Young F mice spontaneously generate cytotoxic T cells against parental targets.

Spleen cells from young (AKR/J female x BALB/c) or (BALB/c female x AKR/J)F1 mice can spontaneously generate effector cytotoxic T lymphocytes (CTL), in a 5-day primary in vitro culture, which lyse target cells from AKR/J and BALB/c but not allogeneic mice. These spontaneous CTL responses first appear when spleen cells are taken from F1 mice at 3 to 4 weeks of age, are maximum at about 5 weeks, and have declined by week 7. The fact that these spontaneous CTL responses are never detectable in the spleen cell cultures from any ages of parental AKR/J and BALB/c mice makes them unique properties of the F1 mice.     

Characterization of porcine T lymphocytes and their immune response against viral antigens.

T lymphocytes play a central role in the antigen-specific immune response against various pathogens. To detect and to characterize porcine T lymphocytes, monoclonal antibodies (mAb) against leukocyte differentiation antigens had been raised and classified for their specificity. Analyses of porcine T lymphocytes with specific mAb against CD4 and CD8 differentiation antigens revealed differences in the composition of the porcine T-lymphocyte population compared to other species. In addition to the known subpopulations, CD4+CD8- T helper cells and CD4-CD8+ cytolytic T lymphocytes, extra-thymic CD4+CD8+ T lymphocytes and a substantial proportion of CD2-CD4-CD8- T cell receptor (TcR)-gamma delta+ T cells could be detected in swine. Functional analyses of porcine T-lymphocyte subpopulations revealed the existence of two T-helper cell fractions with the phenotype CD4+CD8- and CD4+CD8+. Both were reactive in primary immune responses in vitro, whereas only cells derived from the CD4+CD8+ T-helper-cell subpopulation were able to respond to recall antigen in a secondary immune response. With regard to T lymphocytes with cytolytic activities, two subsets within the CD4-CD8+ T-cell subpopulation could be defined by the expression of CD6 differentiation antigens: CD6- cells which showed spontaneous cytolytic activity and CD6+ MHC I-restricted cytolytic T lymphocytes including virus-specific cytolytic T lymphocytes. These results enable now a detailed view into the porcine T-cell population and the reactivity of specific T cells involved in the porcine immune response against pathogens. Furthermore this knowledge offers the possibility to investigate specific interactions of porcine T lymphocytes with virus-specific epitopes during vaccination and viral infections.     

Development in vitro of cytotoxic lymphocytes against murine cytomegalovirus.

Lymphocytes cytotoxic for mouse embryo fibroblasts (MEF) infected with murine cytomegalovirus (MCMV) were produced by in vitro culture of "memory" spleen cells with UV-irradiated, MCMV-infected, MEF. Cytotoxic lymphocytes were developed from spleen cells of mice 10 to 240 days after infection with MCMV. The cytotoxic cells carried the theta and Ly 2 antigens, and were H-2 restricted in the recognition of infected target cells.     

Fetal calf serum-injected F1 mice spontaneously generate specific anti-parental cytotoxic T lymphocytes in in vitro culture

Spleen cells from adult (BALB/c x AKR/J)F1 mice primed in vivo with fetal calf serum (FCS) can spontaneously generate anti-parental AKR/J cytotoxic T cells (CTL) in a 5-day in vitro culture containing 5% FCS. This response is distinguished by the following features: (i) it is anti-parental but not anti-self, and (ii) it has specificity for the Kk parental determinant as shown by mapping studies on a variety of targets and antiserum-blocking experiments. Although specifically elicited by FCS and mediated by FCS-induced T-helper cells, it is ascertained that this cytotoxicity is not directed against Kk components modified by absorbed FCS as shown by cold-target competition studies. Further experiments involved a comparative investigation of the patterns of lysis of allogenically induced CTL, FCS-induced CTL, and natural killer (NK) cytotoxic activities on tumor cell targets. The resistance of BW 5147 tumor targets to NK- and FCS-induced lysis was found to be dramatically overcome by treatment with mitomycin C, and provides circumstantial evidence for a functional relationship between the FCS-induced anti-parental CTL effectors and NK cells based on the observed similarity in lytic patterns of these two effector types. With reference to the work of other authors, the possibility that hybrid resistance and its possible in vitro counterpart, F1 anti-parental CTL cytotoxicity, and NK activity are mediated by similar or common effector mechanisms is discussed.     

The differentiation of cytotoxic T lymphocytes in vitro

Summary Previous neurohistological studies have been extended to include the structures contained solely or mainly within the junctional esophageal segment which may play an important role in the sphincter mechanism. The main findings were: 1) a progressive cranio-caudal thickening of the muscularis mucosae; 2) a conspicuous thickening of the circular muscle layer; 3) abundant and close interconnections between the esophageal striated fibres and gastric smooth muscle cells; 4) presence of annulo-spiral elastic fibres coiled around bundles of striated musculature; 5) increase of the intramural nerve component, particularly Auerbach's plexus, which consisted of a continuous nervous layer containing twice as many neurocytes as found in the upper esophageal segments; 6) presence of numerous interconnected motor endplates often possessing ultraexpansional fibres and secondary endplates. The findings are discussed with emphasis on functional correlations in order to attain a unitary morpho-functional view.Abbreviations used LES lower esophageal sphincter - HPZ high pressure zone; mm: muscularis mucosae - CNS central nervous system - CCK-PZ cholecystokinin-pancreozymin Dedicated to Prof. Dr. Wolfgang Bargmann for his fundamental contributions to Comparative Morphology     

Rotavirus-specific cytotoxic T lymphocytes passively protect against gastroenteritis in suckling mice.

Suckling mice are protected against murine rotavirus-induced gastroenteritis after adoptive transfer of splenic lymphocytes from immunized animals. Adoptive transfer of Thy1(+)-depleted or CD8(+)-depleted lymphocytes abrogated protection against challenge. (We previously found that depletion of Thy1+ or CD8+ lymphocytes from rotavirus-immunized mice decreased rotavirus-specific cytotoxic activity in vitro.) Protection against disease occurred in the absence of rotavirus-specific neutralizing antibodies in the sera of suckling mice. Rotavirus-specific cytotoxic T lymphocytes may be important in either amelioration of acute infection or protection against reinfection.     

Mechanisms of in vivo generation of cytotoxic activity against allograft: 1. Local differentiation of mature cytotoxic T lymphocytes in rejection of allogeneic lymphocytes

Although cytotoxic activity was not detected within the spleen and regional lymph nodes from mice immunized sc with allogeneic lymphocytes, such activity was detected consistently in glass-nonadherent and anti-θ-sensitive peritoneal exudate cells (PE cells) from Day 5 after immunization and reached a maximum by Day 7. Immunized spleen cells developed cytotoxic T lymphocytes (CTLs) earlier and more effectively than normal spleen cells when transferred ip into X-irradiated syngeneic normal mice together with immunizing antigen, while they did not become cytotoxic when transferred without antigen. These results suggest that spleen and lymph node cells which may have differentiated into some transitional state by in vivo immunization may differentiate into mature CTLs, following direct contact with antigen at the site of graft. CTLs generated there appear to be responsible for the rejection of allogeneic lymphocytes. Cytotoxicity of PE cells was also generated in X-irradiated mice and augmented cytotoxicity was generated by treatment with cyclophosphamide.     

Generation of cytotoxic T lymphocytes in vitro. VII. Suppressive effect of irradiated MLC cells on CTL response.

Irradiated cells obtained from MLC at the peak of the CTL response caused profound suppression of generation of CTL when added in small numbers at the initiation of primary MLC prepared with normal spleen cells. The inhibitory activity of the MLC cells was not affected by irradiation (1000 rads) but was abolished by treatment with anti-theta serum and complement. The suppression was immunologically specific. The response of A (H-2a) spleen cells toward C3H (H-2k) alloantigens was suppressed by irradiated MLC cells obtained from MLC prepared with A spleen cells and irradiated C3H-stimulating cells, whereas the response of A spleen cells toward DBA/2 (H-2d) alloantigens was affected relatively little. However, if irradiated C3H X DBA/2 F1 hybrid spleen cells were used to stimulate A spleen cells in MLC, addition of irradiated MLC cells having cytotoxic activity toward C3H antigens abolished the response to both C3H and DBA/2 antigens. The response to DBA/2 antigens was much less affected when a mixture of irradiated C3H and DBA/2 spleen cells was used as stimulating cells. Thus, the presence of MLC cells having cytotoxic activity toward one alloantigen abolished the response to another non-cross reacting antigen only when both antigens were present on the same F1 hybrid-stimulating cells. This suppression of generation of CTL by irradiated MLC cells apparently involves inactivation of alloantigen-bearing stimulating cells as a result of residual cytotoxic activity of the irradiated MLC cells. This mechanism may be active during the decline in CTL activity noted in the normal immune response in vivo and in vitro.     

Regulation of the cytotoxic T lymphocyte response against Qa-1 alloantigens

Spleen cells from B6.Tlaa (Qa-1a) mice primed against C57BL/6 (Qa-1b) splenocytes in vivo generate Qa-1-specific CTL when rechallenged with Qa-1b Ag in vitro. The addition of unirradiated Qa-1b splenocytes to these cultures inhibits the generation of Qa-1-specific CTL. By using highly purified cell populations, we demonstrate that the only cell population in resting spleen capable of causing this inhibition is NK1.1+. Although resting CD8 cells lack inhibitory activity, purified CD8 cells precultured with Con A and IL-2 inhibit anti-Qa-1 CTL. This inhibition is specific for the Qa-1b Ag expressed on the inhibitor cells, is not due to cold target competition, and is thus similar to that ascribed to veto cells. Although NK cells from resting spleen inhibit the generation of Qa-1-specific CTL, NK cells precultured in the presence of Con A and IL-2 show an approximate 30-fold increase in veto activity. Thus, NK cells represent the most likely cell population for down-regulating anti-self class I-reactive CTL.     

Rat cytotoxic T lymphocytes against human T-lymphotropic virus type 1-infected cells recognize gag gene and env gene encoded antigens

T cell immune responses in syngeneic WKA/H rats were analyzed by using lymphoid cell lines, TARS-1, TART-1, and TARL-2, infected with human T-lymphotropic virus type 1 (HTLV-1). Spleen cells of rats in which these cell lines had been rejected were sensitized in vitro with the same cell lines, and cells cytotoxic to these HTLV-1+ cell lines, and cells cytotoxic to these HTLV-1+ cell lines were generated. The effector cells were CTL of the CD5+ CD8+ phenotype and showed restriction of MHC class I Ag. Direct tests as well as cold target cell inhibition tests with an array of cell populations showed that these CTL reacted only with syngeneic HTLV-1+ cell lines. When xenogeneic HTLV-1+ cell lines were similarly utilized for in vitro sensitization, rat CTL specific for syngeneic HTLV-1+ cells were generated. They were not, however, reactive with xenogeneic HTLV-1+ cells used for sensitization. Syngeneic rat cells selectively expressing gag, env, or pX gene coded Ag were prepared by infection of recombinant vaccinia viruses. In cold target cell inhibition tests of anti-HTLV-1 CTL with thus prepared cells, cytotoxicity against the syngeneic HTLV-1+ cells line, TARS-1, was inhibited by syngeneic cells expressing gag gene or env gene coded Ag. Inhibition was, however, more consistent and more dominant by cells with gag gene than those with env gene. Syngeneic cells with pX gene and MHC class I incompatible cells with gag, env, or pX gene did not inhibit cytotoxicity.     

Generation of cytotoxic T lymphocytes in vitro. VI. Effect of cell density on response in mixed leukocyte cultures.

Reexposure of day 14 murine mixed leukocyte culture (MLC) populations to the original irradiated allogeneic stimulating spleen cells has previously been found to result in the ratpid generation of cytolytic T lymphocytes (CTL) associated with a net increase in cultured cell number. Under the experimental conditions used, day 5 MLC cells appeared unable to respond to the allogeneic stimulus. In order to characterize further the development of the potential for anamnestic reactivity during the course of MLC, C57BL/6 spleen cells were incubated with irradiated (1000 rads) DBA/2 spleen cells (primary MLC) for up to 3 weeks. At various time intervals after the onset of the primary MLC, the surviving cells were collected and reexposed, at varying cell concentrations, to irradiated DBA/2 spleen cells (secondary MLC). At daily intervals thereafter, CTL activity was assessed using a quantitative 51Cr-release assay system. A paradoxic effect of responding cell concentration on generation of CTL activity was observed; relatively greater increase in CTL activity was observed as the concentration of responding cells was decreased over a 100-fold range. This effect was more pronounced with responding cells reexposed to antigen after primary MLC for 20 days, but was observed even with normal cells. The apparent unresponsiveness of day 5 MLC cells to alloantigen restimulation could be overcome by simple dilution of responding cells. Cytotoxic activity at the time of restimulation with antigen seems to be a major factor determining the magnitude of the secondary response. Since intact cells bearing alloantigens are required for the generation of CTL in MLC, residual cytotoxic cells reduce the effective antigenic stimulus by destroying stimulating cells. This effect of concentration of responding cells on generation of CTL in MLC complicates interpretation of experiments investigating the role of "inhibitor" and "helper" cell in cell-mediated immune responses occurring in vitro. Under optimal conditions, the highest CTL activity and the largest increase in total cell number was observed 4 days after restimulation of day 10 MLC cells. On a per cell basis, the lytic activity was up to 4 times greater than that observed at the peak of a primary response, and the number of viable cells recovered was nearly 20 times higher than that at the onset. Such secondary MLC are thus a convenient source of lymphoid cells selected primarily on the basis of proliferation induced by alloantigens.     

DC-expressed MHC class I single-chain trimer-based vaccines prime cytotoxic T lymphocytes against exogenous but not endogenous antigens

The poor immunogenicity of many tumors can be partly explained by the inefficiency of the MHC class I peptide presentation pathway. MHC-I-based single-chain trimers (SCT) represent a new class of molecules with the potential to overcome this limitation. We here evaluated the ability of SCT presenting a melanoma antigen peptide (TRP-2) to prime cytotoxic T lymphocyte (CTL) responses in mice when given as DNA vaccines via Gene Gun or when expressed by dendritic cells. The SCT was unable to induce detectable priming or significant anti-tumor activity of CTL using either vaccination strategy, whereas control SCT (with an exogenous peptide) primed strong responses. This study thus provides the first data related to the use of SCT in combination with DC and their application toward self antigens and suggest this potent technology, alone, is insufficient to overcome self tolerance.     

Primary in vitro antibody response from human peripheral blood lymphocytes.

A method for the induction of a primary in vitro antibody response from human peripheral blood lymphocytes is presented. Upon cultivation with trinitrophenyl conjugated polyacrylamide beads (TNP-PAA), an anti-TNP response can be obtained as indicated by the appearance of direct plaque-forming cells from day 5 of culture, with a reproducible peak on day 8. These plaques correspond to cells actively producing antibody of the IgM type, as shown by their inhibition by cycloheximide and by anti-human IgM serum, but not by anti-human Fc gamma serum. Their specificity for the TNP hapten can be demonstrated by the effector cell blockade phenomenon, with highly substituted TNP-human IgG. Although the anti-TNP response induced by TNP-PAA in mouse spleen cell cultures appears T independent the same response in human PBL may involve in addition the participation of T cells, since E-RFC depletion before culture led to a markedly decreased number of plaque-forming cells. A significant response could be obtained from the PBL of all of the 30 normal individuals tested. Importantly, the response was reproducible in its magnitude in the six individuals tested in at least three different experiments. Thus, the in vitro stimulation of human PBL by TNP-PAA can be proposed as a reliable test for the study of human B cell function in a specific primary antibody response.     

Regulation of the activation of cytotoxic T lymphocytes by prostaglandins and antigens

The present study demonstrated the presence of two suppressor circuits in the regulation of the in vitro activation and differentiation of cytotoxic T lymphocytes (CTL); these suppressor circuits were mediated by prostaglandins (PG) and antigens, respectively. In intrinsic suppression, the activation of cytotoxic precursor cells was regulated by the host endogenous production of PG. When the regulation by PG was removed (e.g., using indomethacin), lymphokine-induced cytotoxic cells (LICC) were generated. This activation process can be induced in the absence of antigen or mitogen stimulation. In extrinsic suppression, the presence of antigen induced the generation of antigen-nonspecific suppressor T cells to restrict the expansion of antigen-unrelated cytotoxic lymphocyte clones, whereas the antigen-specific CTL clones were spared. The generation of antigen-specific helper cells further augmented the antigen-specific CTL response. These findings indicate that both antigen specific suppressor T cells and antigen nonspecific suppressor T cells are involved in the regulation of CTL responses. These suppressor circuits not only play an active role in monitoring the activation of CTL clones, they also help to determine the specificity and magnitude of the CTL response.     

Activation of cytotoxic function in T lymphocytes.

The requirements for activation of cytotoxic function in mouse T lymphocytes were investigated. Initial generation of cytotoxicity in normal lymphocytes was equal in magnitude with either Con A or specific alloantigen, and in either case required DNA synthesis. Cytotoxic function in MLC-primed cells could also be regenerated by Con A, the magnitude and target specificity of the cytotoxicity thus generated being indistinguishable from that recalled by specific alloantigen. Cytotoxicity could also be regenerated by third party-stimulating cells; however, the cytotoxicity evoked by third party cells was always specific only for target cells of the original stimulating cell H-2 genotype. The data presented suggest that there are a number of ways to activate cytotoxicity in effector T cells, and are most consistent with a model for T cell triggering that minimizes a strict informational function of antigen-receptor interactions.     

Elicitation from virus-naive individuals of cytotoxic T lymphocytes directed against conserved HIV-1 epitopes

Cytotoxic T lymphocytes (CTL) protect against viruses including HIV-1. To avoid viral escape mutants that thwart immunity, we chose 25 CTL epitopes defined in the context of natural infection with functional and/or structural constraints that maintain sequence conservation. By combining HLA binding predictions with knowledge concerning HLA allele frequencies, a metric estimating population protection coverage (PPC) was computed and epitope pools assembled. Strikingly, only a minority of immunocompetent HIV-1 infected individuals responds to pools with PPC >95%. In contrast, virus-naive individuals uniformly expand IFNγ producing cells and mount anti-HIV-1 cytolytic activity. This disparity suggests a vaccine design paradigm shift from infected to normal subjects.     

Comparative structural analysis of HLA-A3 antigens distinguishable by cytotoxic T lymphocytes: variant E1

Influenza-specific cytotoxic T cells restricted by HLA-A3 recognize differences between HLA-A3 antigens that are serologically indistinguishable. To examine whether this differential recognition had a structural basis, we have compared the structures of HLA-A3 molecules from Epstein Barr virus-transformed peripheral blood lymphocytes of two individuals, E1 and M17. M17 was representative of the majority of HLA-A3-bearing individuals, whereas E1 was a variant distinguished by cytotoxic T cells. Peptide map comparisons revealed a small number of differences when particular amino acids were used to radiolabel the A3 molecules. Sequence analysis and comparison of the results with the prototypic HLA-A3 sequence localized the variability to a tryptic peptide spanning residues 147-157. To obtain the complete amino acid sequence for the E1 and M17 A3 molecules in the 147-157 region, the CNBr fragments beginning at residue 139 were isolated and sequenced. Two amino acid differences were detected between the HLA-A3 molecule of the CTL-defined variant E1 and that of M17. At position 152, Glu in donor M17 had changed to Va1 in donor E1, and at position 156, Leu in donor M17 had changed to Gln in donor E1. The finding that A3 molecules from E1 are altered in the region between residues 147 and 157 is consistent with studies on HLA-A2 variants and Kb mutants showing that this region of class I molecules is important for CTL recognition but not for recognition by serologic reagents.     

Augmentation by serum-induced helper cells of T cell-mediated cytotoxic response against tumor associated antigens and alloantigens

Summary In vitro T cell-mediated cytotoxic responses to tumor associated antigens or alloantigens can be augmented by the addition of small amounts (0.1 to 1%) of syngeneic (mouse) or xenogeneic (rabbit) serum in the standard lymphocyte culture medium. Further studies showed that the augmentation is mediated by helper cells, which are induced by culturing the spleen cells or lymph node cells in the presence of these sera. In the syngeneic system performed with mixed lymphocyte tumor cell cultures (MLTC), the serum-induced helper cells are found to be resistant to the lysis of anti-Thy 1.2 antibody and are radioresistant; thus they have the characteristics of macrophages. In the allogeneic system performed with mixed lymphocyte culture (MLC), the serum-induced helper cells are also found to be resistant to the lysis of anti-Thy 1.2 antibody but are radiosensitive. In the latter case, however, removal of T cells abolishes the helper cell generation and only the T cell-enriched fraction provides for the generation of helper cells, indicating that the helper cells for MLC are probably derived from T cells but lose their susceptibility to anti-Thy 1.2 antibody lysis upon culturing in vitro. A study of the mode of action of the helper cells for MLC showed that they are probably needed at a later stage of cytotoxic response for the amplification of the killing efficiency of the T effector cells whereas the helper cells for MLTC are needed in the early induction phase of the immune response. These results indicate that although serum can augment the cytotoxic responses both in the syngeneic and in the allogeneic systems, the mechanism for the augmentation differs: macrophagelike helper cells are responsible for the augmentation of cytotoxic response to tumor associated antigens, whereas augmentation of cytotoxic response to alloantigens appears to be mediated by a subpopulation of T helper cells. Supported by a grant from the Japan Society for the Promotion of Science (T. I.).