Modulation of natural cytotoxicity by alloantibodies. V. The mechanism of a selective augmenting effect of anti-H-2 antisera on the natural killer activity of mouse spleen cells

Treatment of mouse spleen cells with specific anti-H-2 antisera augments their natural killer (NK) activity against K562 cells but not against YAC target tumor cells. The same population of natural killer cells was found to lyse K562 as well as YAC target cells, since (a) depletion of YAC reactive NK cells by absorption on YAC monolayers resulted in a concomitant depletion of anti-K562 NK activity of mouse spleen cells, and (b) both K562 and YAC cells could inhibit their own as well as each others lysis in a cross-competition assay. Anti-H-2 antiserum could not induce anti-K562 NK activity in spleen cells previously depleted of NK cells by absorption on YAC monolayers, indicating that alloantiserum does not act by recruiting otherwise nonreactive cells to become cytotoxic toward K562 target cells. In a target-binding assay, K562 binding of NK cells (T-cell-, B-cell-, and macrophage-depleted spleen cells) increased five- to eightfold in the presence of anti-H-2 antiserum whereas YAC cells binding of NK cells was not increased. H-2 antigens per se did not appear to be involved in the alloantisera effect since anti-NK antiserum directed against a non-H-2 antigen selectively expressed on NK cells, showed a similar selective NK enhancing effect. Protein A, a reagent which binds to the Fc region of immunoglobulin molecules, completely blocked the alloantiserum induced augmentation of anti-K562 NK activity, but did not alter basal NK activity. Moreover, the F(ab)₂ fraction of alloantibodies failed to enhance anti-K562 cytotoxic activity of mouse spleen cells, indicating a crucial role for the Fc portion of the alloantibodies attached to the NK cells, in NK augmentation. Utilization of several target cell lines with or without membrane Fc receptors (FcR) revealed that alloantiserum enhanced the lysis of only FcR⁺ target cells. It is proposed that alloantibody-coated NK cells, as a result of a secondary interaction between attached alloantibody and Fc receptors on target cells, interact more readily with the target cells and thereby cause a higher level of lytic activity.     

Modulation of natural cytotoxicity by alloantibodies. I. Alloantisera enhancement of cytotoxicity of mouse spleen cells toward a human myeloid cell line.

Natural killer activity of spleen cells obtained from different strains of mice against the human myeloid leukemia cell line, K562, and two mouse cell lines P815 and L1210 was measured by using the 4-hr chromium release assay. The level of cytotoxic activity of spleen cells against the K562 target was usually less than 4% lysis. However, treatment of the spleen cells with a specific anti-H-2 antiserum resulted in a dose-dependent augmentation of the degree of lysis of K562 cells. The augmentation of cytotoxic activity could be obtained by pretreatment of the spleen cells with antisera or by directly adding the antisera to the cytotox-incubation medium. Anti-thy-1 and anti-immunoglobulin antisera had no enhancing effect under similar conditions. The specific alloantisera-treated spleen cells did not show any increase in cytotoxicity against P815 and L1210 target cells. Spleen cells responsible for the alloantiserum-mediated augmentation of cytotoxicity against K562 cells appear to be different from T or B cells as indicated by their resistance to anti-thy-1 and complement treatment and lack of adherence to nylon wool columns.     

T lymphocyte-enriched murine peritoneal exudate cells. III. Inhibition of antigen-induced T lymphocyte Proliferation with anti-Ia antisera.

The recent development of a reliable murine T lymphocyte proliferation assay has facilitated the study of T lymphocyte function in vitro. In this paper, the effect of anti-histocompatibility antisera on the proliferative response was investigated. The continuous presence of anti-Ia antisera in the cultures was found to inhibit the responses to the antigens poly (Glu58 Lys38 Tyr4) [GLT], poly (Tyr, Glu) ploy D,L Ala-poly Lys [(T,G)-A--L], poly (Phe, Glu)-poly D,L Ala-poly Lys [(phi, G)-A--L], lactate dehydrogenase H4, staphylococcal nuclease, and the IgA myeloma protein, TEPC 15. The T lymphocyte proliferative responses to all of these antigens have previously been shown to be under the genetic control of major histocompatibility-linked immune response genes. The anti-Ia antisera were also capable of inhibiting proliferative responses to antigens such as PPD, to which all strains respond. In contrast, antisera directed solely against H-2K or H-2D antigens did not give significant inhibition. Anti-Ia antisera capable of reacting with antigens coded for by genetically defined subregions of the I locus were capable of completely inhibiting the proliferative response. In the two cases studied, GLT and (T,G)-A--L, an Ir gene controlling the T lymphocyte proliferative response to the antigen had been previously mapped to the same subregion as that which coded for the Ia antigens recognized by the blocking antisera. Finally, in F1 hybrids between responder and nonresponder strains, the anti-Ia antisera showed haplotype-specific inhibition. That is, anti-Ia antisera directed against the responder haplotype could completely block the antigen response controlled by Ir genes of that haplotype; anti-Ia antisera directed against Ia antigens of the nonresponder haplotype gave only partial or no inhibition. Since this selective inhibition was reciprocal depending on which antigen was used, it suggested that the mechanism of anti-Ia antisera inhibition was not cell killing or a nonspecific turning off of the cell but rather a blockade of antigen stimulation at the cell surface. Furthermore, the selective inhibition demonstrates a phenotypic linkage between Ir gene products and Ia antigens at the cell surface. These results, coupled with the known genetic linkage of Ir genes and the genes coding for Ia antigens, suggest that Ia antigens are determinants on Ir gene products.     

Blocking of MLC stimulation by anti-Ia sera: studies with the virus plaque assay.

The development of congenic mouse strains identical at the H-2K and H-2D loci but differing by I-region associated (Ia) determinants has permitted an association to be established between Ia determinants and stimulation in mixed lymphocyte culture reactions (MLR). The present experiments were undertaken to establish whether the Ir-coded control of MLR operated at the level of recognition or of stimulation. Reciprocal MLR were established between A.TH and A.TL mouse spleen cells in the presence or absence of anti-Ia sera directed either at determinants of the stimulating or responding cells. The number of T cells responding was assessed by the virus plaque assay. Anti-Ia sera directed against the responding cells were no more inhibitory of the MLR than normal mouse serum. In contrast, anti-Ia sera directed against determinants of the mitomycin-treated stimulating cells markedly inhibited activation of T cells in the MLR.     

Expression of the major AgB histocompatibility complex antigens on different structural components of rat kidney and heart

We have analyzed the expression of the major AgB locus antigens on the parenchymal cell components of rat kidney and heart using (a) heterologous antisera raised against isolated molecules and (b) conventional alloantisera, directed to the serologically detectable AgB and Ia allodeterminants. A modified Staphylococcus aureus Cowan I rosette assay, enabling the characterization of the rosette-forming cell type from stained cytocentrifuged cell smears, was used. The AgB and Ia antigens were detected by both types of antisera on the “passenger” cells of either organ: the anti-Ia reacted especially strongly with a fraction of “passenger” lymphocytes, and anti-AgB also with the “passenger” erythrocytes. All kidney parenchymal components were nearly devoid of both AgB and Ia antigens: only a weak reaction was observed with the vascular endothelial, tubular, or glomerular cells after treatment of either type of antiserum. The heart endothelial cells expressed the AgB and probably some Ia: an intermediary intensive reaction was observed after treatment with heterologous or allospecific anti-AgB, and a weak reaction with anti-Ia. The heart myocardial muscle cells, on the other hand, were nearly devoid of both antigens: no reactivity with heterologous anti-Ia and only a marginal reactivity with heterologous or allospecific anti-AgB was observed.     

Biochemical characterization of murine Ia antigens with xenoantisera. I. Identification of a second la molecule (I-E?) in H-2s haplotype

Rabbit anti-Ia sera was produced by immunization with detergent-solubilized extracts from splenic, lymph-node and thymus cells. The antisera contained activity against H-2 as well as Ia molecules. By a sequential immunoprecipitation assay it was shown that the rabbit anti-mouse H-2s serum precipitated a second Ia molecule in the H-2s haplotype. Previous studies with alloantisera have shown only one Ia molecule associated with this haplotype. Sequential precipitations with alloantiserum against the whole I region were used to show that this second Ia molecule is coded by genes within the I region. Since only I-A- and I-E-region coded molecules are immunoprecipitable in most haplotypes, we presume that the rabbit antiserum could be identifying the I-E-subregion coded molecule in the H-2s haplotype. The rabbit antiserum reacts with an isotypic specificity on the molecule. The studies suggest that the I-E subregion does exist in the H-2s haplotype even though alloantiserum cannot be produced to identify allotypic variants associated with this subregion.     

Ag-B-linked analogue of Ia antigens in the rat.

The reactions of Lewis rat lymphocyte membrane antigens with two alloantisera, BN anti-Lewis and BN anti-Fischer have been studied. Three lines of evidence indicated that these antisera reacted with cell surface antigens homologous to Ia antigens of the mouse. 1) After absorption with Lewis platelets, the antisera killed only 40 to 50% of Lewis spleen cells. The majority of such cells were shown to be Ig-positive B cells by the examination of reaction patterns on lymphocytes after separation on nylon wool into T cell- and B cell-enriched subpopulations. 2) SDS-PAGE analysis of solubilized Lewis spleen cell antigens precipitated with these antisera revealed that the platelet-absorbed antisera reacted with molecules comparable in size to mouse Ia antigens (mw approximately equals 35,000 and 28,000). The unabsorbed sera reacted with these molecules and with additional molecules corresponding in size to mouse K and D antigens (m.w. = 45,000). 3) Neither of these antisera killed significant numbers of spleen cells from the partially congenic strain F.BN (seventh backcross homozygotes), a Fischer rat to which the Ag-B.3 allele is being transferred by repetitive backcrossing, indicating that the genes coding for these Ia-like antigens in the rat are linked to the Ag-B locus.     

Presence of T cell-associated surface antigens on murine NK cells.

The expression of T cell-associated surface antigens on natural killer (NK) spleen cells of C57BL/6 mice was evaluated by cytotoxic depletion experiments with alloantisera prepared against the Thy 1, Ly 1, Ly 2, Ly 5, Ly 6, and NK 1 antigens. The NK activity of these nonimmunized spleen cells for YAC-1 leukemia cells was dramatically reduced by antisera to the Ly 5 and NK 1 antigens. Variable results were obtained with anti-Ly 6 sera--certain pools of this antiserum decreased the NK activity, whereas other pools showed only negligible effects. The NK activity of the same cell suspensions was not affected by antisera to the Thy 1, Ly 1, and Ly 2 antigens. In parallel tests the T cell-associated cell surface antigens of alloimmune T killer cells were similarly evaluated by cytotoxic depletion experiments. In this case, the activity of these cells was consistently diminished by antisera to the Thy 1, Ly 2, Ly 5, and Ly 6 antigens, but not by antisera to the Ly 1 and NK 1 antigens. On this basis it was concluded that the NK cells expressed a restricted subset of T cell-associated alloantigens and therefore may have been derived from the T cell lineage of lymphocytes.     

Detection of at least two distinct mouse I-E antigen molecules by the use of a monoclonal antibody

In an attempt to determine whether the expression of more than a single Ia antigen is determined by the I-E subregion of the mouse major histocompatibility complex (MHC), sequential immunoprecipitation analyses were performed by using a monoclonal antibody and alloantisera reactive with I-E subregion products. 3H-leucine-labeled glycoprotein preparations obtained from H-2d spleen cells were precleared with the monoclonal antibody 14-4-4S and then examined for residual Ia activity precipitable by an alloantiserum detected by SDS-polyacrylamide gel electrophoresis. Residual Ia activity was observed for all three strains of the H-2d haplotype tested. The residual Ia activity could be detected by sera absorbed with B10.A spleen cells, indicating that products of the I-E subregion rather than of the I-C subregion were responsible for this activity. No separable I-Ek molecules were detected in products of B10.A cells with the use of combinations of two monoclonal antibodies (including 14-4-4S) and several appropriate alloantisera. These findings indicate the presence of at least two similar but distinct I-E antigens encoded by the H-2d haplotype.     

Interleukin-2- and mitogen-activated NK-like killer cells from highly purified human peripheral blood T cell (CD3+ N901-) cultures

In this study, highly purified (HP) CD3-positive N901-negative T lymphocytes could be induced to become natural killer (NK)-like in culture in the presence of recombinant interleukin-2 (rIL-2) and phytohemagglutinin (PHA). Thus, purified CD3+ N901- T cells from fresh human peripheral blood were obtained by negative selection using an indirect panning technique. To ensure that T lymphocyte fractions were completely devoid of any detectable NK cells, two additional purification procedures were employed: incubation of post-pan T cells with the NK-cytotoxic lysomotropic agent L-leucinemethylester, and complement-mediated lysis using the NK cell specific NKH1a monoclonal antibody. Purity of CD3+ N901- cells could be confirmed by surface marker analysis, whereby two NK-associated antigens, N901 and H-25, were undetectable, while 94 +/- 1% of cells expressed the CD3 (Leu-4) antigen. On functional analysis, fresh HP CD3+ N901- cells exhibited no cytotoxic activity against the standard NK target K562. When HP NK-depleted T lymphocytes were cultured for 7 days in the presence of rIL-2 (100 U/ml), neither surface antigen expression nor cytotoxic activity against K562 changed significantly. However, significant cytotoxicity against K562 [18 +/- 5% specific lysis at 25:1 effector:target (E/T) ratio] could be induced when HP CD3+ N901- cells were grown for 7 days in the presence of rIL-2 and PHA (0.5% v/v). Concomitantly, antigens N901 and H-25 were found to be coexpressed on a minor proportion (22 +/- 16 and 22 +/- 6%, respectively) of CD3+ (88 +/- 2% on day 7) cells. Four-week long-term culture of HP NK-depleted T cells in the presence of rIL-2 and PHA yielded a continuous increase in cytotoxicity against K562 cells (0 up to 46% specific lysis at 25:1 E/T ratio). Of particular interest was the emergence of cytotoxicity against the NK-resistant Daudi cell target (15 +/- 8% specific lysis at 25:1 E/T ratio on day 21). Expression of antigens N901 and H-25 as well as CD3 remained essentially unchanged in long-term culture. In sorting experiments, the H-25+ cell fraction was significantly enriched for cytotoxicity against K562, when compared to both H-25- and unseparated cell fractions. In summary, our results suggest that a proportion of HP CD3+ N901- T lymphocytes may give rise to cells that exhibit NK-like functional and phenotypic properties.     

Generation of T helper cells in vitro. III. Helper cell culture-derived factors are related to alloantigens coded for the I region of the H-2 major histocompatibility complex.

Thymocyte-macrophage cultures primed with carrier protein release an alloantigen-related factor that enhances the anti-hapten plaque-forming cell response of hapten-primed spleen cells in vitro. Use of immunoadsorbant columns made with a variety of alloantisera indicates that the relevant antigens in this system are coded for the the H-2 major histocompatibility locus in mice. The data indicate that in H-2d and H-2k strains the important genetic regions are in the I region between the K region and the I-C subregion and suggest, based on current understanding of Ia specificities, that the I-A subregion codes for these antigens.     

Nature of the antigenic complex recognized by T lymphocytes. IV. Inhibition of antigen-specific T cell proliferation by antibodies to stimulator macrophage Ia antigens.

We have previously demonstrated that nonimmune guinea pig T lymphocytes could be specifically sensitized with TNP-modified allogeneic macrophages after eliminating the alloreactive T cells with bromodeoxyuridine (BUdR) and light treatment. This procedure allowed the unique opportunity to use anti-Ia sera directed against the Ia antigens of only the stimulator macrophages or responder T cells to determine against which cell type anti-Ia would block TNP-specific stimulation. It was found that the TNP-specific DNA synthetic response of BUdR and light-treated T cells stimulated with TNP-modified allogeneic macrophages was totally eliminated by anti-Ia sera directed solely against the allogeneic stimulator macrophage. In contrast, anti-Ia sera directed only against the responder T cells had no effect on their response to TNP-modified allogeneic macrophages. These findings indicate that macrophage Ia antigens are required for efficient T cell-macrophage interactions and raise the possibility that T cell Ia antigens may not be required for collaboration with macrophages. This latter possibility was substantiated by experiments in which we show that treating T cells with anti-Ia sera and complement to remove the Ia-positive cells either before or after priming, or both, had no effect on their ability to be primed and restimulated with TNP-modified macrophages.     

The distribution of la antigens of the H-2 complex on lymph node cells by immunoferritin labelling

The distribution of Ia antigens on the surfaces of lymph node lymphocytes of several mouse strains was investigated using indirect immunoferritin labeling and electron microscopy. The immunoferritin labeling results agreed with results of cytotoxic tests in strain distribution of reactivity, proportion of cells showing label, and cell populations reacting. Capping was induced by increased incubation temperature but conditions for Ia antigen mobilization varied somewhat between the two anti-Ia antisera employed. Uncapped specimens generally showed a denser, more evenly distributed antigen coating than is the case for H-2 antigens labeled by the same indirect immunoferritin method.     

Nature of the antigenic complex recognized by T lymphocytes. VII. Evidence for an association between TNP-conjugated macrophage membrane components and Ia antigens.

In the present study we examined the effects of anti-sera directed against guinea pig Ia antigens on the ability of TNP-conjugated macrophages to stimulate TNP-specific T lymphocyte proliferation. Treatment of macrophages with anti-Ia sera for 1 hr before, 1 hr immediately after, or as late as 24 hr after TNP-modification resulted in a reduced ability to stimulate the TNP-specific T cell. The inhibition produced by anti-Ia sera was specific and did not result from interference with the ability of macrophages to process TNP-conjugated membrane antigens in a nonspecific manner. Brief treatment with anti-Ia serum did not result in inhibition of Ia-antigen synthesis nor could evidence of carry-over of anti-Ia antibody into the lymphocyte cultures be obtained. These results demonstrate that anti-Ia sera interfere with the development of a TNP-specific immunogen on the macrophage surface and strongly suggest that an association exists between TNP-modified membrane proteins and Ia antigens on the macrophage surface.     

Distinct effects of interferon-gamma and MHC class I surface antigen levels on resistance of the K562 tumor cell line to natural killer-mediated lysis

Various investigators have examined the relationship between tumor cell susceptibility to natural killer (NK) cell lysis and the expression of HLA class I antigens on the tumor cell. There is controversy as to whether or not an inverse relationship exists, and if so, the basis of the relationship between these two phenomena remains undefined. To address these questions, the genomic clones for two HLA antigens were transfected into the erythroleukemia cell line K562, a cell line that is used as the standard to assess human NK and major histocompatibility complex (MHC) nonrestricted cytolysis. Susceptibility to NK lysis was not affected by the de novo expression of HLA antigens on the K562 after DNA mediated gene transfer. Interferon-gamma (IFN-gamma) treatment of K562 induced levels of MHC class I antigen surface expression comparable to those found on the transfected cells; however, the IFN-gamma-treated cells were resistant to NK lysis. When very high levels of surface HLA antigens were induced on the transfectants, a potential effect of class I MHC expression on K562 lysis could be discerned that was distinct from the resistance to NK lysis induced by IFN-gamma-treatment.     

Anti-Ia inhibits the activity of B cells but not a T cell-derived helper mediator

The effects of addition of anti-Ia sera to cultures of B cells responding to a number of different stimuli were studied. Anti-Ia sera inhibited the responses of B cells to thymus-dependent and thymus-independent antigens. The effects of antisera directed at different subregions within theI region were examined, using the same anti-Ia serum and mouse strains congenic atI. There was some indication that antisera directed at theI-C region might be more efficient at inhibiting responses to a thymus-independent antigen than to a thymus-dependent antigen. An antiserum to another B-cell surface component controlled by theH-2 complex, the D glycoprotein, had no effect on the response of B cells to a thymus-dependent antigen. By contrast, anti-Ia serum added to cultures had no effect on the activity of a T cell-derived, nonspecific, B-cell helper mediator (NSM). We concluded that the binding of anti-Ia sera to B-cell surfaces inhibited B-cell responses to antigen, either by competing directly with the binding of signal molecules, or by delivering an inhibitory signal to the B cell, such that it was subsequently refractory to stimulatory signals.     

Anomalous reactions of mouse alloantisera with cultured tumor cells. II. Cytotoxicity is caused by antibodies to leukemia viruses.

Certain alloantisera prepared in mice against H-2 region membrane antigens were found to be unexpectedly cytotoxic for murine sarcoma and leukemia cells in culture. This anomalous cytotoxicity was shown to be the result of antibody in these alloantisera directed against the p15 and gp70 envelope proteins of Mu LV which were present on the surface of the tumor target cells. Sera from aged unimmunized mice of strains used for the preparation of alloantisera also contained antibodies against MuLV protein p15 and gp70 that were cytotoxic for sarcoma and leukemia cells, which indicates that these antibodies occurred naturally in mice. These results independently confirm earlier findings of the widespread occurrence in mouse serum of antibodies reactive with MuLV. The presence of antibody against MuLV in mouse serum which can cause cytotoxic reactions with tumor cells points to the fact that particular caution should be used during the typing of murine sarcomas or leukemias for cell surface antigens, since mouse antisera may yield cytotoxicity (or other serologic reactions) based on anti-MuLV specificities, rather than on anticipated antigens.     

Partial characterization of Ia antigens from murine lymphoid cells

Congenic anti-Ia antisera were used to bind radiolabelled Ia antigens from cells of various strains of mice of knownH-2 haplotype. The results indicate that Ia antigens are proteins of molecular weight 30,000 to 35,000 daltons. The Ia antigens are distinct from known H-2 antigens as judged by independent immunoprecipitation as well as by molecular weight. Ia antigens are synthesized by, and are present on the surface of lymphoid cells as evidenced by incorporation studies using³H-leucine and enzymatic radioiodination of cells, respectively. Tissue distribution of cell surface Ia suggests that Ia antigens are on B cells. Ia antigens were detected in the incubation media of³H-leucine labeled splenocytes suggesting that antigens may be secreted.     

Recognition and lysis by natural killers of tumor cells with participation of laminin

According to the data obtained in the present work, the receptor complex of mouse natural killers (NK) includes laminin, an antibody that blocks NK activity (NKA) regardless of the presence of a complement. Preincubation of mouse splenocytes with antilaminin serum led to a decrease in their NKA towards tumor cell-stargets (CT) with the NKA activity decreasing by a factor of 2 with respect to cultivated cells of rat hepatoma HTC and by a factor of 10 with respect to cultivated cells of human erythroblastosis K562. Pretreatment of splenocytes with normal nonimmune serum did not lead to a change of NKA. The pattern after tumor-cell preincubation with antilaminin serum was quite different; pretreatment of CT K562 led to a twofold decrease in the sensitivity of these cells to NK lysis, whereas the pretreatment of CT K562, on the contrary, made them twice as sensitive to NK lysis. Electrophoretic separation of proteins of CT plasma membranes with subsequent immunoblotting with antilaminin immune serum revealed the presence of laminin on HTC cell plasma membrane, which was identified as laminin 8/9 by the mass-spectrometry method, while no laminin was detected on K562 cells. Preincubation of splenocytes with laminin did not affect NKA with respect to CT K562 and HTC. Pretreatment of CT K562 and HTC with laminin decreased the NKA to zero. The obtained data allow for the suggestion of the doubtless participation of laminin and its receptors in CT cytolysis by NK.     

Role of interferon in natural kill of HSV-1-infected fibroblasts

The production of interferon during natural killer (NK) assays against HSV-1-infected fibroblasts (NK(HSV-1)) was studied to determine whether this interferon was responsible for inducing the preferential lysis of herpes-virus-infected target cells over uninfected target cells. The interferon produced during NK(HSV-1) assays was analyzed and found to have the properties of HU-IFN-alpha. Little or no IFN was produced during NK assays against uninfected fibroblasts (NK(FS)) or K562 (NK(K562)) cells. Although the appearance of interferon in the culture supernatants seemed to parallel the development of cytotoxicity during NK(HSV-1) assays, the levels of cytotoxicity and IFN generated did not correlate, arguing against a strict quantitative dependence of cytotoxicity upon IFN production. NK(K562) and NK(FS) cytotoxicity developed with little or no production of IFN. When IFN-pretreated effector cells were used, there was still a preferential lysis of infected over uninfected target cells. This preferential lysis by IFN-treated effector cells of infected over uninfected targets was seen as early as 2 hr into the assay. Anti-IFN antibodies added to the NK assays, although neutralizing all the IFN produced during the assays, had no effect on NK(FS) or NK(K562) cytotoxic activity and caused a slightly reduction of NK(HSV-1) activity only in one of three experiments. We conclude that although IFN is generated during NK(HSV-1) assays, this IFN cannot solely account for the increased lysis of infected over uninfected cells and that NK(HSV-1) activity is in some other way dependent on the virus infection.