Sustained impairment of IFN-gamma secretion in suppressed HIV-infected patients despite mature NK cell recovery: evidence for a defective reconstitution of innate immunity

The impairment of NK cell functions in the course of HIV infection contributes to a decreased resistance against HIV and other pathogens. We analyzed the proportion of mature and immature NK cell subsets, and measured subsets of IFN-gamma and TNF-alpha-producing NK and T cells in viremic or therapy-suppressed HIV-infected subjects, and noninfected control donors. Viremic HIV(+) individuals had significantly lower proportions of mature CD3(-)/CD161(+)/CD56(+) NK cells and of IFN-gamma-producing NK cells compared with noninfected donors, independent of CD4(+) T cell counts. HIV-infected subjects with undetectable viral load recovered mature CD3(-)/CD161(+)/CD56(+) NK cells and cytotoxicity against tumor (K562) and HSV-infected target cells to percentages comparable with those of uninfected individuals, but their NK cells remained impaired in their ability to produce IFN-gamma. In parallel to these ex vivo findings, in vitro NK cell differentiation of CD34-positive cord blood precursors in the presence of R5 or X4 HIV-1 resulted in the production of NK cells with a normal mature phenotype, but lacking the ability to produce IFN-gamma, whereas coculture of uninfected PBMC with HIV failed to affect mature NK cell properties or IFN-gamma secretion. Altogether, our findings support the hypothesis that mature NK cell phenotype may be uncoupled from some mature functions following highly active antiretroviral therapy-mediated suppression of HIV-1, and indicate that relevant innate immune functions of NK cell subsets may remain altered despite effective viral suppression following antiretroviral treatment.     

Differential production of IFN-gamma, analyzed at the single-cell level, by specific subsets of human NK and T cells from healthy and HIV(+) subjects

BACKGROUND: Interferon gamma is a cytokine that plays a central role in immunity, and is physiologically secreted by T and NK cells under appropriate stimuli during the immune response. By means of flow cytometry, we performed a single cell analysis of interferon gamma producing NK cells and their surface phenotype in normal and HIV(+) individuals that show several defects of cytokine production and cellular immunity. METHODS: PBMC or purified NK cells were stimulated for 1-12 h with PMA/ionomycin in the presence of monensin, subsequently stained for surface CD56 and CD3 or CD8, and for intracytoplasmic IFN-gamma, and analysed by flow cytometry. RESULTS: Our results show that CD56(+) NK cells are more efficient interferon gamma producers than T cells. Moreover, within the CD56(+) NK cell population, those that co-express low density CD8 are the best producers. Finally, we show that NK cells during HIV infection are more massively recruited to interferon gamma production than those from normal subjects. CONCLUSIONS: Both in the normal and HIV(+) subjects, a higher percentage of NK cells than T cells can produce IFN-gamma although differences can be identified within the NK cells subset in terms of IFN-gamma production. The production of IFN-gamma is fully achievable in the HIV(+) subjects, which is consistent with their elevated plasmatic levels of the cytokine. The possibility that NK cells that produce interferon gamma could represent a functionally distinct population committed to the production of this cytokine, is discussed.     

CD48 stimulation by 2B4 (CD244)-expressing targets activates human NK cells

Human NK cells can be activated by a variety of different cell surface receptors. Members of the SLAM-related receptors (SRR) are important modulators of NK cell activity. One interesting feature of the SRR is their homophilic interaction, combining receptor and ligand in the same molecule. Therefore, SRR cannot only function as activating NK cell receptors, but also as activating NK cell ligands. 2B4 (CD244) is the only SRR that does not show homophilic interaction. Instead, 2B4 is activated by binding to CD48, a GPI-anchored surface molecule that is widely expressed in the hemopoietic system. In this study, we show that 2B4 also can function as an activating NK cell ligand. 2B4-expressing target cells can efficiently stimulate NK cell cytotoxicity and IFN-gamma production. Using soluble receptor fusion proteins and SRR-transfected cells, we show that 2B4 does not bind to any other SRR expressed on NK cells, but only interacts with CD48. Lysis of 2B4-expressing target cells can be blocked by anti-CD48 Abs and triggering of CD48 in a redirected lysis assay can stimulate NK cell cytotoxicity. This demonstrates that 2B4 can stimulate NK cell cytotoxicity and cytokine production by interacting with NK cell expressed CD48 and adds CD48 to the growing number of activating NK cell receptors.     

Increased susceptibility to apoptosis of CD56dimCD16+ NK cells induces the enrichment of IFN-gamma-producing CD56bright cells in tuberculous pleurisy

Tuberculous pleuritis is a good model for the study of specific cells at the site of active Mycobacterium tuberculosis (Mtb) infection. We investigated the frequency and phenotype of NK cells in paired samples of peripheral blood and pleural fluid (PF) from patients with tuberculosis (TB) or parapneumonic infection. We demonstrated for the first time a reduction of NK cells in PF from TB with an enrichment in the CD56brightCD16- subset. In agreement, in PF NK cells we observed an increased expression of CD94, NKG2A, CD62L, and CCR7 molecules and lower expression of Bcl-2 and perforin. The activation markers CD69 and HLA-DR were also increased. The enrichment in the CD56bright subset was due to an increased susceptibility to apoptosis of CD56+CD16+ NK cells mediated by heat-labile and stable soluble factors present in tuberculous effusions and not in PF from other etiologies. Furthermore, in TB patients, Mtb-induced IFN-gamma production by PF NK cells was not dependent on the presence of CD3+, CD19+, and CD14+ cells, suggesting a direct interaction of CD56bright cells with Mtb and/or the involvement of other accessory cells present at the site of Mtb infection.     

NK1.1+ thymocytes. Adult murine CD4-, CD8- thymocytes contain an NK1.1+, CD3+, CD5hi, CD44hi, TCR-V beta 8+ subset.

CD4-, CD8- thymocytes were purified from thymi obtained from normal C57BL/6 mice. By flow cytometry analysis, 5 to 10% of these double negative (DN) thymocytes were found to express NK1.1 on their surface. The NK1.1+ DN thymocytes were demonstrated, by two-color fluorescence, to be CD3lo, CD5hi, CD44hi, J11d-, B220-, MEL 14-, IL2R- with 60% expressing TCR-V beta 8 as determined by the mAb F23.1. In contrast, splenic and peripheral blood NK cells were NK1.1+, CD3-, CD5-, TCR-V beta 8- with 40 to 60% being MEL 14+. Unlike peripheral NK cells, fresh DN thymocytes enriched for NK1.1+ cells were unable to kill YAC-1, the classical murine NK cell target. However, these cells were able to mediate anti-CD3 redirected lysis even when they were assayed immediately after purification, i.e., with no culture or stimulation. These data demonstrate that adult murine thymocytes contain NK1.1+ cells which are distinct, both by function and phenotype, from peripheral NK cells. These data also raise the issue of a possible NK/T bipotential progenitor cell.     

NK cell inhibitory receptors prevent tyrosine phosphorylation of the activation receptor 2B4 (CD244)

2B4 is an NK cell activation receptor that can provide a co-stimulatory signal to other activation receptors and whose mode of signal transduction is still unknown. We show that cross-linking of 2B4 on NK cells results in its rapid tyrosine phosphorylation, implying that this initial step in 2B4 signaling does not require coligation of other receptors. Ligation of 2B4 in the context of an NK cell-target cell interaction leads to 2B4 tyrosine phosphorylation, target cell lysis, and IFN-gamma release. Coligation of 2B4 with the inhibitory receptors killer cell Ig-like receptor (KIR)2DL1 or CD94/NKG2 completely blocks NK cell activation. The rapid tyrosine phosphorylation of 2B4 observed upon contact of NK cells with sensitive target cells is abrogated when KIR2DL1 or CD94/NKG2 are engaged by their cognate MHC class I ligand on resistant target cells. These results demonstrate that NK inhibitory receptors can interfere with a step as proximal as phosphorylation of an activation receptor.     

Target cell induced activation of NK cells in vitro: cytokine production and enhancement of cytotoxic function

This study examines the effect of fixed AK-5 tumour cells on rat NK cells. Co-culture of NK cells with fixed tumour cells augmented the cytotoxicity of NK cells against NK-sensitive targets, YAC-1 and AK-5, and induced the secretion of IFN-gamma by NK cells. Antibody against IFN-gamma suppressed the anti-tumour activity of NK cells, whereas the addition of T cells during co-culture enhanced this activity. However, macrophages and B cells had no significant effect when present during co-culture with NK cells. All the inducible cytotoxicity was contained within the NK (CD161+) and NKT (CD3+, CD161+) subsets of lymphocytes. However, in the presence of T cells, the cytolytic potential of NKT cells was higher than that of NK cells alone. The augmentation of cytotoxic activity of NK cells by AK-5 cells in presence of T cells was dependent on IL-2 and IFN-gamma secretion. NK cell activation was blocked by specific antibodies to IL-2 and IFN-gamma in the presence of T cells. Interaction between fixed AK-5 cells with NK and T cell populations induced the expression of Fas-L and perforin in NK cells. These data demonstrate that fixed AK-5 cells initiated cytokine synthesis by NK cells, and the enhanced cytotoxic activity in the presence of T cells was induced as a consequence of the products secreted by activated T lymphocytes. The present observations reflect the possible interactions taking place in vivo after the transplantation of AK-5 tumour in animals. They also suggest direct activation of NK cells after their interaction with the tumour cells.     

Lipopolysaccharide stimulates the proliferation of human CD56+CD3- NK cells: a regulatory role of monocytes and IL-10

NK cells recognize and kill tumor cells and normal cells, and these play an important role in immune defense in cancer, infectious disease, and autoimmunity. NK killing is regulated by positive or negative signals derived from the interaction of surface receptors with ligands on the target cells. However, the mechanisms controlling the proliferation and maintenance of NK cells in normal human individuals are less clearly defined. In this study, using an entirely autologous system, we demonstrate that human peripheral blood CD3-CD56+, killer cell-inhibitory receptor (KIR)-expressing cells proliferate and expand in response to LPS. These responses are enhanced in the presence of anti-IL-10 receptor-blocking Abs or on the removal of CD14+ cells from the cultures. This enhancement is also reflected in substantial increases in cytolytic activity and IFN-gamma production. The negative effect of CD14+ cells may also be IL-10 mediated, IL-10 being lost from the culture supernatants of CD14-depleted PBMC and rIL-10 reversing the effect of this depletion. On the other hand, mRNA for the p35 and p40 subunits of IL-12 is still induced in CD14-depleted cultures. The expansion of CD3-CD56+ cells was also inhibited by CTLA4-Ig, indicating a role for CD80/86. B lymphocytes were not required for the expansion of CD3-CD56+ cells, whereas removal of MHC class II+ cells from CD14-depleted cultures resulted in a complete abrogation of these responses. Expansion of CD3-CD56+ cells was reconstituted in MHC class II-depleted cell cultures by adding back monocyte-derived dendritic cells. These results indicate that the responses of CD3-CD56+ NK cells to LPS may be driven by a MHC class II+ B7+ CD14- peripheral population, most likely blood dendritic cells.     

TGF-beta utilizes SMAD3 to inhibit CD16-mediated IFN-gamma production and antibody-dependent cellular cytotoxicity in human NK cells

TGF-beta can be a potent suppressor of lymphocyte effector cell functions and can mediate these effects via distinct molecular pathways. The role of TGF-beta in regulating CD16-mediated NK cell IFN-gamma production and antibody-dependent cellular cytotoxicity (ADCC) is unclear, as are the signaling pathways that may be utilized. Treatment of primary human NK cells with TGF-beta inhibited IFN-gamma production induced by CD16 activation with or without IL-12 or IL-2, and it did so without affecting the phosphorylation/activation of MAP kinases ERK and p38, as well as STAT4. TGF-beta treatment induced SMAD3 phosphorylation, and ectopic overexpression of SMAD3 resulted in a significant decrease in IFN-gamma gene expression following CD16 activation with or without IL-12 or IL-2. Likewise, NK cells obtained from smad3(-/-) mice produced more IFN-gamma in response to CD16 activation plus IL-12 when compared with NK cells obtained from wild-type mice. Coactivation of human NK cells via CD16 and IL-12 induced expression of T-BET, the positive regulator of IFN-gamma, and T-BET was suppressed by TGF-beta and by SMAD3 overexpression. An extended treatment of primary NK cells with TGF-beta was required to inhibit ADCC, and it did so by inhibiting granzyme A and granzyme B expression. This effect was accentuated in cells overexpressing SMAD3. Collectively, our results indicate that TGF-beta inhibits CD16-mediated human NK cell IFN-gamma production and ADCC, and these effects are mediated via SMAD3.     

Rapidly expanded activated human killer cell clones have strong antitumor cell activity and have the surface phenotype of either T gamma, T-non-gamma, or null cells

Cloned lymphoid cell lines showing cytolytic activity were derived from natural killer (NK) cell-enriched cell fractions obtained by fluorescence-activated cell sorting of cells that reacted with B73 .1, an NK cell-specific monoclonal antibody (MCA). The clones were cultured for more than 30 generations (i.e., more than 10(9) descendants from a single cell). The rapid expansion was achieved by using a special culture system developed for this purpose and based on the use of two types of allogeneic feeder cells. Three phenotypically different types of cytotoxic clones were obtained. These clones showed a broad spectrum of cytolytic activity against several NK-susceptible and NK-nonsusceptible tumor target cells. One of these clones had the following binding pattern to MCA: B73 .1+, T3-, T4-, T8-, HNK1 -, and Lyt-3-. These cells formed rosettes with IgG-coated erythrocytes but not with sheep erythrocytes, and therefore might be null cell-derived. Most of the cytotoxic clones showed the following phenotype: B73 .1+, T3-, T4-, T8-, HNK1 -, Lyt-3+, E+, and EA-gamma +. These clones were probably derived from T-gamma cells. In addition, one clone with cytolytic activity was derived from B73 .1- cells. This had the phenotype B73 .1-, T3+, T4-, T8-, HNK1 -, Lyt-3+, E+, and EA-gamma-, and may be of T-non-gamma cell origin. About 10 noncytolytic clones showed the phenotype B73 .1-, T3+, T4, or T8+, HNK1 -, Lyt-3+, Ia+, E+, and EA-gamma -. An absolute correlation was found between the presence of the B73 .1 antigen, the absence of the T3 marker, and the capacity of the cells to form EA rosettes. Furthermore, all clones except one (Lyt-3-) formed E rosettes. Although the in vitro life span varied from clone to clone, B73 .1- clones generally grew faster and for longer times (greater than or equal to 50 generations) than did B73 .1+ ones (less than or equal to 40 generations). The cytolytic activity, cell surface phenotype as determined with MCA, rosette formation, and target cell specificity spectrum remained stable over the entire culture period. We conclude that the majority of the activated MHC-nonrestricted cytolytic clones obtained in this culture system show a particular phenotype. These cells can be expanded to large numbers. Whether or not these clones might be derived from B73 .1+, HNK1 + NK cells with the morphologic appearance of large granular lymphocytes will be discussed.     

Bystander activation of CD8+ T cells contributes to the rapid production of IFN-gamma in response to bacterial pathogens

The bacterium Burkholderia pseudomallei causes a life-threatening disease called melioidosis. In vivo experiments in mice have identified that a rapid IFN-gamma response is essential for host survival. To identify the cellular sources of IFN-gamma, spleen cells from uninfected mice were stimulated with B. pseudomallei in vitro and assayed by ELISA and flow cytometry. Costaining for intracellular IFN-gamma vs cell surface markers demonstrated that NK cells and, more surprisingly, CD8(+) T cells were the dominant sources of IFN-gamma. IFN-gamma(+) NK cells were detectable after 5 h and IFN-gamma(+) CD8(+) T cells within 15 h after addition of bacteria. IFN-gamma production by both cell populations was inhibited by coincubation with neutralizing mAb to IL-12 or IL-18, while a mAb to TNF had much less effect. Three-color flow cytometry showed that IFN-gamma-producing CD8(+) T cells were of the CD44(high) phenotype. The preferential activation of NK cells and CD8(+) T cells, rather than CD4(+) T cells, was also observed in response to Listeria monocytogenes or a combination of IL-12 and IL-18 both in vitro and in vivo. This rapid mechanism of CD8(+) T cell activation may be an important component of innate immunity to intracellular pathogens.     

Natural cytotoxicity against mouse hepatitis virus-infected cells. II. A cytotoxic effector cell with a B lymphocyte phenotype

The effector cell in mouse spleen which mediates natural cytotoxicity against mouse hepatitis virus (MHV)-infected target cells was characterized. The target cells were MHV-infected BALB/c 3T3, and the assay time was 3 hr. The effector cell, designated virus killer (VK) cell for the purpose of discussion, had the following phenotype: lymphocyte morphology, plastic-nonadherent, nylon wool-adherent, nonphagocytic, cyclophosphamide-sensitive; by antibody plus complement (C) depletion studies, it was asialo GM1-, NK 1.2 alloantigen-negative, Thy-1.2-, Lyt-5-, and macrophage antigen-negative; by rosetting techniques, it was Fc receptor-positive and surface Fab+; by flow cytometry (FACS) analysis, it was Lyt-2-, MAC-1-, Ia+, IgG (gamma)+, IgM (mu)+, IgD (delta)+, and B cell lineage antibody B-220+. NK cells, measured for cytotoxicity on YAC-1 cells, were similarly tested and were found to differ from the VK cell in the following properties: nylon wool-nonadherent, asialo GM1+, NK alloantigen-positive, Lyt-5+, surface Fab-, MAC-1+, Ia-, IgG-, IgM-, IgD-, and B-220-. The VK effector cell had a phenotype highly distinguishable from NK cells, effectors most commonly associated with antiviral natural cytotoxicity. The VK cell had a phenotype identical to that of a B lymphocyte and was identified as such. Although the effector cells displayed cell surface antibody, the antibody did not appear to be involved in lysis, because lysis could not be blocked by F(ab)'2 directed against Fab, mu, or delta. Cytotoxicity was more likely associated with recognition of the B lymphocyte surface by the MHV glycoprotein E2, as shown in the accompanying companion paper. This is the first demonstration that natural cytotoxicity can be mediated by B lymphocytes.     

Lymphokine-activated killer (LAK) cells. VI. NK1.1+, CD3+ LAK effectors are derived from CD4-, CD8-, NK1.1- precursors.

Normal murine splenocytes cultured with IL2 for 6, but not 3, days contained an NK1.1+, CD3+ lytically active subset. These lymphocytes were not derived from NK1.1+ precursors since NK1.1+ cells, purified by flow cytometry, failed to express CD3, as determined by the 145-2C11 mAb, on their surface even after culture with IL2 for 6 days. Instead, the precursors of the NK1.1+, CD3+ effectors were contained in a B cell-depleted CD4-, CD8-, NK1.1- splenic subset. Freshly obtained CD4-, CD8-, NK1.1- splenocytes were mostly CD3+, CD5+, B220-, had no spontaneous lytic activity against YAC-1, and were unable to mediate anti-CD3 directed lysis against FcR-bearing target cells. Culture of the CD4-, CD8-, NK1.1- splenocytes with IL2, for 6 days, resulted in the development of NK1.1+, CD3+, B220+ effectors 40% of which were CD5dim and 20-25% of which expressed TCR-V beta 8 as determined by the F23.1 mAb. The acquisition of NK1.1, B220, and lytic activity by this triple-negative subset was readily inhibited by cyclosporine A (CSA). On the other hand, CSA had no effect on the acquisition of B220 or lytic activity by NK1.1+ precursors obtained by flow cytometry sorting. Moreover, all of the NK1.1+ cells generated by IL2 culture of splenocytes obtained from mice depleted of NK1.1+ lymphocytes (by in vivo injection of anti-NK1.1 mAb) coexpressed CD3 on their surface and were thus distinct from classical NK cells. These findings demonstrate that splenic NK cells do not express or acquire CD3; that the NK1.1+, CD3+ LAK effectors are derived from an NK1.1- precursor; and that CSA is exquisitely selective in its inhibitory effect on LAK generation.     

An atypical population of NK cells that spontaneously secrete IFN-gamma and IL-4 is present in the intraepithelial lymphoid compartment of the rat

The intestinal lymphoid compartment of the rat is large and diverse, but the phenotype and functions of its constituent cell populations are not fully characterized. Using new methodology for the isolation and purification of rat intestinal intraepithelial lymphocytes (IELs), we previously identified a population of alphabeta- and gammadelta-TCR- NKR-P1A+ NK cells. These cells were almost completely restricted to the CD4-CD8- IEL population, and unlike peripheral NK cells in the rat, they were CD2-. We now report that rat intraepithelial NK (IENK) and peripheral NK cells are similar in morphology, in their ability to lyse NK-sensitive targets, and in their ability to suppress a one-way mixed lymphocyte culture. In contrast, however, intraepithelial and splenic NK cells differ markedly in two respects. First, IENK cells express high levels of ADP-ribosyltransferase 2 (a marker of regulatory T cells in the rat) and CD25, whereas peripheral NK cells do not. Second, unlike splenic NK cells, a substantial fraction of IENK cells appear to spontaneously secrete IL-4 and/or IFN-gamma. We conclude that the rat IEL compartment harbors a large population of NKR-P1A+CD3- cells that function as NK cells but display an activated phenotype and unusual cytokine profile that clearly distinguish them from splenic NK cells. Their phenotypic and functional characteristics suggest that these distinctive IENK cells may participate in the regulation of mucosal immunity.     

Development of intestinal intraepithelial lymphocytes, NK cells, and NK 1.1+ T cells in CD45-deficient mice

The transmembrane protein tyrosine phosphatase CD45 is differentially required for the development and function of B, T, and NK cells, with mice partially deficient for CD45 having a significant inhibition of T cell, but not NK or B cell, development. CD45-mediated signaling has also been implicated in the development of intrathymic, but not extrathymic, intestinal intraepithelial T lymphocytes (iIELs) in the CD45ex6(-/-) mouse. As NK1.1(+) CD3(+) (NK-T) cells can also develop through extrathymic pathways, we have investigated the role of CD45 in NK-T cell development. In mice with a complete absence of CD45 expression (CD45ex9(-/-)) the NK-T cell population was maintained in the iIEL compartment, but not in the spleen. Functionally, CD45-deficient NK-T cells were unable to secrete IL-4 in response to TCR-mediated signals, a phenotype similar to that of CD45-deficient iIELs, in which in vitro cytokine production was dramatically reduced. Using the CD45ex9(-/-) mouse strain, we have also demonstrated that only one distinct population of NK-T cells (CD8(+)) appears to develop normally in the absence of CD45. Interestingly, although an increase in cytotoxic NK cells is seen in the absence of CD45, these NK calls are functionally unable to secrete IFN-gamma. In the absence of CD45, a significant population of extrathymically derived CD8alphaalpha(+) iIELs is also maintained. These results demonstrate that in contrast to conventional T cells, CD45 is not required during the development of CD8(+) NK-T cells, NK cells, or CD8alphaalpha(+) iIELs, but is essential for TCR-mediated function and cytokine production.     

The human MHC-restricted cellular response to herpes simplex virus type 1 is mediated by CD4+, CD8- T cells and is restricted to the DR region of the MHC complex

The nature of the in vitro human cytotoxic T-cell responder population to HSV type 1 (HSV-1) was studied. In 5-day HSV-1-stimulated cultures that contained MHC-restricted activity, two phenotypically distinct populations of cells were present that were capable of lysing HSV-1-infected B cell lines in a 5-h 51Cr-release assay. The first was CD4+, CD8-, CD16- cell typical of class II-restricted T cells, whereas the other population bore a CD4-, CD8-, CD16+ NK-cell phenotype. Elimination of the NK cell fraction from bulk cultures by using anti-CD16 plus C frequently resulted in cell populations that killed in an Ag-specific, HLA-DR-restricted fashion. In some cases the anti-CD16-pretreated cultures retained a killing population that was unrestricted to MHC products. In no instance were any cytotoxic T cells that were restricted to class I Ag in evidence. Limiting dilution analysis of precursor frequency indicated that about 1 in 4000 to 1 in 8000 cells from peripheral blood are specific for HSV-1 in seropositive individuals. Comparisons of HLA class I-matched and HLA class II-matched targets with the autologous target by using limiting dilution analysis yielded results entirely consistent with those obtained in the bulk culture assay system.     

B cell induction of IL-13 expression in NK cells: role of CD244 and SLAM-associated protein

NK cells are an important component of the innate immune system that can also interact with B cells in a mutually productive manner. We have previously shown that activated B cells can induce NK cells to up-regulate their secretion of IFN-gamma. In this study, we show that B cells, and, particularly, marginal zone B cells, can, in addition, induce NK cells via direct cell-cell interactions to express mRNA encoding the Th2 cytokine IL-13. The induction of NK cell IL-13 mRNA expression requires the ligation of the CD244 receptor by the CD48 ligand on B cells via signaling pathways that depend upon expression of the X-linked lymphoproliferative disease gene product, SH2D1A/DSHP/SAP (SLAM-associated protein, or SAP) in NK cells. Thus, the positive signals attributed to the B cell activation of CD244 on murine NK cells appears to be more similar to the activity of CD244 on human cells. The induction of IL-13 mRNA by B cells may account for the effect of NK cells on the generation of Th2-type responses in the presence of some adjuvants.     

GP120 specific cellular cytotoxicity in HIV-1 seropositive individuals. Evidence for circulating CD16+ effector cells armed in vivo with cytophilic antibody

Fresh circulating PBMC from HIV-1 seropositive individuals have been found to mediate specific, non-MHC restricted lysis of targets expressing the major envelope glycoprotein of HIV-1, gp120, in 6-h 51Cr release assays. This gp120 specific cell-mediated cytotoxicity (CMC) is broadly reactive against target cells infected with a wide range of viral isolates, is IL-2 augmentable, and is mediated by a CD16+, Leu-7+, CD15-, CD3- population of NK/K cells. The presence of FcR (CD16) on these cells suggested that the lytic specificity for gp120 might be directed by cytophilic antibody bound to the cell surface. Affinity purified F(ab')2 antibody fragments specific for the Fc and F(ab')2 portions of human IgG were used in attempts to block gp120 specific lysis. A 1/50 dilution of these antibodies inhibited gp120 specific cytolytic activity by more than 90% while exhibiting a minimal effect on NK/K cell lysis of K562 targets. The blocking activity of these fragments demonstrates the direct involvement of cytophilic antibody in CMC. In attempts to isolate this cytophilic anti-HIV-1 antibody, short 56 degrees C incubations were used to dissociate antibodies from the surface of PBMC of seropositive individuals. The supernatants generated in this manner exhibited specific gp120 activity in antibody-dependent cellular cytotoxicity assays. The ability of Staphylococcal protein A to remove this activity confirms the presence of cytophilic antibody on freshly isolated PBMC. Selective enrichment of specific cell subpopulations revealed the origin of the cytophilic antibody to be CD16+ NK/K cells and not B cells, T cells, or monocytes/macrophages. These studies show that the gp120-specific CMC seen in HIV-1 seropositive individuals is directed by cytophilic antibody bound to circulating CD16+ NK/K cells and represents a form of direct antibody-dependent cellular cytotoxicity which may provide a primary cytotoxic host defense.     

EBV-specific CD8+ T cell memory: relationships between epitope specificity, cell phenotype, and immediate effector function

EBV infection in humans induces CD8+ T cell memory to viral epitopes derived from both lytic and latent cycle Ags. We have analyzed the relationship between the phenotype and function of the memory pool of T cells specific for these Ags. Lytic epitope-specific populations were heterogeneous in terms of CD45RO/RA and CD28 expression, whereas latent epitope-specific populations were uniformly CD45RO+ and CD28+, consistent with the higher antigenic challenge from lytic epitopes driving some memory cells toward a CD45RA+, CD28- phenotype. However, both types of memory population showed immediate epitope-specific cytotoxicity and type 1 cytokine production in ex vivo assays. Cytotoxic function was not associated with preactivated T cells, as EBV-specific populations were negative for activation markers such as CD69 or CD38, nor could cytotoxic function be ascribed to CD27- or CD56+ subsets, as such cells were not detected in EBV-specific memory. Furthermore, cytotoxicity was not limited to CD45RA+ and/or CD28- fractions, but also was observed in CD45RO+, CD28+ populations in lytic and latent epitope-specific memory. Cytokine (IFN-gamma, TNF-alpha) responses, measured by intracytoplasmic staining after peptide stimulation, also were detectable in CD45RO+ and RA+ subsets as well as CD28+ and CD28- subsets. Of other markers that were heterogeneous in both lytic and latent epitope populations, CCR7 gave the best discrimination of functionality; thus, CCR7+ cells consistently failed to give an IFN-gamma or TNF-alpha response, whereas many CCR7- cells were responsive. Our data are consistent with effector functions having a broad distribution among phenotypically distinct subsets of "effector memory" cells that have lost the CCR7 marker.