Unraveling the complexity of lymphocyte interactions is key to understanding immune network and improving cancer immunotherapy. By designing various adoptive cell transfer protocols in solid tumors established in RAG−/−, RAG−/−gc−/− and RAG−/−GzmB-Tom mice and tracing lymphocytes specific to a self-tumor antigen P1A encoded by an X-linked cancer-germline gene Trap1a, we explored the functional dynamics between T and NK cells. We found that activated CD8+T cells augmented NK cell effector function in a pre-tumor T cell protocol that prevented the development of antigen-escape tumor variants. Using a silica nanofiber 3D matrix to control cellular mobility, confocal imaging showed intercellular contacts of activated CD8+T cells (CD69highCD25high) with multiple naïve NK cells, while naïve CD8+T cells formed single or no contact with NK cells. This interaction led to crossregulation of mitochondrial Ca2+ (mitoCa2+) oscillations in both cells whereby activated CD8+T cells induced differentiation of NK cell effector phenotype and NK cells polarized CD8+T cells towards a central memory phenotype. Intracellularly, CD8+T cells increased JAK1, JAK3, TYK2, STAT2 and STAT6 phosphorylation and oxidative signaling in NK cells. In turn, NK cells restrained IL-2 signaling in CD8+T cells by dampening activation-induced STAT5-dependent expression of IL-2-receptor-a chain. These effects were abrogated following the blockade of mitoCa2+ uptake. Mice deficient in mitoCa2+ handling-regulatory gene Fus1 showed increased incidence of a range of spontaneous sarcomas, lymphomas and leukemia. Data suggest that mitoCa2+ transport-guided intercellular crosstalk between CD8+T and NK cells is critical to prevent tumor development and escape.

CD4(+) T cells develop distinct and often contrasting helper, regulatory, or cytotoxic activities. Typically a property of CD8(+) T cells, granzyme-mediated cytotoxic T cell (CTL) potential is also exerted by CD4(+) T cells. However, the conditions that induce CD4(+) CTLs are not entirely understood. Using single-cell transcriptional profiling, we uncover a unique signature of Granzyme B (GzmB)(+) CD4(+) CTLs, which distinguishes them from other CD4(+)T helper (Th) cells, including Th1 cells, and strongly contrasts with the follicular helper T (Tfh) cell signature. The balance between CD4(+) CTL and Tfh differentiation heavily depends on the class of infecting virus and is jointly regulated by the Tfh-related transcription factors Bcl6 and Tcf7 (encoding TCF-1) and by the expression of the inhibitory receptors PD-1 and LAG3. This unique profile of CD4(+) CTLs offers targets for their study, and its antagonism by the Tfh program separates CD4(+) T cells with either helper or killer functions.

Cancer-germline genes in both humans and mice have been shown to encode antigens susceptible to targeting by cytotoxic CD8 T effector cells (CTL). We analysed the ability of CTL to kill different tumour cell lines expressing the same cancer-germline gene P1A (Trap1a). We previously demonstrated that CTL expressing a T-cell receptor specific for the P1A(35-43) peptide associated with H-2L(d), although able to induce regression of P1A-expressing P815 mastocytoma cells, were much less effective against P1A-expressing melanoma cells. Here, we analysed parameters of the in vitro interaction between P1A-specific CTL and mastocytoma or melanoma cells expressing similar levels of the P1A gene and of surface H-2L(d). The mastocytoma cells were more sensitive to cytolysis than the melanoma cells in vitro. Analysis by video-microscopy of early events required for target cell killing showed that similar patterns of increase in cytoplasmic Ca2+ concentration ([Ca2+]i) were induced by both types of P1A-expressing tumour cells. However, the use of CTL expressing a fluorescent granzyme B (GZMB-Tom) showed a delay in the migration of cytotoxic granules to the tumour interaction site, as well as a partially deficient GZMB-Tom exocytosis in response to the melanoma cells. Among surface molecules possibly affecting tumour-CTL interactions, the mastocytoma cells were found to express intercellular adhesion molecule-1, the ligand for LFA-1, which was not detected on the melanoma cells.

SummaryCD8 T cells contribute to long‐term protection against Listeria monocytogenes infection by differentiating into memory T cells. These rapidly respond to antigen or inflammation upon secondary infection. In this study we used CD8 T cells from OT1 mice and CD4 T cells from OT2 mice expressing a fluorescent chimeric granzyme (GZMB‐Tom) protein to monitor the primary response to infection with ovalbumin‐expressing L. monocytogenes (Lm‐OVA). We show that, unlike poorly responding CD4 T cells, CD8 T cells readily proliferated and expressed high levels of GZMB‐Tom as early as 2 days after infection. FACS analysis showed GZMB‐Tom expression in undivided CD8 T cells, with its level increasing over one to four divisions. OT1 T cells were visualized in the T‐cell zone by confocal microscopy. This showed GZMB‐Tom‐containing granules oriented towards MHCII‐positive cells. Twenty hours later, most OT1 T cells had divided but their level of GZMB‐Tom expression was reduced. Recently divided OT1 cells failed to express GZMB‐Tom. Fourteen hours after secondary infection, GZMB‐Tom was re‐expressed in memory OT1 T cells responding either to Lm‐OVA or L. monocytogenes. Differences in the activation phenotype and in the splenic distribution of OT1 T cells were observed, depending on the challenge. Notably, OTI T cells with polarized granules were only observed after challenge with cognate antigen. This work showed that the GZMB‐Tom knock‐in mice in which GZMB‐Tom faithfully reproduced GZMB expression, provide useful tools to dissect mechanisms leading to the development of anti‐bacterial effector and memory CD8 T cells and reactivation of the memory response to cognate antigen or inflammatory signals.

To evaluate acquisition and activation of cytolytic functions during immune responses we generated knock in (KI) mice expressing Granzyme B (GZMB) as a fusion protein with red fluorescent tdTomato (GZMB-Tom). As for GZMB in wild type (WT) lymphocytes, GZMB-Tom was absent from naïve CD8 and CD4 T cells in GZMB-Tom-KI mice. It was rapidly induced in most CD8 T cells and in a subpopulation of CD4 T cells in response to stimulation with antibodies to CD3/CD28. A fraction of splenic NK cells expressed GZMB-Tom ex vivo with most becoming positive upon culture in IL-2. GZMB-Tom was present in CTL granules and active as a protease when these degranulated into cognate target cells, as shown with target cells expressing a specific FRET reporter construct. Using T cells from mice expressing GZMB-Tom but lacking perforin, we show that the transfer of fluorescent GZMB-Tom into target cells was dependent on perforin, favoring a role for perforin in delivery of GZMB at the target cells’ plasma membranes. Time-lapse video microscopy showed Ca++ signaling in CTL upon interaction with cognate targets, followed by relocalization of GZMB-Tom-containing granules to the synaptic contact zone. A perforin-dependent step was next visualized by the fluorescence signal from the non-permeant dye TO-PRO-3 at the synaptic cleft, minutes before the labeling of the target cell nucleus, characterizing a previously undescribed synaptic event in CTL cytolysis. Transferred OVA-specific GZMB-Tom-expressing CD8 T cells acquired GZMB-Tom expression in Listeria monocytogenes-OVA infected mice as soon as 48h after infection. These GZMB-Tom positive CD8 T cells localized in the splenic T-zone where they interacted with CD11c positive dendritic cells (DC), as shown by GZMB-Tom granule redistribution to the T/DC contact zone. GZMB-Tom-KI mice thus also provide tools to visualize acquisition and activation of cytolytic function in vivo.

An important function of the immune system consists in eliminating infected or transformed cells. Naive CD8+ T lymphocytes differentiate in peripheral lymphoid organs following a first antigen contact. There they acquire the different constituents of the cytolytic machinery and become cytolytic T lymphocytes (CTLs), before migration to the tissues where they meet their specific target. Target cell killing is mediated by the release of granules expressing the Lamp-1 marker 1 and containing effector proteins including perforin 2, 3 and granzymes (granzyme A (GZMA) and B (GZMB) being the main proteases). Effective target cell lysis depends on many factors; so deciphering the mechanisms involved is important, in particular to palliate the failings of the immune system during tumor development. Transient labeling of acidic granules with Lysotracker has elegantly been used to analyze kinetics of granule polarization in CTL/target conjugates. Intracellular staining of fixed and permeabilized cells has allowed elucidation of important steps of CTL granule movements, fusion and degranulation 4-6. In order to develop a fluorescent probe that would stably label the contents of cytolytic granules in living cells, we designed a construct encoding a fusion protein composed of an N-terminal GZMB, a 12 amino-acid linker and a C-terminal tdTomato (tdTom) (excitation: 554 nM, emission: 581 nm, stable at the acidic pH of the granules (pKa 4.7) 7, GZMB-tdTom). This was inserted in the retroviral expression vector MSCV-IRES-HuCD2t (Supporting Information Fig. 1). We first transduced a T-cell hybridoma (HybT) and obtained stable expression of GZMB-tdTom in granules co-expressing GZMB and Lamp-1 (Supporting Information Fig. 2–5). Immunoblots revealed the fusion protein GZMB-tdTom at 85 kDa and tdTom at 55 kDa MW, as expected (Supporting Information Fig. 4). GZMB enzymatic activity could be detected in GZMB-tdTom-HybT cells, albeit at a low level as compared with that in CTLs (Supporting Information Fig. 5D). Whether this results from incomplete processing of the protein in HybT cells requires further investigation (Supporting Information Fig. 5D). To address more physiological conditions, we transduced normal CD8+ CTLs with the GZMB-tdTom construct (Supporting Information Fig. 6). As observed by confocal microscopy, the GZMB-tdTom fusion protein was localized in granules (Fig. 1A). Co-localization between GZMB-tdTom, Lamp-1 and GZMB was observed in granules of CTLs alone (Fig. 1B-i) in CTL/antigenic target conjugates (Fig. 1B-ii) that had re-localized the red granules to the cell–cell contact zone, and in conjugates of CTLs with targets presenting control peptide (Fig. 1B-iii). Redistribution of granules to the CTL/target contact zone was observed in 80% of antigen-specific versus 5% of non-specific conjugates (Supporting Information Fig. 7B). TdTom-transduced cells expressed red tdTom protein spread throughout the cytoplasm (Fig. 1B-iv) and similarly to untransduced CTLs (Supporting Information Fig. 7A) relocalized GZMB-containing granules expressing Lamp-1 to the CTL/target contact zone (Fig. 1B-iv). Mathematical analyses showed that GZMB-tdTom colocalized with Lamp-1 and GZMB (Pearson's Rr coefficient around 0.55) whereas tdTom did not show any colocalization (Rr 0.1) (Supporting Information Fig. 7C). GZMB-tdTom-transduced P14-TCR CTLs express the fusion protein in vesicles/granules that redistribute in CTL/target cell conjugates. Purified Hu-CD2+ GZMB-tdTom-transduced P14-TCR CTLs were used as described in Supporting Information. Targets were RMA-S cells loaded with antigenic (gp33) or control (HY) peptide (10–6 M). (A and B) CTL-target conjugates were analyzed after 30 min at 37°C. Bars = 5 μm. (A) Confocal analysis on fixed GZMB-tdTom-transduced CTLs, alone (left) or conjugated to an antigenic target (t, right). (B) Confocal analysis on fixed and permeabilized cells: GZMB-tdTom-transduced CTLs (i) alone, (ii) conjugated with antigenic or (iii) control targets (t, shown in brightfield); and (iv) TdTom-transduced CTLs conjugated with antigenic targets. Fluorescence is shown for tdTom (red), a-Lamp-1 (green) and a-GZMB (blue). For statistics on colocalization between GZMB-tdTom, anti-Lamp-1 and anti-GZMB fluorescence see Supporting Information Fig. 7A and B. (C) i and ii Antigenic (gp33) or (iii and iv) control (HY) targets were deposited on slides. GZMB-tdTom-transduced TCR-P14 CTLs loaded with Fluo-4 (see Supporting Information) were then deposited. Signals for (i and iii) green Fluo-4 (Ca++ flux) and (ii and iv) red fluorescence (GZMB-tdTom) were recorded for 30–40 min by time lapse video microscopy (Zeiss Meta 560). Images at the indicated time points are shown; for the complete videos see the Supporting Information. For quantification of the images, see Supporting Information Fig. 7D. Analysis and relative quantification of all the images were performed with Image J software. The data are representative of three experiments. Following TCR/antigen engagement, calcium flux and PKC activation are important signals for gene activation and granule migration to the CTL/target contact zone preceding degranulation 4, 8. CTLs preloaded with Fluo-4 were used to monitor by video microscopy the Ca++ fluxes and the redistribution of GZMB-tdTom-containing granules. When GZMB-tdTom-transduced P14-TCR CTLs faced a specific target, an attachment signal preceded a rapid Ca++ flux (10–20 s) and granule translocation to the contact zone occurring at various times (20–480 s) (Fig. 1C-i and ii, Supporting Information Fig. 7D, Video 1). No significant signal was observed when the CTLs were facing control targets (Fig. 1C-iii and iv, Video 2). These kinetics are in agreement with published studies using CTL clones 6, 9. We used the Lamp-1 exposure method to assess CTL degranulation in response to antigenic stimulation and to observe the fate of GZMB-tdTom during that process. GZMB-tdTom-transduced P14-TCR CTLs exposed Lamp-1 in response to gp33-loaded RMA-S, the extent of degranulation being dependent on peptide concentration (Fig. 2A). The percent of GZMB-tdTom fluorescent CTLs markedly decreased (from 20% for non-stimulated or control-peptide stimulated CTLs to 13% for CTLs activated with 10−6 M gp33-loaded RMA-S), with a level of GZMB-tdTom fluorescence much lower in Lamp-1–positive (MRFI 422 (MRFI, mean relative fluorescence intensity)) as compared to Lamp-1–negative (607) CTLs. GZMB expression as measured on fixed and permeabilized cells were also reduced (about 50%) in the antigen-activated CTLs (data not shown). These results suggest that the whole GZMB-tdTom fusion protein was released during degranulation. Similarly, analysis of GZMB-tdTom-transduced OT1-TCR-Gzmb-KO (Gzmb, GZMB-encoding gene) CTLs, in which the only source of GZMB is GZMB-tdTom, showed that expression of GZMB-tdTom as well as GZMB was markedly decreased upon CTL activation with OVA-expressing cells (Supporting Information Fig. 8). We also found that the capacity of GZMB-tdTom-transducted P14-TCR CTLs to kill specific targets was not affected as compared to that of untransduced CTLs (Fig. 2B). Functional characteristics of GZMB-tdTom-transduced CTLs. (A) Release of GZMB-tdTom during CTL/target interaction. GZMB-tdTom-transduced P14-TCR CTLs were analyzed for antigen-induced Lamp-1 exposure when stimulated with RMA-S targets loaded with 10–6, 10–8,10–10 and 10–12 M gp33 antigenic peptide or 10–6 M HY control peptide for 1 h (1/1 CTL/target ratio). FACS analysis was performed on live cells gated on P14-TCR(Vα2)-CD8+ T cells and red fluorescence versus exposed Lamp-1 are shown. Data are representative of five experiments. (B) CTL activity of untransduced or GZMB-tdTom-transduced P14-TCR CTLs. CTL activity was tested in a classical 4 h 51Cr-release assay on RMA-S target cells loaded with 10–6, 10–8 and 10–10 M peptide gp33, as indicated. CTL/target ratio=2.5. Non-specific lysis (on HY-loaded RMA-S) was subtracted. Data are replicates from one experiment. To our knowledge, two attempts at expressing fluorescent GZMB fusion proteins have been reported, but they were not expressed in CTLs 10, 11. Here, we described a new probe that allows visualization of cytolytic granules in living cells during their migration to the CTL/target cell contact zone. It also permits monitoring of GZMB release during antigen-induced degranulation and should be useful to further decipher the various steps leading to CTL activation and cytolytic effector function. This work was supported by institutional funding from «Institut National de la Santé et de la Recherche Médicale» and «Centre National de la Recherche Scientifique», and by grants from «National du Cancer», EC Integrated Project “Cancer Immunotherapy” and CARS Explorer (to A.-M.S.-V.). P.M. and V.G. were supported, respectively, by doctoral fellowships from “Association pour la Recherche sur le Cancer” and “Ministère de la Recherche et de la Technologie”. We thank Bernard Malissen, for his support, Lee Leserman and Stephane Méresse for suggestions and critical reading of the manuscript, Mathieu Fallet and M. Bajénoff for help with video imaging and the personnel of the CIML Imaging and animal facilities for assistance. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The rupture forces and adhesion frequencies of single recognition complexes between an affinity selected peptide/MHC complex and a TCR at a murine hybridoma surface were measured using Atomic Force Microscopy. When the CD8 coreceptor is absent, the adhesion frequency depends on the nature of the peptide but the rupture force does not. When CD8 is present, no effect of the nature of the peptide is observed. CD8 is proposed to act as a time and distance lock, enabling the shorter TCR molecule to bridge the pMHC and have time to finely read the peptide. Ultimately, such experiments could help the dissection of the sequential steps by which the TCR reads the peptide/MHC complex in order to control T cell activation.

Actin polymerization plays a critical role in activated T lymphocytes both in regulating T cell receptor (TCR)-induced immunological synapse (IS) formation and signaling. Using gene targeting, we demonstrate that the hematopoietic specific, actinand Arp2/3 complex-binding protein coronin-1A contributes to both processes. Coronin-1A-deficient mice specifically showed alterations in terminal development and the survival of abT cells, together with defects in cell activation and cytokine production following TCR triggering. The mutant T cells further displayed excessive accumulation yet reduced dynamics of F-actin and the WASP-Arp2/3 machinery at the IS, correlating with extended cell-cell contact. Cell signaling was also affected with the basal activation of the stress kinases sAPK/JNK1/2; and deficits in TCR-induced Ca influx and phosphorylation and degradation of the inhibitor of NF-kB (IkB). Coronin-1A therefore links cytoskeleton plasticity with the functioning of discrete TCR signaling components. This function may be required to adjust TCR responses to selecting ligands accounting in part for the homeostasis defect that impacts abT cells in coronin-1A deficient mice, with the exclusion of other lympho/hematopoietic lineages. Citation: Mugnier B, Nal B, Verthuy C, Boyer C, Lam D, et al. (2008) Coronin-1A Links Cytoskeleton Dynamics to TCRab-Induced Cell Signaling. PLoS ONE 3(10): e3467. doi:10.1371/journal.pone.0003467 Editor: Jean Kanellopoulos, University Paris Sud, France Received July 17, 2008; Accepted September 20, 2008; Published October 21, 2008 Copyright: 2008 Mugnier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Inserm, CNRS, the ‘Association pour la Recherche sur le Cancer’ (ARC), the ‘Fondation Princesse Grace de Monaco’, and the Commission of the European Communities (to PF); and from the ‘Ministère de l’Education Nationale et de la Recherche’ (ACI #108) (to PF and AA). BN was supported by fellowships from the ‘Ligue Nationale Contre le Cancer’ and ARC. BM was supported by a fellowship from ARC. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ferrier@ciml.univ-mrs.fr . These authors contributed equally to this work.

Actin polymerization plays a critical role in activated T lymphocytes both in regulating T cell receptor (TCR)-induced immunological synapse (IS) formation and signaling. Using gene targeting, we demonstrate that the hematopoietic specific, actin- and Arp2/3 complex-binding protein coronin-1A contributes to both processes. Coronin-1A-deficient mice specifically showed alterations in terminal development and the survival of alpha beta T cells, together with defects in cell activation and cytokine production following TCR triggering. The mutant T cells further displayed excessive accumulation yet reduced dynamics of F-actin and the WASP-Arp2/3 machinery at the IS, correlating with extended cell-cell contact. Cell signaling was also affected with the basal activation of the stress kinases sAPK/JNK1/2; and deficits in TCR-induced Ca2+ influx and phosphorylation and degradation of the inhibitor of NF-kappaB (I kappa B). Coronin-1A therefore links cytoskeleton plasticity with the functioning of discrete TCR signaling components. This function may be required to adjust TCR responses to selecting ligands accounting in part for the homeostasis defect that impacts alpha beta T cells in coronin-1A deficient mice, with the exclusion of other lympho/hematopoietic lineages.

Cbl proteins have been implicated in ligand-induced TCR/CD3 down-modulation, but underlying mechanisms are unclear. We analyzed the effect of mutation of a cbl-binding site on ZAP-70 (ZAP-Y292F) on dynamics, internalization, and degradation of the TCR/CD3 complex in response to distinct stimuli. Naive CD8 T cells expressing the P14 transgenic TCR from ZAP-Y292F mice were selectively affected in TCR/CD3 down-modulation in response to antigenic stimulation, whereas neither anti-CD3 Ab-, and PMA-induced TCR down-modulation, nor constitutive receptor endocytosis/cycling were impaired. We further established that the defect in TCR/CD3 down-modulation in response to Ag was paralleled by an impaired TCR/CD3 internalization and CD3 zeta degradation. Analysis of T/APC conjugates revealed that delayed redistribution of TCR at the T/APC contact zone was paralleled by a delay in TCR internalization in the synaptic zone in ZAP-Y292F compared with ZAP-wild-type T cells. Cbl recruitment to the synapse was also retarded in ZAP-Y292F T cells, although F-actin and LFA-1 redistribution was similar for both cell types. This study identifies a step involving ZAP-70/cbl interaction that is critical for rapid internalization of the TCR/CD3 complex at the CD8 T cell/APC synapse.

Class I molecules of the major histocompatibility complex (MHC)are expressed on the cell surface as a complex of two noncovalently associated units, a 4045,000 dalton subunit called the heavy chain and an 11,000 dalton subunit called 32 microglobulin (/32m) (1). In the mouse this appears to be true for molecules encoded in the Qa-Tla region as well as for molecules encoded by the K, D, or L loci (2, 3). In contrast to the 45,000 dalton class I chain, O2m is not integrated into the membrane. Similarly, it is believed that the heavy chains of class I molecules are not expressed in the absence of/32m. Evidence for this comes primarily from the study of two cell lines. The Burkitt iymphoma line, Daudi, expresses neither class I H L A antigens nor B2m on the cell surface (4). In this cell line the primary defect is the inability to synthesize /32m, as shown by biochemical studies and by somatic cell hybridization experiments in which the expression of Daudi HLA antigens could be rescued either by mouse or human fl2m, provided by the normal par tner in the hybrid (4). Essentially the same kind of data have been obtained by Hyman and his collaborators (5, 6) using a somatic cell variant of the C3H (H-2 k) tbymoma, R 1. These studies led to the conclusion that class I proteins have to be associated with 32m for expression on the cell surface. We report here on a spontaneous variant of the B6 lymphoma EL4, which in contrast to the wild-type cell line, expresses neither H-2K b nor 32m but does express H-2D b, which can be detected by serological reactivity as well as by cytotoxic T lymphocytes (CTL).

The origin of autoreactive CD4-CD8- T cells is largely unknown. In TCR transgenic (Tg) mice expressing the cognate class I MHC antigen, CD4-CD8- T cells differed depending on characteristics of Tg-TCR/antigen interaction. Tg-TCR/CD3lo CD4-CD8- T cells expressing the NK1.1 marker were observed only for a Tg-TCR whose stimulation by antigen was independent of CD8. Unlike normal T cells, which have essentially TCR-associated zeta homodimers, these cells had a high proportion of TCR-associated zeta-Fc epsilon RI gamma heterodimers. They were also characterized by an unusually high content of Fc epsilon RI gamma mRNA and low content of mRNA encoding CD3 epsilon, CD3 gamma, CD3 delta, and zeta. Based on their phenotype and selection requirements, it is proposed that CD4-CD8- thymic precursor cells can be driven along the CD4-CD8-NK1.1+ pathway following coreceptor-independent TCR signaling at an intrathymic stage when Fc epsilon RI gamma and CD3 components are coexpressed.

As shown previously, a given cytotoxic T lymphocyte (CTL) clone (KB5.C20) could be induced to express the Fas ligand (FasL) by either T cell receptor (TCR) engagement or phorbol 12‐myristate 13‐acetate (PMA)/ionomycin stimulation. In contrast, another CTL clone (BM3.3) has now been found to exert Fas‐based cytotoxicity only after TCR engagement, but not after PMA/ionomycin stimulation. This suggested the existence of a PMA‐insensitive, antigeninduced pathway leading to FasL expression. The inability of PMA to promote Fas‐based cytotoxicity in BM3.3 cells was correlated with a defect in expression of the classical protein kinase C (PKC) isoforms α and βI. In KB5.C20 cells depleted of PMA‐sensitive PKC isoforms and thus no longer responsive to PMA, Fas‐based cytotoxicity could still be induced via the TCR/CD3 pathway. On the other hand, a requirement for phosphatidylinositol‐3 kinase (PI3K) selectively in this TCR/CD3‐induced pathway was demonstrated by specific inhibition with wortmannin. These results suggest that FasL expression when induced via the TCR/CD3 involves PI3K, and when induced by PMA/ionomycin requires the expression of PMA‐sensitive PKC isoforms absent in clone BM3.3. Additional data suggest that in neither case was NF‐χB activation implicated in FasL expression.

To study the interactions between T cells and class I MHC products, we developed in vitro a T-cell line reactive to H-2Kb stimulating cells and derived T-cell clones from it. Although the T-cell line could proliferate in the absence of exogeneous T-cell growth factors when stimulated with H-2Kb spleen cells, each of the derived T-cell clones required both H-2Kb stimulating cells and an external source of T-cell growth factor for its propagation. Each of the T-cell clones was also cytolysic for H-2Kb target cells. Such T-cell clones allowed the comparison of the antigenic requirements for proliferation and cytolysis. By using H-2K b mutant mice, we found that while the original anti-H-2Kb T-cell line reacted with each of the six mutants tested, the individual T-cell clones could be distinguished in terms of their reactivity pattern. Similar fine specificity patterns were found when H-2K b mutant cells were used as stimulating or target cells for any given T-cell clone. Each of the three monoclonal H-2Kb-specific antibodies reacting with different epitopes of the H-2Kb molecule totally inhibited H-2Kb-induced proliferation and lysis by the T-cell clones. Further blocking studies involved use of Fab antibody fragments and definition of their reactivity on cells from the H-2K b mutants. We concluded that: (1) blocking with a monoclonal antibody does not prove identity of alloantigens recognized by the T-cells and the antibody; (2) a monoclonal antibody could either block or not block H-2Kb-CTL interactions depending on structural variations of the H-2Kb molecule not affecting the CTL-H-2Kb functional interaction; (3) blocking one type of H-2Kb-T-cell interaction (induction of proliferation) always affects the other type (cytolysis).