The blocking activity of antisera raised to either tumor antigens or alloantigens in cytotoxicity assays using syngeneic or allogeneic killer cells on the P815 murine mastocytoma target

Methods have been published whereby a tumor-specific antigen associated with membranes of the P815 mastocytoma of DBA/2J mice was purified. Antiserum, raised in rabbits, to this material demonstrated specificity for P815 as opposed to other cells or materials of DBA/2J origin when tested by either complement-mediated target cell lysis or the enzyme-linked immunosorbent assay ELISA. This antiserum was tested for its ability to block killing by in vitro raised syngeneic lymphocytes cytotoxic for P815. It was found that this antiserum as well as antiserum raised in rabbits to normal DBA/2J membrane components and anti-H-2^d antiserum (raised in congenic mice) were all able to block killing when ⁵¹Cr-labeled P815 targets were pretreated with these antisera. On the other hand, only the anti-DBA/2 serum and the anti-H-2^d serum were capable of slightly blocking syngeneic killing of L1210 cells. Similarly, C57B1/6 cytotoxic lymphocytes raised against DBA/2 cells were blocked by pretreatment of ⁵¹Cr-labeled P815 targets with the rabbit anti-DBA/2 serum and the anti-H-2^d serum but not by the anti-P815 serum. The implications of these observations are discussed.     

Lectin-dependent cell-mediated cytotoxicity: induction of a unique effector cell population.

Spleen cells from C57BL/6 (H-2^b) mice were assayed for their ability to mediate lectin-dependent (Con A, PHA) cell-mediated cytotoxicity (LDCC), following immunization with erythrocytes, bovine serum albumin, Bacillus Calmette Guerin, and allogeneic (H-2^d) P815 cells. Sensitization with viable, but not formaldehyde-fixed, P815 cells resulted in lectin-dependent lysis of syngeneic EL-4 cells. All other sensitization procedures failed to produce LDCC. Spleen cells from mice challenged with high (10⁸) doses of P815 cells were capable of mediating both direct (anti-P815) cytotoxicity and LDCC, while challenge with low (10⁴) doses of P815 cells produced strong LDCC reactivity in the apparent absence of direct cytotoxicity (DCMC). Characterization of the effector cells indicated that LDCC reactivity was mediated by an activated, non-adherent T cell population. The effector cells appear to be unique in that LDCC could be induced in the absence of DCMC, LDCC activity appeared prior to DCMC, and DCMC could be removed by adsorption on P815 monolayers without depleting LDCC reactivity.     

A study of the ability of H-2Kk-Iak containing subcellular fractions to elicit primary anti-H-2 cytotoxic T lymphocytes

In order to identify an antigen required for elicitation of anti-H-2 cytotoxic T lymphocytes (CTLs), we have purified the H-2-K^k glycoprotein, incorporated it into a lipid vesicle, and tested it for its ability to elicit anti-H-2K^k CTLs. The results indicate that a lipid vesicle containing purified H-2K^k can elicit specific secondary anti-H-2K^k CTLs. In addition it was shown that in association with a partially purified Ia^k fraction the primary anti-H-2K^k CTL response was enhanced. It was also shown that Ia^k antigens alone could elicit an anti-H-2^k CTL response. Although a low primary response was found with purified H-2K^k, it was observed that lipid vesicles containing both H-2K^k and Ia^k glycoproteins could elicit a significantly enhanced primary anti-H-2K^k CTL response. Lipid vesicles containing H-2K^k-Ia^k were tested for their enhanced capacity to elicit anti-H-2 CTLs as well as for their ability to elicit anti-H-2K^k CTLs in the presence of supernatants from concanavalin A-stimulated spleen cells.     

Arrest of the cell cycle reduces susceptibility of target cells to perforin-mediated lysis

Cytotoxic T lymphocytes secrete a pore-forming cytolysin, perforin, that damages membranes of target cells. They also ligate Fas receptors on target cells and provoke apoptotic death. A20 (B lymphoma) and P815 (mastocytoma) cell lines were examined for their susceptibility to perforin-mediated lysis and to Fas-induced apoptosis after blockade of the cell cycle at the G₁/S interface. Cells were arrested at the G₁/S interface by inhibition of DNA synthesis with thymidine or aphidicolin. Subsequently, the treated cells were incubated either with CTL cytotoxic granules or the Fas-specific monoclonal antibody Jo-2. We show that arrest of the cell cycle at the G₁/S interface markedly reduced the susceptibility of target cells to perforin-mediated lysis. In contrast, growth arrest with thymidine or aphidicolin increased susceptibility of A20 and P815 cells to Fas-mediated apoptosis. Susceptibility to lysis by intact CTLs was not affected significantly by blockade of target cells with aphidicolin or thymidine. When cells surviving exposure to perforin-containing granules were isolated on Ficoll density gradients and cell-cycle profiles were examined by flow cytometry, the ratio of G₁ to G₂cells increased among the survivors exposed to granules in contrast to controls incubated with buffer alone. The data suggest that cells in G₁ phase of the cell cycle are less susceptible to the perforin pathway than cells in G₂and S phases but are more susceptible to the Fas pathway. J. Cell. Biochem. 69:425–435, 1998. © 1998 Wiley-Liss, Inc.     

The spatial relationship of the viral and H-2 antigens recognized by anti-viral CTLs

With the use of monospecific rabbit anti-G protein and mouse monoclonal anti-H-2K^k, we have analyzed the spatial relationship of the serologically defined H-2K^k antigens and the major surface glycoprotein (G protein) of vesicular stomatitis virus (VSV) to those antigens recognized by B10.A (k, d) anti-VSV cytotoxic T lymphocytes (CTLs). The ability of monoclonal anti-H-2K^k or rabbit anti-G protein to inhibit specifically the cytolytic activity of B10.A anti-VSV CTLs indicates that the G protein and the H-2K^k molecules are in close proximity to the viral and H-2K^k antigens recognized by the anti-VSV (CTLs. By the method of sequential immunoprecipitation, we also demonstrated that only 10–30 percent of the serologically defined G and H-2K^k molecules are in theG-H-2K ^k complexes.Abbreviations used in this paper Con A Concanavalin A - cpm counts per minure - CTLs cytotoxic T lymphocytes - E: T ratio effector: target ratio - G major surface glycoprotein of VSV - MHC major histocompatibility complex - MOI multiplicity of infection - NP40 Nonidet-P40 - PAGE polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - SaCI Staphylococcus aureus, Cowan I strain - SDS sodium dodecyl sulfate - UV ultraviolet light     

Two Distinct Populations of Primary Cytotoxic Cells Infiltrating into Allografted Tumor Rejection Sites: Infiltration of Macrophages Cytotoxic against Allografted Tumor Precedes That of Multiple Sets of Cytotoxic T Lymphocytes with Distinct Specificity to Alloantigens

It has been reported that the rejection of tumor allografts is mainly mediated by cytotoxic T lymphocytes (CTLs). Here, we characterized the cytotoxic effector cells of C57BL/6 (B6; H-2^b) mice infiltrating into the rejection site of the i.p. allografted Meth A fibrosarcoma (or P815 mastocytoma) cells of H-2^d origin. Two types of cytotoxic cells (i.e., CD8⁺ CTLs and macrophages (Mφs)) were identified by flow cytometric fractionation of the infiltrates or by specific in vitro elimination of cells either with antibody (Ab)-coated beads or with an Ab-plus complement. Of particular interest, these effector cells showed distinct and unique target specificities. First, the CTLs were inactive against transplanted tumor (e.g., Meth A) cells, whereas they were cytotoxic against donor-related concanavalin A (Con A) blasts as well as CTLL-2 (H-2^b) cells transfected with a class I gene of H-2^d origin. A cold target competition assay suggested that the CTLs were composed of multiple sets of T cells, each of which specifically recognized different allo-antigens. Second, the Mφs lysed the allografted tumor cells but were inert toward the Con A blasts and the CTLL-2 transfectants. Unexpectedly, the infiltration of Mφs preceded the infiltration of CTLs by several days during the course of rejection. These results indicate that two distinct populations of unique cytotoxic cells (i.e., CTLs and Mφs) are induced in the allografted tumor rejection site, and that the infiltration of cytotoxic Mφs responsible for rejection precedes that of the CTLs cytotoxic against cells expressing donor-related allo-antigens.     

H-2 haplotype specificity of molecular requirements for trinitrophenyl recognition by anti-hapten cytotoxic T lymphocytes

The requirement of direct covalent association of trinitrophenyl (Tnp) groups with cell surface components for functional interactions with anti-Tnp cytotoxic T lymphocytes (CTLs) was analyzed. The question was approached by comparing three different methods of modifying target cells with Tnp groups and analyzing the ability of three anti-Tnp effector populations with different H-2 haplotypes (H-2^k, H-2^d, and H-2^b) to lyse the syngeneic Tnp-modified cells. All effector cell populations were able to lyse in an H-2 restricted manner the appropriate target cell modified with 2,4,6-trinitrobenzenesulfonic acid. As previously shown, H-2^k anti-Tnp CTLs exhibited true H-2 restriction while H-2^d anti-Tnp and H-2^b anti-Tnp CTLs lysed the haptenated syngeneic target cell preferentially but not exclusively. Cells modified by either trintrophenylated bovine serum albumin (Tnp₃₅-BSA) or trinitrophenylated Sendai virus (Tnp-SV) were rendered susceptible to lysis depending upon the H-2 haplotypes of the target cells and the anti-Tnp effector cells. H-2^k anti-Tnp CTLs were able to lyse H-2^k target cells modified with either Tnp₃₅-BSA or Tnp-SV; however, H-2^d anti-Tnp or H-2^b anti-Tnp CTLS did not significantly lyse the H-2^d or H-2^b target cells modified by either Tnp₃₅-BSA or Tnp-SV. The results suggest that Tnp groups not covalently linked with cell surface specific components can be recognized by H-2^k anti-Tnp CTLs, but not by H-2^d or H-2^b anti-Tnp CTLs.     

H-2 antigens incorporated into phospholipid vesicles interact specifically with allogeneic cytotoxic T lymphocytes

Subcellular fractions containing different H-2 antigens were tested for their ability to inhibit specific T cell-target cell conjugate formation. H-2-containing membrane vesicles, lentil-lectin-purified H-2 antigens solubilized with detergent (referred to in the text as high-density fraction) or incorporated into lipid vesicles, inhibited T cell-target cell conjugate formation effectively and specifically. However, two- to threefold more protein was required to inhibit T cell-target cell conjugate formation when detergent-solubilized lentil-lectin-purified H-2 antigens were tested. This suggests that a lipid matrix is advantageous for interaction with anti-H-2 T-cell receptors. Experiments were also undertaken to demonstrate specific binding of liposomes containing ¹²⁵I-labeled H-2 antigen to anti-H-2-specific cytotoxic T lymphocytes (CTLs). The binding of the ¹²⁵I-labeled H-2-containing liposomes was saturable and was specifically inhibited by unlabeled H-2 antigens. Monospecific anti-H-2 sera specifically inhibited the binding of liposomes containing H-2 antigen to the CTLs. The results suggest that a specific interaction can occur between serologically defined H-2 antigens and the receptor of anti-H-2 CTLs.     

Inhibition of prostaglandin synthesis in human endothelial cells treated with metabolic inhibitors

Endothelial cell injury is often associated with increased synthesis of prostaglandin (PG)I₂. We observed, however, that endothelial cells treated with metabolic inhibitors which reduce cellular ATP content develop an injury pattern characterized by reduced PGI₂ synthesis. This study examined the relationship between cell injury, arachidonic acid metabolism and ATP content in human umbilical vein endothelial cells treated with 2-deoxyglucose (2DG), a glycolytic inhibitor, and oligomycin (OG), a respiratory chain inhibitor. Either inhibitor alone significantly reduced cellular ATP concentrations, but only OG reduced basal PG synthesis. The combination of 2DG and OG, however, was more effective than either agent alone in reducing cellular ATP content (≥ 50% of control) and inhibiting basal and agonist-stimulated PGI₂ synthesis. This reduced PGI₂ synthesis preceded ⁵¹ chromium release, lactic dehydrogenase release and was not associated with a net release of arachidonic acid from cell membranes. Histamine, A23187 and bradykinin stimulated PGI₂ synthesis in untreated but not in 2DG and OG treated cells. Exogenous arachidonic acid increased PGI₂ synthesis to a similar extent in both 2DG and OG treated and untreated cells. Therefore, reduced PG synthesis in 2DG and OG treated endothelial cells is not due to inhibition of cyclooxygenase. Furthermore, reduced PG synthesis in these cells occurs prior to cell injury and is not strictly associated with cellular ATP depletion.     

In vivo anti-melanoma efficacy of allo-restricted CTLs specific for melanoma expanded by artificial antigen-presenting cells

Cytotoxic CD8⁺ T cells are key effectors in the immunotherapy of malignant and viral diseases. However, autologous T cell responses to tumor antigens presented by self-MHC are usually weak and ineffective. Allo-restricted T cells represent a potent source of tumor-specific T cells for adoptive immunotherapy. This study reports in vivo anti-melanoma efficacy of the pTRP2-specific allo-restricted CTLs expanded from the BALB/c splenocytes by multiple stimulations with aAPCs made by coating H-2K^b-Ig/pTRP2 dimeric complexes, anti-CD28 antibody, 4-1BBL molecules and CD83 molecules to cell-sized latex beads. The induced allo-restricted CTLs exhibited specific lysis against RMA-S cells pulsed with the peptide pTRP2 and H-2K^b+ melanoma cells expressing TRP2, while a murine Lewis lung carcinoma cell line 3LL could not be recognized by the CTLs. The peptide-specific activity was inhibited by anti-H-2K^b monoclonal antibody Y3. Adoptive transfer of the allo-restricted CTLs specific for malignant melanoma expanded by the aAPCs can mediate effective anti-melanoma response in vivo. These results suggested that the specific allo-restricted CTLs expanded by aAPCs coated with an MHC-Ig/peptide complex, anti-CD28 antibody, 4-1BBL and CD83 could be a potential option of specific immunotherapy for patients with malignant melanoma. X.-l. Lu and X.-b. Jiang have contributed equally to this work. An erratum to this article can be found at     

Spontaneous autocytotoxicity against an unexpected H-2 d haplotype in MRL/lpr (H-2 k) autoimmune disease-prone mice

The MRL/lpr (H-2 ^k) inbred strain, a model for the autoimmune disease systemic lupus erythematosus, differs from the healthy inbred strain MRL +/+ (H-2 ^k) by only 0.1 % of its genome. Southern blot analysis using class I and class II probes confirmed the H-2 ^k genotype of both strains. Among the Ia^k-positive peritoneal cells, cells with an unexpected expression of Ia^d specificities were detected in a radioimmunoassay using several monoclonal antibodies and one conventional antiserum. This was only found in aged (6- to 9-month-old) mice both in the MRL/lpr strain (32 % Ia^d-positive mice) and in the MRL +/+ strain (42% Ia^d-positive mice). Furthermore, 24% of aged MRL/lpr mice exhibited strong spontaneous cytotoxic T lymphocyte (CTL) activities against P815 (H-2 ^d) target cells, and 57% had a weaker but still detectable level of cytotoxicity. In contrast, such a CTL activity has never been found in the MRL +/+ strain. These results suggest that the anti-H-2^d d CTL plays a role in the onset of the autoimmune process in MRL/lpr mice.     

Failure of specific adoptive immunotherapy owing to survival and outgrowth of variant cells

Summary Adoptive immunotherapy, the transfer of spleen cells from immunized mice to mice with a small tumor, was usually curative for mice with the P815 mastocytoma provided that steps were taken to prevent the generation of tumor-induced suppressor cells in the recipient animal. However, failure of adoptive immunotherapy of the P815 tumor, resulting in regrowth of either the primary intradermal or a metastatic tumor, was observed in 10 out of 112 animals receiving graded doses of 7.5×10⁷ to 3.0×10⁸ immune spleen cells. Examination of the ten tumors in mice that failed to respond to therapy revealed that seven of them were significantly less susceptible than the original P815 tumor to rejection in vivo by transferred anti-P815-specific effector cells. In addition, nine of the ten therapy-failure tumors were also less susceptible than the original P815 tumor to lysis in vitro by P815-specific, but not DBA/2-specific, cytotoxic T lymphocytes. Sensitivity to lysis by tumor-specific cytotoxic T cells was not, however, strongly correlated with sensitivity to rejection in vivo by P815-specific effector spleen cells. Neither in vivo sensitivity to rejection, nor sensitivity to cytotoxic T cells, was correlated with alterations in class I major histocompatibility complex antigen expression. These results suggest that the survival and outgrowth of variant tumor cells was frequently the cause of failure of specific adoptive immunotherapy of the P815 tumor, and that selection for cells with a reduced sensitivity to killing by cytotoxic T cells was only one mechanism that might lead to an immunotherapeutic failure.This work was supported by a grant from RJR Nabisco Inc., a grant from the J. M. Foundation, and by USPHS grant CA-40597 awarded by the National Cancer Institute     

H-2 antigens incorporated into phospholipid vesicles elicit specific allogeneic cytotoxic T lymphocytes

Different H-2 antigen-containing subcellular fractions were tested for their ability to elicit specific anti-H-2 cytotoxic T lymphocytes (CTLs). Intact cells and membrane vesicles were capable of eliciting strong anti-H-2 primary and secondary CTL responses. However, detergent-solubilized H-2 antigens partially purified with lentil lectin were greatly reduced in their capacity to elicit secondary anti-H-2 (CTLs) and at all amounts tested did not elicit a primary CTL response. Lentil lectin-purified H-2 antigens incorporated into egg lecithin plus cholesterol (30% w/w) vesicles elicited a strong secondary anti-H-2 CTL response and a low but significant primary anti-H-2 CTL response. The results also indicate that T-cell-defined specificities are closely associated with serologically defined private and public specificities.     

Evidence for differentiation of NK1+ cells into cytotoxic T cells during acute rejection of allogeneic bone marrow grafts

The ability of lethally irradiated C57BL/6 mice to acutely reject H-2^d bone marrow is due to a lymphocyte population that is NK1⁺, ASGM1⁺, CD4^–, CD8^–, CD3⁺. Transfer of spleen cells from C57BL/6 mice expressing these antigens into nonresponder 129 mice adoptively transfers the ability to reject H-2^d marrow grafts. The specificity of this rejection maps to the H-2D major histocompatibility complex (MHC) region. Transplantation of high doses of H-2^d marrow into C57BL/6 overrides the acute rejection mechanism leading to graft survival. During growth of the graft, a cytolytic activity develops that is due to ASGM1⁺, CD8⁺ cytolytic T lymphocytes (CTLs) with H-2L^d specificity. The possibility that the ASGM1⁺, CD8⁺ CTLs are descendents of the CD3⁺, NK1⁺, ASGM1⁺, CD8^– cells responsible for acute rejection is investigated by adoptive cell transfer experiments. We show that beige mice that lack NK1⁺ cells as well as the ability to acutely reject H-2^d marrow fail to generate specific CTLs after transplantation with a high dose of H-2^d marrow. Transfer of highly purified NK1⁺ cells from B6.PL-Ly-2 ^a /Ly-3 ^a (Lyt-2.1) into beige mice together with H-2^d marrow leads to generation of Lyt-2.1 CTLs from donor NK1⁺ cells. These results show that specific CTLs are generated from NK1⁺ cells during acute marrow graft rejection. Offprint requests to: G. Dennert.     

Molecular heterogeneity of D-end products detected by anti-H-2.28 sera

In capping experiments with peripheral T lymphocytes, two anti-H-2.28 sera (AKR anti-AKR.L, anti-K^b, and C3H anti-0H.B10, k anti-b) that do not contain any Qa-2-specific antibodies are able to redistribute not only the H-2.28-positive H-2 molecules, but also Qa-2 molecules. This is due to the capacity of these sera to react with Qa-2 molecules because on cells where all known molecules of the H-2 ^d haplotype were capped (K1^d, K2^d, D^d, M^d, L^d, L2^d), both antisera still reacted when the cells came from a Qa-2 positive D^d strain (B10.A) but not when the cells were of Qa-2 negative strain (BALB/cByA). The reaction with la and non-H-2 antigens was excluded in these experiments. These data show that Qa-2 and H-2 antigens share some specificities of the H-2.28 family. Other anti-private and anti-public anti-H-2 sera failed to react with the Qa-2 molecules.     

Activation of specific suppressor cells with heat-treated allogeneic tumor cells

The ability of heat-treated allogeneic cells to induce suppressor cells was examined. The tumor cell lines EL-4 (H-2^b) and P815-X2 (H-2^d), were heated to 56 °C for 10 min and injected intravenously into mice of the DBA/2J (H-2^d) and C57BL/6J (H-2^b) strains, respectively. After 4 days, the splenocytes of the treated mice were mixed with normal spleen cells and cultured for 5 days with allogeneic tumor cells. The cytotoxic T-cell response was reduced in cultures of these cell mixtures. An allogeneic difference was required to induce suppression because the syngeneic combination did not induce suppressor cell activity. Furthermore, the induction of cytotoxic T cells to the C118 cell line (H-2^k) was not suppressed by this procedure, which suggests that the suppression was haplotype specific. These suppressor cells were sensitive to anti-Thy 1.2 and complement, cortisone, and cyclophosphamide, but insensitive to irradiation. These are characteristics similar to suppressor cells activated by intact cells. Heat treatment abrogated the tumor cell's ability to induce a proliferative and a primary, but not a secondary, cytotoxic T-cell response. The heat-treated cells also lost their ability to function as cold target inhibitor cells, but retained the same quantity of serologically detected antigens as the intact cells. These results suggest that the serologically detected antigens are responsible for the activation of the suppressor cells of the cytotoxic T-cell response.     

Stabilization and functional studies of high-molecular-weight murine lymphotoxins

High levels of lymphotoxin-like activity (LT) were found in supernatants from secondarily stimulated immune mouse splenocytes activated with concanavalin A (Con A) in vitro. Splenocytes obtained from C57Bl/6 mice immune to the P815 mastocytoma were restimulated in vitro with mitomycin C-treated P815 cells, and then stimulated with Con A. High levels of unstable LT activity are rapidly (2–4 hr) released by these lectin-stimulated splenocytes. The introduction of a crosslinking agent, glutaraldehyde, was found to stabilize this LT activity and allowed us to perform more defined biochemical studies and to examine the functional activities of the LT classes. The lytic activity in these supernatants resided in the high-molecular-weight classes, termed Complex (Cx > 200,000 daltons) and alpha-heavy (α_H 130,000–160,000 daltons). It was found that the Cx and α_H LT classes from the secondarily stimulated immune splenocytes cause lysis of allogeneic target cells, P815 and EL-4, in a 16-hr ⁷⁵Semethionine release assay, and in some cases, this lysis was specific for the sensitizing target cell.     

Immunological properties of Fc receptor on lymphocytes. 6. Characterization of suppressive B-cell factor (SBF) released from Fc receptor-bearing B cells.

EA, i.e., antigen-antibody complexes are able to induce an antigen-nonspecific suppressive factor(s) from FcR⁺ B cells by binding on FcR. This factor, termed “suppressive B-cell factor (SBF)” was only effective on H-2 compatible, but not on H-2 incompatible spleen cells in an adoptive cell transfer system. Furthermore, SBF, prepared from B10.A (H-2^a) splenic FcR⁺ B cells, suppressed the adoptive primary response of B10.D2 mice (H-2^d), in addition to A/J mice (H-2^a) against DNP-DE, by the pretreatment of cells with SBF in vitro. Absorption with affinity columns demonstrated that active components) of SBF from C3H/He mice (H-2^k) was eliminated by both B6 anti-CBA (H-2^b anti-H-2^k) and B10.D2 anti-B10.BR (H-2^d anti-H-2^k), but not B10 anti-B10.A (H-2^b anti-H-2^a). In contrast, the suppressive activity of SBF was eliminated neither by anti-mouse Ig nor by a heat-aggregated human γ-globulin column. These results indicate that SBF contains a product coded by the right-hand side of H-2 gene complex, but does not contain Ig determinants nor FcR. Thus, it is conceivable that a compatibility of the right-hand side of H-2 gene complex is required for inducing effective suppression of spleen cells by SBF. SBF was considered to be a trypsin-resistant and heat-labile substance with a molecular weight of 30,000–63,000. The target cells for SBF were FcR^? B precursors, but not helper T cells.     

Ultrastructure of an influenza virus-specific cytotoxic T-cell clone and its interaction with P815 and macrophage targets

The morphology of a cloned, H-2^d-restricted, type A influenza virus-cross-reactive cytotoxic T-cell line (L4) and its interaction with histocompatible P815 and macrophage targets was studied by transmission electron microscopy. Most characteristic features of L4 cells were organelle-free, filament-rich pseudopods, by which contact was made with target cells, tightly stacked Golgi saccules near the nuclear cleft, and large (1–3 μm) multivesiculate cytoplasmic bodies of unknown function. L4 cells contacted both type A influenza virus-infected and control (uninfected or type B virus-infected) targets, but tight contact involving interdigitating pseudopodal projections was prominent only with type A virus-infected target cells. Target cell lysis was characterised by zeiosis; however, no degranulation, membrane damage, or transfer of ultrastructurally identifiable material from killers to targets was ever observed.     

Isolation and characterization of an isoantigenic variant from a heterozygous mouse lymphoma in culture

A cell line from a mouse lymphoma heterozygous at the chromosome region for the H-2^d and H-2^k alleles was originally obtained from a transplantable lymphoma in the (C3H × DBA/2)F₁ hybrid (H-2^d/H-2^k) and cultured in vitro. The original cultured line, termed parent line, was susceptible to the cytotoxic action of antibodies directed against antigenic components of both the d and k alleles. The parent line also absorbed hemagglutinins from both anti-d anti-k antisera. A resistant, variant subline was selected from the original population by immunoselection in vitro with anti-H-2^d antibody and complement in a cytotoxic system. After one year in continuous culture in the absence of selecting antisera, the variant subline was still resistant to the cytotoxic action of anti-H-2^d antibody. Serologic analysis of the variant indicated that it had lost the D antigenic component of the d allele, had a reduced amount of the H component, controlled by both the d and k alleles, and had retained the K component of the k allele. Possible genetic mechanisms that might account for the emergence of the variant line are discussed. While the results do not necessarily support an analysis based on mitotic recombination, ascribing other mechanisms is also difficult because of aneuploidy in the cell line. Finally, the experiments point out the advantages of using in vitro immunoselective methods in the genetics of mammalian somatic cells.