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Sensitization of mouse splenic lymphocytes in vitro with sodium borohydride, suggesting that the biologic effects of sodium periodate are-treated autologous spleen cells stimulated a one-way mixed lymphocyte reaction and led to the generation of thymus-derived cytotoxic effector cells. These effectors were capable of lysing in 4 hr periodate-treated syngeneic and, to a lesser extent, periodate-treated allogeneic target cells. These results suggest that sensitization by periodate-treated autologous cells could result either from a specific reaction to modified self components or from a nonspecific mitogenic stimulation. Effector cells generated by allogeneic sensitization were detected on periodate-modified targets, irrespective of the H-2 antigens expressed by the targets. The effects of periodate modification on both stimulator and target cells were reversible by sodium periodate are dependent on the formation of a free aldehyde group on cell surface glycoproteins. Pretreatment of stimulator cells with neuroaminidase prevented the effect of periodate treatment, suggesting that the sensitization involves oxidized sialic acid residues. During the 4-hour 51Cr-release assay periodate-treated targets could be used to detect cytotoxic effector cells of any specificity. Fresh spleen cells and lymphocytes cultured for 5 days without antigen or in the presence of lipopolysaccharide did not lyse periodate-treated targets. An increasing level of cytotoxicity was detected on periodate-treated targets when the effector cells were generated, respectively, by stimulation with concanavalin A, by sensitization with periodate-modified autologous cells. Although the lysis of periodate-treated targets is itself nonspecific, effector cell specificity could be determined by selective blocking of the lytic phase with cells syngeneic to the stimulators. These results indicate that a nonspecific interaction can occur between lymphocytes and periodate-treated target cells, but that this interaction leads to lysis only when the lymphocytes were activated to become cytotoxic effectors.

Publisher Summary This chapter highlights the generation of trinitrophenyl (TNP) specific H-2-restricted murine cytotoxic cells as a function of TNP-cell surface presentation. The generation of the cytotoxic effectors sensitized by the addition of trinitrobenzene sulfonate-modified cells on TNP proteins is dependent on the presence of both T lymphocytes and a population of glass-adherent, radio resistant cells. The chemically modified H-2-restricted CML model may not depend on actual covalent conjugation of the TNP group to H-2-coded cell surface products and, therefore, could be similar to the H-2 -restricted viral and minor transplantation antigen models. These findings could also raise the possibility that H-2 -restricted cytotoxic immune reactions involving autoimmunity may be generated by soluble components associated with autologous cells. The analysis of the antigenicity and the immunogenicity of TNP-presenting cells as a target or stimulator for TNP-specific, H-2-restricted T-cell mediated cytotoxicity is summarized as a function of the mode of presentation of the TNP moiety on the cell surface.

Publisher Summary This chapter highlights positive and negative liposome-based immunoselection techniques. Liposomes are vesicles composed of one or several phospholipid bilayers surrounding a closed aqueous compartment. They are useful for marking, killing, or rescuing cell populations. The use of liposomes as fluorescent reagents, notably for the cell sorter, offers high signal with low background. Liposomes are formed with the compound of interest encapsulated within the enclosed space or as part of the component phospholipids. After liposome formation, the liposomes are coupled to the freshly activated protein at room temperature, usually by mixing the protein and liposomes and dialyzing against buffered saline at pH 8–8.5 for several hours. High specificity of action is the case for the use of liposomes for negative selection, which requires that the target determinant is endocytosed by the cell, in contrast to the action of antibody and complement, for which expression of the molecule in question is sufficient for the cell to be killed. Positive selection with liposome-encapsulated protective reagents, though studied in a small number of model systems, is seriously challenged only by the fluorescence-activated cell sorter, which has been used for this application in only a few laboratories highly experienced in its use.

Specificity of the T-cytotoxic effector cells generated in cell-mediated lympholysis (CML) to TNP-modified autologous cells is such that the target cells: (a) must be modified by the same agent as that used for sensitization; and (b) must express the same haplotype at the K and/or D regions of the murine major histocompatibility complex (MHC) as the stimulating and/or responding cells (1–4). However, the involvement of products of the mouse MHC in H-2 -associated “hapten modified” syngeneic immune responses need not necessarily be restricted to products of the K and D regions. Products of the I region may also be important. In fact, dominant, H-2 -linked immune response (Ir) genes appear to control CML response potential to the H-2D d -TNP specificity. At least two such genes affect this CML--one which maps between I-A and I-J and the other which maps to the left of 1-A. Furthermore, products of the I region appeal to be important in generating secondary in vitro mixed lymphocyte reactions to TNP-modified syngeneic cells, since proliferative responses can be generated when there is H-2 homology at the K, D , or I regions between the secondary and primary stimulating cells.

Murine spleen cells were fractionated over nylon wool or Sephadex G-10 columns, and the cell types involved in the generation of trinitrophenyl (TNP)-specific, H-2 restricted (TNP-self) cytotoxic effector cells were studied from cultures stimulated with trinitrobenzene sulfonate (TNBS)-modified syngeneic cells, TNP-conjugated soluble proteins such as bovine gamma-globulin (TNP-BGG), or bovine serum albumin (TNP-BSA). Unfractionated or nylon nonadherent responding cells generated such effectors, irrespective of whether the cultures were stimulated with TNBS-modified cells or TNP-conjugated proteins. TNP-modified T lymphocytes, B lymphocytes, and phagocyte-enriched spleen cells were all capable of stimulating TNP-self effectors. TNP-self effectors. TNP-self as well as allogeneic cytotoxic responses were dependent on the presence of a radioresistant non-T cell that was removed by Sephadex G-10 fractionation and was replaced by irradiated, Thy 1.2-negative, glass adherent spleen cells, enriched in phagocytic cells. Results obtained by using glass adherent cells that were allogeneic or semi-syngeneic to the responding cells indicated that H-2 homology was not required for efficient glass adherent cell function, and that the H-2 restriction of TNP-self effectors is not determined by these glass adherent cells.

Publisher Summary The major histocompatibility complex (MHC) has been found to code for cell surface antigens involved in allograft rejection. This genetic region has also been shown to control immune response potential in a number of animal species. In the mouse, the MHC, known as the H-2 complex, has been divided into four major regions: K, I , S, and D. The K and D regions determine the strong serologically detectable transplantation antigens of the mouse, which are important for tissue graft rejection. The chapter also discusses the different ways in which the respective antigenic determinants (e.g., virus, hapten, or minor antigen) can be presented in association with H-2-coded products on the cell surface. Moreover, the chapter also discusses CML model. One advantage that the chemically modified CML model has over the viral and weak transplantation antigen models is the potential for investigating the antigenic contribution made by the modifying agent to the fine specificity of the T-cell recognition. The fact that some of the H-2-restricted syngeneic CML models are under the control of H-2-linked Ir genes as well as being restricted with K and D region products, suggests multiple functional roles for H-2-coded gene products in cell-mediated immunity. Finally, the chapter concludes that MHC products may be simultaneously important for recognition by T lymphocytes and for the H-2-restricted structures which they recognize the K, I, or D region products associated with the infectious agent.

Eleven long-term cytotoxic T lymphocyte (CTL) clones derived from C57BL/10 T cells sensitized in vivo and in vitro with trinitrobenzene sulfonate- (TNBS) treated syngeneic cells were all restricted to the K end of H-2b. The fine specificity of these CTL clones was analyzed by using H-2Kbm mutant target cells and H-2Kb-specific monoclonal antibodies (mAb). Seven distinct patterns of reactivity of the T cell clones could be observed with the use of six H-2Kbm mutant target cells. Further heterogeneity could be detected in terms of the ability of anti-Lyt-2 mAb to inhibit CTL activity. Cross-reactivity between H-2Kb + TNP and H-2Kbm + TNP was observed for all clones tested for bm5 and bm6, but less frequently for bm3 (8/11), bm8 (7/10), bm4 (4/11), and bm1 (3/11). It was further observed that amino acid substitutions located in the first domain only (one clone), or in the second domain only (six clones), or in either the first or the second domain (three clones) of the H-2Kb molecule could affect target cell recognition by a given T cell clone. the latter type of reactivity suggested that some clones recognized "conformational" determinants of the H-2 molecule, or that amino acid substitutions in one domain might influence the structure of the next domain. One H-2Kb + TNP-reactive clone exhibited a heteroclitic behavior with decreasing avidities for target cells expressing H-2Kbm8 + TNP, H-2Kb + TNP, and H-2Kbm8, which further extends the various patterns of T cell cross-reactions observed within a given class of MHC products. The use of H-2Kb-specific mAb in blocking studies as an attempt to define further the H-2Kb epitopes recognized by CTL clones indicated that: a) TNBS treatment may affect the antigenicity of the H-2Kb molecule as assessed by some mAb; and b) that the T cell clone-target cell interaction may or may not be inhibited by a given mAb, depending on structural variations of the H-2Kb molecule (use of H-2Kbm mutants) that do not affect the interaction itself. These results indicate that this type of analysis does not permit correlation of serologic- and T cell-defined epitopes.

The mechanism(s) of T lymphocyte “functions” such as cytolysis (1–4) or proliferation involve those cell surface structures that insure specific recognition and may involve other cell surface structures as well. Detection of these may be via the use of monoclonal antibodies (mAb) selected for their ability to inhibit lymphocyte functions. Indeed, anti-Lyt-2 mAb have been extensively studied as to their inhibitory effect on mouse T cell-mediated cytolysis, with repeated suggestions that Lyt-2 itself may be related to the T cell specific receptor (5–10). We have developed a range of xenogeneic rat anti-mouse mAb selected for their ability to inhibit T cell-mediated cycolysis (11,12). Three of them will be used in the present report: H35-17.2 mAb, which is most probably an anti- Lyt-2 mAb, as an experimental counterpoint to the two other mAb; H35-27.9 mAb, which differs from an anti-Lyt-2 mAb at least by the tissue distribution of the structures it recognizes; and H35-89.9 mAb, which immunoprecipitates from lymphoid cell surfaces two polypeptides of 180K and 94K molecular weight.

SCOPUS: ar.j, info:eu-repo/semantics/published

Monoclonal antibodies (mAb) were prepared from the spleen of a BALB.K mouse immunized with a H-2Kb-specific alloreactive cytolytic T cell (CTL) clone of B10.BR origin, KB5-C20. Six of these mAb were selected for specific binding to the immunizing clone. Only one of these mAb, Désiré-1, inhibited the CTL activity of clone KB5-C20. Functional and biochemical evidence showed mAb Désiré-1 to be anti-clonotypic (anti-Ti). This mAb immunoprecipitated from lysates of clone KB5-C20 a protein that migrated at 80 KD in nonreducing conditions and at 40 to 45 KD in reducing conditions. Analysis in NEPHGE resolved the latter into an acidic chain of 43 KD and a basic chain of 40 KD. An additional protein was coprecipitated at 25 KD in nonreducing and 27 KD in reducing conditions, which was distinct from the Thy-1 antigen. Expression of the clonotype defined by Désiré-1 was found on clone KB5-C20 and on clone KB5-A7 originating from the same B10.BR T cell population. These two clones exhibited a similar pattern of reactivity on H-2Kbm mutant target cells. Another clone (KB5-C1) from the same B10.BR T population showed a distinct reactivity pattern on the H-2Kbm mutant target cells and did not express the Désiré-1 clonotype, nor did Con A-activated B10.BR blast cells or H-2Kb-specific clone BM3.3 of CBA/J origin. Among the five other selected mAb, one reacted with the three Kb-specific CTL clones of B10.BR, but not with the Kb-specific clone of CBA origin, whereas the last four mAb bound only to clone KB5-C20. None of these mAb could immunoprecipitate the Ti alpha/beta chains. Ti modulation induced by mAb Désiré-1 on clone KB5-C20 did not induce comodulation of the structures bound by the other five mAb on clone KB5-C20, thus indicating that these structures were not part of the Ti-T3 complex. These results characterize a clonotypic mAb reacting with an H-2Kb-specific CTL clone that will be useful in future studies. They also indicate that screening for clone-specific mAb by selective binding assays may select for mAb against as yet undefined structures, some of which exhibit an apparently clone-specific distribution.

We investigated the basis for partial reactivity of naive CD8 T cells expressing an alloreactive transgenic TCR in response to a mutant alloantigen. When unstimulated APCs were used, IFN-gamma as well as IL-2 and cell proliferation were observed in response to wild-type Ag, whereas mutant Ag induced only IFN-gamma. DNA binding and reporter gene assays showed that the response to mutant Ag involved NF-kappaB, but not AP-1 activation, whereas wild-type Ag activated both transcription factors. Increasing the contribution of costimulatory signals by using LPS-activated APCs partially corrected the activation by mutant Ag, because proliferation and weak IL-2 production could be measured. This also led to AP-1 activation, albeit with delayed kinetics, in response to mutant Ag. To explain how engagement of the same TCR by distinct ligands results in different T cell responses, it may be proposed, in line with models stressing the importance of the kinetics of Ag/TCR interaction, that two types of signals be distinguished: a "fast" short-lived signal is sufficient to activate NF-kappaB; whereas a "slow" signal obtained after prolonged TCR engagement is required for AP-1 activation. Failure to activate AP-1 in limiting conditions (unstimulated mutant APC) was partially corrected by increasing costimulation.

In many models of cell-cell adhesion, it was reported that some cell membrane molecules might be redistributed into contact areas. However, this phenomenon was not subjected to precise quantification. In the present work, fluorescence microscopy, immunolabelling and digital image processing were combined to analyse quantitatively the spatial organization of specific or nonspecific conjugates made with a cytolytic T (CTL) lymphocyte clone (BM3.3) and target cells (EL4 or RDM4). Binding was achieved under calcium-free conditions to study the earliest steps of cell interaction, preceding CTL activation. Fluorescent antibodies were used to label class I histocompatibility molecules on both killer and target cells, and T cell receptor, CD3, CD8 and LFA-1 (CD18/CD11a) on the killer cells. Membrane bilayers were stained with a fluorescent phospholipid, glycoconjugates were labelled with periodic oxidation and Lucifer Yellow uptake, and polymerized actin was revealed with a fluorescent phallacidin derivative. Also, the fine geometry of killer-target interaction area was studied with electron microscopy and computer-assisted contour analysis. It is concluded that: (1) qualitative examination of fluorescence photomicrographs cannot permit accurate comparison between different fluorescence densities. (2) The cell-cell contact area was about fourfold higher in specific conjugates than in non-specific ones. (3) The surface density of adhesion molecules exhibited similar increases (between 30 and 80%) in the contact areas of both specific and nonspecific conjugates. (4) However, the amount of redistributed surface molecules was higher when cell-cell interaction was enhanced either by specific immunological recognition (in specific conjugates) or periodate oxidation. (5) Since redistribution did not require extracellular calcium and it was detected on nonspecific conjugates, this did not require full lymphocyte activation. Spatial reorganization of cell surface molecules may thus be a general consequence of adhesion, cell surface mobility and intermolecular forces.

We analyzed the phosphorylation and the dynamics of TCR/CD3, CD8 and MHC class I molecules during the activation of a CD8+ cytotoxic T lymphocyte clone and of CD8- T helper hybridomas transfected with the gene coding for the native (J. Gabert, C. Langlet, R. Zamoyska, J.R. Parnes, A.M. Schmitt-Verhulst, and B. Malissen. 1987. Reconstitution of MHC class I specificity by transfer of the T cell receptor and Lyt-2 genes. Cell 50:545) or truncated CD8 alpha molecule. The CD3 components gamma and epsilon and the CD8 alpha subunit were phosphorylated after activation of the CTL clone with the protein kinase C activator PMA. Class I MHC molecules were phosphorylated irrespective of PMA activation. Constitutive phosphorylation of the MHC class I products was found to be intrinsic to the transmembrane/cytoplasmic portion of the molecules because it was transferred to the CD8 alpha hybrid molecules composed of extracellular CD8 and MHC class I transmembrane and intracytoplasmic domains (CD8-e/MHC-t-i). Measurements of the dynamics of these cell surface molecules by using radiolabeled mAb revealed distinct behaviors: TCR/CD3 complex ligand internalization was increased (around 50% after 40 to 60 min) after PMA activation, whereas the ligand of class I MHC molecules was internalized at constant rate irrespective of PMA activation. Ligand bound to native CD8 molecules was poorly internalized, irrespective of the activation of the T cells with PMA. The same ligand bound to the CD8-e/MHC-t-i hybrid molecule was internalized at the same rate as a class I MHC molecule ligand, indicating that the behavior of the hybrid molecule was characteristic of the transmembrane/cytoplasmic portion of MHC class I molecules.

The present studies have made use of in vitro derived H-2Kb mutants to analyze the fine specificity of alloreactive cytotoxic T lymphocytes (CTL). The variants were derived by negatively selecting mutagenized tumor cells with a monoclonal anti-H-2Kb antibody and positively selecting for residual cells expressing serologically altered H-2Kb molecules. Details of this procedure are described in the companion paper. Selected populations of bulk alloreactive and cloned CTL were examined for recognition of the variants. In contrast to the serologic findings presented in the companion paper, there does not appear to be a correlation between the monoclonal antibody used to select the R8 variant and the CTL specificities recognized. In several instances, CTL clones could discriminate between variants having identical serologic profiles. Therefore, it would appear that the CTL have a large repertoire of allorecognition, even when generated across a mutant anti-Kb combination reflecting only a few amino acid differences. In addition, a diverse set of epitopes can be recognized on the Kb molecule. Finally, in some instances a change in what would appear to be a single amino acid resulted in a profound alteration of CTL recognition even though the Kb mutant molecule expressed limited serologic changes. These results support the idea that small changes in the H-2Kb molecule can have dramatic effects on CTL even though there are relatively little effects on serologic recognition of the target molecule.

We previously showed that thymocytes express high levels of c-ets-1 protein (Ets1) that can be rapidly phosphorylated following mitogenic stimulation using lectins. We demonstrate here that T cell receptor (TCR) specific stimulation with monoclonal antibodies of mature CD8+ or CD4+ T cells also results in the rapid phosphorylation of Ets1, reinforcing the hypothesis of a possible role for Ets1 in T cell activation. In addition to the major Ets1 product (mu-p63c-ets-1), we identify in mouse thymocytes and mature T cells a distinct 52 Kd Ets1 related protein (mu-p52c-ets-1). In contrast to the major Ets1 protein, mu-p52c-ets-1 is poorly phosphorylated in unstimulated cells. Furthermore, mitogenic stimulation of thymocytes and T cells failed to induce in mu-p52c-ets-1 the Ca2(+)-dependent phosphorylation events which are known to drastically affect the migration of the major Ets1 protein in SDS polyacrylamide gels. Mu-p52c-ets-1, like mu-p63c-ets-1, is a nuclear-chromatin associated protein which exhibits DNA binding activity in vitro. However, in contrast to the major Ets1 protein, the association of mu-p53c-ets-1 with chromatin and its ability to bind to DNA in vitro are unaffected by activation stimuli resulting in an increase in [Ca2+]i. Finally, we present indications suggesting that mu-p52c-ets-1 might be the murine equivalent of the translation product of an alternatively c-ets-1 spliced mRNA described in human cells by others.

The use of specific T helper cells (TH) has allowed the detection of anti-hapten plus self cytotoxic T-lymphocyte (CTL) precursors in cell populations unable to generate autonomous reactions (and considered as 'immature'). This led us to reevaluate the ontogeny of CTL precursors in different tissues in the presence of adult TH. It was found that thymocytes from newborn mice contained levels of CTL precursors comparable to those of adults and were dependent on TH throughout the donor's life. In the spleens, however, 3 periods could be distinguished: (a) from birth to 4 days after birth, no CTL precursors could be detected even in the presence of TH; (b) from the 5th day to the 2nd week after birth, CTL responses could be obtained only in the presence of TH; (c) after the 2nd week, autonomous responses could be obtained. No evidence for a suppressive mechanism was found in the spleen of the newborn mice, nor could lack of responsiveness be attributed to newborn accessory-cell malfunction. The data indicate that a limiting cell in the development of anti-hapten plus self CTL is the TH from the 4th to the 16th day after birth. The absence of CTL precursors in the spleen, but not in the thymus of neonates would indicate differential T-cell maturation in the two organs or late migration to the spleen.

CD4+ and CD8+ T cells emerge from thymic selection expressing a TCR restricted by MHC class II (TCRII) and MHC class I (TCRI), and upon Ag stimulation develop respectively into Th and CTL effector cells. The influence of thymic differentiation and antigenic stimulation on the determination of T cell functions was studied, with CD4+ T cells expressing a transgenic TCRI that reacts with the class I alloantigen H-2K(b) in a CD8-independent fashion. Such T cells additionally express a TCR, probably TCRII, in which the transgenic TCR beta-chain is associated with endogenously rearranged TCR alpha-chains. Upon in vitro stimulation with H-2K(b)-expressing cells, both CD8+ and CD4+ transgenic TCR+ T cells developed into CTL capable of killing Ag-expressing target cells through a perforin-dependent mechanism, and secreted IL-2 and IFN-gamma. Fas ligand-dependent killing could also be induced in both CD8+ and CD4+ in vitro stimulated T cells. The capacity to secrete IL-4 was restricted to the CD4+ T cells, however, suggesting that both CD8/CD4-shared and CD4-unique programs can be elicited by stimulation of CD4 T cells through a TCRI. Acquisition of CTL function was also induced upon class II alloantigen stimulation through the endogenously rearranged TCRII, which represents a polyclonal set of TCRs. IL-2, IFN-gamma, and after restimulation, IL-4, were also produced. Thus: 1) events associated with intrathymic selection influence the gene program activated in response to the same TCRI/APC interaction; and 2) CD4+ T cells expressing a TCRI and a TCRII can activate the same gene program after engagement of either one of these TCRs.