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At least 19 recordsLinked to original sources
PubMed1985

Studies on the cytolytic attack mechanism of the cytotoxic T lymphocyte (CTL): preparation of antisera against cellfree cytosolic extracts of a CTL clone capable of blocking the lethal hit stage of CTL cytolysis and analysis of the cytolytic structure.

Rat antiserum (as well as purified IgG and F(ab')2 fragments) raised against cellfree cytosolic extracts (CFE) of an alloimmune cytotoxic T lymphocyte (CTL) clone (B6.1.SF.1) is a potent inhibitor of CTL-mediated cytotoxicity. Inhibition by this antiserum (termed alpha CTLL) occurred during the postbinding lethal hit stages of cytolysis, because it did not inhibit target cell binding, nor did it prematurely dissociate CTL-target cell conjugates; inhibition was observed regardless of the H-2 haplotype of the target cell or CTL employed; inhibition was reversible when pretreated, and washed CTL were used as effectors; and in Ca++ pulse experiments alpha CTLL inhibited cytolysis beyond the Ca++-dependent (lethal hit) stage of cytolysis. This antiserum did not inhibit lysis of P815 cells by activated murine macrophages or by human cytotoxic cells, and extensive absorption of the antiserum on viable thymocytes, normal spleen cells, or CTL did not reduce its blocking activity. CFE prepared from several sources of CTL, including in vivo elicited peritoneal exudate lymphocytes (PEL), secondary MLC-generated CTL, alloimmune splenic T cells, and CTL clones, contained material(s) that inhibited the ability of alpha CTLL to block CTL-mediated cytolysis. The inhibitory activity was not detected in CFE from a variety of noncytotoxic cell sources, including thymocytes, normal C57BL/6 spleen cells, EL4 or P815 tumor cells, macrophages, and helper T cell clones. It was also absent in CFE prepared from human CTL cells. Furthermore, although alpha CTLL neutralizing activity was not detectable in CFE prepared from memory CTL, it rapidly appeared in CTL parallel to the development of cytolytic activity during secondary MLC cultures. The inhibitory material in CTL-CFE appeared to be specific for alpha CTLL antibody, as it did not affect the CTL blocking activity of anti-Lyt-2 or anti-target cell antisera. Finally, CTL-CFE did not contain proteases that degraded the alpha CTLL antibody. By the use of a soluble-phase immunoabsorbent assay, the biochemical properties of materials present CFE derived from CTL and reactive with alpha CTLL antibody were examined. CTL cytosolic material(s) reactive with alpha CTLL IgG was unstable to brief heating (50 degrees C) or acidic pH, but not to high ionic strength buffers. The material was inactivated by treatment with pronase but not by DNase, collagenase, or trypsin. Gel filtration chromatography of CTL-CFE revealed multiple peaks of alpha CTLL neutralizing activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Class II antigen-specific murine cytolytic T lymphocytes (CTL). II. Genuine class II specificity of Lyt-2+ CTL clones.

Class II-specific allogeneic cytolytic T lymphocytes (CTL) consist of two types of cells, i.e., Lyt-2+L3T4- and Lyt-2-L3T4 T cells. The Lyt-2+L3T4- class II-specific CTL population constitutes a conspicuous exception to the general correlation observed between the class of major histocompatibility complex antigen recognized and the type of accessory molecules expressed by T cells. In order to examine the specificity of such an exceptional T cell population, CTL clones were established by limiting dilution of a bulk CTL line developed in an I region incompatible combination of mouse strains, B10.QBR anti-B10.MBR. These CTL lines showed single genetic specificity indicating their clonal nature with respect to CTL activities. Lyt-2+L3T4- (2+4-), Lyt-2-L3T4+ (2-4+) and Lyt-2-L3T4- (2-4-) clones were obtained. Among many CTL clones showing a spectrum of genetic specificities, 2+4- and 2-4+ clones with apparent I-Ak-specificity, were studied further and four lines of evidence confirmed their class II specificity: 1) genes encoding the target antigen for these CTL clones were mapped within the I-A subregion by simple genetics; 2) an I-Ak-specific monoclonal antibody readily blocked specific cytolysis by these clones; 3) the clones failed to react with cells expressing mutated I-Ak antigens; and 4) a B cell tumor transfected with alpha- and beta-chain genes of I-Ak was specifically lysed by these CTL clones. These data therefore establish the existence of Lyt-2+ CTL with genuine class II specificity. All 2-4+ CTL were sensitive to the blocking effect of an antibody to L3T4, whereas none of the 2+4- class II-specific CTL were sensitive to blocking by an anti-Lyt-2 antibody, indicating that class II-specific CTL with "wrong phenotype" is not dependent on the function of the accessory molecule. Besides true class II-specific CTL clones, 2+4- clones with a spectrum of genetic specificities were obtained, including clones recognizing a combination of an I-Ak product and the Kb molecule. Two 2-4- clones were also specific for the combination of Kb + I-Ak. These clones most likely recognize an allogeneic class II antigen in the context of a class I antigen and therefore would more appropriately be included in the class I-restricted T cell population.

Animals↗
PubMed1987

CTL adhesion and antigen recognition are discrete steps in the human CTL-target cell interaction.

Th initial step in cytolytic T lymphocyte (CTL)-mediated cytolysis involves target cell adhesion and antigen recognition. To investigate these initial events in the CTL-target interaction, we used HLA-A2- and HLA-B7-specific human CTL clones and HLA-typed B lymphoblastoid target cells. By using two different adhesion assays, we demonstrated antigen nonspecific CTL-target cell adhesion. To more precisely define the contribution of the antigen-specific receptor to CTL-target cell adhesion, we used the HLA-A2, HLA-B7, and mock transfected RD target cells. Consistent with the results when using B lymphoblastoid target cells, the CTL clones demonstrated equivalent adhesions to the RD target cells whether or not they expressed HLA-A2 or HLA-B7. These results suggested that CTL-target cell adhesion occurred independent of the T cell receptor. By using the calcium-sensitive dye Indo-1 and flow cytometry, we assessed CTL-target cell adhesion and CTL activation. Simultaneous measurement of adhesion and intracellular free calcium demonstrated that CTL-target cell adhesion alone did not activate CTL clones. Both CTL-target cell adhesion and the presence of the appropriate HLA target molecule were necessary for the efficient activation of human CTL. MAb inhibition studies indicated that antigen nonspecific adhesion is largely regulated by the LFA-1, CD2 (LFA-2/T11), and LFA-3 cell surface molecules. These antigen nonspecific cell-cell interaction molecules appear to play an important role in facilitating antigen recognition and subsequent target cell lysis.

Antibodies, Monoclonal↗
PubMed1989

Molecular analyses of a five-amino-acid cytotoxic T-lymphocyte (CTL) epitope: an immunodominant region which induces nonreciprocal CTL cross-reactivity.

The cytotoxic T-lymphocyte (CTL) response to lymphocytic choriomeningitis virus infection determines the outcome of infection. Here we show that this response in BALB/c mice (H-2d), when analyzed both at the primary CTL level and using CTL clones, is predominantly monospecific. The vast majority of CTL have a common specificity for a single epitope in the virus nucleoprotein, which can be minimally identified by amino acids GVYMG. This epitope is presented by the Ld class I glycoprotein. We used these data to design a subunit CTL vaccine, whose effectiveness is demonstrated in the accompanying report (L. S. Klavinskis, J. L. Whitton, and M. B. A. Oldstone, J. Virol. 63:4311-4316, 1989). Further analysis indicates that, while CTL clones share a common minimal epitope, they differ in their ability to recognize cells infected with a related but distinct strain of lymphocytic choriomeningitis virus. Studies on the molecular nature of CTL cross-reactivity indicate that CTL induced by similar sequences may cross-react in a unidirectional manner. These novel observations suggest that CTL vaccines, to achieve optimal effectiveness, should not simply include virus sequences which will yield a CTL response; the immunizing sequences should also be selected to ensure that the fine specificities of the induced CTL are such that they maximize the chance of recognizing serotypically diverse strains.

Amino Acid Sequence↗
PubMed1991

The CD45RA molecule is expressed in naive murine CTL precursors but absent in memory and effector CTL.

We have studied the expression of the CD45RA molecule in murine cytotoxic T lymphocytes (CTL) specific for the allogeneic H-2Kb molecule at different stages of differentiation. The CD45RA phenotype of naive H-2Kb-specific CTL precursors has been determined using primary in vitro CTL responses. For the analysis of memory CTL we have immunized mice in vivo followed by restimulation in vitro. We have also determined the CD45RA expression at the CTL effector stage. Our results show that among naive CD8+ T cells both the CD45RA+ and the CD45RA- subpopulations can mount Kb-specific CTL responses. In contrast, memory CTL responses are mediated only by the CD8+ CD45RA+ T cell subpopulation. Similarly, effector CTL are CD8+ CD45RA- while the CD8+ CD45RA+ subpopulation does not exhibit specific cytolytic activity. The data indicate that CD45RA expression changes during CTL differentiation and that memory as well as effector CTL lack this marker.

Animals↗
PubMed1992

Cytotoxic T lymphocytes (CTL) against a transforming gene product select for transformed cells with point mutations within sequences encoding CTL recognition epitopes.

The 94-kD large tumor (T) antigen specified by simian virus 40 (SV40) is sufficient to induce cell transformation. T antigen contains four H-2Db-restricted cytotoxic T lymphocyte (CTL) recognition epitopes that are targets for CTL clones Y-1, Y-2, Y-3, and Y-5. These epitopes have been mapped to T antigen amino acids 207-215 (site I), 223-231 (sites II and III), and 489-497 (site V), respectively. Antigenic site loss variant cells that had lost one or more CTL recognition epitopes were previously selected by coculturing SV40-transformed H-2Db cells with the site-specific Db-restricted CTL clones. The genetic bases for T antigen CTL recognition epitope loss from the variant cells were identified by DNA amplification and direct sequencing of epitope-coding regions from variant cell DNAs. Cells selected for resistance to CTL clone Y-1 (K-1; K-1,4,5; K-3,1) carry deleted SV40 genomes lacking site I, II, and III coding sequences. Point mutations present within the site II/III coding region of Y-2-/Y-3-resistant cell lines specify the substitution of asparagine for lysine as T antigen amino acid 228 (K-2) or phenylalanine for tyrosine at position 230 (K-3). Point mutations identified within independently selected Y-5 resistant populations (K-5 and K-1,4,5) direct the substitution of isoleucine for asparagine at position 496 (K-5) or the substitution of phenylalanine for isoleucine at position 491 (K-1,4,5) of T antigen. Each substitution causes loss of the relevant CTL recognition epitope, apparently by compromising CTL T cell receptor recognition. These experiments identify specific amino acid changes within a transforming protein that facilitate transformed cell escape from site-specific CTL clones while allowing maintenance of cellular transformation. This experimental model system provides unique opportunities for studying mechanisms of transformed cell escape from active immunosurveillance in vivo, and for analysis of differential host immune responses to wild-type and mutant cell-transforming proteins.

Amino Acid Sequence↗
PubMed1988

Frequency of human alloantigen-reactive T lymphocytes. III. Evidence that cyclosporine has an inhibitory effect on human CTL and CTL precursors, independent of CsA-mediated helper T cell dysfunction.

We have used limiting dilution analysis to study the behavior of alloantigen-reactive cytolytic T lymphocytes derived from human peripheral blood. During these studies, we found that the presence of cyclosporine in limiting dilution microcultures significantly impairs the subsequent development of alloantigen-reactive cytolytic T cell activity. As a result, CsA reduces the estimate of CTL precursor frequency by limiting dilution analysis. CTL frequency estimates are reduced by CsA in a dose-dependent manner, and concentrations of CsA that are readily achieved in human peripheral blood (100-1000 ng/ml) are capable of reducing estimates of CTL frequency by 90% to 100%. Further studies revealed that (1) human CTL derived either from fresh peripheral blood or from primary mixed lymphocyte cultures are sensitive to the suppressive effects of cyclosporine in limiting dilution microcultures, indicating that CsA influences both alloantigen-primed CTL and CTL precursors; (2) CsA impairs an immunologic event or events, that occurs for at least the first four days of limiting dilution microculture incubation; (3) CsA-mediated suppression is eliminated by separation of CTL from cyclosporine; (4) CsA blocks development of CTL generation, but not cell proliferation in limiting dilution microcultures; and (5) the CsA-mediated suppression is not reversed by supraoptimal concentrations of IL-2, high concentrations of gamma-IFN, or supplementation with the multiple lymphokines present in MLC supernatants. These data suggest that CsA may have a direct inhibitory influence on the differentiation of human CTL precursors that is independent of helper T cell dysfunction.

Cell Differentiation↗
PubMed1976

Antigenic specificity of the cytolytic T lymphocyte (CTL) response to murine sarcoma virus-induced tumors. I. Preferential reactivity of in vitro generated secondary CTL with syngeneic tumor cells.

Incubation of spleen cells from mice having rejected a Moloney sarcoma virus (MSV)-induced tumor with syngeneic irradiated lymphoma or sarcoma cells bearing MSV-associated antigens in secondary mixed leukocyte-tumor cell cultures (MLTC) resulted in the generation of highly active cytolytic T lymphocytes (CTL) specifically directed against syngeneic target cells bearing MSV-associated antigens. When MSV-immune spleen cells from C57BL/6 (H-2b) and BALB/c(H-2d) mice were compared with respect to their ability to generate CTL in syngeneic secondary MLTC, it was found that both lymphoid cell populations were equally able to mount an anamnestic CTL response to MSV-associated antigens as assessed by a short-term 21Cr release assay. However, quantitative analysis of the activity of both CTL populations on either H-2b or H-2d tumor cells indicated that target cells sharing the same major histocompatibility complex (MHC) as the effector cells were lysed 10- to 100-fold more efficiently than allogeneic target cells. As suggested by the results of inhibition experiments using mixtures of 51Cr-labeled and unlabeled target cells, preferential lysis of syngeneic versus allogeneic tumor cells might be related to the establishment of effective adhesions between the former and CTL. Direct evidence for the role of MHC in determining the antigenic specificity of CTL directed against MSV-associated antigens was provided by results obtained using MSV-immune spleen cells from congenic resistant mice. Furthermore, studies of the response of F1 (H-2b/d) hybrid mice showed that stimulation of immune spleen cells with tumor cells from one parental strain or the other in secondary MLTC resulted in the generation of CTL capable of lysing tumor target cells of the same perental strain as the stimulating cells, but not of the other. The results thus suggested the presence of two sets of CTL precursor cells in F1 MSV-immune spleens, each set responding exclusively to tumor antigens associated with only one of the two parental phenotypes.

Animals↗
PubMed1988

Mechanism of target cell lysis by cytotoxic T lymphocytes (CTL) employing RSV-induced H-2 congenic and recombinant mouse tumor cells: demonstration of soluble mediators released from H-2-restricted CTL clones.

The secretion and the specificity of cytotoxic mediators from H-2-restricted cytotoxic T lymphocytes (CTL) were examined using non-virus-producing target tumor cells induced by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) in B10 congenic and recombinant mice. By using rat concanavalin A supernatant, two H-2-restricted CTL clones were established from cytotoxic effector cells of B10.A(5R) mice primed with SR-RSV-induced syngeneic tumor Cell-free supernatants from the H-2-restricted CTL clones cocultured with syngeneic tumor cells had selectively high cytotoxic activity for syngeneic and H-2-compatible tumor cells, but not for H-2-incompatible tumor cells. YAC-1 cells, and B10.A(5R) blasts as defined in the 5-hr 51Cr-release assay. The cytotoxic activity was detected in the cell-free supernatants from the CTL clones cocultured with the CTL-sensitive syngeneic and H-2-compatible tumor cells, but not with the CTL-insensitive tumor cells and YAC-1 cells. The cytotoxic activity of the cell-free supernatant could be adsorbed by the syngeneic tumor cells, but not by YAC-1 and L(s) cells. Thus, the H-2-restricted CTL clones against SR-RSV-induced tumor cells were capable of releasing cytotoxic mediators by coculturing with syngeneic or H-2-compatible tumor cells, and the cytotoxic mediators showed a certain H-2-restricted manner in killing the target cells. These results suggest that the lysis of RSV-induced tumor cells by H-2-restricted CTL can at least in part be mediated by cytotoxic factors.

Adsorption↗
PubMed1988

Virus-lymphocyte interactions. IV. Molecular characterization of LCMV Armstrong (CTL+) small genomic segment and that of its variant, Clone 13 (CTL-).

Immunocompetent adult mice mount a vigorous cytotoxic T lymphocyte (CTL) response against the Armstrong (ARM) 53b strain of LCMV after primary inoculation. In contrast, the Clone 13 variant of ARM 53b, originally isolated from the spleen of a persistently infected mouse, suppresses LCMV-specific CTL responses (R. Ahmed et al. (1984) J. Exp Med 60, 521). The induction and generation of CTL maps to the short (S) RNA segment and not the long (L) RNA segment of LCMV (Y. Riviere et al. (1986) J. Immunol. 136, 304). The CTL recognition epitope, expressed in virus-infected target cells, also maps to the S segment of the LCMV ARM genome, and is structurally and functionally intact in Clone 13-infected target cells. Here we report the S RNA sequences of both ARM 53b and its variant Clone 13. Comparison reveals a single amino acid difference. However, sequence divergence at this position also occurs among other strains of LCMV (Pasteur, Traub, WE) which do elicit CTL responses. Hence, (1) the amino acid difference is unrelated to the phenotypic divergence of Clone 13, (2) suppression of the CTL response by Clone 13 is not linked to the CTL recognition epitope, and (3) the structure or function responsible for CTL immunosuppression by Clone 13 most likely maps to the L RNA segment. Further, the availability of the complete S RNA sequence for LCMV ARM and ARM Clone 13 variant allows a detailed comparison with WE (V. Romanowski et al. (1985) Virus Res. 3, 110-114), the only other LCMV S RNA so far sequenced.

Amino Acid Sequence↗
PubMed1990

Age-related decrement in cytotoxic T lymphocyte (CTL) activity is associated with decreased levels of mRNA encoded by two CTL-associated serine esterase genes and the perforin gene in mice.

The age-related decline in cytotoxic T lymphocyte (CTL) activity has been recognized for many years. Age-related alterations in several immunologic events have been suggested to be partly or completely responsible for this decline. We had previously demonstrated (Bloom et al., Cell. Immunol. 1988. 144: 440) in mice that a deterioration in the lytic mechanism may be at least in part responsible for the decline in CTL activity. We now report that this decline correlates with an age-related decrease in serine esterase activity released into the supernatant medium in the process of generating CTL. Northern analyses were then used to examine the effect of age on expression of genes encoding for perforin and two CTL-associated serine esterases. The products of all three of these genes have all been postulated to play roles in CTL-mediated lysis. We show that the expression of all three of these genes appears to decline with age in the process of generating allogeneic CTL. These alterations in gene expression correlated both with diminished cytolytic and released esterase activities generated by mixed leukocyte culture in spleen cells of old mice compared to young. The age-related decline in gene expression could not be attributed to shifts in T cell subsets, but CD8+ cells generated by allogeneic stimulation of nylon wool-passed spleen cells from old mice expressed significantly less cytolytic activity than those from young. This report is the first demonstration of an age-related decrease in expression of a functionally related group of genes. In addition, these findings are compatible with the suggested roles for perforin and serine esterase release in CTL-mediated target cell lysis.

Aging↗
PubMed1986

Expression of CTL-defined, AKR/Gross retrovirus-associated tumor antigens by normal spleen cells: control by Fv-1, H-2, and proviral genes and effect on antiviral CTL generation.

In previous studies we have characterized H-2-restricted cytolytic T lymphocytes (CTL) type specific for Gross cell surface antigen-positive tumor cells induced by AKR/Gross leukemia viruses. The generation of such CTL was shown to be controlled by at least three genetic loci including H-2 and Fv-1. The Fv-1n phenotype was able to negate positive immune response gene effects of the H-2b haplotype. Fv-1n-mediated inhibition appeared to operated by allowing the early expression by normal cells of N-ecotropic leukemia virus-related antigens recognized by the antiviral CTL, perhaps via tolerance induction. In the present study, the expression of CTL-defined viral antigens by normal cells is further considered. Possible gene dosage effects by H-2 as well as Fv-1 and the other virus-related (V) genes, including proviral structural loci, were examined by comparison of a panel of congenic and F1 mice. These experiments indicated that the quantitative level of expression of CTL-defined viral antigens was primarily controlled by the Fv-1 genotype. Gene dosage effects were also observed for the V genes and, in some situations, for H-2. The importance of the early display of viral antigens by normal cells was underscored by the inability of those mice to generate specific antiviral CTL responses. Even strains expressing low levels of viral antigens, such as responder X nonresponder (AKR.H-2b:Fv-1b X AKR.H-2b)F1 mice, failed to respond. These results are discussed with respect to the inability of mice of the AKR background to respond with specific antiviral CTL generation and in light of their high incidence of spontaneous leukemia.

AKR murine leukemia virus↗
PubMed1987

Class II antigen-specific murine cytolytic T lymphocytes (CTL). I. Analysis of bulk populations and establishment of Lyt-2+L3T4- and Lyt-2-L3T4+ bulk CTL lines.

Murine allogeneic cytolytic T lymphocytes (CTLs), including long-term bulk CTL lines, were induced in I-region-incompatible combinations of strains in vitro in order to study the phenotypes of class II major histocompatibility complex (MHC) antigen-specific CTLs, as well as the possible functional involvement of accessory cell interaction molecules such as Lyt-2 and L3T4. This report shows that class II-specific allogeneic CTL populations consist of two types of T cells. Lyt-2+L3T4- (2+4-) and Lyt-2-L3T4+ (2-4+), in variable proportions depending on the strain combination, that in vitro bulk CTL lines with each of these phenotypes can be established, that the killing function of 2-4+ CTL is sensitive to the blocking effect of anti-L3T4 antibodies, suggesting functional involvement of this molecule in the CTL-target interaction, that anti-Lyt-2 antibodies fail to block killing by 2+4- cells, suggesting that such CTLs do not utilize this molecule in their killing function, and that while I-A-specific CTLs of both phenotypes are detectable, 2-4+ cells could not be detected among I-E-specific CTL populations.

Animals↗
PubMed1985

Signal requirements for the in vitro differentiation of cytotoxic T lymphocytes (CTL): distinct soluble mediators promote preactivation of CTL-precursors, clonal growth and differentiation into cytotoxic effector cells.

We describe here that the requirement of accessory cells for the polyclonal activation of high-density (resting) murine T lymphocytes can be bypassed by soluble mediators present in culture supernatants of concanavalin A (Con A)-activated murine spleen cells. Induction of responsiveness is confined to Lyt-2+ T cells; GK 1.5+ T helper cells require signals provided by accessory cells. Using this system the lymphokine signal requirements for the polyclonal activation of Lyt-2+ cytotoxic T lymphocyte (CTL) precursors could be defined. We show that Con A fails to trigger in Lyt-2+ responder T cells the expression of interleukin 2 (IL 2) receptors and assume that this explains why recombinant (rec) DNA-derived IL 2 fails to induce proliferative responses. Complementation of rec IL 2 with an IL 2 receptor-inducing factor (RIF) induces proliferative responses. RIF alone triggers IL 2 receptor expression in 10-12% of Lyt-2+ T cells exposed to Con A. This lymphokine appears to be distinct from colony-stimulating factor 1, IFN-gamma and IL 1. Resting Lyt-2+ T cells cultured in limiting numbers in the presence of Con A, RIF plus rec IL 2 do proliferate, yet exhibit no cytolytic activity. Differentiation into CTL can be brought about by addition of cytotoxic T cell differentiation factor (CTDF). We conclude that the polyclonal activation pathway of CTL from resting CTL precursors can be subdivided into three stages: preactivation, clonal growth and CTL differentiation. Each of these stages appears to be controlled by a distinct lymphokine, RIF, IL 2 and CTDF, respectively.

Animals↗
PubMed1987

Recognition of Rous sarcoma virus-induced tumor antigens by cytotoxic T lymphocytes (CTL): studies on specificity of killing by CTL employing H-2 congenic and recombinant mouse tumor cells.

The specificities of cytotoxic T lymphocytes (CTL) were studied for the analysis of CTL against tumor-specific cell surface antigen(s) (TSSA) of non-virus-producing tumor cells induced by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) in B10 congenic and recombinant mice. Eight CTL clones were established from immune spleen cells of B10.A(5R) mice. These clones demonstrated six patterns of cytotoxic reactivity in vitro: Two clones showed H-2 restriction in tumor cell lysis. Two other clones had the capacity to lyse syngeneic, H-2K-compatible B10 and H-2-incompatible B10.A(4R) tumor cells, but not YAC-1 cells. One clone had cytotoxic activity against syngeneic, H-2D-compatible B10.D2 tumor cells and YAC-1 cells, but not against H-2-incompatible tumor cells. One clone had cytotoxic activity against syngeneic and YAC-1 tumor cells, but not against either H-2-compatible or H-2-incompatible tumor cells. One clone had lytic activity to syngeneic, H-2-compatible, H-2-incompatible, and YAC-1 tumor cells. Another clone killed H-2-incompatible B10.A(4R) tumor and YAC-1 cells, but not syngeneic or H-2-compatible tumor cells. All these clones strongly expressed surface Thy-1.2 antigens, whereas the expression of Lyt-1.2 and Lyt-2.2 antigens was different from clone to clone. These results demonstrate heterogeneity of both lytic specificity and phenotype of CTL against RSV-induced mouse tumor cells, suggesting the existence of multiple antigenic sites on the RSV TSSA recognized by CTL populations.

Animals↗
PubMed1991

Cytotoxic T lymphocyte (CTL)-mediated cytolysis proceeds in the absence of Na+/H+ antiport activity: regulation of cytosolic pH by the Na+/H+ antiport in a cloned CTL.

Cytotoxic T lymphocyte (CTL)-mediated cytolysis of specifically bound target cells (TC) is thought to be triggered by cross-linking the T-cell antigen receptor (TcR). Biochemical events associated with TcR cross-linking include increased intracellular calcium levels [Ca2+]i, hydrolysis of phosphatidylinositol (PI), and an increase in intracellular pH [pH]i. Whereas CTL-mediated cytolysis of some TC is calcium-dependent, and PI hydrolysis is speculated to trigger the CTL lethal hit via activation of PKC, little is known about changes in [pH]i relating to activation of the lethal hit stage. We report regulation of [pH]i in a cloned CTL by the electroneutral Na+/H+ antiport during activation with PMA and specific antigen-bearing TC. Furthermore, using 5-(N-methyl-N-isobutyl) amiloride (MIBA), a potent antiport inhibitor, we demonstrate that Na+/H+ exchange is not required for activation of CTL cytolytic activity.

Acid-Base Equilibrium↗
PubMed1988

Correlations of in vivo growth of CTL-susceptible and -resistant variant tumor cell lines in CTL-responder AKR.H-2b:Fv-1b and -nonresponder AKR.H-2b mice.

Spontaneously occurring lymphoma/leukemias in AKR and AKR.H-2b mice are characterized by their expression of the Gross cell surface antigen (GCSA) and their weak immunogenicity. Although of a responder H-2 type, AKR.H-2b mice could not raise cytolytic T lymphocytes (CTLs) against a syngeneic GCSA+ tumor (AKR.H-2bSL1). In contrast, AKR.H-2b:Fv-1b mice served as a source for "antiviral" CTLs specific for GCSA+ tumors such as AKR.H-2bSL1, but not for CTLs against the cl.18-5 variant tumor, an antiviral CTL-resistant subclone derived from AKR.H-2bSL1. In the present study in vivo tumor challenge experiments demonstrated that both the ability of the recipient strain to raise CTLs and the sensitivity of the tumor to the CTLs were critical factors which determine tumor growth and recipient mortality. Furthermore, the ability to raise protective immunity against AKR.H-2bSL1 and cl.18-5 tumor challenge by preimmunization was investigated. It was not possible to raise protective immunity in CTL-nonresponder AKR.H-2b mice. In the case of AKR.H-2b:Fv-1b mice, immunization with allogeneic GCSA+ E male G2 tumor cells leads to complete protective immunity--not only against parental AKR.H-2bSL1 but, somewhat surprisingly, also against cl.18-5 variant, tumor challenge. Consistent with these findings and at the same time with an in vivo role for antiviral CTL, however, CTLs directed to the E male G2, AKR.H-2bSL1, and cl.18-5 tumors could be generated from the spleens of mice which had rejected cl.18-5 tumor cells. Interestingly, immunization of AKR.H-2b:Fv-1b mice with syngeneic AKR.H-2bSL1 tumor cells failed to raise any protective immunity. Thus, the data suggested that the concurrent recognition of allogeneic components with tumor-associated transplantation antigens (TATA) might be important in the induction of sufficient protective immunity against syngeneic GCSA+ tumors. Finally, the possible relationship of TATA and retroviral antigens, such as gp70 and p30 or as defined by CTL clones, is discussed.

AKR murine leukemia virus↗
PubMed1989

Effect of IL-2 in vitro on CTL generation in spleen cells of young and old mice: asialo GM1+ cells are required for the apparent restoration of the CTL response.

The role of asialo GM1+ (ASGM1+) cells and exogenous IL-2 in the age-related decline in allospecific CTL activity was evaluated. Primary CTL were generated in mixed leukocyte culture (MLC) [BALB/cANN (H-2d) anti C57BL/6N (H-2b)] and tested for allospecific lytic activity against the EL-4 (H-2b) cell culture line, and for non-MHC-restricted activity against WEHI-3 (H-2d) and YAC-1 (H-2a). Cultures included responder cell populations which had been treated with antibody to ASGM1 plus complement or complement alone, and irradiated stimulator cells, in the presence or absence of rIL-2 or crude IL-2-containing supernatants. The amount of rIL-2 used to accommodate the age-related decline in IL-2 production was determined empirically to be 500 U by assessing IL-2 production in MLCs containing responder cells from young versus old animals. rIL-2 appeared to restore the allospecific CTL activity generated by spleen cells of old mice to the level of that of young. However, treatment with anti-ASGM1 antibody revealed that this restoration was due to an effect of the IL-2 on ASGM1+ cells. The allospecific target cells, EL-4, were not sensitive to lymphokine-activated killer (LAK) cells induced by IL-2 alone under the conditions used. It is suggested that the apparent restoration was due to increased LAK-like (or MHC-nonrestricted) activity mediated by an ASGM1+ cell in the CTL precursor population.

Age Factors↗