytes in fresh human melanomas enriches for tumor-reactive T cells

CD8+ tumor-infiltrating lymphocytes (TILs) in human melanomas express high levels of PD-1 and are functionally impaired. However, adoptive cell therapy using in vitro-expanded TIL can be a highly effective therapy for patients with advanced melanoma. This discrepancy led us to further analyze the CD8+PD-1+ TILs. We found that the percentage of PD-1-expressing CD8+ T cells was higher in the tumor digests that generate tumor-reactive TILs after in vitro culture in interleukin-2 (P=0.0007). Also sorted and expanded CD8+PD-1+ T cells in tumor digests showed much higher tumor-specific interferon-γ production compared with CD8+PD-1? T cells. These results suggested that tumor-specific CD8+ T cells in melanoma tumor digests are largely PD-1, and this population can recover function after culturing in interleukin-2. PD-1 has been reported as an inhibitory receptor on T cells. We found that the in vitro functional suppression of cultured-TILs from native levels of PD-L1 expression on melanomas was minimal, and moreover expression level of PD-1 on CD8+ tumor-specific TILs decreased during the culture. As a consequence, the PD-1 receptor can be a useful biomarker for enriching tumor-specific T cells from fresh melanomas.     

Fas antigen stimulation induces marked apoptosis of T lymphocytes in human immunodeficiency virus-infected individuals

Apoptosis (programmed cell death) of T lymphocytes has been proposed as a mechanism which plays an important role in the pathogenesis of human immunodeficiency virus (HIV) disease. Activation of Fas (CD95) can either result in costimulation of proliferation and cytokine production or in the induction of apoptosis of T lymphocytes. This raises the possibility that Fas is involved in the observed T cell apoptosis during HIV disease. In this report we show that peripheral blood CD4+ and CD8+ T lymphocytes from HIV-infected individuals undergo apoptosis in vitro in response to antibody stimulation (cross-linking) of Fas at a much higher frequency than from uninfected controls. This anti-Fas- induced T cell apoptosis is markedly higher than spontaneous T cell apoptosis in HIV-infected individuals. Antibodies against other members of the tumor necrosis factor (TNF)/nerve growth factor receptor family such as CD27, CD30, CD40, 4-1BB, p55 TNF receptor, p75 TNF receptor, and TNF receptor-related protein did not result in any increase of T cell apoptosis above that spontaneously observed in HIV+ individuals. Anti-Fas-induced apoptosis was much higher in symptomatic HIV-infected individuals; and the magnitude of anti-Fas-induced CD4+ T cell apoptosis correlated inversely with peripheral blood CD4+ T cell absolute counts. Surface expression of Fas on T cells was also found to be higher in HIV-infected individuals. Resting and activated CD4+ and CD8+ T cells both underwent apoptosis in response to anti-Fas antibody. L-Selectin positive memory CD4+ T cells were especially susceptible to anti-Fas-induced apoptosis. These findings show that CD4+ and CD8+ T lymphocytes in HIV-infected individuals are primed in vivo to undergo apoptosis in response to Fas stimulation, suggesting that Fas signaling may be responsible for the T lymphocyte functional defects and depletion observed in HIV disease.     

CD4+ T helper cell response is required for memory in CD8+ T lymphocytes induced by a poly(I:C)-adjuvanted MHC I-restricted peptide epitope

It was known that double-stranded RNA (dsRNA) has vaccine adjuvant activity by acting through the dsRNA/toll-like receptor (TLR)3 signaling. More recently, it was reported that immunization of mice with a poly(I:C)-adjuvanted major histocompatibility (MHC) class I-restricted peptide epitope derived from a tumor-specific antigen protein induced strong tumor-killing CD8 CTL responses. However, it remains unclear whether the poly(I:C) can help an MHC class I-restricted peptide epitope to induce a strong and functional memory CD8 CTL response in the absence of a T helper (TH) response. By using in vivo CTL and direct tumor challenging assays, we demonstrated that, although poly(I:C) as a vaccine adjuvant significantly enhanced the immune responses induced by an MHC I-restricted peptide epitope, MHC class II-restricted TH cell responses were still required to generate memory CTL responses. The MHC class II-restricted TH epitopes can be cognate, noncognate, or even from the necrotic bodies of un-related tumor cells. Thus, future efforts of using synthetic dsRNA as an adjuvant to induce specific and long-lasting memory CTL immune responses should include the provision of an appropriate TH cell response.     

Transfusion practices in human immunodeficiency virus-infected patients

BACKGROUND: The reported immunomodulatory effects of transfusion raise concern about the potential for virus activation and tumor growth in human immunodeficiency virus (HIV)-infected patients. In the absence of “standards” of transfusion practice for such patients, a survey of transfusion policies among institutions specializing in the care of HIV- infected patients was performed to delineate current practices. STUDY DESIGN AND METHODS: A survey developed by the Transfusion Practices Committee of the American Association of Blood Banks was sent to 47 AIDS clinical trial units and 14 regional hemophilia centers in North America. RESULTS: Forty-three percent of centers completed the survey. Most centers observed more than 200 HIV-infected patients each. The key findings were that 1) 81 percent of centers used identical red cell transfusion criteria for HIV-infected and noninfected patients; 2) 52 percent used recombinant human erythropoietin as initial treatment for zidovudine-induced anemia, while 46 percent used recombinant human erythropoietin for anemia not associated with zidovudine; 3) 35 percent of centers used white cell-reduced blood components in lieu of cytomegalovirus (CMV)-seronegative components when administering transfusion(s) to CMV-seronegative patients; 4) 27 percent gamma- radiated cellular components, but no case of graft-versus-host disease had been observed; 5) > 85 percent of centers used monoclonal factor VIII for pediatric and adult hemophiliacs infected with HIV; 6) approximately one-third of centers routinely white cell-reduced cellular components; and 7) the most common reasons for white cell reduction included reduction of febrile reactions and CMV risk, reduction of platelet alloimmunization, and delay of immunomodulatory consequences of transfusion. CONCLUSION: There is marked heterogeneity in transfusion practice for HIV-infected patients. Modification of cellular components to achieve different objectives is routine in many centers.     

CD27 expression promotes long-term survival of functional effector-memory CD8+ cytotoxic T lymphocytes in HIV-infected patients

Human immunodeficiency virus (HIV)-specific CD8(+) T cells persist in high frequencies in HIV-infected patients despite impaired CD4(+) T helper response to the virus, but, unlike other differentiated effector cytotoxic T lymphocytes, most continue to express the tumor necrosis factor receptor family member CD27. Because the ligand for CD27 (CD70) is also overexpressed in HIV-infected hosts, we examined the nature of expression and potential functional consequences of CD27 expression on HIV-specific CD8(+) T cells. Analysis of CD27(+) and CD27(-) T cells derived from the same HIV-specific clone revealed that retention of CD27 did not interfere with acquisition of effector functions, and that after T cell receptor stimulation, CD27(+) cells that concurrently were triggered via CD27 exhibited more resistance to apoptosis, interleukin 2 production, and proliferation than CD27(-) T cells. After transfer back into an HIV-infected patient, autologous HIV-specific CD27(-) T cells rapidly disappeared, but CD27(+) T cells derived from the same clone persisted at high frequency. Our findings suggest that the CD27-CD70 interaction in HIV infection may provide CD27(+) CD8(+) T cells with a survival advantage and compensate for limiting or absent CD4(+) T help to maintain the CD8 response.     

Human CD3+ T lymphocytes that express neither CD4 nor CD8 antigens

CD3+ T lymphocytes expressing neither CD4 nor CD8 antigens exist in normal human peripheral blood in low frequency (approximately 3% of lymphocytes). The CD3+,4-,8- phenotype was stably maintained after in vitro culture in IL-2. Culture of CD3+,4-,8- cells in only rIL-2 generated cytotoxic T cells that lysed NK-sensitive and NK-insensitive tumor cell targets without MHC restriction. These experiments clearly show that phenotypically and functionally competent T cells expressing neither CD4 nor CD8 are present in normal peripheral blood.     

Dramatic rise in plasma viremia after CD8(+) T cell depletion in simian immunodeficiency virus-infected macaques

To determine the role of CD8(+) T cells in controlling simian immunodeficiency virus (SIV) replication in vivo, we examined the effect of depleting this cell population using an anti-CD8 monoclonal antibody, OKT8F. There was on average a 99.9% reduction of CD8 cells in peripheral blood in six infected Macaca mulatta treated with OKT8F. The apparent CD8 depletion started 1 h after antibody administration, and low CD8 levels were maintained until day 8. An increase in plasma viremia of one to three orders of magnitude was observed in five of the six macaques. The injection of a control antibody to an infected macaque did not induce a sustained viral load increase, nor did it significantly reduce the number of CD8(+) T cells. These results demonstrate that CD8 cells play a crucial role in suppressing SIV replication in vivo.     

Influenza virus-infected dendritic cells stimulate strong proliferative and cytolytic responses from human CD8+ T cells.

Antigen-specific, CD8+, cytolytic T lymphocytes (CTLs) could potentially provide resistance to several infectious and malignant diseases. However, the cellular requirements for the generation of specific CTLs in human lymphocyte cultures are not well defined, and repetitive stimulation with antigen is often required. We find that strong CD8+ CTL responses to influenza virus can be generated from freshly isolated blood T cells, as long as dendritic cells are used as antigen presenting cells (APCs). Small numbers of dendritic cells (APC:T cell ratio of 1:50-1:100) induce these CTL responses from most donors in 7 d of culture, but monocytes are weak or inactive. Whereas both dendritic cells and monocytes are infected with influenza virus, the former serve as effective APCs for the induction of CD8+ T cells while the latter act as targets for the CTLs that are induced. The strong CD8+ response to influenza virus-infected dendritic cells is accompanied by extensive proliferation of the CD8+ T cells, but the response can develop in the apparent absence of CD4+ helpers or exogenous lymphokines. CD4+ influenza virus-specific CTLs can also be induced by dendritic cells, but the cultures initially must be depleted of CD8+ cells. These findings should make it possible to use dendritic cells to generate human, antigen-specific, CD8+ CTLs to other targets. The results illustrate the principle that efficient T cell-mediated responses develop in two stages: an afferent limb in which dendritic cells are specialized APCs and an efferent limb in which the primed T cells carry out an immune response to many types of presenting cells.