Lysis of cells infected with HIV-1 by human lymphocytes targeted with monoclonal antibody heteroconjugates

The present study was undertaken to determine whether human PBL can be specifically focused to lyse cells infected with HIV-1 by mAb heteroconjugates that can bridge target and effector cells. A mAb directed against the central portion of HIV-1 glycoprotein gp110 was chemically cross-linked to a mAb directed against the CD3/TCR complex or to a mAb directed against the CD16 Fc gamma-R expressed on large granular lymphocytes (LGL). HIV-1-infected cells, but not uninfected cells, were found to be lysed to a greater extent by PBL in the presence of the gp110 X CD3 or the gp110 X CD16 antibody heteroconjugate than in the presence of the single antibodies or a mixture of the mAb comprising the heteroconjugates. Pretreatment of PBL with anti-CD3 or IL-2 augments their ability to lyse HIV-1-infected cells in the presence of the heteroconjugates. Lysis by anti-CD3-activated PBL in the presence of the gp110 X CD3 heteroconjugate was found to be mediated by CD8+-enriched T cells, whereas lysis by IL-2-treated PBL in the presence of the gp110 X CD16 heteroconjugate is mediated by PBL enriched for CD16+ cells, which are primarily LGL. Furthermore, PBL from asymptomatic, HIV-1-infected seropositive donors were found to be functional in lysing HIV-1-infected cells in the presence of the antibody heteroconjugates. Such antibody heteroconjugates, which can target T cells or LGL to lyse HIV-1-infected cells, may be of prophylactic or therapeutic value in HIV-1-infected individuals.     

The anti-idiotypic antibody 1F7 selectively inhibits cytotoxic T cells activated in HIV-1 infection

Circulating CD8+ T lymphocyte numbers rise substantially following infection with HIV-1. This expanded CD8+ T cell population includes HIV-specific CTL and CTL that kill activated uninfected CD4+ lymphocytes. Experimental, epidemiological and clinical evidence supports the possibility that expansion of CD8+ CTL contributes to CD4+ T cell depletion and disease progression in human HIV infection. Therefore, modulation of CD8+ T cell numbers or of certain CD8+ CTL activated in HIV-infected individuals may be beneficial. It was found that 1F7, a mAb against an idiotype common to anti-HIV and anti-simian immunodeficiency virus (SIV) antibodies, selectively inhibited both anti-HIV CTL and CTL against uninfected CD4+ T cells. Alloantigen-specific CTL and NK cells from either HIV-infected individuals or controls were unaffected by 1F7. Prolonged incubation of CD8+ T cells from HIV-infected individuals with 1F7 induces apoptosis, which was shown to be reflected functionally in reduced total CTL activity and in especially reduced CTL activity against uninfected CD4+ lymphocytes. The selective reactivity of 1F7 with certain CD8+ CTL could be applied towards the modulation of CD8+ T cell responses involved in AIDS pathogenesis.     

Induction of lysis by T cell receptor gamma delta+/CD3+ T lymphocytes via CD2 requires triggering via the T11.1 epitope only

The requirements for activation of the lytic machinery through CD2 of TCR gamma delta+/CD3+ cells were examined, by utilizing bispecific heteroconjugates containing anti-CD2 mAb cross-linked to anti-DNP. Contrary to the CD2 activation requirements in TCR alpha beta+/CD3+ cells, cytotoxic activity in TCR gamma delta+/CD3+ clones and TCR-/CD3- NK cell clones can be induced by heteroconjugates containing a single anti-CD2 (OKT11.1) mAb. Activation of TCR gamma delta+/CD3+ cells via CD2 is independent of heteroconjugates binding to CD16 (Fc gamma RIII), because heteroconjugates prepared from Fab fragments induced equal levels of lysis. Moreover, anti-CD16 mAb did not inhibit triggering via CD2 in TCR gamma delta+/CD3+ cells. In TCR-/CD3- NK cells, however, induction of cytotoxicity via CD2 is co-dependent on interplay with CD16. Anti-CD3 mAb blocked the anti-CD2 x anti-DNP heteroconjugate-induced cytotoxicity of TCR gamma delta+/CD3+ cells, indicating a functional linkage between CD2 and CD3 on these cells. We conclude that induction of lysis via CD2 shows qualitatively different activation requirements in TCR gamma delta+/CD3+, TCR alpha beta+/CD3+ CTL and TCR-/CD3- NK cells.     

NK-like cytotoxicity of allospecific gamma delta TCR+ T cell clones]

We recently generated a series of human alloantigen-specific, CD3+, gamma delta- TCR+ clones by stimulating CD3+, CD4-, CD8- T cells from normal individuals with allogeneic lymphoblastoid cell lines (LCL). These clones display cytotoxic activity against their specific stimulators but not against irrelevant LCL. Most but not all of these clones express the NK cell associated marker, CD57, and kill NK-sensitive targets such as the K562 and Molt 4 lines, but not NK-resistant line, Raji. Gamma delta clones which lacked expression of CD57 had no detectable NK activity. The allospecific cytotoxicity of CD57+ and CD57- clones was inhibited by mAb to CD3 or the TCR delta- chain. In contrast, the NK-like activity of the CD57+ clones was enhanced by these antibodies over a wide range of antibody concentration. An HLA class I framework-specific mAb had no effect on NK-like cytolysis but did inhibit allospecific killing, suggesting that the target structures on the surface of allospecific and NK-sensitive cells are distinct. The receptors utilized by the gamma delta- TCR+ clones to recognize NK-sensitive and allospecific targets are also distinct, since killing of NK-sensitive targets was blocked by the presence of cold (unlabeled) NK-sensitive cells but not by cold allospecific targets, whereas allospecific cytolysis was inhibited by cold allospecific targets but not by NK-sensitive cells. We conclude that some CD3+, TCR- gamma delta+ clones exhibit NK-like as well as allospecific killing and that these two activities are mediated by distinct receptor-ligand interactions.     

Virus-specific cytotoxic T lymphocytes in human immunodeficiency virus type 1-infected chimpanzees.

Chimpanzees have been important in studies of human immunodeficiency virus type 1 (HIV-1) pathogenesis and in evaluation of HIV-1 candidate vaccines. However, little information is available about HIV-1-specific cytotoxic T lymphocytes (CTL) in these animals. In the present study, in vitro stimulation of peripheral blood mononuclear cells (PBMC) from infected chimpanzees with HIV-1 Gag peptides was shown to be a sensitive, reproducible method of expanding HIV-1-specific CD8(+) effector CTL. Of interest, PBMC from two chimpanzees had CTL activity against Gag epitopes also recognized by major histocompatibility complex class I-restricted CTL from HIV-1-infected humans. The use of peptide stimulation will help to clarify the role of CTL in vaccine-mediated protection and HIV-1 disease progression in this important animal model.     

Leucoagglutinin induces differential proliferation of lymphocyte subsets

In this study we have established culture conditions that allow the preferential and rapid expansion of either T cell receptor (TCR)+/CD3+16- T lymphocytes or TCR-/CD3-16+ natural killer (NK) cells, or the non-selective outgrowth of both subsets. Optimal proliferation of lymphocytes was obtained using a combination of irradiated allogeneic peripheral blood lymphocytes (PBL) and irradiated Epstein Barr virus (EBV) transformed lymphoblastoid B cell lines (B-LCL). Addition of 1 microgram/ml leucoagglutinin to the culture medium induced a preferential outgrowth of TCR+/CD3+16- T lymphocytes. The proportion of TCR-/CD3-16+ NK cells was decreased to 5% or less, although still a 2000-fold multiplication of TCR-/CD3-16+ NK cells was obtained at day 13. Without leucoagglutinin a 1000-fold increase of about 70% pure TCR-/CD3-16+ NK cells was obtained at day 13. Intermediate concentrations of leucoagglutinin (0.1-0.3 micrograms/ml) resulted in a non-selective expansion of both NK cells and T cells. Irrespective whether leucoagglutinin was added or not, the number of TCR+/CD3+8+ lymphocytes increased more rapidly relative to the TCR+/CD3+4+ lymphocytes resulting in an increased TCR+/CD3+8+ population size. Also under limiting dilution conditions leucoagglutinin increased the frequency of proliferating cells. In contrast to the preferential outgrowth of TCR+/CD3+8+ lymphocytes in bulk cultures, approximately 80% of the clones generated was TCR+/CD3+4+, demonstrating a growth promoting effect of TCR+/CD3+4+ lymphocytes on TCR+/CD3+8+ lymphocytes in PBL bulk cultures.     

Patterns of cytokine production in human immunodeficiency virus type 1 (HIV-1)-specific human CD8+ T cells after stimulation with HIV-1-infected CD4+ T cells

Although human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells can produce various cytokines that suppress HIV-1 replication or modulate anti-HIV-1 immunity, the extent to which HIV-1-specific CD8+ T cells produce cytokines when they recognize HIV-1-infected CD4+ T cells in vivo still remains unclear. We first analyzed the abilities of 10 cytotoxic T-lymphocyte (CTL) clones specific for three HIV-1 epitopes to produce gamma interferon, macrophage inflammatory protein 1beta, and tumor necrosis factor alpha after stimulation with epitope peptide-pulsed cells. These CTL clones produced these cytokines in various combinations within the same specificity and among the different specificities, suggesting a functional heterogeneity of HIV-1-specific effector CD8+ T cells in cytokine production. In contrast, the HIV-1-specific CTL clones for the most part produced a single cytokine, without heterogeneity of cytokine production among the clones, after stimulation with HIV-1-infected CD4+ T cells. The loss of heterogeneity in cytokine production may be explained by low surface expression of HLA class I-epitope peptide complexes. Freshly isolated HIV-1-specific CD8+ T cells with an effector/memory or memory phenotype produced much more of the cytokines than the same epitope-specific CTL clones when stimulated with HIV-1-infected CD4+ T cells. Cytokine production from HIV-1-specific memory/effector and memory CD8+ T cells might be a critical event in the eradication of HIV-1 in HIV-1-infected individuals.     

Natural killer cells in perinatally HIV-1-infected children exhibit less degranulation compared to HIV-1-exposed uninfected children and their expression of KIR2DL3, NKG2C, and NKp46 correlates with disease severity

NK cells play an integral role in the innate immune response by targeting virally infected and transformed cells with direct killing and providing help to adaptive responses through cytokine secretion. Whereas recent studies have focused on NK cells in HIV-1-infected adults, the role of NK cells in perinatally HIV-1-infected children is less studied. Using multiparametric flow cytometric analysis, we assessed the number, phenotype, and function of NK cell subsets in the peripheral blood of perinatally HIV-1-infected children on highly active antiretroviral therapy and compared them to perinatally exposed but uninfected children. We observed an increased frequency of NK cells expressing inhibitory killer Ig-like receptors in infected children. This difference existed despite comparable levels of total NK cells and NK cell subpopulations between the two groups. Additionally, NK cell subsets from infected children expressed, with and without stimulation, significantly lower levels of the degranulation marker CD107, which correlates with NK cell cytotoxicity. Lastly, increased expression of KIR2DL3, NKG2C, and NKp46 on NK cells correlated with decreased CD4+ T-lymphocyte percentage, an indicator of disease severity in HIV-1- infected children. Taken together, these results show that HIV-1-infected children retain a large population of cytotoxically dysfunctional NK cells relative to perinatally exposed uninfected children. This reduced function appears concurrently with distinct NK cell surface receptor expression and is associated with a loss of CD4+ T cells. This finding suggests that NK cells may have an important role in HIV-1 disease pathogenesis in HIV-1-infected children.     

Peripheral blood cytotoxic gammadelta T lymphocytes from patients with human immunodeficiency virus type 1 infection and AIDS lyse uninfected CD4+ T cells,and their cytocidal potential correlates with viral load

Progression of human immunodeficiency virus type 1 (HIV-1) infection in humans is marked by declining CD4+-T-cell counts and increasing virus load (VL). Cytotoxic T lymphocytes (CTL) play an important role in the lysis of HIV-infected cells, especially during the early phase of asymptomatic infection. CTL responses in the later phase of disease progression may not be as effective since progressors with lower CD4+-T-cell counts have consistently higher VL despite having elevated CTL counts. We hypothesized that, apart from antiviral effects, some CTL might also contribute to AIDS pathogenesis by depleting CD4+ T cells and that this CTL activity may correlate with the VL in AIDS patients. Therefore, a cross-sectional study of 31 HIV-1-infected patients at various clinical stages was carried out. Purified CTL from these donors as well as HIV-seronegative controls were used as effectors against different human cell targets by using standard 51Cr release cytolytic assays. A direct correlation between VL and CTL-mediated, major histocompatibility complex (MHC)-unrestricted lysis of primary CD4+-T-cell, CEM.NKR, and K562 targets was observed. CD4+-T-cell counts and duration of infection also correlated with MHC-unrestricted cytolytic activity. Our data clearly show that gammadelta CTL are abnormally expanded in the peripheral blood of HIV-infected patients and that the Vdelta1 subset of gammadelta T cells is the main effector population responsible for this type of cytolysis. The present data suggest that gammadelta CTL can contribute to the depletion of bystander CD4+ T cells in HIV-infected patients as a parallel mechanism to HIV-associated immunopathogenesis and hence expedite AIDS progression.     

Loss of CD4+ T Cells in Human Immunodeficiency Virus Type 1-Infected Chimpanzees Is Associated with Increased Lymphocyte Apoptosis

Supportive evidence that apoptosis contributes to loss of CD4⁺ lymphocytes in human immunodeficiency virus type 1 (HIV-1)-infected humans comes from an apparent lack of abnormal apoptosis in apathogenic lentivirus infections of nonhuman primates, including HIV-1 infection of chimpanzees. Two female chimpanzees were inoculated, one cervically and the other intravenously, with HIV-1 derived from the LAI/LAV-1b strain, which was isolated from a chimpanzee infected with the virus for 8 years. Within 6 weeks of infection, both recipient chimpanzees developed a progressive loss of CD4⁺ T cells which correlated with persistently high viral burdens and increased levels of CD4⁺ T-cell apoptosis both in vitro and in vivo. Lymph nodes from both animals also revealed evidence of immune hyperactivation. Intermediate levels of T-cell apoptosis in both peripheral blood and lymph nodes were seen in a third chimpanzee that had been infected with the LAI/LAV-1b strain for 9 years; this animal has maintained depressed CD4/CD8 T-cell ratios for the last 3 years. Similar analyses of cells from 4 uninfected animals and 10 other HIV-1-infected chimpanzees without loss of CD4⁺ cells revealed no difference in levels of apoptosis in these two control groups. These results demonstrate a correlation between immune hyperactivation, T-cell apoptosis, and chronic loss of CD4⁺ T cells in HIV-1-infected chimpanzees, providing additional evidence that apoptosis is an important factor in T-cell loss in AIDS. Furthermore, the results show that some HIV-1 strains are pathogenic for chimpanzees and that this species is not inherently resistant to HIV-1-induced disease.     

Spontaneous human T-cell cytotoxicity against murine hybridomas expressing the OKT3 monoclonal antibody: comparison with natural killer cell activity

Human peripheral blood lymphocytes (PBL) exhibited spontaneous cytotoxicity against OKT3 monoclonal antibody (mAb)-expressing murine hybridoma cells (OKT3 hybridomas). In contrast, other murine hybridomas expressing OKT4, OKT8, anti-HLA DR, and anti-HLA A, B, and C mAb were not lysed. PBL showed much lower levels of cytotoxicity (3 folds) against OKT3 hybridomas as compared with NK activity against the K562 targets. Lymph node (LN) cells exhibited the inverse relationship of cytotoxicity levels. The addition of OKT3 mAb to the effector cells totally blocked both the binding and the lysis of OKT3 hybridoma targets, indicating that the CD3 antigen on the effector cells may be involved in recognition of the targets. The addition of concanavalin (Con A) also inhibited the cytotoxicity of OKT3 hybridomas. OKT4 mAb-expressing hybridomas became susceptible to lysis after chemical attachment of OKT3 mAb with CrCl3. The kinetics of lysis of OKT3 hybridomas resembled that of NK activity. Both cytotoxicities were detectable after 1 to 2 hr and reached plateau levels by 4 to 6 hr. Effector cells responsible for lysis of OKT3 hybridomas expressed T3, T8, and Leu 7 antigens, but lacked T4 and Leu 11b antigens, and were sensitive to the treatment with L-leucine methyl ester. These results indicate that T3+, T8+, Leu 7+ and T4-, and Leu 11- granular lymphocytes have a spontaneous cytotoxic activity against OKT3 hybridomas which is different from classic NK activity. These findings may provide a method for the assessment of T-cell cytotoxicity mediated presumably by in vivo generated cytotoxic T lymphocytes in blood and the other immune organs.     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. I. Lysis restricted by HLA class II MB and DR antigens

In contrast to general findings that mouse and human cytotoxic T lymphocytes (CTL) are restricted in cytotoxic activity by major histocompatibility complex (MHC) class I antigens, we previously found that some herpes simplex virus (HSV) type I-infected cells that shared no HLA class I antigens with the HSV-1-stimulated lymphocytes were lysed. In this study, we addressed the question of the role of HLA antigens in human T cell-mediated lysis of HSV-1-infected cells by generating clones of HSV-1-directed CTL from two HSV-1-seropositive individuals. CTL clones that lysed autologous HSV-1-infected lymphoblastoid cell lines (LCL), but not natural killer-sensitive K562 cells or uninfected or influenza virus-infected LCL, were tested for cytotoxicity against a panel of allogeneic HSV-1-infected LCL. Clone KL-35 from individual KL lysed only HSV-1-infected LCL sharing the HLA class II MB1 antigen with KL. With all four CTL clones isolated from individual PM, only HSV-1-infected LCL sharing DR1 with PM were lysed. Monoclonal antibody s3/4 (directed against MB1 ), but not TS1/16 or B33 .1 (directed against a DR framework determinant), blocked lysis of autologous HSV-1-infected cells by KL-35. In contrast, B33 .1, but not s3/4, blocked lysis of autologous HSV-1-infected cells by the PM CTL clones but not by KL-35. Together, these results indicate that our five human CTL clones which are directed against HSV-1-infected cells, and which are all OKT3+, OKT4+, OKT8-, are restricted in lytic activity by HLA class II MB and DR antigens. These results suggest that the HLA D region-encoded class II antigens may be important in the recognition and destruction of virus-infected cells by human CTL.     

Different effects of Nef-mediated HLA class I down-regulation on human immunodeficiency virus type 1-specific CD8(+) T-cell cytolytic activity and cytokine production

A previous study using a Nef-defective human immunodeficiency virus type 1 (HIV-1) mutant suggested that Nef-mediated down-regulation of HLA class I on the infected cell surface affects the cytolytic activity of HIV-1-specific cytotoxic T-lymphocyte (CTL) clones for HIV-1-infected primary CD4(+) T cells. We confirmed this effect by using a nef-mutant HIV-1 strain (NL-M20A) that expresses a Nef protein which does not induce down-regulation of HLA class I molecules but is otherwise functional. HIV-1-specific CTL clones were not able to kill primary CD4(+) T cells infected with a Nef-positive HIV-1 strain (NL-432) but efficiently lysed CD4(+) T cells infected with NL-M20A. Interestingly, CTL clones stimulated with NL-432-infected CD4(+) T cells were able to produce cytokines, albeit at a lower level than when stimulated with NL-M20A-infected CD4(+) T cells. This indicates that Nef-mediated HLA class I down-regulation affects CTL cytokine production to a lesser extent than cytolytic activity. Replication of NL-432 was partially suppressed in a coculture of HIV-1-infected CD4(+) T cells and HIV-1-specific CTL clones, while replication of NL-M20A was completely suppressed. These results suggest that HIV-1-specific CD8(+) T cells are able to partially suppress the replication of HIV-1 through production of soluble HIV-1-suppressive factors such as chemokines and gamma interferon. These findings may account for the mechanism whereby HIV-1-specific CD8(+) T cells are able to partially but not completely control HIV-1 replication in vivo.     

Lysis of endogenously infected CD4+ T cell blasts by rIL-2 activated autologous natural killer cells from HIV-infected viremic individuals

Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK) cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However, it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous, endogenously HIV-1-infected CD4+ T cells. Here, we stimulate primary CD4+ T cells, purified ex vivo from HIV-1-infected viremic patients, with PHA and rIL2 (with or without rIL-7). This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that, subsequent to the selective down-modulation of MHC class-I (MHC-I) molecules, HIV-1-infected p24(pos) blasts become partially susceptible to lysis by rIL-2-activated NK cells, while uninfected p24(neg) blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However, the degree of NK cell cytolytic activity against autologous, endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and -B alleles and against heterologous MHC-I(neg) cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs) and with the high frequency of the anergic CD56(neg)/CD16(pos) subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively, our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24(pos) blasts derived from primary T cells.     

Modulation of lymphokine release and cytolytic activities by activating peripheral blood lymphocytes via CD2

We had previously shown that the signal of activation delivered via CD2 varies according to the mitogenic pair of CD2 mAb used. We had selected two typical mAb pairs, D66 + T11(1) and GT2 + T11(1), the former delivering the "richest" signal, the latter the poorest. Here we analyzed the cytolytic activities generated within PBL-stimulated by these two pairs. When purified CD2+,3- cells were cultured with either one of these two pairs, no significant lymphokine-activated killer (LAK) activity--namely the activity exerted on NK-resistant malignant cell lines or fresh tumor cells--was detected, thereby demonstrating the inability of CD2 mAb pairs to directly trigger the LAK precursors. By contrast, when purified CD2+,3+ cells were cultured, only D66 + T11(1) was able to trigger a potent CTL activity, as judged by targeting their activity, at the effector phase, with a bridging CD3 mAb on a FcR+ target cell or by using heteroaggregates on FcR- malignant cells. When whole PBL were used, a similar and moderate LAK activity was generated after culture with either one of the 2 CD2 mAb pairs. This, in fact, masked quite different events. The amounts of endogeneous IL-2 released in PBL cultures with GT2 + T11(1) was rather low, although it was sufficiently high in PBL cultures with D66 + T11(1) to generate a potent LAK activity. Yet, PBL stimulated with D66 + T11(1) released concomitantly a high amount of IL-4 which inhibited the development of the LAK activity, as demonstrated by unmasking this activity with an anti-IL4 antiserum and which did not inhibit the T CTL activity; this IL-4 secretion was not seen with GT2 + T11(1). Therefore, stimulation by these two typical CD2 mAb pairs induce a striking different pattern of IL synthesis.     

Heat Shock Cognate Protein 71-Associated Peptides Function as an Epitope for Toxoplasma gondii-Specific CD4+ CTL

HLA-DR-restricted CD4⁺ cytotoxic T-lymphocyte (CTL) lines specific for Toxoplasma gondii (T. gondii)-infected melanoma cells have been established from peripheral blood lymphocytes (PBLs) of a patient with chronic toxoplasmosis. The role of heat shock cognate protein (HSC) 71 in antigen (Ag) processing and presentation of T. gondii-infected melanoma cells to these CD4⁺ CTL lines was investigated. A human melanoma cell line (P36) pulsed with T. gondii-infected P36 cell-derived HSC71 was lysed by a T. gondii-specific CD4⁺ CTL line (Tx-HSC-1). The Tx-HSC-1 also killed T. gondii-infected P36 cells. The lytic activity of Tx-HSC-1 against P36 cells pulsed with T. gondii-infected P36 cell-derived HSC71 was inhibited by monoclonal antibodies (mAbs) against HSC71. Anti-human leukocyte antigen (HLA)-DR mAb also partially blocked the lytic activity, whereas anti-HLA-A,B,C mAb did not block the lytic activity. In addition, a flow cytometric analysis with these specific mAbs against HSC71 showed HSC71 to be expressed on the cell surface of T. gondii-infected P36 cells as well as uninfected P36 cells. These data indicate that HSC71 molecules are expressed on human melanoma cell line P36, and that HSC71 may play a potential role in Ag presentation and processing of T. gondii-infected P36 cells to CD4⁺ CTL.     

NK cell lysis of HIV-1-infected autologous CD4 primary T cells: requirement for IFN-mediated NK activation by plasmacytoid dendritic cells

In vivo, several mechanisms have been postulated to protect HIV-1-infected cells from NK surveillance. In vitro, previous research indicates HIV-1-infected autologous CD4(+) primary T cells are resistant to NK lysis. We hypothesized that NK lysis of HIV-1-infected target cells would be augmented by the presence of accessory cells and/or accessory cell factors. In this study, we show that stimulation of plasmacytoid dendritic cells (PDC) with the TLR9 agonist, CpG ODN 2216, triggered NK lysis of HIV-1-infected autologous CD4(+) primary T cells. PDC-stimulated NK lysis was dependent upon MHC class I (MHC-I) down-regulation on infected cells, and primary HIV-1 isolates that exhibited enhanced MHC-I down-regulation were more susceptible to NK-mediated lysis. PDC-stimulated NK lysis of HIV-1-infected autologous CD4(+) primary T cells was blocked by neutralizing Abs to type 1 IFN and was perforin/granzyme dependent. Overall, our data suggest that HIV-infected cells are not innately resistant to NK lysis, and that exogenous NK stimulation derived from PDC can trigger NK cytotoxicity against HIV-1-infected autologous CD4(+) primary T cells.     

HIV-1 transmission and function of virus-infected monocytes/macrophages.

Monocyte/macrophages (MM) were isolated from HIV-1 seronegative individuals, infected with HIV-1 and examined for their ability to infect autologous T lymphocytes with and without concomitant presentation of exogenous Ag. HIV-1-infected MM presented tetanus toxin (TT) and streptokinase to T cells (as measured by [3H]thymidine incorporation) comparable to presentation by uninfected MM. In these studies, it was observed that HIV-1-infected MM without additional exogenous Ag stimulated autologous T lymphocytes, however, to a lesser degree than with TT and streptokinase. Virus production in T cells appeared to be relative to the degree of stimulation with the highest levels of stimulation and infection observed when T cells were exposed to HIV-1-infected TT-presenting MM. Studies were carried out to examine some of the restricting elements in MM-mediated infection of T lymphocytes with and without TT presentation. Antibodies to CD4, as well as soluble immunopurified gp120, blocked cell-mediated infection indicating that infection of T cells was through the CD4 molecule as has been demonstrated with cell-free virus. In addition, soluble gp120 inhibited Ag presentation by HIV-1-infected and uninfected MM. mAb to MHC class II Ag HLA-DR and -DP blocked T cell infection by HIV-1-infected MM with and without presentation of TT. No effect was observed with mAb to MHC class I Ag. These results indicate that virus transmission to T lymphocytes can be mediated by HIV-1-infected MM and that these cells maintain their function as APC. Activation of T cells appears to be important in the process of T cell infection in this system inasmuch as antibodies that block Ag presentation and thus a T cell proliferative signal inhibit infection.     

Differences in surface phenotype between cytolytic and non-cytolytic CD4+ T cells. MHC class II-specific cytotoxic T lymphocytes lack Leu 8 antigen and express CD2 in high density

A number of cell surface molecules are differentially expressed on functionally distinct subsets of CD4+ T cells. However, to date CD4+ T cells capable of becoming CTL have not been shown to be phenotypically distinct from other CD4+ T cells, and in the current study we examined the ability of Leu 8+ and Leu 8- CD4+ subpopulations to become cytotoxic effectors after their stimulation with allogeneic lymphoblastoid cell lines. Although CD4+, Leu 8+ cells proliferated more vigorously than CD4+, Leu 8- cells in primary cultures stimulated with allogeneic LCL, the CD4+, Leu 8- population was the major source of cytotoxic effectors, killing targets with specificity for their class II MHC alloantigens. In most subjects, CD4+ precursors of CTL were distinguished not only by their lack of Leu 8 expression but also by their relatively high density of CD2, LFA-1, and LFA-3, molecules known to mediate non-specific cell-to-cell adhesion and postulated to be markers of immunologic memory. The absence of Leu 8 does not appear to be a reliable memory cell marker, however, because Leu 8+ as well as Leu 8-, CD4+ cells from PPD skin test positive subjects responded to the recall Ag, PPD. During 3 mo of continuous culture with allogeneic stimulators, Leu 8- cells retained their cytolytic activity and remained unreactive with anti-Leu 8 mAb, whereas Leu 8+ cells remained non-cytolytic and reactive with anti-Leu 8, suggesting that under the conditions used the Leu 8 phenotype is relatively stable. PHA or anti-CD3 mAb enhanced non-specific killing by alloantigen-stimulated CD4+,Leu 8- lines but failed to unmask any cytolytic potential in CD4+,Leu 8+ lines. We conclude that MHC class II-specific cytolytic CD4+ T cells can be distinguished from non-cytolytic CD4+ cells on the basis of their surface phenotype, and that most CD4+ CTL are contained within the Leu 8- subpopulation.     

Contact of human immunodeficiency virus type 1-infected and uninfected CD4+ T lymphocytes is highly cytolytic for both cells.

Individuals infected with the human immunodeficiency virus (HIV) experience a marked loss of CD4+ T lymphocytes, leading to fatal immunodeficiency. The mechanisms causing the depletion of these cells are not yet understood. In this study, we observed that CD4+ T lymphocytes from HIV type 1 (HIV-1)-infected and uninfected individuals rapidly lysed B lymphoblasts expressing the HIV-1 envelope glycoprotein on the cell surface and Jurkat cells expressing the complete virus. Contact of uninfected CD4+ T cells with envelope glycoprotein-expressing cells also resulted in the lysis of the uninfected CD4+ T cells. Cytolysis did not require priming or in vitro stimulation of the CD4+ T cells and was not restricted by major histocompatibility complex molecules. Cytotoxicity was inhibited by soluble CD4 and anti-CD4 monoclonal antibodies that block binding of CD4 to gp120. In addition, neutralizing anti-CD4 and anti-gp120 monoclonal antibodies which block postbinding membrane fusion events and syncytium formation also inhibited cell lysis, suggesting that identical mechanisms in HIV-infected cultures underlie cell-cell fusion and the cytolysis observed. However, cytotoxicity was not always accompanied by the formation of visible syncytia. Rapid cell lysis after contact of uninfected and HIV-1-infected CD4+ T cells may explain CD4+ T-cell depletion in the absence of detectable syncytia in infected individuals. Moreover, because of its vigor, lysis of envelope-expressing targets by contact with unprimed CD4+ T lymphocytes may at first glance resemble antigen-specific immune responses and should be excluded when cytotoxic T-lymphocyte responses in infected individuals and vaccinees are evaluated.