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The aim of this study was to evaluate the utility of donor-specific antibodies (DSA) and flow cytometry crossmatch (FCCM) as tools for predicting antibody-mediated rejection (AMR) in desensitized kidney recipients. Sera from 44 patients with DSA at the time of transplant were reviewed. Strength of DSA was determined by single antigen Luminex bead assay and expressed as mean fluorescence intensity (MFI). T- and B-cell FCCM results were expressed as mean channel shift (MCS). AMR was diagnosed by C4d deposition on biopsy. Incidence of early AMR was 31%. Significant differences in the number of DSAs (p = 0.0002), cumulative median MFI in DSA class I (p = 0.0004), and total (class I + class II) DSA (p0.0001) were found in patients with and without AMR. No significant difference was seen in MCS of T and B FCCM (p = 0.095 and p = 0.307, respectively). The three-yr graft survival in desensitized patients with DSA having total MFI9500 was 100% compared to 76% with those having total MFI9500 (p = 0.022). Desensitized kidney transplant recipients having higher levels of class I and total DSA MFI are at high risk for AMR and poor graft survival. Recipient DSA MFI appears to be a more reliable predictor of AMR than MCS of FCCM.

Fas ligand (FasL)-mediated cytotoxicity is initiated in natural killer (NK) cells through ligation of their activating receptors. The CD16 receptor has been shown to induce FasL expression and cytotoxicity in NK cells. In this study, we made the novel observation that FasL expression was upregulated in NKL cells stimulated through 2B4 and LFA-1 activating receptors, implying a role for FasL-mediated cytotoxicity early in the immune response. Coligation with CD94/NKG2A human leukocyte antigen (HLA) class I inhibitory receptor did not block the induced FasL expression; therefore, these opposing pathways appear to function independently. We also showed, however, that FasL-mediated cytotoxicity was downregulated in CD94/NKG2A-expressing LAK cells in response to the HLA-E ligand, suggesting a mechanism by which aberrant cells expressing class I may evade FasL-mediated cytotoxicity. Thus we show for the first time that 2B4, LFA-1, and CD94/NKG2A receptors are involved in modulating FasL expression and, therefore, cytotoxicity mediated by NK cells.

Major histocompatibility complex (MHC) class II antigens are expressed on human foreskin keratinocytes (HFKs) following exposure to interferon gamma. The expression of MHC class II proteins on the cell surface may allow keratinocytes to function as antigen-presenting cells and induce a subsequent immune response to virus infection. Invariant chain (Ii) is a chaperone protein which plays an important role in the maturation of MHC class II molecules. The sequential degradation of Ii within acidic endocytic compartments is a key process required for the successful loading of antigenic peptide onto MHC class II molecules. Since human papillomavirus (HPV) 16 E5 can inhibit the acidification of late endosomes in HFKs, the E5 protein may be able to affect proper peptide loading onto the MHC class II molecule. To test this hypothesis, HFKs were infected with either control virus or a recombinant virus expressing HPV16 E5 and the infected cells were subsequently treated with interferon-γ. ELISAs revealed a decrease of MHC class II expression on the surface of E5-expressing cells compared with control virus-infected cells after interferon treatment. Western blot analysis showed that, in cells treated with interferon gamma, E5 could prevent the breakdown of Ii and block the formation of peptide-loaded, SDS-stable mature MHC class II dimers, correlating with diminished surface MHC class II expression. These data suggest that HPV16 E5 may be able to decrease immune recognition of infected keratinocytes via disruption of MHC class II protein function.

Natural killer cytotoxicity is down-regulated by HLA Class I-specific inhibitory receptors classified as killer inhibitory receptors (KIRs) or C-type lectins. The regulation of their inhibitory signaling pathways is not completely understood. The YTINDY NK-like cell line was transfected to express p58.2 KIR (YT/C143 transfectant) or CD94/NKG2A C-type lectin (YT/CD94 transfectant); and YT/C143, but not YT/CD94, cytotoxicity was down-regulated by Class I. YT/C143 and YT/CD94 expressed equally low p56(lck) levels, suggesting that p56(lck) is not absolutely required for p58.2 signaling but may be required for CD94/NKG2A signaling. Lower SHP-1 levels and activity were observed in YT/CD94 compared to YT/C143. However, increasing SHP-1 to equivalent levels in YT/C143 did not restore inhibition in YT/CD94. Our results suggest that the combination of low p56(lck) and SHP-1 levels may be responsible for the absent inhibitory signal in YT/CD94. In addition, the possible expression of CD94/NKG2C activating receptor may override inhibitory signals transduced through CD94/NKG2A.

Umbilical cord blood (UCB) is now widely accepted as a source of stem cells in patients with malignant hematologic and genetic disorders. We have recently reported that in a series of 30 pediatric UCB transplant recipients comparable outcome to that anticipated with other unrelated stem cell sources. In our series, however, the probability of GVHD for grade III-IV was 9% and no UCB recipient developed chronic GVHD. The reason for the low incidence of GVHD after UCB transplantation is not fully understood. Because functional NK cells are among the first population of lymphocytes to be detected in UCB transplant recipients, 2 months post-transplant on average, we wanted to establish whether NK cells could be implicated in reducing the risk of GVHD. Here, we confirm that early NK cells detected in UCB transplant recipients activate the granzyme/perforin lytic pathway and, in addition, they can mediate Fas/Fas ligand (FasL) activity, a finding not previously reported. Both pathways develop simultaneously and are detectable months before the other lymphocytes, notably CD8 are fully functional. Our contention, therefore, is that the low GVHD observed in UCB recipients may be partially due to early NK cells.

Cell death by apoptosis is an efficient mechanism of eliminating unwanted or aberrant cells. Triggering of Fas, a member of the tumor necrosis factor (TNF) receptor superfamily, by anti-Fas antibodies or by the Fas ligand (FasL), has been shown to cause cell death by apoptosis. A recent study from our laboratory has demonstrated that Fas crosslinking leads to the dephosphorylation of the tumor suppressor retinoblastoma protein (Rb) and that this dephosphorylation is inhibited by calyculin A, a serine/threonine phosphatase inhibitor. In this investigation, we compared the effect of Fas crosslinking by CH11, an anti-Fas mAb, with two cyclin-dependent kinase (CDK) inhibitors, a peptide that specifically inhibits CDK2 (cdk2 inh) and roscovitine, which inhibits CDK2, CDC2, and CDK5. We illustrate that roscovitine induced DNA fragmentation, whereas cdk2 inh did not. In contrast to Fas-induced apoptosis, roscovitine-induced apoptosis was resistant to calyculin A. Both cdk2 inh and roscovitine induced cleavage of poly (ADP-ribose) polymerase (PARP) within 2 h. Roscovitine, however, led to the degradation of Rb, whereas cdk2 inh did not. Furthermore, both CH11 and roscovitine caused cell cycle arrest in S phase. In contrast, cdk2 inh did not have any effect on Jurkat cell cycle progression. Taken together, our results strongly suggest that the maintenance of Rb in its hyperphosphorylated form during S phase may be necessary for cell survival and that Rb dephosphorylation during S phase may constitute a crucial step in Fas-induced apoptosis.

Objective: The presence of small numbers of cells of donor origin in the circulation of recipients of organ transplants (microchimerism) may correlate with immunologic tolerance. As part of our ongoing studies on microchimerism, we evaluated the utility of seven PCR-based assays for the detection of the less abundant DNA in paired mixtures (100 ng total DNA). Design and methods: DNA samples were screened to identify pairs informative for one or more PCR assays. DNA mixtures from the informative pairs were then analyzed using at least one assay. The assays were based on the X-Y homologous region; a Y chromosome microsatellite locus; three autosomal microsatellite loci; the D1S80 minisatellite locus; and sequence specific oligonucleotide probe (SSOP) analysis of the HLA DRB1 locus. Results: About 0.1% of male DNA against a background of female DNA was detectable using primers for the X-Y homologous region, but the sensitivity was increased to 0.0001% using nested primers for the Y chromosome microsatellite marker. Analysis of the minor DNA component was difficult with the three autosomal microsatellite assays because of the presence of shadow bands. Similar problems with the D1S80 assay were resolved using more stringent PCR conditions, and the sensitivity was 0.1%. Using the DRB1 locus, we were able to detect 1% DNA in the mixed samples. Conclusions: These studies show that: (a) nested PCR for the Y chromosome is the most sensitive assay for the detection of microchimerism; (b) D1S80 is a useful marker for microchimerism; (c) additional optimization of analytical conditions is required if autosomal microsatellite markers and the SSOP assay are to be used for microchimerism analysis.

We and others have recently shown that human NK cells express the Fas ligand (FasL) constitutively and that they can trigger the lysis of Fas positive (Fas+) target cells (TC) by apoptosis. We have also previously demonstrated that NK cells exposed to sensitive TC temporarily lose their ability to lyse sensitive TC via the granule-mediated pathway and that this loss is recovered when inactivated NK cells (NKi) are incubated in medium supplemented with IL-2, IL-12 or IL-15. In this study, we investigated the fate of the Fas-lytic pathway in NK cells exposed to either Fas+ or Fas− TC. To this end, we exposed NK cells to Jurkat (Fas−) or Jurkat (Fas+) TC for up to 6 h, separated NK cells from the TC and assessed the residual lytic activity against K562, a traditional human NK cell target, Jurkat Fas+ and Jurkat Fas− TC. Fas lytic activity was determined in calcium free medium, in the presence or absence of two distinct Fas-blocking monoclonal antibodies and a Fas.Fc fusion protein. In parallel experiments, the extent of DNA fragmentation in the three TCs was also assayed by the JAM test. Our results indicate that: (i) NK cells exposed to susceptible Fas+ TC temporarily lose most of their lytic potential due to the granule-mediated pathway, while only partially losing the Fas-lytic pathway. They also partially lose their ability to fragment DNA. (ii) NK cells exposed to Fas+ TC completely recover the Fas lytic pathway and the ability to fragment DNA via the Fas/Fas ligand when incubated in medium supplemented with IL-2 for 18 h.

Several investigators have recently examined the effect of Fas (CD95)-mediated apoptotic cell death on target cells (TC). The effect of Fas-mediated death on viral RNA within the TC, however, has not been explored. In this study, we investigated the ability of the Fas pathway to mediate pre-lytic degradation of vesicular stomatitis virus (VSV) RNA and TC RNA. We show that engagement of Fas antigen on VSV-infected Jurkat cells induces pre-lytic degradation of VSV RNA transcripts, whereas full-length VSV genome RNA, known to be tightly associated with viral proteins, is not degraded. Cellular RNA, including beta-actin and glyceraldehyde-3-phosphate-dehydrogenase mRNAs, is also degraded by Fas-mediated cytotoxicity. In addition, Fas-mediated cytotoxicity reduced the yield of VSV plaque-forming units (PFU) from Jurkat by an average of 82.0%. An anti-Fas blocking Ab inhibited the RNA degradation and restored the number of VSV PFU to near control levels. These data indicate that the Fas lytic pathway could play a role in the elimination of viruses through degradation of intracellular viral RNA. reserved

We have previously shown that in cytolytic cells exposed to sensitive targets the mRNA of the cytolytic protein perforin undergoes rapid downregulation. We now demonstrate that perforin message undergoes accelerated turnover in NK3.3 cells exposed to sensitive TC. This inducible mRNA decay phenomenon is specific for cytolytic protein messages, as levels of the constitutive message β-actin are unchanged. This TC-induced perforin mRNA turnover cannot be attributed to a blockage of RNA synthesis, or to a rapid half-life ( t 1 2 ). To determine the region(s) within the perforin transcript responsible for governing this TC-mediated turnover event, various segments of the perforin cDNA were cloned and inserted into the 3′ UTR of rabbit beta globin (RG). Constructs containing perforin coding region cDNA, but not 3′ UTR cDNA, mediated TC-induced mRNA turnover. These data indicate that multiple elements governing perforin mRNA stability reside within the coding region, a novel type of mRNA regulation not previously described.

Lysis of target cells (TC) by cytolytic lymphocytes involves the secretion of cytoplasmic granules containing perforin and serine esterases by the effector cell (EC). Recently, a granule-independent cytolytic mechanism involving the interaction of the apoptosis-triggering Fas antigen (CD95) with Fas ligand (FasL) has been revealed in T cells. However, whether the Fas lytic pathway also functions in NK cells has not been established. We purified human peripheral NK cells (> 98% CD56+) and found that PMA and ionomycin treatment upregulated FasL message and stimulated the NK cells to lyse a Fas+ TC. This lysis was partially inhibited by the anti-Fas-blocking antibody M3 or by Fas.Fc fusion protein. We also found that FasL is constitutively expressed on the human NK-like leukemia cell line YT-INDY and that YT-INDY utilizes a Ca(2+)-independent Fas lytic pathway, as well as the granule pathway. We have previously shown that CD28/B7 interactions are involved in TC recognition by YT-INDY. K562 cotransfected with Fas and B7-1 (K562/Fas/B7) was lysed by YT-INDY at a higher level than a vector-transfected K562 line, whereas K562 transfected with Fas alone was not. Lysis of K562/Fas/B7 cotransfectants was partially Fas-mediated, as indicated by the presence of Ca(2+)-independent, M3-inhibitable lysis. Ca(2+)-independent, Fas-mediated lysis of several TC by YT-INDY was inhibited by anti-CD28 antibody. Anti-LFA-1 also inhibited Fas-mediated cytotoxicity in YT-INDY. Thus, fresh human NK cells and the human NK-like cell line YT-INDY are capable of using the Fas lytic pathway. In YT-INDY, CD28/B7 and LFA-1/ICAM interactions appear to influence the Fas lytic pathway.

Cell-mediated cytotoxicity (CMC) has traditionally been thought to involve the release of granule components, including perforin and granzymes, from the effector cell (EC) onto the target cell (TC) membrane. Recently, a granule-independent cytolytic mechanism involving the interaction of Fas antigen (CD95) with Fas ligand has been described. We have generated antisense perforin (YT-xP1) and granzyme B (YT-xGrB) transfectants of the human NK-like cell line YT-INDY. These transfectants have greatly reduced cytolytic ability when compared to the vector-transfected control cell line (YT-neo). In this study, however, we demonstrate that the antisense transfectants retain the ability to lyse Fas+ TC. Fas-mediated lysis is Ca(2+)-independent and is inhibited by a monoclonal anti-Fas blocking Ab, M3. By RT-PCR, we detect message for FasL in unstimulated YT-xP1 and YT-xGrB transfectants, as well as in unstimulated YT-neo. By flow cytometry, we show that YT-neo, YT-xGrB, and YT-xP1 constitutively express surface FasL. These data indicate that in a human NK-like cell line, similar to the murine system, the granule and Fas pathways of cytotoxicity function independently of one another. At least with the TC tested, our data also indicate that the granule and Fas pathways together account for nearly 100% of the cytolytic ability of YT-INDY.

Our laboratory has previously demonstrated that natural killer (NK) cell-mediated cytotoxicity is protein kinase C (PKC)-dependent and that PKC is translocated from the cytoplasm to the plasma membrane during NK cell activation. Furthermore, exposuring NK cells to a sensitive target cell for 4-6 hr at 37 degrees C rendered NK cells functionally inactive and these inactivated effector cells (i) do not turn over PI in response to K562 stimulation and (ii) lose mRNA for perforin and granzyme A and B less than 30 min after contact with K562. In this study, we first confirmed earlier findings that the interaction of sensitive target cells with human NK cells triggers an influx of extracellular calcium into NK cells. In addition, using flow cytometry we demonstrated that there was a delayed maximum uptake of extracellular calcium into functionally inactive NK cells when these cells were reexposed to fresh K562. Finally, we demonstrated that exposuring NK cells to K562 for 4 hr leads to a loss of NK cytotoxic activity and to the maximal expression of CD69.

Unrelated cord blood (UCB) hematopoietic stem cells were serially transplanted into two human leukocyte antigen (HLA)-identical siblings with T cell, B cell, natural killer cell severe combined immunodeficiency. Brother A received a 4/6-matched, HLA DRbeta1-identical but class I-disparate UCB graft after myeloablative dosages of busulfan, melphalan, and antithymocyte globulin. He experienced complete donor chimerism, severe acute gastrointestinal graft-versus-host disease (GVHD), and limited chronic skin GVHD that resolved with treatment. Two years later, brother B received unfractionated marrow from brother A after reduced-intensity conditioning with cyclophosphamide and antithymocyte globulin. Brother B experienced mixed-donor (i.e. original UCB) chimerism and no histologically documented GVHD. Both brothers are clinically well; brother A is in a fully immunologically reconstituted state. The uneventful course and progressive increase in donor chimerism after the second transplantation indicates that hematopoietic cells derived from the older brother's marrow engrafted without causing GVHD, suggesting that acquired tolerance to disparate unrelated HLA antigens was achieved.

Objective The primary function of chemokines is the regulation of leukocyte trafficking by stimulating directional chemotaxis. The chemokine CXCL14 (BRAK) is highly expressed in all normal tissues, but is not expressed in most malignant tissues. The chemotactic activity of CXCL14 has been difficult to characterize. Recently it was reported that CXCL14 is a chemoattractant for activated monocytes and immature dendritic cells. Given that CXCL14 is downregulated upon transition to malignancy, we sought to characterize whether CXCL14 might play a role in NK cell chemotaxis. Methods Human natural killer (NK) cells were isolated from buffy coats obtained from normal volunteers and were activated with lymphocyte conditioned media, IL-2, and ionomycin. Standard transwell chemotaxis assays, proliferation assays, and chromium release cell cytotoxicity assays were performed. Results CXCL14 was found to stimulate migration of activated human NK cells in transwell chemotaxis assays by 1.4-fold. Similarly, it increased migration of an IL-2-dependent natural killer leukemia (NKL) cell line by 1.9-fold. Antisera against CXCL14 or pertussis toxin blocked this chemotactic effect. However, CXCL14 did not affect the proliferation or cytotoxic activity of normal human NK cells. CXCL14 also stimulated the chemotaxis of immature monocyte-derived dendritic cells. Conclusions CXCL14 may play a role in the trafficking of NK cells to sites of inflammation or malignancy. In addition, the downregulation of the expression of CXCL14 might be an important step in successful oncogenesis to prevent NK immune surveillance of the malignancy.

Infection with vesicular stomatitis virus (VSV), a rhabdovirus and economically significant animal pathogen, was previously demonstrated to induce apoptosis. The mechanism of induction and the role of apoptosis in the VSV-host response have not been completely elucidated. Previous data from our laboratory have suggested that caspase-3 is required for the induction of apoptosis but not viral replication in VSV-infected cells. However, these studies used inhibitors that are selective but not specific for caspase-3. To circumvent this difficulty, we infected both MCF-7 cells which do not express caspases-3 (null), and stable transfectants which express caspase-3 (C3+). When caspase-3 null cells were infected, significant PARP cleavage did not occur, but when C3+ cells were infected, PARP cleavage did occur efficiently. Studies in null and C3+ also suggest that: (1) caspases-3 and -7 are activated sequentially after VSV infection; (2) cell shrinkage and detachment are caspase-3 dependent, but cell rounding is not; and (3) the viral titers were similar between caspase-3 null and C3+ cells suggesting that activation of caspases-3 and -7 are not required for viral replication. Taken together, these results strongly support that the activation of caspase-3 by VSV infection is required for efficient apoptosis induction but not viral replication in vitro. Apoptosis mediated by caspase-3, then, is likely either a host cell response to viral replication or perhaps may be required for in vivo viral replication and spread.

Infection with vesicular stomatitis virus (VSV), the prototype rhabdovirus, causes apoptotic DNA fragmentation, but the role of apoptosis in the VSV-host interaction remains unclear. Apoptosis is the gene-regulated mechanism triggered by a wide variety of stimuli that lead to cell death in a choreographed manner. In the present study, infection of the Jurkat T cell line with VSV led to activation of caspase-3 and caspase-7, with subsequent apoptotic events involving poly (ADP ribose) polymerase (PARP) cleavage, DNA fragmentation, and membrane damage. Caspase activation was correlated with viral protein expression suggesting a link between viral replication and apoptosis. We hypothesized that VSV replication might depend on apoptosis and that the inhibition of apoptosis would lead to significant decreases in viral titers. When various inhibitors of apoptosis in VSV-infected cells were used, PARP cleavage and DNA fragmentation were inhibited but the production of infectious progeny was not affected. In addition, we demonstrated that the activation of caspase-3-like proteases is required for VSV-induced apoptosis but not in vitro viral replication. Apoptosis following VSV infection is likely to be either a host-cell attempt to control viral replication or may be a ploy used by the virus to facilitate its in vivo replication and spread.

Apoptosis is triggered by a number of different stimuli including the activation of Fas antigen, a member of the TNF family, by the Fas ligand. The signal transduction events implicated in apoptosis are complex and remain only partially understood. In this study, we used calyculin A, a potent inhibitor of serine/threonine (ser/thr) phosphatases types 1 and 2A, to investigate the role of ser/thr phosphatases in Fas-induced apoptosis. We showed that calyculin A inhibited Fas-induced DNA fragmentation and cytolysis in Jurkat cells and that this inhibition was not due to the modulation of Fas. Okadaic acid also inhibited Fas-induced apoptosis of Jurkat cells, but at much higher concentrations (μM level), thus implicating that type 1 phosphatases rather than type 2A are inhibited at nM concentrations. Cross-linking Fas led to the dephosphorylation of the retinoblastoma gene product (Rb) within 5 min, and to PARP cleavage within 2 h. Both events were inhibited by calyculin A indicating that apoptotic death triggered by Fas cross-linking involves the activation of type 1 ser/thr phosphatases.

We and other workers have recently isolated three novel CC chemokines termed Exodus-1/LARC/Mip-3alpha, Exodus-2/6Ckine/SLC/TCA4, and Exodus-3/Mip-3beta/CKbeta11/ELC. These chemokines share an amino terminal Asp-Cys-Cys-Leu sequence, unique among all chemokines. They also selectively regulate migration of adult T cells. Indeed, there is evidence that Exodus-2 and -3 are critical for adult T-cell adhesion to high endothelial venules in lymph nodes, a rate-limiting step for T-cell trafficking through nodal tissue. Less is known of the factors controlling migration of naive human fetal T cells. We tested whether these chemokines could regulate chemotaxis in cord blood T-cell populations, and compared that efficacy with normal peripheral blood adult T cells. The findings indicated that naive CD45RA+ cord blood T-cell migration is stimulated by Exodus-2 and -3, and CD4+ cord blood T cells are attracted preferentially by Exodus-2 or -3 as compared with CD8+. Exodus-2 and -3 are likely to be critical in regulating the flux of naive CD4 + fetal T-cell population of secondary lymphoid tissue.

Multidrug resistance (MDR) in tumor cells is commonly associated with the over-expression of P-glycoprotein (Pgp), the product of the MDR1 gene. In this study, we investigated whether over-expression of Pgp in natural killer (NK) cells would influence their granule- as well as fas-mediated cytolytic activities. YT-INDY, a human NK-like cell line, was transfected with the MDR1 gene, then tested for Pgp activity the presence of various concentrations of R-verapamil, a potent Pgp inhibitor. We showed that, unlike control YT-INDY, the Pgp activity of the transfectants (YT-mdr(+)) was only partially inhibited by R-verapamil. We also showed that Fas lytic activity was unaltered and that the loss of granule-mediated cytotoxicity was not due to reduced LFA-1 expression or to a decrease in target cell (TC) binding. Our data indicate that Pgp may be involved in the release of cytotoxic molecules.

Cytotoxic cells provide a crucial defense against DNA and RNA viral infections. Here we describe an in vitro model to study the fate of vesicular stomatitis virus (VSV) RNA in cells undergoing apoptosis. Using the [3H]uridine release assay, we show that human LAK cells induce the degradation of RNA in infected U937 cells in addition to inhibiting the production of infectious virions. LAK cell-mediated RNA degradation was blocked by the serine protease inhibitor, 3,4-dichloroisocoumarin. Purified human granzyme B but not inactivated granzyme B, granzyme A, or perforin rapidly induced degradation of RNA in VSV-infected U937 cells in a dose- and time-dependent manner without lysing the cells and suppressed viral production. Northern analysis of RNA extracted from infected cells with a VSV full-length cDNA probe confirmed that levels of viral transcripts were reduced by treatment with granzyme B. Nevertheless, the amount of host beta-actin mRNA was also reduced in infected cells, suggesting that treatment with granzyme B induced apoptosis. Consistent with this notion, infected cells exposed to granzyme B rapidly developed DNA strand breakage. Taken together, the data suggest that granzyme B in the absence of perforin reduced VSV production by activating a mechanism that degraded viral transcripts in infected U937 cells.

We have recently reported that Ser/Thr phosphatases play a key role in regulating natural killer (NK) cell lytic activity and that calyculin A and okadaic acid affect this activity differently [Bajpai and Brahmi (1994) J. Biol. Chem. 269, 18864–18869]. Here, we investigate a mechanism that might account for this differential action of calyculin A and okadaic acid on NK cells. Calyculin A specifically inhibited the lytic activity of YT-INDY, an NK-like cell line, and hyperphosphorylated 60 and 78 kDa proteins. The kinetics of appearance of these two proteins was correlated with the loss of lytic activity. In contrast, okadaic acid did not significantly affect either of these activities. The 78 kDa protein is localized in the cytosolic compartment whereas the 60 kDa protein is distributed equally between the membrane and the cytosolic fractions. Both proteins display a kinase activity and are phosphorylated mainly at serine and threonine residues but not at tyrosine residues. The activation of these kinases is specific to calyculin A treatment; it is independent of protein kinase C, protein kinase A, Ca2+, phosphotyrosine phosphatase and protein synthesis de novo. In conclusion, we have demonstrated that calyculin A, but not okadaic acid, hyperphosphorylates two proteins with Ser/Thr kinase activity, thus explaining the differential regulation of NK cells by these two Ser/Thr phosphatase inhibitors.

In human NK cells and CTL it has been shown that release of lytic molecules is, at least in part, responsible for the lysis of target cells (TC). Of the various types of molecules thought to be involved in cell-mediated cytotoxicity (CMC), perforin and the serine proteases (granzymes A and B) are the best described. Using mammalian expression vectors (pRSV-neo and pSV 2 -neo), antisense constructs for perforin and granzyme B were independently electroporated into YT-INDY, a human non-MHC-restricted, IL-2-independent, cytotoxic lymphocyte. Transfected YT-INDY was then selected for expression of the plasmid by antibiotic G418 resistance. The presence of plasmid was confirmed by detection of the integrated plasmid G418 resistance gene using PCR. The presence of antisense perforin in YT-INDY (YT-xPFP) inhibited lytic ability by >95% compared to YT-INDY transfected with plasmid alone or plasmid with unrelated antisense (YT-neo, YT-ctrl, respectively). Likewise, the presence of antisense GrB (YT-xGrB) inhibited the lytic ability of YT-INDY by >95%. Western analysis revealed a 30% decrease in the level of perlatin and a 55% decrease in granzyme B protein levels compared to YT-neo. Northern analysis using oligo probes complementary to perforin and granzyme B mRNA showed a decrease in their respective message levels. In conclusion, stably transfected antisense constructs for perforin and granzyme B essentially eliminated the lytic ability of YT-INDY. These results strongly indicate that both perforin and granzyme B are required by this human cytotoxic lymphocyte for effective TC lysis.

The original human NK-like line YT was reported to lyse K562 and several B- and T-cell lines. The YT subline we are investigating, YT-INDY, does not lyse K562 or the T-cell line Molt-4. It does, however, lyse the EBV+ Burkitt lymphoma (BL) B-cell line Raji and EBV-immortalized B-cell lines. Several EBV- BL lines and an EBV- pre-B-cell leukemia line that we tested were not appreciably lysed by YT-INDY. To determine if EBV plays a role in TC susceptibility to lysis by YT-INDY, we compared YT-INDY's ability to lyse the EBV- BL line BL41 to its ability to lyse an EBV-infected derivative of BL41. The EBV-infected cell line was lysed, on average, at twice the level of the uninfected line. CD28/B7 interactions appeared to be involved in TC recognition by YT-INDY. Therefore, we examined the level of expression of B7 molecules on the infected and uninfected BL41 lines. An average of 15% of the uninfected BL41 cells expressed B7-1/B7-3, compared to 79% of the infected. B7-2 expression was similar in the two cell lines. Lysis of EBV-infected BL41 was reduced by anti-B7-1/B7-3 (BB1) or anti-CD28 antibodies (Abs) to the level of lysis of the uninfected line, indicating that upregulation of B7-1/B7-3 by the virus may be responsible for the enhanced susceptibility. We attempted to determine the particular EBV latent protein responsible for B7-1/B7-3 upregulation by analyzing BL41 clones expressing LMP1, EBNA-2/EBNA-LP, or EBNA-1. All of the high-expressing clones showed a higher level of B7-1/B7-3 expression than the vector-transfected control cell line, with LMP1-expressing clones expressing the highest amount. EBNA-1 clones and a high-expressing EBNA-2/EBNA-LP clone had a slightly higher density of B7-2 on their surface than the remaining clones. The increased expression of molecules of the B7 family correlated with increased susceptibility of the clones to lysis by YT-INDY. Anti-CD28 or a combination of anti-B7-1/B7-3 and anti-By-2 did not inhibit lysis of the clones to the level of lysis of the vector-transfected control cell line in all cases. We conclude that intact EBV enhances susceptibility to YT-INDY lysis by upregulating B7-1/B7-3. EBV proteins expressed individually also enhance susceptibility to lysis and upregulate members of the B7 family.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytolytic T lymphocyte (CTL) and natural killer cells (NK) lose their lytic potential after interaction with sensitive target cells that can be restored upon incubation with interleukin-2. In this study we observed that preincubation with Ser/Thr phosphatase inhibitor calyculin A inhibited both CTL and NK cell-mediated cytotoxicity (CMC) in a dose-dependent manner. In contrast, okadaic acid inhibited only CTL-CMC without significantly affecting NK-CMC. Incubation of CTL and NK cells with their sensitive TC inhibited both CTL-CMC by 74% and NK-CMC by > 80%. This loss in lytic activity was accompanied by a loss of 60% and > 80% in the cellular p-nitrophenyl phosphate phosphatase (pNPPase) activity in CTL and NK cells, respectively. When treated with 100 units/ml interleukin-2 for 16-18 h at 37 degrees C, inactivated CTL and NK cells recovered 70% and 100% of their lytic activity and approximately 60% and 100% of phosphatase activity, respectively. Analysis revealed that > 80% of the pNPPase activity was associated with membrane-bound CD45, and it is this phosphatase activity that was reversibly affected by target cell-induced inactivation/reactivation of CTL and NK cells. These results suggest that Ser/Thr phosphatases and CD45 play a key role in modulating the lytic activity of effector cells exposed to sensitive target cells.

We have previously shown that CTL and NK cells rapidly down regulate perforin mRNA and become functionally inactive within 4-6 hr after exposure to sensitive target cells (TC). We report here for the first time that CTL also down regulate perforin mRNA upon exposure to resistant, but binding, TC. When three separate human MHC-restricted CTL lines were exposed to resistant TC, perforin mRNA was rapidly degraded. Removal of both extracellular Ca++ and Mg++ prevented perforin message down regulation, whereas removal of Ca++ alone did not, indicating that CTL:TC binding was required. Unlike the response of CTL exposed to sensitive TC, resistant TC did not trigger serine esterase (SE) release, suggesting distinct signalling pathways for perforin mRNA down regulation and granule exocytosis. Moreover, using western analysis, we showed that there was limited (< 10%) perforin protein release after CTL:TC interaction, suggesting that CTL loss of lytic activity after exposure to sensitive TC is not due to massive depletion of perforin. Treatment of CTL with mAb to CD2, CD3, CD2 + CD3, CD8, Class I and LFA-1 did not induce perforin mRNA down regulation. Furthermore, mAb to CD2, CD3, CD8, Class I, Class II, CD54 and LFA-1 did not block TC-mediated perforin mRNA down regulation although lysis of TC was inhibited.

The specific role of sulfhydryl groups in cell-mediated cytoxicity (CMC) is still unknown. Here we demonstrate that natural killer cells and lymphokine-activated killer cells, when incubated with phenylarsine oxide (PAO), an organoarsenic compound showed a dose- and time-dependent inhibition of CMC and antibody-dependent cellular cytotoxicity (ADCC). PAO interacted directly with the effector cells (EC) without affecting the target cells (TC) or EC:TC conjugate formation. The loss of cytotoxicity was not due to lack of degranulation or to inhibition of serine esterases in PAO-treated cells. However, PAO inhibited the target-induced down regulation of phosphatidylinositol (PI) level in NK cells indicating that PAO blocked the cytolytic cascade at an early stage, upstream of PI. In addition, PAO also did not affect the disulfide link of the zeta-chain dimers, implicated in signal transduction in cytotoxic lymphocytes but did cause the rapid phosphorylation of the zeta chain. Finally, the effect of PAO on CMC was competitively blocked by dithiothreitol, a dithiol, but not by β-mercaptoethanol, a mono-thiol. Taken together, these results indicate for the first time how sulfyhydryl groups may regulate CMC and ADCC.

In this investigation we studied the modulation of human NK- and CTL-mediated cytotoxicity in response to extracellular nucleotides. NK cell-mediated cytotoxicity (CMC) was inhibited in a dose-dependent manner by ATP/ADP, GTP/GDP, and by pentasodium triphosphate (PST), whereas MHC-restricted CTL were inhibited by GTP/GDP and PST, but not by ATP/ADP. Triphosphates were the most potent inhibitors, followed by diphosphates and monophosphates which were the least effective, suggesting that the inhibition was not due to the sugars nor adenosine and guanosine nucleotides, but rather to the increasing negative charges. Cultured CTL, fresh NK cells that had been incubated with IL-2 for 18 hr and IL-2-dependent NK 3.3 cells were all inhibited by GTP, but not by ATP. This differential regulation of fresh NK cells and CTL by exogenous nucleotides is dependent upon the presence of IL-2, but IL-4, IL-6, and IL-8 did not have any effect. Mouse CTL are resistant to ATP presumably because they contain high levels of ecto-ATPases. Different levels of ecto-ATPase activity in human CTL and NK cells may therefore explain the difference in the responses of these effector cells to extracellular nucleotides. To test this possibility we determined the levels of ecto-ATPases in human CTL and NK cells and showed that CTL contained five times more ecto-ATPases than NK cells. Incubation of NK cells with IL-2 or IL-4 did not significantly change the level of ecto-ATPase activity on NK cells. Treatment of NK cells with IL-2 also did not significantly change the substrate specificity of NK-ecto-ATPases toward the extracellular ATP and GTP. Furthermore, treatment of CTL and NK cells with a potent ecto-ATPase inhibitor, 5'-fluorosulfonylbenzoyladenosine (FSBA), did not significantly alter the effect of exogenous nucleotides on the lytic potential of CTL and NK cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Recent evidence from our laboratory has demonstrated that NK/LAK cell activation of human lymphocytes is protein kinase C (PKC)-dependent. Here, we have investigated the translocation of PKC in human NK cells exposed to sensitive targets or to PMA, a phorbol ester. In NK cells exposed to K562 for 6 hr, we observed a weak translocation of PKC whereas in NK cells exposed to PMA more than 90% of cytosolic PKC was translocated to the membrane in less than 5 min. Stimulation of NK cells with an NK-resistant target, however, did not translocate PKC even after 6 hr. Translocation of PKC to the membrane was followed by the appearance of PKM, the cytosolic calcium/phospholipid ( Ca 2+ PL )-independent form of PKC. The conversion of PKC to PKM was mediated by calpain, an intracellular calcium-dependent thiol proteinase. When we used two inhibitors of calpain, calpain inhibitor I (CI-I) and calpain inhibitor II (CI-II), both caused a dose-related enhancement of NK-CMC when the inhibitors were present throughout the 3-hr chromium release assay. This enhancement could be circumvented by PMA or by the PKC inhibitor H-7. CI-I and CI-II added together caused a greater increase in NK-CMC than when each was added alone. CI-I and CI-II also enhanced antibody-dependent cell-mediated cytotoxicity (ADCC), substantiating further our previous contention that the activation of both NK-CMC and ADCC may involve a common lytic pathway. Activation of NK cells with IL-2 for 18 hr at 37 °C was inhibited in the presence of CI-I. To investigate a possible feedback inhibition mechanism due to the buildup of PKC, we examined phosphatidylinositol (PI) metabolism in NK cells activated by IL-2 in either the presence or the absence of CI-I. We observed a significant decrease in PI turnover when NK cells, activated in the presence of IL-2 and CI-I, were stimulated with K562 as compared to NK cells activated by IL-2 alone, then stimulated with K562.

A monoclonal antibody (MoAb 11-4) was raised against K562, a human erythroleukemia cell line sensitive to natural killer cell-mediated cytotoxicity (NK-CMC). Immunological analysis revealed MoAb to be IgG2b. Alone, the MoAb was not cytotoxic for K562 and did not bind to the effector cells, but the addition of this antibody to macrophage-depleted human peripheral blood lymphocytes increased killing of K562 in a 4-hr NK-CMC assay. The maximum increase in NK-CMC was observed when MoAb 11-4 was added to target cells prior to the formation of effector/target cell conjugates. This effect was dose dependent, was specific for K562, and, contrary to conventional antisera, occurred at very low concentrations of MoAb. When MoAb was added either to Percoll-purified large granular lymphocytes (LGL) or to LGL-depleted lymphocytes, only the latter demonstrated a significant increase in the killing of K562 in a 4-hr chromium release assay. Kinetics studies revealed that although the overall LGL-mediated lysis was only slightly increased at 4 hr, the maximum lytic activity was reached within 2 hr. These studies suggest that (1) human LGL and LGL-depleted cell populations bear Fc receptors for mouse IgG2b and (2) although the cytotoxic activities of both cell populations are increased by treatment with MoAb 11-4, the kinetics of this increase are different.

In a recent study, we evaluated the functional status of human natural killer (NK) cells after their interaction with the NK-sensitive tumor target cell (TC), K562. We demonstrated that effector cells (EC), after treatment with K562 for 4 hr, lost greater than 90% of their original lytic activity. In this investigation, we examined whether this functional loss of NK cell activity represented an irreversible event in the NK lytic mechanism. Initial studies focused on the ability of K562-inactivated EC (ECi), which had been separated from their TC, to recover cytolytic activity following an 18-hr incubation. Our results indicated that ECi recovered 28% of their lytic activity in complete medium (CM) alone, 64% in CM containing interferon-beta (IFN-beta), and 91% in CM supplemented with interleukin 2 (IL-2). Analysis of the data revealed, however, that neither IFN-beta nor IL-2 simply boosted the lytic capacity of NK cells which initially escaped inactivation, but also, each cytokine affected the lytic capabilities of EC that were either truly inactivated by K562 or precursor NK (pre-NK) cells. Thus, to evaluate further the basis of IFN-beta and IL-2-induced ECi augmentation, we first treated the EC with IFN-beta or IL-2 prior to their interaction with K562 so that pre-NK cell subsets would be promoted to fully competent NK cells. Both pretreated EC preparations, after interacting with K562 for 4 hr, lost greater than 90% of their original lytic activities. NK inactivation did not result from cell death nor reflect alterations in conjugate formation or the percentages of Leu-7- and Leu-11-positive EC. IL-2-pretreated ECi, as did ECi, regained some lytic activity after incubation in CM alone, but recovered significantly more activity in CM containing IFN-beta or IL-2. In contrast to the restimulation profiles obtained for ECi and IL-2-pretreated ECi, IFN-pretreated ECi regained lytic function after incubation with IL-2, but not appreciably with IFN-beta or in CM alone. Overall, these findings suggest that EC, either untreated or pretreated with IFN-beta or IL-2, significantly lose their lytic capabilities following interaction with K562 while retaining their ability to bind to the TC; IFN-beta acts predominantly on pre-NK cells, but not on ECi; and IL-2 appears to play an important role in restoring lytic potential to functionally inactive NK cells.

We report the results of immunologic studies in a family in which the father (III-5) and his two daughters (IV-7 and IV-8) had the hyper-IgM syndrome (IHIS). Repeated immunoglobulin levels done on III-5 showed a typical IHIS pattern: low IgG, traces of IgA, and high IgM. IV-7, who also had stage IIA Hodgkin's disease, had a similar pattern except after irradiation therapy to sites of disease, when IgM dropped to normal range while IgG and IgA remained low. IV-8, on the other hand, had normal IgG and IgA and moderately elevated IgM until age 18 months, when she gradually developed the IHIS pattern. All three patients had normal numbers of B cells (sIg) and of T cells, although IV-7 had increased suppression. Finally, all three patients shared the A3,B7 haplotype and none was blood type O. IHIS is not necessarily X linked, is not associated with blood type O, and appears to be heterogeneous even within the same family. Inheritance in this family is apparently autosomal dominant and the father may represent a new mutation.

This study examined the role of phospholipid metabolism in human natural killer (NK) cells upon activation by tumor target cells (TC). The effector cell (EC) population consisted of peripheral blood lymphocytes enriched for NK cells. Upon a 5-min exposure of EC to the NK-sensitive tumor TC K562 and U937, nearly four- and threefold increases in the incorporation of 32 P into Phosphatidylinositol (PI) occurred, respectively. In contrast, no increase in 32 P incorporation into PI was seen when two NK-resistant TC were used. In addition, little or no change in the incorporation of 32 P into phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine took place with any of the above TC. Depletion of Leu 11b-positive cells abolished the increase in 32 P incorporation into PI when K562 were used in the phospholipid assay. Furthermore, labeling kinetics of this phospholipid turnover showed that it occurred less than 5 min following exposure to NK-sensitive TC and that phosphatidic acid, a breakdown product of phosphoinositides, was produced during this 5-min period. These results indicated that metabolism of a phosphoinositide took place and that it occurred in association with early activation events in NK cells. Quercetin and dibutyryladenosine-cyclic monophosphate (dbcAMP) plus theophylline exerted profound inhibitory effects on both NK activity and PI metabolism, suggesting a linkage between the two events. The inhibitors had no effect on target cell-binding capacity, indicating that the inhibition occurred postbinding. PI metabolism took place in the absence of extracellular calcium even though NK activity was completely abolished under the same conditions. Thus, we have shown PI metabolism, but not other phospholipids, to occur in human NK cells upon exposure to NK-sensitive TC, in association with early activation events. This event was independent of extracellular calcium and could be inhibited by quercetin or dbcAMP plus theophylline.

We have investigated the kinetics of inhibition and regeneration of human natural killer (NK) cell-mediated lysis of K562, a human erythroleukemia cell line, by the potent tumor-promoting agent phorbol-12-myristate-13-acetate (PMA). It is shown that PMA inhibits NK cell-mediated cytotoxicity (CMC) in a dose-dependent manner whether the compound is present throughout the 4-hr cytotoxic assay or the effector cells (EC) are pretreated with PMA. Pretreatment of the target cells (TC) with PMA produced a different profile of NK activity suggesting that PMA inhibition of NK-CMC is primarily due to the inactivation of EC. PMA-induced inhibition of NK-CMC does not affect TC binding and is not circumvented by compounds that enhance intracellular levels of cyclic guanosine monophosphate (cGMP) or calcium. Furthermore, and contrary to a recent report, PMA-induced inhibition of NK-CMC is independent of monocytes. Finally, kinetic studies revealed that PMA-induced inhibition of NK-CMC occurs rapidly and is fully reversible provided that "regenerated EC" are thoroughly washed, prior to the cytotoxic assay, to rid the cell suspension of residual PMA. The potential implications of these results to the currently accepted theory of TC destruction by NK cells, the stimulus-secretion model, are discussed.

Immunosuppression characterizes many human diseases including leukemia and AIDS. Friend virus (FV)-induced murine leukemia is a useful model for studying both malignancy and immunosuppression. In a previous series of experiments, we have demonstrated that untreated FV-infected mice died within 40 days post-infection, whereas infected mice given 150 cGy total body irradiation (TBI) on days 5 and 12 exhibited long-term survival. In this report, we show that no leukemic cells or type C virus particles are found in the spleens of mice treated with TBI. In addition, both NK activity as well as bone marrow cell's proliferative responses to PHA and Con A were fully restored. This treatment produces long term control of FV-induced murine leukemia, and thus might have relevance for the treatment of a number of immunosuppressive diseases including AIDS.

The possible involvement of soluble cytotoxic factors (SCF) in the lytic mechanism of natural killer (NK) cell-mediated cytotoxicity (CMC) was investigated. Tumor target cells (TC) were examined for their ability to stimulate the release of SCF from human peripheral blood lymphocytes (PBL). SCF were detected when effector cells (EC) were stimulated with mycoplasma-contaminated (MC)-TC and with concanavalin A (Con A), but mycoplasma-free (MF)-TC were unable to induce SCF. Pretreatment of EC with beta-interferon augmented the production of SCF with MC-K562 and Con A, but not with MK-K562. However, MF-K562 once infected with the culture supernatant from MC-K562, as well as mycoplasmas concentrated from culture supernatant, were able to induce SCF. Only Con A was capable of generating cytotoxic supernatants from EC that had been inactivated with MF-K562 and from EC depleted of Leu 11b positive cells. Moreover, when EC were evaluated for NK activity after coculture with MF-K562, their ability to kill fresh MF-K562 in the standard NK-CMC assay was greatly reduced. This loss of lytic potential was not accompanied by SCF release. Collectively, these data suggest that mycoplasma organisms play a crucial role in the induction of human SCF, and the implication that SCF, including NK cytotoxic factors (NKCF), are the lytic mediators in NK-CMC needs to be re-evaluated.

The effect of acute exercise on natural killer (NK) activity and on the distribution of phenotypic characteristics of peripheral blood lymphocytes was examined. Trained and sedentary individuals underwent a standard progressive exercise test on a cycle ergometer using an incremental work load of 15 W (90 kpm), increased every minute. Each subject was encouraged to exercise to exhaustion, and total ventilation and mixed expired O2 and CO2 were measured every 30 sec. All subjects reached the “anaerobic” threshold as judged by the deflection of ventilation at a work load nearVO2max. NK activity against K562 reached maximum levels immediately after exercise, dropped to a low point 120 min later, then slowly came back to preexercise levels within 20 hr. No significant differences were observed between the trained and the sedentary groups. Furthermore, immediately after exercise the proportion of OKT-3+ and OKT-4+ cells was reduced by 29.8±3.6 and 33.6±5.4%, respectively; the percentage Leu-7+ and Leu-11a+ cells was increased by 53.9±1.7 and 57.3±2.9%, respectively. The percentage OKT-8+ cells was not significantly altered. When the percentage binding of effector to target cells was examined, it was highest at 0 min post-exercise (19±6.2%) and lowest at 120 min postexercise (7±3.9%), but the absolute number of NK cells remained unchanged. The source of serum used in the lytic assay had no effect on the NK activity, as fetal calf serum and autologous sera drawn at different time intervals during exercise gave similar results. Epinephrine suppressed and interferon enhanced the NK activity of all cells tested, which indicates that during and after exercise NK cells did not go through a refractory stage. Our results show that, contrary to other lymphocyte subpopulations, NK cells are resistant to the depletive and modulatory effects of acute exercise and the increase in NK activity is not due to an increase in the absolute number of NK cells.

Three DR9 cell lines were examined in the Workshop study: DKB, HID, and KT12. Serologic typing in the Workshop indicates that only DKB is completely homozygous throughout the HLA region. KT12 is clearly not consanguinous, being heterozygous at the A and B loci, but appears to be homozygous in the D region, as indicated in Table 1. HID appears to be heterozygous at the DP locus by cellular typing and at the C locus by Work shop serology and showed additional DQ reactivity with several DQw1-related mAbs, including 77.34 and KS6 and KS7, which were negative with the DKB and KT12 cell lines in the immunofluorescence analysis (4). All three cells lines were serologically DQw9 in the Workshop serology analysis and reacted with the broad DQw3-related mAbs 17.15, IVD12, and PLM12, as well as IIB3, which does not react with DQw7(TA10+) cells in the immunofluorescence analysis. Together, these preliminary serologic results suggested that either HID was not homozygous in the HLA-DQ region or it carried a unique DQ allele reactive with all of these antibodies.

We investigated hydroxyl radical (OH) production by human natural killer (NK) cells, using electron spin resonance (ESR) spectroscopy and 5,5 dimethyl-1-pyrroline-N-oxide (DMPO), a spin trap specific for OH production. We confirmed that hydroxyl radical scavengers, n-propyl gallate and catechin, inhibited NK cell-mediated cytotoxicity (NK-CMC) in a dose-dependent manner and demonstrated that DMPO also inhibited NK-CMC. Polymorphonuclear leukocytes (PMNL) activated by opsonized zymosan (2.4 mg/ml) and mixed with DMPO (0.14 M) showed an early increase in hydroxyl radical production, leading to a net production of free radical of almost 400 pMol/106 cells. We then mixed NK cells with K562, an NK-sensitive tumor cell, at a 1:1 ratio and added DMPO (0.14 M). We pelleted the cells to increase EC to TC binding before taking the sample readings. Activated NK cells showed no increase in OH production, leading to a net production of free radicals less than 1% that of activated PMNL. These data strongly suggest that hydroxyl radical production does not play a role in the early events of NK cell activation; they indicate a need to reevaluate the mechanism of inhibition of NK-CMC by OH scavengers.