Antigen-bearing Target Cells Research Articles

Geometry of a TLR2-Agonist-Based Adjuvant Can Affect the Resulting Antigen-Specific Immune Response.

Targeted delivery of otherwise nonimmunogenic antigens to Toll-like receptors (TLRs) expressed on dendritic cells (DCs) has proven to be an effective means of improving immunogenicity. For this purpose, we have used a branched cationic lipopeptide, R4Pam2Cys, which is an agonist for TLR2 and enables electrostatic association with antigen for this purpose. Here, we compare the immunological properties of ovalbumin formulated with different geometrical configurations of R4Pam2Cys. Our results demonstrate that notwithstanding the presence of the same adjuvant, branched forms of R4Pam2Cys are more effective at inducing immune responses than are linear geometries. CD8+ T-cell-mediated responses are particularly improved, resulting in significantly higher levels of antigen-specific cytokine secretion and cytolysis of antigen-bearing target cells in vivo. The results correlate with the ability of branched R4Pam2Cys conformations to encourage higher levels of DC maturation and facilitate superior antigen uptake, leading to increased production of proinflammatory cytokines. These differences are not attributable to particle size because both branched and linear lipopeptides associate with antigen-forming complexes of similar size, but rather the ability of branched lipopeptides to induce more efficient TLR2-mediated cell signaling. Branched lipopeptides are also more resistant to trypsin-mediated proteolysis, suggesting greater stability than their linear counterparts. The branched lipopeptide facilitates presentation of antigen more efficiently to CD8+ T cells, resulting in rapid cell division and upregulation of early cell surface activation markers. These results as well as cognate recognition of Pam2Cys by TLR2 indicate that the adjuvant's efficiency is also dependent on its geometry.

Fcγ receptors enable anticancer action of proapoptotic and immune-modulatory antibodies

Antibodies have important roles in controlling cellular immunity through interaction with activating or inhibitory Fcγ receptors (FcγRs). FcγR engagement can facilitate receptor cross-linking on target cells, or induce retrograde FcγR signals to stimulate or suppress antibody-dependent, cell-mediated depletion of antigen-bearing target cells. Recent studies uncover unexpectedly important roles for FcγRs in the anticancer action of antibodies designed to trigger tumor cell apoptosis or enhance antitumor immunity. Here, we outline a conceptual framework for understanding these findings and discuss their mechanistic and translational implications.

Distinct roles of cytolytic effector molecules for antigen‐restricted killing by CTL in vivo

Cytotoxic T lymphocytes (CTLs) represent one of the front lines of defense for the immune system, killing virus-infected and tumor-transformed cells. CTL use at least two mechanisms to induce apoptosis in their targets, one mediated by perforin and granzymes, and the other triggered by the death ligand, CD95 ligand (CD95L). Here, we used an in vivo cytotoxicity assay to measure specific clearance of antigen-bearing target cells in mice that had previously been immunized with noninfectious cell-associated antigens. We found that perforin was dispensable for efficient clearance of antigen-bearing cells from immunized mice, but only if CD95/CD95L was functional; however, there was a delay in target cell clearance in the absence of perforin. In addition, we observed ∼35% target cell clearance in the absence of both perforin and CD95L, which was only slightly abrogated in the presence of a neutralizing anti-tumor necrosis factor (TNF) antibody. The presence of a dominant negative Fas-associated death domain (FADD) did not block target cell clearance and therefore cannot be attributed to known death receptors. Taken together, these data suggest that perforin- and CD95L-dependent killing are complementary at early time points, each can compensate for the absence of the other at later time points, and that there is an additional component of antigen-restricted CTL killing independent of perforin, CD95L, and TNFα.

T Cell Immunotherapy for Adenoviral Infections of Stem Cell Transplant Recipients.

Adenoviruses remain a major cause of mortality and morbidity in hematopoietic stem cell transplant (HSCT) recipients. To date, no effective anti-adenoviral treatment is available, presenting a potential role for adoptive T cell therapy, as an increased risk of infection may be correlated with lack of endogenous T cell immunity. Therefore the goal of this project was to evaluate the use of adenovirus-specific cytotoxic T lymphocyte (CTL) lines for the prophylaxis and treatment of adenoviral infections post-HSCT. CTLs are initiated by stimulation with donor-derived mononuclear cells transduced with a recombinant adenovirus type 5 vector pseudotyped with a type 35 fiber (MOI 200 virus particles (vp) per cell). Responder cells are expanded by weekly restimulation with autologous EBV-transformed B lymphoblastoid cell lines (LCL) transduced with the same vector using an MOI of 500 vp. Nine of the 11 CTL lines manufactured for clinical use were predominantly CD4+ (range 12–95%), but all had a CD8+ component (range 3.5–79%). In chromium release assays, all CTL lines showed specific killing of both adenovirus and EBV antigen-bearing target cells. Recognition of adenovirus and EBV targets was not significantly different; at 20:1 effector:target ratio there was 18.5 ± 14% specific lysis of adenovirus targets and 23 ± 15% lysis of EBV targets while uninfected recipient target cells were not lysed (1.5 ± 2%). We identified the hexon as the immunodominant target antigen in the CTL lines. To test the hypothesis that these polyclonal CD4+ and CD8+ hexon-specific T cells can provide protection against adenovirus infections in HSCT recipients, we administered donor-derived bi-virus-specific CTL lines to pediatric recipients of allogeneic stem cell transplant. So far, we have treated 7 patients in this phase I dose escalation study (using the continual reassessment model); 2 patients on dose level 1 (5x106/m2), 2 on dose level 2 (1.5x107/m2) and 2 on dose level 3 (4.5x107/m2). Of 6 patients who received CTL as prophylaxis, all remain negative for adenovirus and EBV and there were no adverse events attributable to CTL infusion. Post-infusion, samples for immune monitoring were collected for at least 3 months and analyzed by pentamer and IFN-g ELISPOT assays in batches to minimize inter-assay variablility. An increase in the frequency of EBV-specific T cells could be detected in the blood by 2 weeks post infusion in all evaluated patients, likely due to the presence of this latent virus in all patients. However, there was no significant rise in the adenovirus-specific T cell frequency post-infusion. The seventh patient received the bi-virus-specific CTL at a dose of 5x106/m2 as treatment for adenovirus disease. This patient had adenoviral pneumonia requiring ventilatory support. Following the CTL infusion the patient made a dramatic clinical recovery, was weaned off mechanical ventilation, and discharged a month after the infusion. In contrast to the prophylaxis group, this patient had a substantial rise in adenovirus-specific T cells detected in the peripheral blood by 4 weeks post-CTL infusion, which coincided with his clinical recovery and clearance of the virus from the tracheal aspirate, suggesting that the infused cells were effective in vivo. Adoptive immunotherapy for adenovirus appears to be safe but expansion of adenovirus-specific CTL in vivo may require the presence of antigen to stimulate the infused cells.

Distinct role of lymphocyte function-associated antigen-1 in mediating effective cytolytic activity by cytotoxic T lymphocytes

Lymphocyte function-associated antigen-1 (LFA-1) interaction with intercellular adhesion molecules (ICAMs) facilitates T cell antigen receptor (TCR)-mediated killing. To dissect TCR and LFA-1 contributions, we evaluated cytolytic activity and granule release by cytotoxic T lymphocytes (CTL) as well as intracellular granule redistribution and morphology of CTL stimulated with natural TCR ligand in the presence or absence of LFA-1 engagement. Although other adhesion mechanisms, e.g., CD2-CD58 interaction, could substitute for LFA-1 to trigger CTL degranulation, productive LFA-1 ligation was indispensable for effective target cell lysis by the released granules. LFA-1-mediated adhesion to glass-supported bilayers containing intercellular adhesion molecule-1 was characterized by a much larger junction area, marked by LFA-1 segregation, and a more compact cell shape compared with those observed for CD2-mediated adhesion to bilayers containing CD58. A larger contact induced by intercellular adhesion molecule 1 determined a unique positioning of granules near the interface. These data provide evidence that LFA-1 delivers a distinct signal essential for directing released cytolytic granules to the surface of antigen-bearing target cells to mediate the effective destruction of these cells by CTL.

A novel tool for B-cell tolerance research: Characterization of mouse alloantibody development using a simple and reliable cellular ELISA technique

In animal-based transplantation research, the measurement of anti-donor antibodies in transplant recipients is limited by lack of an appropriate technique. We have developed a novel immunoassay capable of quantifying antibody bound to cell-surface major histo- compatability complex (MHC) and non-MHC antigens, using splenocytes from wild-type and MHC-deficient mice as antigen-bearing target cells. We utilized our “cellular ELISA” (CELISA) technique to study the development of tolerance versus immunity in the B-cell compartment in response to neonatal exposure to allogeneic fetal liver cells (FLC). This neonatal tolerance protocol typically induces permanent acceptance of donor-type and third-party cardiac allografts, but rejection of both donor-type and third-party skin grafts occurs. C3H/He (C3H; H-2 k) mice were injected as neonates with BALB/c (BALB; H-2 d) FLC and transplanted as adults with C57BL/6 (B6; H-2 b) cardiac grafts. Despite long-term acceptance of third-party B6 cardiac grafts, serum contained increased anti-B6 IgG and IgM levels as measured by CELISA; IgM production was elevated by 2 weeks posttransplant and remained stable, while IgG production increased rapidly between 2 and 5 weeks posttransplant. In another experimental setting, CELISA assays were able to detect that neonatal injection of C3H mice with FLC from wild-type B6 mice or from MHC class II-deficient or class I/II-deficient (B6 background) mice (CI +CII +, CI +CII −, CI −CII −, respectively) prevented sensitization to B6 antigens by subsequent skin transplants but did not induce graft acceptance, whereas FLC from MHC class I-deficient–only (CI −CII +) did not prevent B6 sensitization. The CELISA technique is a simple and sensitive means for quantifying alloantibodies in mice and will assist in further delineating the role of the B-cell compartment in neonatally induced cardiac allograft acceptance.

Viral-Specific Cytotoxic T Lymphocytes Lyse Human Immunodeficiency Virus–Infected Primary T Lymphocytes by the Granule Exocytosis Pathway

Cytotoxic T lymphocytes (CTL) lyse antigen-bearing target cells by two distinct pathways. Whereas granule exocytosis targets any antigen-bearing cell, fas-mediated cytotoxicity kills only fas-expressing cells and does not require antigen expression. Fas pathway activation can potentially lead to lysis of uninfected bystander cells. We examined the relative usage of the two pathways by CTL clones and cell lines directed against four different human immunodeficiency virus (HIV) proteins in lysing primary HIV-infected targets. Although fas was expressed on HIV-infected primary CD4+ T cells, their lysis by antigen-specific CD8+ CTL was only by the granule pathway. Fas ligand (fasL) was not detectable on antigen-specific CD8 clones, T-cell lines, or circulating HIV-specific CD8 T cells from HIV-infected donors, stained with a tetrameric HLA-A2-HIV-peptide complex. FasL expression by HIV-specific CTL clones was not activated by exposure to HIV-presenting cells, but was after unphysiological stimulation with phorbol myristate acetate (PMA). CTL clones did not lyse bystander Jurkat cells, but HIV-infected primary CD4+ T cells lysed uninfected bystander cells by the fas-mediated pathway. These results suggest that HIV-specific CD8+ CTL do not cause HIV immunopathology by lysing bystander cells. On the contrary, fas-mediated lysis of uninfected cells by HIV-infected cells may contribute to CD4 decline.

Viral-Specific Cytotoxic T Lymphocytes Lyse Human Immunodeficiency Virus–Infected Primary T Lymphocytes by the Granule Exocytosis Pathway

AbstractCytotoxic T lymphocytes (CTL) lyse antigen-bearing target cells by two distinct pathways. Whereas granule exocytosis targets any antigen-bearing cell, fas-mediated cytotoxicity kills only fas-expressing cells and does not require antigen expression. Fas pathway activation can potentially lead to lysis of uninfected bystander cells. We examined the relative usage of the two pathways by CTL clones and cell lines directed against four different human immunodeficiency virus (HIV) proteins in lysing primary HIV-infected targets. Although fas was expressed on HIV-infected primary CD4+ T cells, their lysis by antigen-specific CD8+ CTL was only by the granule pathway. Fas ligand (fasL) was not detectable on antigen-specific CD8 clones, T-cell lines, or circulating HIV-specific CD8 T cells from HIV-infected donors, stained with a tetrameric HLA-A2-HIV-peptide complex. FasL expression by HIV-specific CTL clones was not activated by exposure to HIV-presenting cells, but was after unphysiological stimulation with phorbol myristate acetate (PMA). CTL clones did not lyse bystander Jurkat cells, but HIV-infected primary CD4+ T cells lysed uninfected bystander cells by the fas-mediated pathway. These results suggest that HIV-specific CD8+ CTL do not cause HIV immunopathology by lysing bystander cells. On the contrary, fas-mediated lysis of uninfected cells by HIV-infected cells may contribute to CD4 decline.

Ly49A inhibitory receptors redistribute on natural killer cells during target cell interaction.

When T effector cells meet antigen-bearing target cells, there is a specific accumulation of T-cell receptors, co-receptors and structural proteins at the point of cell-cell contact. Ly49 inhibitory receptors bind to murine major histocompatibility complex (MHC) class I molecules and prevent natural killer-(NK) cell cytotoxicity. In this study we have tested whether inhibitory receptors accumulate at the point of cell-cell contact when NK cells encounter target cells bearing MHC class I ligands for those inhibitory receptors. We have used RNK-16 effector cells that express Ly49A receptors and have found that there was a specific accumulation of Ly49A receptors at the point of NK cell-target cell contact when the target cells expressed H-2Dd. We also observed that engagement of Ly49A on NK cells resulted in an altered redistribution of potential triggering receptors CD2 and NKR-P1. These data indicate that inhibitory receptors, like activating receptors, may specifically aggregate at the point of cell-cell contact which may be necessary for them to mediate their full inhibitory effect.

T-cell help exposed

Cytotoxic T cells (CTLs) recognizing peptide antigens bound to major histocompatibility complex (MHC) class I molecules are involved in the lysis of antigen-bearing target cells. Differentiation from naive CD8+ T cells to CTLs (CTL priming) is thought to often be dependent on help from CD4+ T helper (Th) cells. The nature of the help is to activate dendritic cells (DCs), which are known to be potent antigen-presenting cells, but not to directly activate CTLs (Ridge et al.[ 1 Ridge J.P. et al. A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature. 1998; 393: 474-478 Crossref PubMed Scopus (2025) Google Scholar ]). CD40 ligand expressed on the surface of activated Th cells stimulates DCs via CD40 signalling to increase antigen presentation and co-stimulatory capacity, resulting in CTL priming. This requirement for Th cells to prime CTLs can be replaced by anti-CD40 monoclonal antibody (mAb) (Ridge et al.[ 1 Ridge J.P. et al. A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature. 1998; 393: 474-478 Crossref PubMed Scopus (2025) Google Scholar ]; Bennett et al.[ 2 Bennett S.R.M. et al. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature. 1998; 393: 478-480 Crossref PubMed Scopus (1769) Google Scholar ]; Schoenberger et al.[ 3 Schoenberger S.P. et al. T-cell help for cytotoxic T lymphocytes is mediated by CD40–CD40L interactions. Nature. 1998; 393: 480-483 Crossref PubMed Scopus (2154) Google Scholar ]). In MHC class II-deficient mice, which possess no Th cells, anti-CD40 mAb is capable of helping the induction of CTL responses. Thus, anti-CD40 mAb provides an opportunity for vaccination under circumstances that were previously difficult, either because of a lack of available class II-restricted antigenic determinants (as may occur with some tumour antigens) or because of a deficiency in CD4+ Th cells (as occurs in AIDS patients), as shown in MHC class II-deficient mice.

Help for cytotoxic-T-cell responses is mediated by CD40 signalling.

Cytotoxic T lymphocytes (CTLs) which carry the CD8 antigen recognize antigens that are presented on target cells by the class I major histocompatibility complex. CTLs are responsible for the killing of antigen-bearing target cells, such as virus-infected cells. Although CTL effectors can act alone when killing target cells, their differentiation from naive CD8-positive T cells is often dependent on 'help' from CD4-positive helper T (TH) cells. Furthermore, for effective CTL priming, this help must be provided in a cognate manner, such that both the TH cell and the CTL recognize antigen on the same antigen-presenting cell. One explanation for this requirement is that TH cells are needed to convert the antigen-presenting cell into a cell that is fully competent to prime CTL. Here we show that signalling through CD40 on the antigen-presenting cells can replace the requirement for TH cells, indicating that T-cell 'help', at least for generation of CTLs by cross-priming, is mediated by signalling through CD40 on the antigen-presenting cell.

Antigen binding and cytotoxic properties of a recombinant immunotoxin incorporating the lytic peptide, melittin

Background: The majority of immunotoxins studied to date incorporate toxins that act in the cytosol and thus need to be endocytosed by the target cell. An alternative strategy for immunotoxin development is the use of membrane active toxins, such as the pore-forming proteins. Melittin, a 26 amino acid cytolytic peptide from bee venom, is such a protein. Objectives: We report here the construction, production and functional analysis of a recombinant immunotoxin obtained by fusion of genes which encode an antibody fragment (scFv) with an oligonucleotide encoding melittin. Study design: The antibody fragment was derived from a murine monoclonal antibody, K121, which recognises a specific epitope (KMA) expressed on the surface of human κ myeloma and lymphoma cells, and on human free κ Bence Jones protein (BJP). Melittin is a 26-amino acid, membrane-lytic peptide which is a major component of bee venom. The scFv of K121 was constructed by PCR to link VH and VL genes via an oligonucleotide which encodes a flexible, hydrophilic peptide. An oligonucleotide encoding melittin and the peptide marker sequence FLAG was fused to the scFv construct using a similar linker peptide. The gene construct (scFv-mel) was inserted into the secretion vector pPOW and expressed in Escherichia coli (TOPP2). Results: Expression of the recombinant scFv-mel gene and purification of the protein product was monitored by Western blot analysis. Following purification by anti-FLAG affinity chromatography, the recombinant immunotoxin (scFv-mel) was assessed for antigen binding and for cytotoxic activity by flow cytometry using antigen-expressing and non-expressing cell targets. The scFv-mel was found to exhibit binding and killing properties consistent with the specificity of the original K121 antibody. Moreover, the cytolytic activity of the scFv-mel was significantly greater on a molar basis than that of native melittin alone. Conclusion: The data presented here constitute the first report of a melittin-based recombinant immunotoxin and demonstrate that such a membrane active immunotoxin can be synthesised in a bacterial expression. Linking of melittin to an antibody fragment overcame the non-specific toxicity of melittin as the recombinant immunotoxin exhibited specific toxicity towards antigen-bearing target cells. The observation that the immunotoxin exhibited enhanced cytotoxic activity over the free toxin indicates the potential of this approach for the development of an effective therapeutic agent.

Molecular Interactions in the Activation of Effector and Precursor Cytotoxic T Lymphocytes

Cell-cell interactions are influenced by parameters that cannot readily be studied using either intact cells or soluble molecules. Replacing one of the pair of interacting cells with an artificial cell surface construct allows novel insights to be gained into some of these parameters. Application of this approach to the study of CTL has helped to clarify the contrasting roles of some of the various receptors that are involved in recognition, adhesion and activation. In addition, it has revealed features of these receptor ligand interactions that help to explain how CTL are able to carry out effective immune surveillance and elimination of virus-infected or tumor cells. Although not discussed in this review, artificial cell surface constructs have also been effectively employed to study the interaction of TH cells with class II bearing surfaces. Class I protein and peptide antigen can be sufficient to mediate adhesion and activate CTL effector function through the TCR and CD8. In addition, interactions of other co-receptors with their ligands can act along with TCR and CD8 in a cascade of activated adhesion and co-stimulatory signal generation to allow adhesion and response when antigen and/or class I surface densities are too low to be sufficient by themselves to initiate response. The relative contributions of the various receptor/ligand interactions to a given CTL/target encounter will depend upon the affinity of the TCR for antigen and on the densities and types of ligands, including antigen, displayed on the target cell surface. It appears that the CTL has the ability to accomplish its task in a variety of ways, providing it with considerable flexibility in recognizing and eliminating antigen-bearing target cells. Thus, downregulation of any one particular ligand on a virus-infected or tumor cell does not allow escape from CTL surveillance provided that at least a low level of class I antigen remains present. The CTL is able to employ several co-receptors specific for ligands common to many cell types without being diverted from effective immune surveillance, since these receptors only become activated to mediate high-avidity adhesion when antigen is detected by the TCR. Cloned effector CTL are most amenable to studies of the kind reviewed here, since large numbers of homogenous cells can be obtained, antigen-specific adhesion can be readily measured and response is rapid and easily quantitated.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional heterogeneity of murine intestinal intraepithelial lymphocytes: studies using TCR-αβ + IEL lines and fresh iel isolates reveal multiple cytotoxic subsets differentiated by CD5, CD8α/α, and CD8α/β expression

Three T-cell lines, isolated from murine small intestine epithelia, have been studied with respect to phenotypic properties and cytotoxic activity. All lines were TCR-αβ +, Thy-1 +, CD3 +, CD4 −, CD 8+ but differed in that one line was CD8α/α +, CD5 −; one line was CD8α/α +, CD5 +; and one line was CD8α/β +, CD5 +. Both the CD8α/α +, CD5 −, and the CD8α/β +, CD5 + lines lysed antigen-bearing target cells; however, the latter line also spontaneously lysed natural killer (NK)-sensitive target cells. The CD8α/α +, CD5 + IEL line was nonlytic for antigen-bearing target cells, for NK-sensitive target cells, and in assays that detect lytic activity regardless of specificity. Three-color flow cytometric analyses of intestinal intraepithelial lymphocytes (IEL) in freshly-extracted preparations indicated that cells with the phenotypes of the three IEL lines are normally present in the murine intestine epithelium, and revealed considerable variability in the distribution and density of CD5 expression on murine IEL. In freshly extracted IEL depleted of CD5 or CD8β by cell sorting, cytotoxicity was found to reside both within the CD5 + and the CD5 − IEL subsets, as well as in the CD8β-depleted (i.e., CD8α/α +) subset. These findings demonstrate: that cytotoxicity of murine IEL resides among multiple phenotypic subsets; that the distribution and density of CD5 on IEL is more complex than previously described; and that T-cell lines of IEL origin are valuable for dissecting functional properties of specific IEL subsets, particularly those that constitute a small proportion of the total IEL.

Cytotoxicity of a recombinant ricin-A-chain fusion protein containing a proteolytically-cleavable spacer sequence

Chimeric proteins composed of ricin toxin A chain (RTA) and staphylococcal protein A (PA) have been produced in E. coli. Constructs consisting of N-terminal RTA and C-terminal PA (RTA-PA) or N-terminal PA and C-terminal (PA-RTA) were capable of binding to immunoglobulin G (via PA) and of specifically depurinating 28 S ribosomal RNA (via RTA). However, neither fusion protein was cytotoxic to antigen-bearing target cells in the presence of an appropriate monoclonal antibody presumably because the RTA could not be released from the PA within the cytosol where the ribosomal substrate of RTA is located. The overcome this, a short amino acid sequence from diphtheria toxin was engineered between the RTA and PA to produce a disulfide-linked loop containing a trypsin sensitive cleavage site. Cleavage of this fusion protein with trypsin converted the RTA-DT-PA to the two chain form consisting of RTA linked by a disulfide bond to PA. The cleaved fusion protein was highly toxic to Daudi cells coated with anti-immunoglobulin antibody suggesting that the RTA could be released from the PA by reduction within the cytosol.

T cell receptor-mediated signaling occurs in the absence of inositol phosphate production.

Murine cytotoxic T lymphocytes (CTL) can lyse certain target cells in the presence or absence of extracellular Ca2+ (Ostergaard, H. L., Kane, K. P., Mescher, M. F., and Clark, W. R. (1987) Nature 330, 71-72). We have examined the effect of extracellular Ca2+ on the inositol phosphate response to antigen by CTL. In Ca2+-containing medium relevant antigen-bearing target cells and the mitogen concanavalin A induce a rapid accumulation of inositol phosphates in CTL. In the presence of 4 mM EGTA the antigen- and mitogen-stimulated increases in inositol mono-, bis-, and tris-phosphates cannot be detected, even with 10 mM LiCl added. Abrogation of the inositol phosphate response occurs whether CTL are preincubated in EGTA or EGTA is added with the stimulus. The results indicate that the killing of certain target cells by murine CTL may be independent of the involvement of the phosphatidylinositol pathway. Furthermore, since Ca2+-independent cytolysis remains antigen-specific, the data strongly support the existence of additional T cell receptor-mediated second messenger pathway(s) in CTL.

HIV infection does not require endocytosis of its receptor, CD4

The T cell surface molecule CD4 interacts with class II MHC molecules on the surface of target cells as well as with the envelope glycoprotein of human immunodeficiency virus (HIV). Internalization of CD4 molecules is observed after exposure of CD4+ T cells to either phorbol esters or appropriate antigen-bearing target cells. To determine whether HIV entry proceeds via receptor-mediated endocytosis or direct viral fusion with the cell membrane, we have constructed two mutants in the cytoplasmic domain of the CD4 protein that severely impair the ability of CD4 molecules to undergo endocytosis. Quantitative infectivity studies reveal that HeLa cell lines expressing wild-type or mutant CD4 molecules are equally susceptible to HIV infection. In addition, HIV binding does not lead to CD4 endocytosis. These studies indicate that although the CD4 molecule can be internalized, HIV entry proceeds via direct fusion of the viral envelope with the cell membrane.

A secretable serine proteinase with highly restricted specificity from cytolytic T lymphocytes inactivates retrovirus-associated reverse transcriptase

TSP-1, a murine T cell specific proteinase, is expressed in cytolytic T lymphocytes and secreted upon their interaction with antigen bearing target cells. In searching for possible extracellular substrates of the enzyme in the physiological environment of cytolytic effector cells, we have investigated the proteolytic activity of TSP-1 on retroviral proteins. It is shown that reverse transcriptase derived from the retrovirus Moloney murine leukemia virus is inactivated by TSP-1 via limited proteolysis. The data suggest the possibility that cytolytic T lymphocytes are able to interfere with retroviral replication by secreting a serine proteinase which degrades viral proteins.

Antigen receptor-triggered secretion of a trypsin-type esterase from cytotoxic T lymphocytes.

Exocytosis of cytolysin-containing granules from cytotoxic T lymphocytes (CTL) was studied with the use of granule enzyme (BLT esterase) as a convenient biochemical marker. Using cloned CTL, we demonstrate here that BLT esterase secretion into the supernatant is specifically triggered by antigen-bearing target cells and that this secretion is inhibited by soluble monoclonal antibodies against the antigen-specific T cell receptor (TcR). Immobilized anti-receptor antibodies induced efficient enzyme secretion in the absence of target cells, thus implying a direct involvement of TcR complex in triggering exocytosis of granules. These results support the role of the granule exocytosis in CTL functions and provide a quantitative and direct assay of a rapid CTL functional response to antigenic stimulation.

Measurement of tumour reactive antibody and antibody conjugate by competition, quantitated by flow cytofluorimetry

Binding of unlabelled monoclonal antibody preparations has been assessed by cometition at saturation with fluorochrome labelled homologous antibody for binding to antigen bearing target cells. The extent of competition was measured by quantitative flow cytofluorimetry, and simple mathematical procedures have been developed to allow the interpretation of competition data in terms of antibody binding activity. In the system studied, non-specific (non-competitive) fluorescence was minimal, but an iterative method to calculate its contribution to the measured signal is given. This approach has the advantage that the antibody preparation to be tested does not need to be labelled or modified; this is particularly important when evaluating the binding activity of therapeutic antibody conjugates. Comparison with a well characterized standard antibody preparation provides a rapid, sensitive and accurate quality control procedure. This test is also simple to perform, requiring only the mixing of labelled and unlabelled antibodies with target cells, a single incubation, followed by analysis without washing of the target cells.