TB/HIV integration at primary care level: A quantitative assessment at 3 clinics in Johannesburg, South Africa.

BACKGROUND: In 2004 the World Health Organization WHO) released the Interim Policy on Collaborative TB/ HIV activities. According to the policy, for people living with HIV (PLWH), activities include intensified case finding, isoniazid preventive therapy (IPT) and infection control. For TB patients, activities included HIV counselling and testing HCT), prevention messages, and cotrimoxazole preventive therapy (CPT), care and support, and antiretroviral therapy ART) for those with HIV-associated TB. While important progress has been made in implementation, targets of the WHO Global Plan to Stop TB have not been reached. OBJECTIVE: To quantify TB/HIV integration at 3 primary healthcare clinics in Johannesburg, South Africa. METHODS: Routinely collected TB and HIV data from the HCT register, TB 'suspect' register, TB treatment register, clinic files and HIV electronic database, collected over a 3-month period, were reviewed. RESULTS: Of 1 104 people receiving HCT: 306 (28%) were HIV-positive; a CD4 count was documented for 57%; and few received TB screening or IPT. In clinic encounters among PLWH, 921 (15%) had documented TB symptoms; only 10% were assessed by smear microscopy, and few asymptomatic PLWH were offered IPT. Infection control was poorly documented and implemented. HIV status was documented for 155 (75%) of the 208 TB patients; 90% were HIV-positive and 88% had a documented CD4 count. Provision of CPT and ART was poorly documented. CONCLUSION: The coverage of most TB/HIV collaborative activities was below Global Plan targets. The lack of standardised recording tools and incomplete documentation impeded assessment at facility level and limited the accuracy of compiled data.

A Ran-independent pathway for export of spliced mRNA.

All major nuclear export pathways so far examined follow a general paradigm. Specifically, a complex is formed in the nucleus, containing the export cargo, a member of the importin-beta family of transporters and RanGTP. This complex is translocated across the nuclear pore to the cytoplasm, where hydrolysis of the GTP on Ran is stimulated by the GTPase-activating protein RanGAP. The activity of RanGAP is increased by RanBP1, which also promotes disassembly of RanGTP-cargo-transporter complexes. Here we investigate the role of RanGTP in the export of mRNAs generated by splicing. We show that nuclear injection of a Ran mutant (RanT24N) or the normally cytoplasmic RanGAP potently inhibits the export of both tRNA and U1 snRNA, but not of spliced mRNAs. Moreover, nuclear injection of RanGAP together with RanBP1 blocks tRNA export but does not affect mRNA export. These and other data indicate that export of spliced mRNA is the first major cellular transport pathway that is independent of the export co-factor Ran.

Squid, Cup, and PABP55B function together to regulate gurken translation in Drosophila.

During Drosophila melanogaster oogenesis, the proper localization of gurken (grk) mRNA and protein is required for the establishment of the dorsal-ventral axis of the egg and future embryo. Squid (Sqd) is an RNA-binding protein that is required for the correct localization and translational regulation of the grk message. We show that Cup and polyA-binding protein (PABP) interact physically with Sqd and with each other in ovaries. We show that cup mutants lay dorsalized eggs, enhance dorsalization of weak sqd alleles, and display defects in grk mRNA localization and Grk protein accumulation. In contrast, pAbp mutants lay ventralized eggs and enhance grk haploinsufficiency. PABP also interacts genetically and biochemically with Encore. These data predict a model in which Cup and Sqd mediate translational repression of unlocalized grk mRNA, and PABP and Enc facilitate translational activation of the message once it is fully localized to the dorsal-anterior region of the oocyte. These data also provide the first evidence of a link between the complex of commonly used trans-acting factors and Enc, a factor that is required for grk translation.

The patient impact of point-of-care vs. laboratory placement of Xpert(®) MTB/RIF.

BACKGROUND: The Xpert(®) MTB/RIF assay can diagnose tuberculosis (TB) rapidly and with great accuracy. The effect of Xpert placement at point of care (POC) vs. at an off-site laboratory on patient management remains unknown. DESIGN: At a primary care clinic in Johannesburg, South Africa, we compared TB diagnosis and treatment initiation among 1861 individuals evaluated for pulmonary TB using Xpert performed either at POC or offsite. RESULTS: When Xpert was performed at POC, a higher proportion of Xpert-positive individuals started treatment (95% vs. 87%, P = 0.047) and time to treatment initiation was shorter (median 0 vs. 5 days, P < 0.001). In contrast, among Xpert-negative TB cases, a higher proportion (87% vs. 72%, P = 0.001) started treatment when the sample was sent to the laboratory, with a shorter time to treatment (median 9 vs. 13 days, P = 0.056). While the overall proportion of presumed TB patients starting treatment was independent of Xpert placement, the proportion started based on a bacteriologically confirmed diagnosis was higher when Xpert was performed at POC (73% vs. 58%, P = 0.006). CONCLUSIONS: Placement of Xpert at POC resulted in more Xpert-positive patients receiving treatment, but did not increase the total number of presumed TB patients starting treatment. When samples were sent to a laboratory for Xpert testing, empiric decision making increased.

Interleukin-2 inhibits HIV-1 replication in human macrophages by modulating expression of CD4 and CC-chemokine receptor-5.

OBJECTIVE: To determine the effect of recombinant human interleukin (IL)-2 on HIV-1 replication and macrophage colony stimulating factor (M-CSF) production by HIV-1-infected monocyte-derived macrophages (MDM). DESIGN: Therapeutic use of IL-2 increases the number and function of CD4+ T cells. IL-2 also increases M-CSF production and M-CSF receptor expression by human monocytes, but the subsequent effects on HIV-1 replication in MDM have yet to be determined. MDM from HIV-1-seronegative donors were cultured in the presence and absence of IL-2 and infected with HIV-1. Harvested supernatants were monitored for reverse transcriptase activity and M-CSF production. RESULTS: Reverse transcriptase activity was significantly lower when MDM cultures were treated with IL-2 for 10 days prior to infection with HIV-1. IL-2 did not stimulate production of inhibitory chemokines or cytokines, but FACS analysis revealed that expression of CD4, the primary HIV-1 receptor, and CC-chemokine receptor-5, a coreceptor used by macrophage-tropic viruses, are down modulated after treatment with IL-2. CONCLUSION: IL-2 may not only be of benefit in restoring immune function in AIDS patients, but may also help to prevent the infection of healthy macrophages by decreasing their expression of HIV-1 receptors.

Human astrocytes inhibit HIV-1 expression in monocyte-derived macrophages by secreted factors.

OBJECTIVE: To determine the effects of primary human fetal and adult astrocytes on HIV-1 replication in monocyte-derived macrophages (MDM). DESIGN: HIV-1 can infect the brain in the early stage of systemic infection. The HIV-1-associated cognitive/motor complex develops later in the course of the disease, suggesting that brain cells may inhibit the early productive infection and the development of neurological disease. In this study, we established an in-vitro coculture system to determine whether astrocytes can modulate HIV-1 replication in MDM. METHODS: Elutriated human monocytes were differentiated in culture, then infected with monocyte tropic HIV-1. One day after infection, MDM were co-cultured with primary astrocytes. Reverse transcriptase (RT) activity was used to monitor virus replication. RT-polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and bioassay were used to assess cytokine production. RESULTS: Primary human astrocytes suppressed HIV-1 replication in MDM via the production of soluble factors. Cytokine inhibitors of HIV-1, such as IFN-gamma, IL-4, IL-10 and IL-13, were not detectable, whereas transforming growth factor beta (TGF-beta) was constitutively produced only in its latent form. Paraformaldehyde-fixed astrocytes, unable to secrete cytokines, failed to inhibit HIV-1. These cells caused enhanced virus replication, however, which correlated with an increase in macrophage colony stimulating factor (M-CSF) production. CONCLUSIONS: Human astrocytes can increase and decrease HIV-1 expression in MDM. An imbalance between the positive and negative effects of astrocytes may contribute to the expression of virus in the brain, and the development of HIV-1-associated cognitive/motor complex.

Rapid method for purification of human T lymphocytes for further functional studies.

We have evaluated a commercially available reagent, T Lympho-kwik, for ability to provide enriched, functional human T cell populations. We have found that this method is more rapid, reliable and efficient than other available methods of T cell isolation and/or macrophage depletion. Although the T Lympho-kwik-treated peripheral blood mononuclear cells (PBMC) are enriched for T cells, devoid of macrophages, and are not activated by the T Lympho-kwik reagent, they can be induced to proliferate, secrete lymphokines and/or develop alloreactive cytolytic T cell activity under appropriate experimental conditions. Hence, T Lympho-kwik treatment of PBMC is a highly effective technique for providing enriched T cells that are free of accessory cells and that can be used for further functional studies.

Selenium supplementation suppresses tumor necrosis factor alpha-induced human immunodeficiency virus type 1 replication in vitro.

Selenium is a nutritionally essential trace element that is important for optimal function of the immune system. It is incorporated into selenoproteins as the amino acid selenocysteine and it is known to inhibit the expression of some viruses. In this study, we show that selenium supplementation for 3 days prior to exposure to tumor necrosis factor alpha (TNF-alpha) partially suppresses the induction of human immunodeficiency virus type 1 (HIV-1) replication in both chronically infected T lymphocytic and monocytic cell lines. In acute HIV-1 infection of T lymphocytes and monocytes in the absence of exogenous TNF-alpha, the suppressive effect of selenium supplementation was not observed. However, selenium supplementation did suppress the enhancing effect of TNF-alpha on HIV-1 replication in vitro in acutely infected human monocytes, but not in T lymphocytes. Selenium supplementation also increased the activities of the selenoproteins, glutathione peroxidase (GPx) and thioredoxin reductase (TR), which serve as cellular antioxidants. Taken together, these results suggest that selenium supplementation may prove beneficial as an adjuvant therapy for AIDS through reinforcement of endogenous antioxidative systems.

Recombinant CD4-Pseudomonas exotoxin hybrid protein displays HIV-specific cytotoxicity without affecting MHC class II-dependent functions.

The present study describes several in vitro activities of CD4(178)-PE40, a recombinant protein containing a portion of human CD4 linked to active regions of Pseudomonas aeruginosa exotoxin A. Using assays for cell viability, we demonstrate that the hybrid toxin displays highly selective cytotoxicity for HIV-infected T lymphocytes. In a latently infected human T-cell line which is inducible for HIV expression, toxin sensitivity is observed only upon virus induction. At concentrations which readily kill HIV-infected T cells, CD4(178)-PE40 has no observable cytotoxic effects on uninfected human cell lines expressing surface major histocompatibility complex (MHC) Class II molecules, and does not interfere with cellular responses known to be dependent on functional association between CD4 and MHC Class II molecules.

Thrombin is a regulator of astrocytic endothelin-1.

Endothelin-1, a potent vasoconstrictor of cerebral vessels, is produced by rat primary astrocytes and is subject to autostimulatory regulation in these cells. In this study we examined the effect of thrombin on astrocytic endothelins and report that endothelin-1 is released into the culture fluid in response to thrombin treatment. However, increased production of endothelin-1 is not accompanied by a concomitant increase in steady-state levels of endothelin-1 mRNA as assessed by reverse transcriptase-polymerase chain reaction, even though thrombin stimulation leads to increased inositolphospholipid turnover and activation of the nuclear factor AP1. Thus, astrocytic production of endothelin-1 may be mainly post-transcriptionally regulated in response to thrombin stimulation. In addition, two endothelin receptor genes (ET(A) and ETB) were found to be transcribed simultaneously in primary astrocyte cultures, and both thrombin and endothelin-1 stimulation result in a distinct temporary decrease in ET(A) mRNA. These studies suggest a role for thrombin in the regulation of brain perfusion through astrocytic endothelin-1 expression.

Source and cause of endothelin-1 release into cerebrospinal fluid after subarachnoid hemorrhage.

Despite years of research, delayed cerebral vasospasm remains a serious complication of subarachnoid hemorrhage (SAH). Recently, it has been proposed that endothelin-1 (ET-1) mediates vasospasm. The authors examined this hypothesis in a series of experiments. In a primate model of SAH, serial ET-1 levels were measured in samples from the perivascular space by using a microdialysis technique and in cerebrospinal fluid (CSF) and plasma during the development and resolution of delayed vasospasm. To determine whether elevated ET-1 production was a direct cause of vasospasm or acted secondary to ischemia, the authors also measured ET-1 levels in plasma and CSF after transient cerebral ischemia. To elucidate the source of ET-1, they measured its production in cultures of endothelial cells and astrocytes exposed to oxyhemoglobin (10 microM), methemoglobin (10 microM), or hypoxia (11% oxygen). There was no correlation between the perivascular levels of ET-1 and the development of vasospasm or its resolution. Cerebrospinal fluid and plasma levels of ET-1 were not affected by vasospasm (CSF ET-1 levels were 9.3 +/- 2.2 pg/ml and ET-1 plasma levels were 1.2 +/- 0.6 pg/ml) before SAH and remained unchanged when vasospasm developed (7.1 +/- 1.7 pg/ml in CSF and 2.7 +/- 1.5 pg/ml in plasma). Transient cerebral ischemia evoked an increase of ET-1 levels in CSF (1 +/- 0.4 pg/ml at the occlusion vs. 3.1 +/- 0.6 pg/ml 4 hours after reperfusion; p < 0.05), which returned to normal (0.7 +/- 0.3 pg/ml) after 24 hours. Endothelial cells and astrocytes in culture showed inhibition of ET-1 production 6 hours after exposure to hemoglobins. Hypoxia inhibited ET-1 release by endothelial cells at 24 hours (6.4 +/- 0.8 pg/ml vs. 0.1 +/- 0.1 pg/ml, control vs. hypoxic endothelial cells; p < 0.05) and at 48 hours (6.4 +/- 0.6 pg/ml vs. 0 +/- 0.1 pg/ml, control vs. hypoxic endothelial cells; p < 0.05), but in astrocytes hypoxia induced an increase of ET-1 at 6 hours (1.5 +/- 0.6 vs. 6.4 +/- 1.1 pg/ml, control vs. hypoxic astrocytes; p < 0.05). Endothelin-1 is released from astrocytes, but not endothelial cells, during hypoxia and is released from the brain after transient ischemia. There is no relationship between ET-1 and vasospasm in vivo or between ET-1 and oxyhemoglobin, a putative agent of vasospasm, in vitro. The increase in ET-1 levels in CSF after SAH from a ruptured intracranial aneurysm appears to be the result of cerebral ischemia rather than reflecting the cause of cerebral vasospasm.

Point-of-care Xpert® MTB/RIF for smear-negative tuberculosis suspects at a primary care clinic in South Africa.

OBJECTIVE: To assess the clinical utility and cost of point-of-care Xpert® MTB/RIF for the diagnosis of smear-negative tuberculosis (TB). DESIGN: Cohort study of smear-negative TB suspects at a South African primary care clinic. Participants provided one sputum sample for fluorescent smear microscopy and culture and an additional sample for Xpert. Outcomes of interest were TB diagnosis, linkage to care, patient and provider costs. RESULTS: Among 199 smear-negative TB suspects, 16 were positive by Xpert, 15 by culture and 7 by microscopy. All cases identified by Xpert began anti-tuberculosis treatment the same or next day; only one of five Xpert-negative culture-positive cases started treatment after 34 days. Xpert at point of care offered similar diagnostic yield but a faster turnaround time than smear and culture performed at a centralized laboratory. Compared to smear plus culture, Xpert (at US$9.98 per cartridge) was US$3 less expensive per valid result (US$21 vs. US$24) and only US$6 more costly per case identified (US$266 vs. US$260). CONCLUSION: Xpert is an effective method of diagnosing smear-negative TB. It is cost saving for patients, especially if performed at point of care, but it is costly for health care providers. Data-driven studies are needed to determine its cost-effectiveness in resource-poor settings with diverse diagnostic practices.

Augmentation of virus secretion by the human immunodeficiency virus type 1 Vpu protein is cell type independent and occurs in cultured human primary macrophages and lymphocytes.

The human immunodeficiency virus type 1-specific Vpu protein is a small integral membrane phosphoprotein that induces degradation of the virus receptor CD4 in the endoplasmic reticulum and, independently, increases the release of progeny virions from infected cells. To address the importance of Vpu for virus replication in primary human cells such as peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM), we used three different sets of monocyte-tropic molecular clones of human immunodeficiency virus type 1: a primary isolate, AD8+, and two chimeric variants of the T-cell-tropic isolate NL4-3 carrying the env determinants of either AD8+ or SF162 monocyte-tropic primary isolates. Isogenic variants of these chimeric viruses were constructed to express either wild-type Vpu or various mutants of Vpu. The effects of these mutations in the vpu gene on virus particle secretion from infected MDM or PBMC were assessed by determination of the release of virion-associated reverse transcriptase into culture supernatants, Western blot (immunoblot) analysis of pelleted virions, and steady-state or pulse-chase metabolic labeling. Wild-type Vpu increased virus release four- to sixfold in MDM and two- to threefold in PBMC, while nonphosphorylated Vpu and a C-terminal truncation mutant of Vpu were partially active on virus release in primary cells. These results demonstrate that Vpu regulates virus release in primary lymphocyte and macrophage cultures in a similar manner and to a similar extent to those previously observed in HeLa cells or CD4+ T-cell lines. Thus, our findings provide evidence that Vpu functions in a variety of human cells, both primary cells and continuous cell lines, and mutations in Vpu affect its biological activity independent of the cell type and virus isolate used.

Enumeration of viral antigen-reactive helper T lymphocytes in human peripheral blood by limiting dilution for analysis of viral antigen-reactive T-cell pools in virus-seropositive and virus-seronegative individuals.

A limiting-dilution analysis technique was developed which enumerates human T cells with the capacity to secrete T-cell growth factors such as interleukin 2 after contact with herpes simplex virus type 1 (HSV-1) or cytomegalovirus (CMV) antigens (operationally defined as virus-reactive helper T cells [HTL]). By using this limiting-dilution analysis technique, the peripheral blood of HSV-seropositive individuals was analyzed for the frequency of HSV antigen-reactive HTL and for the ability either to proliferate or to secrete detectable T-cell growth factors in conventional HSV antigen-stimulated lymphocyte cultures. We found that the magnitudes of the latter two responses did not correlate directly with the frequency estimates of HSV antigen-reactive HTL. The study was expanded to analyze both HSV and CMV reactivities within individuals. Those who were seropositive for HSV or CMV were found to have relatively high HTL frequencies for the viral antigens to which they were sensitized. However, those who were seronegative for one of the viruses often had HTL reactive with that virus in their peripheral blood. These latter HTL frequencies were highly variable and ranged from undetectable to quite prominent, even within the same individual at different times. In general, it was found that viral antigen-reactive serologic activity does not necessarily reflect the status of viral antigen-reactive cell-mediated immunity in humans and that viral antigen-induced T-cell responses may be unexpectedly complex, rather than absent, in individuals who are seronegative for a particular virus.

Detection of HSV-1-induced lymphokine production in human cells by a direct indicator cell addition assay.

These studies present an efficient and sensitive method for detection of T cell growth factor (TCGF) activity in human lymphocyte cultures and illustrate that T cell growth factors are associated with T lymphocyte-mediated anti-HSV-1 responses. Secretion of TCGF is induced after stimulation of human peripheral blood mononuclear cells ( PBMNC ) with herpes simplex virus type 1 (HSV-1). Lymphokine activity is detected in a simple, sensitive method by studying [3H]thymidine incorporation after the addition of murine CTLL -20 cells to cultures of gamma-irradiated (4000 R), virus-stimulated PBMNC . By using this assay, we find that PBMNC from seropositive but not seronegative individuals produce detectable TCGF activity in a dose-dependent manner after incubation with HSV-1. Maximum activity is detected between 24 to 48 hr of incubation and correlates with in vitro proliferation of nonirradiated PBMNC in response to the virus. In addition, gamma-irradiated (1000 to 3000 R) PBMNC , which are frequently used as a source of antigen-presenting cells (APC), can secrete TCGF after contact with HSV-1. Lymphokine production by the APC-containing population is eliminated by gamma-irradiation (5000 R); such APC can still present UV-inactivated HSV-1 to HSV-1-responsive lymphoblasts, indicating that lymphokine production by T cells residing in the APC population is not essential for antigen presentation.

Hrb27C, Sqd and Otu cooperatively regulate gurken RNA localization and mediate nurse cell chromosome dispersion in Drosophila oogenesis.

Heterogeneous nuclear ribonucleoproteins, hnRNPs, are RNA-binding proteins that play crucial roles in controlling gene expression. In Drosophila oogenesis, the hnRNP Squid (Sqd) functions in the localization and translational regulation of gurken (grk) mRNA. We show that Sqd interacts with Hrb27C, an hnRNP previously implicated in splicing. Like sqd, hrb27C mutants lay eggs with dorsoventral defects and Hrb27C can directly bind to grk RNA. Our data demonstrate a novel role for Hrb27C in promoting grk localization. We also observe a direct physical interaction between Hrb27C and Ovarian tumor (Otu), a cytoplasmic protein implicated in RNA localization. We find that some otu alleles produce dorsalized eggs and it appears that Otu cooperates with Hrb27C and Sqd in the oocyte to mediate proper grk localization. All three mutants share another phenotype, persistent polytene nurse cell chromosomes. Our analyses support dual cooperative roles for Sqd, Hrb27C and Otu during Drosophila oogenesis.

Viral antigen stimulation of the production of human monokines capable of regulating HIV1 expression.

We have previously described model systems for cytokine-induced regulation of chronically HIV-infected promonocyte and T cell clones. Using these systems, we have shown that monokines contained in supernatants from LPS-stimulated human monocyte/macrophages (MO) up-regulate HIV expression, reflected by an increase in reverse transcriptase activity, viral RNA levels, and expressed viral proteins. Current studies were designed to determine whether viral Ag can interact with MO and secondarily affect HIV1 expression by stimulating monokine production. We found that certain herpes-group viruses, including CMV and EBV, augment HIV1 expression by inducing monokine production, whereas others, such as HSV1, HSV2, varicella-zoster virus, and human herpes virus 6 were unable to function in this capacity. The HSV1 and HSV2 Ag which failed to stimulate monokine production did not interfere with MO stimulation by CMV Ag, suggesting that failure to induce HIV expression was not attributable to MO suppression. When nonherpes group viruses were tested, we found that human adenovirus, hepatitis B virus, and vaccinia virus all failed to stimulate the production of monokines capable of activating HIV in the chronically infected cell lines. In contrast, HIV1 can augment its own expression by inducing the secretion of monokines which up-regulate HIV expression in the infected cells. The viral Ag-induced MO supernatants capable of up-regulating HIV expression did so in a dose-dependent manner, whereas viral Ag alone produced no significant change. Monokine production mediated by viral Ag was not attributable to contaminating endotoxin. These studies provide a model to determine whether other opportunistic infections may induce the expression of HIV by indirect mechanisms, such as the stimulation of cytokine production.

Soluble tumor necrosis factor receptor: inhibition of human immunodeficiency virus activation.

The inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) has been shown to stimulate human immunodeficiency virus type 1 (HIV-1) replication in both chronically and acutely infected T lymphocytes and monocytes. Transcriptional activation of the HIV long terminal repeat and subsequent increase in virus production are linked to TNF activation of the cellular transcription factor NF-kappa B. Here we report the use of two forms of soluble recombinant type 1 (p80) TNF receptor to inhibit TNF-induced HIV activation in vitro. One receptor form is a monomer containing the entire 236 residues of the extracellular (ligand-binding) portion of p80. A second receptor form is a chimeric homodimer containing these residues fused to a truncated human IgG1 immunoglobulin heavy chain and, thus, resembles a bivalent antibody without light chains. These recombinant receptor proteins were tested for their ability to inhibit TNF-alpha-induced expression of HIV-1 in chronically infected human cell lines. We also examined the ability of the soluble receptors to limit the activation of the HIV-long terminal repeat transcription. The soluble TNF receptor dimer was most effective at blocking TNF-alpha-induced HIV-1 expression in both monocytoid and lymphoid cells. The molar ratio of TNF-receptor dimer to TNF-alpha found to be most effective was, at least, 5:1. We conclude that at specific TNF/soluble TNF-receptor dimer ratios, TNF-alpha-induced HIV-1 transcription and expression can be limited in vitro.

Stimulation with autologous lymphoblastoid cell lines: lysis of Epstein-Barr virus-positive and -negative cell lines by two phenotypically distinguishable effector cell populations.

Human lymphocytes, stimulated in vitro for 6 days with x-irradiated or glutaraldehyde-treated autologous Epstein-Barr (EB) virus-transformed lymphoblastoid cell lines (LCL), are cytotoxic for autologous and allogeneic EB+ LCLs as well as for several EB- cell lines that are also susceptible to lysis by interferon-activated natural killer (NK) cells. To determine whether the apparent nonspecific lysis mediated by LCL-stimulated cells is due to a mixture of effector cells directed against different target cells, advantage was taken of our recent finding that monoclonal antibody OKT8 reacts with human cytotoxic T lymphocytes but not with NK cells or NK-like cells generated in mixed leukocyte cultures. The depletion of OKT8+ cells from LCL-stimulated cultures by treatment with OKT8 and complement abolished or markedly depleted cytotoxicity against all EB+ target cells tested, whereas cytotoxicity against EB-, NK-sensitive cell lines including K562, MOLT-4 and HSB-2 was not or only minimally reduced. These results indicate that stimulation with autologous LCL results in the generation of OKT8+ cytotoxic T lymphocytes that lyse EB virus-transformed LCL and OKT8- NK-like cells that lyse EB-, NK-sensitive cells.

Phenotypes of human natural killer cell populations detected with monoclonal antibodies.

In our recent studies, human natural killer (NK) cell activity was found to be decreased 2- to 4-fold after treatment of monocyte-depleted peripheral mononuclear cells with monoclonal antibody OKM1 and complement (C). The present study was undertaken to determine whether there is an additional population of NK cells that is OKM1-, since treatment with OKM1 and C decreased, but did not eradicate, NK cell activity. Treatment of lymphocytes with monoclonal antibody OKT11A, which reacts with all sheep red blood cell rosetting lymphocytes, and C also decreased NK cell activity. Although approximately 90% of OKT11A+ cells are OKT3+, NK cell activity resides within the OKT11A+ cell population, which is OKT3- since OKT3-cell depletion fails to decrease NK cell activity. Double fluorescence analysis of OKT3-depleted lymphocytes revealed that 54% of the OKM1+ cells are OKT11A- and 45% of the OKT11A+ cells are OKM1-, thus demonstrating that within the OKT3-depleted population, approximately one-half the OKM1+ cells are OKT11A- and vice versa. Treatment of lymphocytes with OKM1 together with OKT11A and C decreased NK cell activity against 3 NK-sensitive leukemia lines--K562, MOLT-4, and HSB-2--more than did treatment with either antibody alone; virtually no lytic activity was retained after elimination of OKM1+ and OKT11A+ cells. The results thus provide strong evidence that there is at least 2 populations of human NK cells; one is OKM1+ and the other is OKT11A+