Lytic Assay Research Articles

Analysis of vesicle immunolysis assays: The direct binding model

Assays based on lysis of lipid vesicles have shown high sensitivity. However, little as yet is known about the quantitative relationships among the various assay parameters, due in part to the lack of a predictive theoretical model. This paper presents the derivation of the equations that describe a simple model assay system in terms of the total fraction of vesicles with bound antibodies and the distribution of vesicles with one, two, or more antibodies bound. The equations show how the binding of antibodies to vesicles is affected by such variables as: vesicle concentration, antigen density on vesicle surfaces, antibody concentration, and antibody affinity. With the distribution functions, experiments can be designed to determine the minimum number of antibodies needed to lyse a vesicle. In addition, it is shown how estimations of the ultimate sensitivity of lipid vesicle lytic assays can be made. The model can be used to optimize vesicle lysis assay systems.

Cytolytic and biochemical properties of cytoplasmic granules of murine lymphokine-activated killer cells.

The incubation of murine spleen cells in the lymphokine interleukin 2 (IL 2) gives rise to lymphokine-activated killer (LAK) cells capable of lysing fresh tumor cells in short-term lytic assays. During the course of cultures used to generate LAK cells, cytoplasmic granules were prepared and were analyzed for the presence of the cytolysin previously described in large granular lymphocytes (LGL) and cytotoxic T lymphocytes (CTL). Such cytolysin activity is initially undetectable, appears after 2 days of culture, and continues to increase until day 7. The LAK cytolysin has properties similar to those of previously described cytolysins with respect to nonspecific killing of various target cells, rapid kinetics, and absolute dependence on calcium. Antibodies raised against purified LGL tumor granules neutralized the activity of the LAK cytolysin. The precursors of both the LAK cells and the cells bearing the cytolysin are eliminated by treatment with anti-asialo-GM1 and complement, strongly suggesting that the actual LAK effector cells and the cytolysin-bearing cells are identical. Biochemical analysis of the LAK granules indicate that they contain the lysosomal enzyme arylsulfatase. The protein content of granules isolated from various days of culture with r-IL 2 undergoes a dramatic change, with major protein bands around 30,000 daltons becoming prominent, as well as the cytolysin protein band at 70,000 daltons. These data suggest that the mechanism of cell lysis by LAK cells is similar to that of CTL and natural killer-mediated lysis, and each of these forms of lymphocyte-mediated cytolysis is based on a granule exocytosis mechanism.

The effects of hexose 6-O-sulfate esters on human natural killer cell lytic function.

Natural cell-mediated cytotoxicity (NCMC) is inhibited by some neutral hexoses and hexose phosphates at 25 to 100 mM concentrations. In this study we describe the effects of hexose 6-O-sulfate esters on NCMC against K-562 target cells. Mannose 6-sulfate, galactose 6-sulfate, N-acetylglucosamine 6-sulfate, and N-acetylgalactosamine 6-sulfate inhibit NCMC in a dose-dependent manner at concentrations of 10 mM and below. Inhibitory effects of mannose 6-sulfate and galactose 6-sulfate were evident at concentrations as low as 1.25 mM. The neutral forms of these sugars, glucose and glucose 6-sulfate, did not inhibit NCMC over this range of concentrations. Comparison of the inhibitory effects of sulfated and phosphorylated forms of mannose and galactose indicated that the sulfated forms are much more potent inhibitors. Formation of effector cell:target cell conjugates was unaffected by the presence of sugar sulfates. Calcium pulse experiments demonstrated that inhibitory effects of sugar sulfates were exerted after the Ca++-dependent triggering step in the NK lytic process. Kinetic studies showed that addition of sugars as long as 60 min after initiation of cultures yielded potent inhibitory effects. Sugar sulfates were not toxic for effector cell populations and effectors were not refractory for lytic function after removal of sugars. Sugar sulfates were inhibitory against multiple tumor types in both human and murine NK lytic assays. These results suggest that the sugar sulfates inhibit NK cells at a postconjugation, posttriggering step involving lectin-like receptors or lectin-like molecules.

Trypanosoma cruzi: Isolate dependence in the induction of lytic antibodies in the mouse and rabbit

Lytic antibodies able to interact with the live trypomastigotes of Trypanosoma cruzi have been associated with both protection and active infection. There are T. cruzi isolates unable to induce lytic antibodies despite their capacity in eliciting agglutinins and precipitins for immunofluorescent labeling. The host's spontaneous cure was ruled out as being responsible for negative results. The test performed either at 4 C or in the presence of sodium azide proved that negative lytic assays could not be attributed to capping phenomena. The classification of the parasites as T. cruzi was confirmed by their behavior in tissue culture and in the vector, as well as by the cross-protection exhibited by chronically infected mice against other lethal T. cruzi isolates. Cross-resistance achieved by these mice also suggests that the host's protection during chronic infection is independent of the lytic antibody titer.

Clonal analysis of T lymphocytes in spleens from patients with Hodgkin's disease. Frequent occurrence of unusual T4-positive cells which co-express cytolytic activity and production of interleukin-2.

T-lymphocyte populations isolated from spleens of untreated patients with Hodgkin's disease (HD) were grown by combining limiting dilution techniques and an assay system that allows clonal proliferation of virtually all human T cells. Under these conditions, high proportions of splenic T lymphocytes (50-80%) underwent clonal expansion, so that the set of clones obtained could be considered representative of the starting T-cell population. A total number of 229 clones from 6 HD spleens (3 uninvolved and 3 histologically involved by the disease) and 133 clones from 3 control spleens (obtained from otherwise healthy individuals, who underwent post-traumatic splenectomy) were examined for surface markers and tested in functional assays. One hundred and seventy-five clones from HD spleens and 75 clones from control spleens expressed the "helper/inducer" (T3+ T4+ T8-)phenotype, whereas as 54 clones from HD spleens and 58 control clones expressed the "cytotoxic/suppressor" (T3+T4-T8+) phenotype. As assessed by a non-specific lectin-dependent lytic assay, the proportion of HD clones displaying cytolytic activity was higher than that of cytolytic clones derived from control spleens. The majority of T4+ clones obtained from HD spleens (either uninvolved or histologically involved by the disease) were cytolytic, whereas only a small proportion (less than 10%) of T4+ clones derived from normal peripheral blood or spleens displayed cytolytic activity. The cytolytic potential of T4+ clones obtained from HD spleens did not reflect the activity of natural killer (NK) cells, since a minority of these clones exerted NK activity on K562 target cells. In addition, most of the T4+ cytolytic clones derived from HD spleens produced particularly high amounts of interleukin-2 (IL-2). These data indicate that T lymphocytes which concentrate in spleens of patients with HD consist at least in part of an infrequent T4+ cell subset co-expressing cytolytic activity and production of IL-2. These cells may reflect a cytotoxic reaction against unknown antigens associated with self class-II histocompatibility antigens.

Frequent T4-positive cells with cytolytic activity in spleens of patients with Hodgkin's disease (a clonal analysis).

Purified T lymphocytes isolated from spleens of untreated patients with Hodgkin's disease (HD) were cloned by using a microculture system previously shown to allow clonal expansion of virtually all peripheral blood T lymphocytes. Cells were plated under limiting conditions with irradiated feeder cells and PHA. Interleukin 2 (IL 2)-containing supernatants were added 48 hr later. The phenotypic and functional characteristics of a total number of 221 clones derived from six different HD spleens were investigated and compared with those of 133 clones obtained from three spleens of otherwise healthy individuals who underwent posttraumatic splenectomy. The majority of T cell clones derived from HD spleens expressed the T4+ (helper/inducer) phenotype. However, further functional characterization showed that as much as 50% of these T4+ clones displayed cytolytic activity in a lectin-dependent lytic assay allowing detection of cytolytic cells of any specificity. In contrast, less than 10% T4+ clones derived from control spleens were cytolytic, as assessed by the same lectin-dependent lytic assay. The cytolytic potential of T4+ and T8+ clones established from spleens of patients with HD did not reflect the induction of lymphokine-activated killer cells, because only a minority of them displayed natural killer (NK) activity against NK-sensitive K562 and MOLT-4 cell lines. These findings indicate that T lymphocytes found in the spleens of patients with HD may represent, at least in part, the expansion of a subset present in small percentages among normal peripheral blood or spleen T lymphocytes, which is involved in a cytotoxic reaction.

Complement-dependent cellular cytotoxicity: lymphoblastoid lines that activate complement component 3 (C3) and express C3 receptors have increased sensitivity to lymphocyte-mediated lysis in the presence of fresh human serum.

Lymphocyte-mediated lysis of cells of the Raji, Daudi, Jijoye, and Bjab lines was elevated when fresh human serum was added to the assay. A higher proportion of effector-target conjugates was observed in the presence of human serum. In similar experiments lysis of 1301, Rael, and P3HR-1 cells was unaltered. All cell lines activated the alternative pathway of complement but they varied in the expression of receptors for complement component 3 (C3) and in the ability to fix the C3 cleavage products on their membrane. The enhancement of lysis in the presence of human serum occurred only with those cells that bound C3. This characteristic was correlated to the expression of C3 receptors. Analysis of the nature of the deposited C3 was performed with Raji cells. Raji cells exposed to human serum bound C3b as indicated by the immunoadherence test. The C3b was further processed to C3bi, because the immunoadherence declined with time and conjugate formation increased with Daudi cells, which carry the C3 receptors CR2 and CR3. This suggests that in the lytic assay lymphocytes with C3bi receptors are recruited in the presence of human serum. We assume that the bridge of C3 molecules between targets and effectors increases the avidity of their interaction.

Down-Regulation of K Cell Activity by Neutrophils

Abstract Human neutrophils, activated by phorbol-myristate acetate (PMA), (A- neutrophils), were found to suppress lymphocytic killer (K) cell- mediated antibody-dependent cellular cytotoxicity (ADCC). Resting (R) neutrophils, ie, PMA-untreated cells, were completely ineffective. Suppression was optimal when A-neutrophils were added at the beginning of the ADCC assay. Furthermore, A-neutrophils were found to cause an approximately 80% reduction in the number of Raji target cell-bound lymphocytes. These data indicate that A-neutrophils inhibit K cell activity by interfering with the target cell recognition. A-neutrophils were capable of reducing the percentage of Fc receptor (FcR)-bearing lymphocytes with a half-time of 7.2 minutes, through a process preventable by the serine-protease inhibitors tosyl-lysine-chloromethyl ketone (TLCK) and lima bean trypsin inhibitor (LBTI). Conversely, A- neutrophils caused a very slow decrease in the amount of Raji cell- bound antibodies, as detected by the complement-mediated lytic assay. Thus, only lymphocyte FcR structures seem to be highly susceptible to neutrophil-derived TLCK- and LBTI-inhibitable proteases. Furthermore, supernatants from A-neutrophils were found to inhibit K cell ADCC and lymphocyte binding to Raji target cells. In addition, LBTI prevented the A-neutrophil-dependent and the supernatant-dependent inhibition of both K cell ADCC activity and lymphocyte-target cell conjugate formation. Together these data suggest that A-neutrophils suppress K cell function through a protease-mediated impairment of the FcR binding capacity. The results provide evidence that human neutrophils are endowed with mechanisms to regulate K cell ADCC activity.

Down-regulation of K cell activity by neutrophils

Human neutrophils, activated by phorbol-myristate acetate (PMA), (A-neutrophils), were found to suppress lymphocytic killer (K) cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Resting (R) neutrophils, ie, PMA-untreated cells, were completely ineffective. Suppression was optimal when A-neutrophils were added at the beginning of the ADCC assay. Furthermore, A-neutrophils were found to cause an approximately 80% reduction in the number of Raji target cell-bound lymphocytes. These data indicate that A-neutrophils inhibit K cell activity by interfering with the target cell recognition. A-neutrophils were capable of reducing the percentage of Fc receptor (FcR)-bearing lymphocytes with a half-time of 7.2 minutes, through a process preventable by the serine-protease inhibitors tosyl-lysine-chloromethyl ketone (TLCK) and lima bean trypsin inhibitor (LBTI). Conversely, A-neutrophils caused a very slow decrease in the amount of Raji cell-bound antibodies, as detected by the complement-mediated lytic assay. Thus, only lymphocyte FcR structures seem to be highly susceptible to neutrophil-derived TLCK- and LBTI-inhibitable proteases. Furthermore, supernatants from A-neutrophils were found to inhibit K cell ADCC and lymphocyte binding to Raji target cells. In addition, LBTI prevented the A-neutrophil-dependent and the supernatant-dependent inhibition of both K cell ADCC activity and lymphocyte-target cell conjugate formation. Together these data suggest that A-neutrophils suppress K cell function through a protease-mediated impairment of the FcR binding capacity. The results provide evidence that human neutrophils are endowed with mechanisms to regulate K cell ADCC activity.

Assignment of human natural killer (NK)-like cells to the T cell lineage. Single allospecific T cell clones lyse specific or NK-sensitive target cells via distinct recognition structures.

The aim of the present study was to define the cell lineage of mixed lymphocyte culture (MLC)-induced natural killer (NK) effector cells. Human MLC cells were plated under limiting microculture conditions in the presence of irradiated spleen cells and interleukin 2-containing supernatant. After 18 days, microcultures were scored for proliferation and for cytolytic activity against specific lymphoblasts and NK-sensitive K562 target cells. About 1 in 7 and 1 in 5 proliferating microcultures had specific or NK-like cytolytic activity, respectively. Moreover, several microcultures exhibited dual (specific and NK-like) cytolytic activity, even when they had been established at relatively low numbers of responding cells/well (0.5-0.25) to ensure a high probability of monoclonality. Direct evidence for the existence of cytolytic effector cells with dual activity was achieved by using clones derived from single MLC T cells by micromanipulation. Out of 26 cytolytic clones so derived, 16 exhibited specific cytolytic activity, whereas 22 lysed K562 target cells. More interestingly, 12 of these 26 clones were active against both types of target cells. Only one of these clones was able to lyse autologous or unrelated target cells. In contrast, all such clones lysed the NK-sensitive cell lines G11, MOLT-4, Raji, Daudi, Chang and T-24. Addition of saturating amounts of B9-4 monoclonal antibody in the lytic assays resulted in the inhibition of the specific cytolysis, but not the NK-like activity of clones with dual cytolytic activity. It thus appears that (a) alloreactive cytotoxic T lymphocytes can mediate both specific and NK-like cytolysis and (b) two independent recognition structures are involved in this dual activity.

Target determinants for F1 hybrid anti-parental H-2d cell-mediated lympholysis: self antigens controlled by the D end.

The immunogenetic specificity and reactivity with syngeneic target cells were determined for (C57BL/6 x DBA/2)F1 anti-parental DBA/2 cytotoxic T lymphocytes (CTL) elicited in primary mixed spleen cell cultures. A panel of peritoneal exudate cells (PEC) from mice bearing nonrecombinant and recombinant H-2-Tla haplotypes and their F1 hybrids was used as the source of target and inhibitor cells for direct lytic and competitive inhibition assays. PEC of H-2d and several recombinant haplotypes bearing the d alleles at the D region, including H-2u, were as susceptible to lysis as parental DBA/2 PEC, irrespective of non-N-2 genetic constitution. Those of H-2b, H-2k, H-2s, and recombinant haplotypes not carrying the d allele in the D region were not lysed above the background levels. Syngeneic F1 PEC were not lysed by CTL, but were nevertheless capable of inhibiting the lysis of DBA/2 PEC, albeit slightly less effectively than homozygous Dd-positive PEC inhibitors. Mapping by inhibition assays indicated that the presence of at least one copy of the H-2Dd allele was necessary and was sufficient to confer inhibitory capacity to H-2 heterozygous PEC. These results extend previous observations on primary F1 anti-parental H-2b and H-2k cell-mediated lympholysis (CML) and indicate generality of autoreactive F1 anti-parent CML responses against surface structures coded for or controlled by H-2K or H-2D region genes.

Effect of interferon on cell proliferation and generation of cytotoxic potential in mixed autologous and allogeneic lymphocyte cultures.

Autologous and allogeneic mixed lymphocyte cultures (AMC and MLC) were assayed for blastogenesis, generation of cytotoxic potential, and the effect of interferon (IFN-alpha) on these features. The cells of the mixed cultures lysed K562, Daudi, and autologous and allogeneic phytohaemagglutinin blasts. Stimulator-specific cytotoxicity was observed only in MLC. B blasts induced with Staphylococcus aureus were only affected in a stimulator-specific manner. Short-term IFN treatment of the MLC-derived effectors before the lytic assay enhanced the nonspecific component of cytotoxicity. Cell proliferation was considerably lower in AMC than in MLC. This was decreased when IFN-alpha was added at the initiation of the cultures. The presence of IFN influenced the generation of lytic potential. Comparison of the lysis of the different targets exerted by MLC-activated cells suggested that the specific component was more substantially elevated than the nonspecific one. It is likely that the IFN induced such modifications in the culture conditions that favoured the proliferation of the specific clone. Re-exposure of lymphocytes cultured in the presence of IFN to another dose of IFN before the assay had no influence on their lytic potential.

IFN-α-induced modulations of the events in human mixed lymphocyte cultures

Autologous mixed lymphocyte cultures (AMC) with T-enriched subset of human blood lymphocytes as responders and B-cells or plastic adherent cells as stimulators and allogeneic mixed lymphocyte cultures (MLC) were assayed for blastogenesis and generation of cytotoxic potential. The activated cells lyzed K562 and Daudi, autologous and allogeneic PHA-blasts. The AMC population affected the autologous and allogeneic blasts at a similar strength and there was no indication for selective effects. B-Blasts induced with Staphylococcus aureus were not lyzed. The MLC populations had a stimulation-specific cytotoxic component. This was revealed by the stronger effect against the stimulator PHA-blasts and by the lysis of the stimulator B-blasts. Short-time interferon (IFN) treatment prior to the lytic assay enhanced the anti-Daudi and anti-K562 lytic activity of the AMC and MLC populations. With AMC the lytic efficiency against the autologous and allogeneic PHA-blasts were not changed while with MLC they were also elevated. This increase was confined to the non-specific component of the cytotoxicity. The proliferation of lymphocytes was suppressed when interferon was added at the initiation of the mixed cultures. On a per-cell basis the cytotoxic potential of these cultures were stronger. In the MLC the stimulation-specific component increased more substantially than the effect against the non-specific targets. It is possible that the IFN-induced modification of the culture conditions such as suppression of the initial proliferation favored the growth of the specific clone. Re-exposure of these cells to another dose of interferon prior to the lytic assay had no effect on the lytic potential.

Enhanced binding and functional activity of human natural killer cells after interferon treatment

Much attention has recently been directed toward natural cytolysis and its augmentation. Interferon is a well-documented natural killing enhancer, albeit its mechanism of modulation is still controversial. Utilizing a fluorochrome-labeled binding assay in a batch system correlated with a chromium release lytic assay, we examined binding and NK function against the NK susceptible target K562. We found not only percentage cytolysis increased after incubation with interferon, but also percentage effector binding cells increased substantially. Thus, we conclude that effector incubation with interferon increases the number of NK susceptible target binding cells.

Activation of human blood lymphocyte subsets for cytotoxic potential

Blood lymphocytes of individuals differ in the spontaneous cytotoxic potential exerted in vitro against certain cell lines (natural killing, NK). In the low NK donors, the activity can be enhanced by short-term IFN pretreatment of the effectors (interferon activated killing, IAK) and by addition of PHA to the short-term assay (lectin-dependent cellular cytotoxicity, LDCC). Lymphocyte subpopulations fractionated on the basis of nylon adherence, SRBC, and EA rosette formation differ in their response to these measures. The results obtained with IFN-treated lymphocytes of low NK donors were similar in strength to the spontaneous activity of the high NK donors. Therefore, the distinction between NK and IAK is only operational. The nylon passed E receptor-negative and low-avidity E receptor-positive cells had the strongest NK activity. These subsets can be triggered for enhanced activity by IFN. In the majority of the cases the high-activity E-receptor-positive subset which did not sediment with EA indicators had low NK effect and was not triggered by IFN. Addition of PHA to the lytic assay, however, induced activity in the subset. Realization of DNA synthesis was not necessary for the lytic performance. The PHA-imposed triggering event was not dependent on IFN production nor on induction of the competence for IFN response. The results showed that all non-B lymphocyte subsets separated on the basis of nylon wool adherence, SRBC, and EA rosetting contain cells with lytic potential if the appropriate stimulus is used. The relative activities of the subsets against K562 and Daudi differed. Cells which rosetted readily with EA indicators had weak effect against Daudi.

F1 hybrid-anti-parental H-2b cell-mediated lympholysis: genetic control of target antigens by the H-2D region.

The immunogenetic specificity of (C57BL/6 X DBA/2)F1 anti-parental C57BL/6 cytotoxic T lymphocytes (CTL) induced in primary mixed spleen cell cultures was determined in direct lytic and competitive inhibition assays. A large panel of peritoneal exudate cells (PEC) bearing nonrecombinant and recombinant H-2-Tla haplotypes was the source of target and inhibitor cells. All PEC of H-2b, H-2bc, H-2j, and H-2ja types, irrespective of background genetic constitution, were as susceptible to direct lysis as C57BL/6 PEC, but PEC of H-2a, H-2d, H-2k, H-2q, H-2s, and H-2u types were not. The possible involvement of the Tla region in controlling target antigens was excluded by testing PEC obtained from 4 H-2/Tla or intra-Tla recombinant mouse strains. The genes controlling target antigens were mapped to the D region with the aid of 9 intra-H-2 recombinants; for target PEC to be lysed it was necessary and sufficient that Db antigens be part of the H-2 phenotype. These results were confirmed by competitive inhibition assays. Resident peritoneal cells not exposed to fetal bovine serum were also lysed by F1 anti-parental H-2b CTL, a demonstration that target antigens are expressed on normal cells.

Radioimmunoassay for serum and urinary lysozyme.

We describe a radioimmunoassay for lysozyme (EC 3.2.1.17, mucopeptide N-acetylmuramohydrolase) in plasma and urine. It involves use of an antibody, raised in rabbits, to lysozyme from urine from a patient with monocytic leukemia. This antiserum, diluted 4000-fold, is incubated with radiolabeled lysozyme for 2 h and antibody-bound lysozyme is separated from free lysozyme with dextran-coated charcoal. Validation of the assay included precision and parallelism studies with serum and urine samples from patients with monocytic leukemia and analgesic nephropathy. Results of the radioimmunoassay and those obtained with a Micrococcus lysodeikticus lytic assay correlated well (r = 0.94). Sensitivity of the assay was 3.5 micrograms/L for both serum and urine. The main advantages of the assay are its good precision and reproducibility and its high sensitivity.

Inhibition of H-Y cell-mediated cytolysis by monoclonal H-Y specific antibody.

Assays of H-Y specific, cell-mediated cytolysis (CMC) in vitro were carried out with B6 female effector cells and B6 male target cells. Monoclonal H-Y antibody was added to the lytic assay to test whether the antigenic determinant(s) involved in H-Y-specific CMC was distinct from the serologically detected H-Y antigen. Significant blocking was observed, suggesting that the H-Y antigen detectable serologically is similar to H-Y antigen recognized by cytotoxic T cells.

Target Cell Recognition by Cytolytic T Cells: Different Requirements for the Formation of Strong Conjugates or for Proceeding to Lysis

Abstract Conjugates between nonadherent peritoneal exudate lymphocytes from alloimmune mice, and neutral redlabeled or unlabeled tumor cell targets, were assayed by a procedure involving co-sedimentation of lymphocytes and target cells, vigorous resuspension, then visualization under phase-contrast or normal optics. The requirements for obtaining such conjugates, and for maintaining them in a state resistant to shear force, were compared to those for cytolysis in the usual 51Cr release procedure. The following discrepancies were noted between the requirements for formation and visualization of such conjugates, and the requirements for the recognition stage of the cytolytic assay: 1) although in both cases divalent cations were required, more Mg++ was necessary to obtain suspended conjugates than to obtain lysis; 2) although cytochalasin A added before cell contact was inhibitory in both cases, the post-recognition steps leading to lysis were not affected, whereas preformed conjugates were affected; 3) glucose was required to obtain suspended conjugates, whereas the hexose requirement in the 51Cr release assay was restricted to a post-recognition state leading to lysis. The discrepancies between the two assays were explained by the observation that under all the inhibitory conditions tested (addition of EDTA, deoxyglucose, or cytochalasin A) preformed conjugates, although not immediately dissociated, were rendered labile to shear force. The resuspension step, necessary to visualize conjugates, imposed more stringent requirements on the T cell-target cell interaction than did the lytic assay using pelleted cells. The results suggest that the establishment of a tenacious linke between a cytolytic T lymphocyte and a target cell requires secondary, nonimmunologic interactions that follow the initial specific recognition event, and that these strengthening interactions are not necessary for cytolysis. The T cell receptor itself might only have a weak affinity, or be readily detached from the membrane, so that it has more of a “reading” than a “binding” role. A practical conclusion is that the metabolic requirements for T cell “recognition” are not fixed, but are imposed by the experimental conditions under which recognition is tested.

Monoclonal anti-rat MHC (H-1) alloantibodies.

We wish to report the isolation of 6 clones of hybrid myelomas secreting rat antibodies with specificity for alloantigens of the rat major histocompatibility complex H-1 (AgB). An account of the isolation of the first such clone including details of the immunization and fusion techniques has been published (1). Briefly two AO (H-1w, AgB2) rats were hyperimmunized against DA (H-1a, AgB4) lymphoid cells and finally boosted with an intravenous injection of DA cells 2.5 days or 4 days before fusion. Spleen cells were fused with P3-X63-Ag8 BALB/c mouse myeloma cells in the presence of Polyethylene Glycol (PEG) 1500. We used the intravenous route to boost before fusing to favour localization of the antibody response in the spleen (2). After fusion, aliquots initially containing 2 x 106 spleen cells were grown in the wells of 6 x 4 Linbro trays in HAT medium for two weeks. Active growth of presumptive hybrids was seen in all wells. Supernatants obtained at this stage were assayed for specific antibody by 51Cr release from AO and DA RBC in a complement dependent lytic assay. 106/113 wells contained significant lytic activity against DA RBC while no autoantibody activity against AO RBC was detected.