Early Quantification of Hematoma Hounsfield Units on Noncontrast CT in Acute Intraventricular Hemorrhage Predicts Ventricular Clearance after Intraventricular Thrombolysis.

BACKGROUND AND PURPOSE: Thrombolytic efficacy of intraventricular rtPA for acute intraventricular hemorrhage may depend on hematoma composition. We assessed whether hematoma Hounsfield unit quantification informs intraventricular hemorrhage clearance after intraventricular rtPA. MATERIALS AND METHODS: Serial NCCT was performed on 52 patients who received intraventricular rtPA as part of the Clot Lysis Evaluation of Accelerated Resolution of Intraventricular Hemorrhage trial and 12 controls with intraventricular hemorrhage, but no rtPA treatment. A blinded investigator calculated Hounsfield unit values for intraventricular hemorrhage volumes on admission (t0), days 3-4 (t1), and days 6-9 (t2). Controls were matched uniquely to 12 rtPA-treated patients for comparison. RESULTS: Median intraventricular hemorrhage volume on admission for patients treated with intraventricular rtPA was 31.9 mL (interquartile range, 34.1 mL), and it decreased to 4.9 mL (interquartile range, 14.5 mL) (t2). Mean (±standard error of the mean) Hounsfield unit for intraventricular hemorrhage was 52.1 (0.59) at t0 and decreased significantly to 50.1 (0.63) (t1), and to 45.1 (0.71) (t2). Total intraventricular hemorrhage Hounsfield unit count was significantly correlated with intraventricular hemorrhage volume at all time points (t0: P = .002; t1: P < .001; t2: P < .001). On serologic and CSF analysis at t0, only higher CSF protein was positively correlated with intraventricular hemorrhage Hounsfield units (P = .03). In 24 matched patients treated with rtPA and controls, total intraventricular hemorrhage Hounsfield units were significantly lower in patients treated with rtPA at t2 (P = .02). Higher Hounsfield unit quantification of fourth ventricle hematomas independently predicted slower clearance of this ventricle (95% CI, 0.02-0.14; P = .02), along with higher intraventricular hemorrhage volume (95% CI, 0.02-0.41; P = .03) and lower CSF protein levels (95% CI, -0.003 to -0.002; P < .001). CONCLUSIONS: Intraventricular hemorrhage Hounsfield unit counts decrease significantly in the acute phase and to a greater extent with intraventricular rtPA treatment. Intraventricular hemorrhage Hounsfield units are correlated significantly with CSF protein and not with serum erythrocyte or platelet concentrations. Hounsfield unit counts may reflect intraventricular hemorrhage clot composition and rtPA sensitivity.

CD9 expression enhances the susceptibility of myeloma cell lines to cell-mediated cytolysis.

Myeloma tumor cells, both freshly excised and cultured, are extremely resistant to cell-mediated cytolysis. As evidence suggests that B-cell susceptibility to lysis is dependent upon its state of differentiation and activation, we tested the ability of a variety of B-cell proliferation and differentiation agents, including pokeweed mitogen (PWM), to enhance the sensitivity of myeloma cells to cell-mediated lysis. PWM was found to significantly enhance the susceptibility of myeloma cell lines and freshly isolated myeloma cells to interleukin-2 (IL-2)-activated cell-mediated cytolysis. This effect was seen with the use of both IL-2-stimulated natural killer (NK) cells and T cells as effectors. The enhanced sensitivity of myeloma cells to cytolysis correlated with an increase in their cell surface expression of CD9, a pre-B cell marker and member of the transmembrane 4 superfamily. Incubation of PWM-stimulated myeloma cells with either monoclonal antibodies or antisense oligonucleotides directed against CD9 abrogated the effect of PWM. In order to determine whether there was a direct relationship between the expression of CD9 and enhanced sensitivity to cytolysis, myeloma cell lines that lacked CD9 expression were transfected with the CD9 gene. The level of cell surface CD9 expression correlates with enhanced susceptibility to lysis. Therefore, CD9 appears to be an important component in enhancing the sensitivity of myeloma cells to lysis mediated by IL-2-activated T cells and NK cells.

Gene structure of human and mouse NKLAM, a gene associated with cellular cytotoxicity.

A novel gene involved in cytotoxicity, NKLAM [for "natural killer (NK) lytic-associated molecule"], has recently been identified in human NK cells. Its expression correlates with the cytolytic activity of both NK cells and cytotoxic T cells (CTLs); treatment of these cells with NKLAM-specific antisense oligonucleotides inhibits their cytotoxic function. NKLAM encodes a zinc finger protein that resides in NK cytolytic granules. Here, we identified a second human NKLAM transcript that differs from the original only at the 3' end, extending the open reading frame, and therefore encoding a significantly longer protein (731 residues compared with 587). The genomic structure of human NKLAM indicates that these two isoforms are products of alternative splicing. Both forms of NKLAM protein are expressed in NK cells, with the larger protein predominant. Human NKLAM cDNA was used to isolate mouse NKLAM from a C57BL/6 spleen cDNA library. There is a single NKLAM isoform in the mouse which shares over 89% nucleotide and 94% amino acid homology with the larger human form of NKLAM. The genomic organization of NKLAM is similar in both mouse and human. As with human NKLAM, mouse NKLAM mRNA is selectively expressed in CD8+ T cells NK cells, and activated peritoneal macrophages and further induced by cytokines that enhance the cytotoxic activity of these cells. These results indicate that human and mouse NKLAM are highly conserved both structurally and functionally, reinforcing the premise that this gene plays an important role in cellular cytotoxicity.

NK lytic-associated molecule: a novel gene selectively expressed in cells with cytolytic function.

NK cells are most effective in killing a broad spectrum of primary tumor cells after stimulation with cytokines. We have cloned a novel gene, designated NKLAM (for NK lytic-associated molecule), whose expression is associated with this cytokine-enhanced process. NKLAM expression is up-regulated in NK cells by IL-2 and IFN-beta. NKLAM is also selectively expressed by activated macrophages and CTL. Treatment of NK cells and CTL with NKLAM antisense oligonucleotides specifically decreases their cytolytic activity, while having no effect on cell growth. The NKLAM gene encodes a 62-kDa ring finger-containing protein that localizes to the cytoplasmic granules in NK cells. Further study of this gene may add to our understanding of cytotoxic processes common to NK cells, CTL, and activated macrophages.

Thymidine kinase (TK) gene-transduced human lymphocytes can be highly purified, remain fully functional, and are killed efficiently with ganciclovir.

A graft-versus-leukemia (GVL) effect has been considered a major factor responsible for cures in patients with hematologic malignancies undergoing allogeneic bone marrow transplantation; however, associated graft-versus-host disease (GVHD) results in significant morbidity and mortality. T-cell depletion reduces the incidence and severity of GVHD but eliminates, at least partially, the GVL effect. Reinfusion of donor T lymphocytes at relapse posttransplantation can induce a potent antitumor response, but GVHD still occurs in the majority of patients. Prior transduction of T lymphocytes with the suicide gene, the viral thymidine kinase (TK), permits specific cell kill on administration of ganciclovir (GCV). Therefore, infusion of TK-transduced T lymphocytes may induce GVL effect and allow for their subsequent selective elimination in case GVHD develops. To evaluate the efficacy and feasibility of this promising approach, anti-CD3-stimulated primary human lymphocytes cultured in interleukin-2 were TK-transduced by a retroviral vector carrying both TK and neomycin-resistance genes. After selection in G418, more than 90% of the cells contained the TK gene as shown by a semiquantitative polymerase chain reaction. In addition, 1 to 5 days of GCV exposure, at clinically achievable concentrations of 20 to 50 micromol/L, induced > or = 90% killing of G418-selected cells without affecting nontransduced cells. Correlation of the extent of T-cell kill and the proportion of TK-gene-transduced cells is consistent with the absence of a bystander effect. Transduced cells were CD3+ and either CD8+ or CD4+ and retained functional properties of untransduced cells. In vivo administration of GCV prevented tumor development after subcutaneous injection of TK-transduced murine myeloma cells (MOPC-11), whereas such an effect was not observed on injection of untransduced cells into the opposite flank. Our studies provide critical information that (1) adequate numbers of TK-transduced lymphocytes can be selected efficiently with > or = 90% purity, (2) selected cells remain functional, (3) 24 hours of exposure to GCV at clinically achievable concentration effects > or = 90% killing of selected cells, and (4) GCV is effective in vivo in killing TK-transduced cells. Based on these data, a clinical study has been initiated in patients with multiple myeloma with persistent or relapsing disease after T-cell-depleted allogeneic transplants.

Glutamine suppresses PGE2 synthesis and breast cancer growth.

Reduced natural killer (NK) activity found in tumor-bearing hosts has been associated with high levels of prostaglandin E2 (PGE2) produced by monocytes in vitro. We have previously demonstrated a dependence of NK cell activity on glutamine (GLN) levels in vitro and in vivo. Further, glutathione (GSH) is antagonistic to PGE2 synthesis. We hypothesized that GLN, through increased GSH production, leads to decreased PGE2 synthesis and upregulation of NK cytotoxic activity. To test this, we examined the effects of oral GLN on GSH and PGE2 concentrations, NK activity and tumor growth in a rat breast cancer model. Starting on the day of MTF-7 tumor implantation 18 Fisher 344 rats were pair-fed chow and gavaged with 1 g/kg/day GLN (n = 9) or an isonitrogenous amount of Freamine (FA) (n = 9). Seven weeks after tumor implantation rats were sacrificed. Tumors were measured, weighed, and processed for tumor morphometrics. Spleens were removed, lymphocytes isolated and assayed for NK activity. Blood GLN, GSH, and PGE2 concentrations were measured. Over the 7-week study period tumor growth was decreased by approximately 40% in the GLN-supplemented group. This decrease in growth was associated with a 2.5 fold greater NK activity in the GLN-fed rats vs FA-fed rats. This correlated with a 25% rise in GSH concentration and a proportional decrease in PGE2 synthesis. Decreased tumor volume in rats fed GLN was not associated with changes in morphometrics. Oral GLN supplementation enhances NK activity resulting in decreased tumor growth. The enhanced NK activity seen with oral GLN supplementation in the tumor-bearing host is associated with GSH mediated suppression of PGE2 synthesis.

Biological aspects of multiple myeloma.

Although considerable progress has been made towards understanding many aspects of myeloma, the myeloma stem cell and the factors that drive the disease remain elusive. Recent developments in the molecular analysis of clonality have helped to confirm the presence of pre-switch B cells that are of the same clone as the myeloma plasma cells. The role of these cells in myelomagenesis has not been demonstrated, and the isotypic heterogeneity of the clonally-relevant cells suggests that the pre-switch B cells are pre-malignant progenitors of the tumour cells. Thus, the circulating clonal B cells appear to be the earliest progenitors of the mature, monoclonal plasma cells. IL-6, and possibly other cytokines, are involved in driving this process. The role of IL-6 in myeloma is complex and more involved than its proposed growth factor function. In the absence of IL-6, dependent cells become apoptotic. Increased IL-6 signalling also leads to apoptosis of myeloma cells, possibly as a result of terminal differentiation. In the presence of exogenous IL-6, the IL-6 receptor appears to be the rate-limiting factor in the pathway's activity. IL-6 may regulate the survival of myeloma cells by stimulating c-myc transcription, possibly from the P0 promoter. The high levels of c-myc transcripts and protein could regulate myeloma cell proliferation and apoptotic death by controlling p53 expression and, through it, the expression of the Rb and BAX genes. Proliferative signalling in myeloma cells is likely to be intrinsic, within the tumour cell compartment. Molecules such as CD28 and B7, both expressed by less mature myeloma cells, could represent one such self-self stimulatory mechanism, with IL-6, possibly through stimulation of c-myc expression, providing the signals for survival and differentiation.

Autoregulatory circuits in myeloma. Tumor cell cytotoxicity mediated by soluble CD16.

BACKGROUND: Multiple myeloma remains an incurable malignancy due to marked resistance of the tumor to standard doses of chemotherapy. Treatment approaches, using chemotherapeutic dose escalation and hematopoietic stem cell support have resulted in significant augmentation of tumor mass reduction such that complete remissions are effected in approximately 50% of patients. These remissions are however, often not durable. In the setting of minimal residual disease, therefore, adjunctive immunotherapy may be useful. METHODS: Peripheral blood mononuclear cells were studied from 28 untreated patients with multiple myeloma (MM). Mononuclear cell CD16 (FcR gamma III) expression was determined by flow cytometry. The effect of lymphocyte-derived soluble CD16, isolated by affinity chromatography, on MM cell growth and differentiation was assessed. MM cell proliferation, viability, immunoglobulin production and gene expression was studied. RESULTS: Data are presented indicating that cells expressing CD16 are increased in untreated patients with IgG-secreting myeloma. The predominant phenotype of these cells is CD8+ or CD56+. These CD16+ cells can produce a soluble form of the Fc receptor (sFcR, sCD16) that can bind to surface Ig on cultured human IgG-secreting myeloma cells and effect suppression of tumor cell growth and Ig secretion. This effector function is accompanied by concomitant suppression of c-myc as well as IgH and IgL gene transcription. Finally, prolonged exposure to sCD16 causes myeloma tumor cell cytolysis. CONCLUSIONS: sCD16 and possibly other soluble FcR are candidate molecules for adjunctive immunotherapy of myeloma, once complete responses have been effected by intensive cytotoxic therapy, now possible in up to 50% of newly diagnosed patients.

Harry M. Vars Research Award. Glutamine enhances immunoregulation of tumor growth.

BACKGROUND: It is known that tumor progression is associated with a depletion in host glutamine (Gln) stores and a depression of natural killer (NK) cell activity. After demonstrating an in vitro dependence of NK cell activity on Gln and glutathione concentration, this study evaluated the effects of oral Gln on Gln and glutathione metabolism, NK cell activity, and tumor growth in the tumor-bearing rat. METHODS: Two days before tumor implantation, rats (n = 32) were randomized to receive Gln (1 g/kg/d) or an isonitrogenous amount of glycine by gavage and pair-fed food. On day 21 after tumor implantation, rats were killed, and tumors were measured and processed for glutaminase activity, glutathione content, and tumor morphometrics. Splenic lymphocytes were assayed for NK cell activity via a chromium (51Cr) release assay using YAC (NK-cell-sensitive mouse tumor cell line) target cells. Blood Gln and glutathione were measured. A second set of rats (n = 16) were treated similarly except that ketamine was given twice weekly to suppress NK cell activity. RESULTS: During the 3-week study period, tumor growth was decreased by 40% in the Gln group. This decrease in growth was associated with a 30% increase in NK cell activity. Administration of ketamine to rats completely reversed the higher NK cell activity and decreased the tumor growth seen in the Gln-treated group. CONCLUSIONS: These data indicate that oral Gln supplementation, through support of host Gln stores and glutathione production, may decrease tumor growth by enhancing NK cell activity.

Immunoregulation of murine and human myeloma.

The studies presented in this review address the issue of immunoregulation of growth and differentiation of myeloma tumor cells by host effector cells (Fig. 1). In the murine myeloma system, substantial, direct evidence is presented that myeloma tumor cell growth and differentiation can be modulated by host idiotype-, antigen-, and isotype-specific immunoregulatory cells. Only isotype-specific immunoregulatory cells, however, represent an endogenous, host-generated response, whereas idiotype- and antigen-specific responses require exogenous manipulation (immunization) of the host. In patients with multiple myeloma, however, little direct evidence is available showing regulation of growth and differentiation of tumor cells. Instead, a substantial body of literature suggests a complex interaction between the host immune system and the tumor that, in principle, may result in regulation of tumor cell growth and/or differentiation, although at best the regulation is clinically ineffective. Changes in the phenotype and functions of T cells, B cells, macrophages, and NK/LAK cells have been demonstrated in patients with multiple myeloma and, at least in vitro, each of these cell populations has been shown to modulate growth/differentiation or survival of human myeloma cells. The future challenge in our studies of the immunobiology of multiple myeloma will be to better understand the mechanisms controlling growth of myeloma tumor cells and the interactions between the tumor cells and the host immune system so that we might develop appropriate strategies to augment antitumor immune responses.

The role of IL-4 in proliferation and differentiation of human natural killer cells. Study of an IL-4-dependent versus an IL-2-dependent natural killer cell clone.

The role of IL-4 in proliferation and differentiation of human NK cells was studied using newly established sublines of an IL-4-dependent NK cell clone (IL4d-NK cells) and an IL-2-dependent NK cell clone (IL2d-NK cells) derived from a parental conditioned medium-dependent NK cell clone (CM-NK cells). IL-4 induced the higher proliferation of CM-NK cells, but abolished their NK activity and decreased CD16 and CD56 Ag expression. In contrast, IL-2 induced the higher NK activity and increased CD16 and CD56 Ag expression. Addition of anti-IL-4 antibody to the culture of CM-NK cells with CM inhibited the proliferation, but slightly increased NK activity, and largely increased CD56 Ag expression. Addition of anti-IL-2 antibody to the culture of CM-NK cells with CM inhibited both proliferation and cytotoxicity. Proliferation of IL4d-NK cells, which is totally dependent on rIL-4, is greater than that of IL2d-NK cells, which was greater than parental CM-NK cells. Morphologically, IL4d-NK cells are small and round, whereas IL2d-NK cells are large and elongated. Anti-IL-4 antibody inhibited proliferation of IL4d-NK but not IL2d-NK cells, whereas anti-IL-2 antibody inhibited that of IL2d-NK but not IL4d-NK cells. IL-2 was not detected in the supernatant from IL4d-NK cells, nor was IL-2-mRNA expressed in IL4d-NK cells. In contrast, IFN-gamma production and protein expression in IL4d- and IL2d-NK cells were detected. NK cell activation markers (CD16 and CD56) were expressed on IL2d-NK cells but not IL4d-NK cells. IL4d-NK cells were not cytotoxic to any tumor cells tested, whereas IL2d-NK cells displayed potent NK activity and lymphokine-activated killer activity. IL4d-NK cells failed to bind K562 tumor cells, whereas one-third of the IL2d-NK cells did. IL4d-NK cells responded to rIL-2, proliferated, and differentiated into cytotoxic NK cells, whereas IL2d-NK cells failed to respond to rIL-4 and died. These results raise a possibility that IL4d-NK cells or IL2d-NK cells primarily represent the immunologic properties of immature or activated types of human NK cells, respectively. Our results provide the first evidence of the capability of IL-4 to support continuous proliferation of a lymphocyte clone with immature NK cell characteristics and to stimulate IFN-gamma production in the clone. IL-4 is suggested as a potential growth factor for certain types of human NK cell progenitors.

In vitro infection of natural killer cells with different human immunodeficiency virus type 1 isolates.

Natural killer (NK) cells are a discrete subset of leukocytes, distinct from T and B lymphocytes. NK cells mediate spontaneous non-MHC-restricted killing of a wide variety of target cells without prior sensitization and appear to be involved in initial protection against certain viral infections. Depressed NK cell-mediated cytotoxicity, one of the many immunological defects observed in AIDS patients, may contribute to secondary virus infections. Here we report that clonal and purified polyclonal populations of NK cells, which expressed neither surface CD4 nor CD4 mRNA, were susceptible to infection with various isolates of human immunodeficiency virus type 1 (HIV-1). Viral replication was demonstrated by detection of p24 antigen intracellularly and in culture supernatants, by the presence of HIV DNA within infected cells, and by the ability of supernatants derived from HIV-infected NK cells to infect peripheral blood mononuclear cells or CD4+ cell lines. Infection of NK cells was not blocked by anti-CD4 or anti-Fc gamma RIII monoclonal antibodies. NK cells from HIV-infected and uninfected cultures were similar in their ability to lyse three different target cells. Considerable numbers of cells died in HIV-infected NK cell cultures. These results suggest that loss of NK cells in AIDS patients is a direct effect of HIV infection but that reduced NK cell function involves another mechanism. The possibility that NK cells serve as a potential reservoir for HIV-1 must be considered.

Kinetics of cellular cytotoxicity mediated by a cloned human natural killer cell line.

The kinetics of natural killer (NK) cytotoxicity mediated by the cloned interleukin 2 (IL 2)-dependent human natural killer cell line NK3.3 has been investigated. This cloned cell line exhibits strong cytotoxic activity that is restricted to NK target cells. The initial rate of lysis of K-562 target cells by these cloned NK cells was, as anticipated, substantially greater than that previously reported for human peripheral blood NK cell preparations. However, in contrast to the kinetics of NK cytolysis mediated by fresh peripheral NK cells, the rate of 51Cr-release declined substantially within 1 to 3 h after initiation of assays involving NK3.3 cells and reached a plateau value in experiments conducted for longer periods. The data obtained suggest that NK3.3 cells cannot readily recycle and kill multiple target cells. Based on a model involving one lethal hit per active NK3.3 cell, estimates for the frequency of cytolytic cells in NK3.3 cultures were computed and compared to estimates obtained by the application of a kinetic model previously described. The cytotoxic activity of the NK3.3 cells was also found to be highly dependent on the conditions used for propagation and assay of these cells and, when cultured under "standard" conditions, only a fraction of the cloned NK3.3 cells was capable of effecting lysis of K-562 target cells. However, for the most optimal conditions developed to date, each NK cell killed an average of 1 to 2 target cells before inactivation. Although no significant differences in viability or growth rate were observed for the three different conditions described, up to 300-fold differences in lytic activity were observed. The observed strong dependence of the lytic function of NK3.3 cells on culture conditions should prove valuable for further investigations of mechanisms governing the regulation and function of human NK cells.

Susceptibility to natural killer cell-mediated cytolysis is independent of the level of target cell class I HLA expression.

To test the hypothesis that susceptibility to NK cell-mediated cytolysis varies inversely with the levels of target cell class I HLA expression, NK-susceptible K562 and MOLT-4 target cells have been transfected via electroporation with cloned human class I HLA-A2 and HLA-B7 genes. Stably transfected cells expressing varying levels of cell-surface class I HLA have been selected by fluorescent activated cell sorting and tested for susceptibility to NK-mediated cytolysis by freshly isolated peripheral blood NK cells from nine normal volunteers as well as by cloned human NK effectors and tumor cells from a patient with an NK cell lymphoproliferative disorder. Expression of class I HLA did not alter the susceptibility of K562 or MOLT-4 target cells to NK-mediated cytolysis by any of the effectors tested. In addition, the class I HLA-expressing transfectant cells were identical to mock transfected cells in their ability to compete for lysis in cold target inhibition assays. Treatment of both mock-transfected and class I HLA-transfected K562 cells with IFN-gamma resulted in decreased susceptibility to NK-mediated cytolysis which was independent of the total level of class I HLA expression. These results demonstrate that the level of target cell class I HLA expression is not sufficient to determine susceptibility or resistance to NK-mediated cytolysis of the classical NK targets K562 and MOLT-4.

Changes in gene expression associated with IFN-beta and IL-2-induced augmentation of human natural killer cell function.

NK function can be augmented by a variety of agents, including the cytokines IL-2 and IFN. The mechanisms associated with IL-2- and IFN-mediated augmentation of NK function are largely unknown. In order to learn more about the regulation of NK activity, we have studied changes in gene expression that occur upon treatment of a cloned line of NK cells (NK 3.3) with rIL-2 and rIFN-beta. Both IL-2 and IFN-beta induced rapid augmentation of lysis mediated by NK 3.3, which was significant within 1 h, peaked at 6 h of treatment, and declined by 12 h. This enhancement of lytic function was independent of proliferation and associated with a corresponding increase in steady state levels of RNA coding for both the nuclear proto-oncogene c-myb and for the IL-2R. These changes were specific in that RNA levels of another nuclear proto-oncogene, c-myc, were increased by IL-2 but not by IFN-beta, whereas HLA class I RNA levels were relatively unchanged by either IL-2 or IFN-beta treatment. Treatment of NK 3.3 with the combination of IL-2 and IFN enhanced both lysis and c-myb expression in an additive fashion. These findings suggest that c-myb may play a regulatory role in the cytolytic activity of NK cells.

Natural killer-cell activity in cyclosporine-treated renal allograft recipients.

We prospectively studied natural killer (NK)-cell activity in 16 cyclosporine-treated renal transplant recipients. NK function remained intact in the group as a whole in the initial 6 months following transplantation. The percentage of CD16-positive cells within the peripheral blood mononuclear-cell population was highly correlated with NK activity both prior to and following transplantation in the absence of rejection. During rejection, the correlation was poor. A marked increase in NK activity occurred during 9 of 12 rejection episodes; similar increases in NK activity were rarely observed in the absence of rejection. Significant infiltrates of NK cells, as determined by expression of CD16, were not demonstrated in stained biopsy specimens obtained from rejecting allografts. Pretransplant NK activity did not predict clinical outcome of the allograft. Our results indicate that NK cells are activated during allograft rejection in cyclosporine-treated patients, but their exact role in the rejection process is unknown.

At least two loci encode polymorphic class I MHC antigens in the horse.

Six monoclonal antibodies and ten alloantisera were used to precipitate cell surface molecules of approximately 44 kDa (class I MHC antigens) from radiolabelled equine peripheral blood lymphocytes. All ten antisera were raised against antigens of a single donor horse (horse 0834, ELA-A2,-A2). Four methods of producing antisera were compared: one or two pregnancies, skin allografting, and skin grafting followed by pregnancy. Immunization by pregnancy appeared to produce antibodies against class I products only, while skin grafting raised antibodies to class II antigens as well. Nine of the antisera were raised across an entire MHC haplotype barrier, while one recipient carried the ELA-A2 antigen of the donor. The pregnancy antiserum raised across this barrier probably identifies a second polymorphic class I locus in the horse. Sequential immunoprecipitation using this antiserum in the first stage and an anti-MHC haplotype antiserum or monoclonal antibody reagent in the second stage supported this hypothesis. Gene products of this second ELA class I locus are immunogenic in pregnancy.

Lymphocyte function-associated antigen 1 (LFA-1) and natural killer (NK) cell activity: LFA-1 is not necessary for all killer: target cell interactions.

A panel of five monoclonal antibodies detecting human lymphocyte function-associated antigen 1 (LFA-1) was generated and shown by competitive binding studies to react with at least four distinct epitopes on this molecule. The antibodies were then tested for their ability to inhibit the lytic activity of a variety of different human natural killer (NK) populations on a panel of four NK-susceptible target cells (K562, MOLT-4, HSB-2, and Jurkat). When heterogeneous NK populations derived from fresh peripheral blood and mixed-lymphocyte culture (MLC)-generated lines were used, these anti-LFA-1 monoclonal antibodies (MAbs) inhibited lysis of all four NK targets; this finding supports the notion that LFA-1 molecules play an important role in NK-mediated lysis. When tested on a cloned line of NK cells (NK 3.3), lysis of K562 was inhibited by these MAbs, but lysis of the other three targets was not affected. This represents an instance where a MAb specific for LFA-1 inhibits the lytic activity of NK cells against some but not all targets; thus the LFA-1 molecule cannot be considered under all circumstances to be an absolute requirement in NK-mediated lysis.

Anti-HLA class I antibodies inhibit the T cell-independent proliferation of human B lymphocytes.

The role of major histocompatibility complex-encoded class I molecules in the proliferation of human B lymphocytes is presently unclear. This question was addressed by investigating the effect of three individually derived anti-HLA class I monoclonal antibodies (mAb) on purified human B cells (less than 1.5% T cells) stimulated by either the T-independent mitogen Staphylococcus aureus or the phorbol ester, phorbol-12-myristate-13-acetate. The three anti-HLA class I antibodies, whether specific for gene products of the HLA-A locus (mAb 131), HLA-B locus (mAb 4E), or HLA-A, -B, and -C locus (mAb W6/32), inhibited S. aureus-induced proliferation by 70 to 90%. This inhibition was significant over a 5-day culture period, was not altered by the addition of exogenous interleukin 2 or B cell growth factor, and was not due to nonspecific cytotoxicity. In addition, the inhibition of proliferation was unchanged when the mAb were added 12 hr after the initiation of culture. The proliferative response was not affected by either of the control antibodies OKB7 and R3-367. In contrast with S. aureus-stimulated B cells, phorbol-12-myristate-13-acetate-induced proliferation was resistant to the inhibitory activity of HLA class I-specific antibodies. These results suggest that HLA class I molecules are involved in human B lymphocyte proliferation and may regulate a critical event preceding the upregulation of protein kinase C activity.