Characterization of the increased cytotoxicity of gelonin anti-T cell immunoconjugates compared with ricin A chain immunoconjugates.

Ribosomal inactivating proteins such as gelonin (Gel) and ricin A chain (RTA) conjugated to MoAbs bind to specific target cells, and upon internalization inhibit protein synthesis, ultimately resulting in cell death. We report here that Gel anti-T cell MoAb conjugates are more cytotoxic than RTA conjugates when tested against human peripheral blood mononuclear cells (PBMC). This increased cytotoxicity is observed whether Gel is conjugated to the anti-T cell MoAb or to an anti-mouse immunoglobulin Fab' fragment which then binds to the murine anti-human T cell MoAb. Gel conjugates are not only effective at lower concentrations, but also produce a greater extent of inhibition of cellular proliferation. Moreover, a 10 min exposure to a Gel conjugate is as effective as a 90 h exposure to an RTA conjugate. When part of anti-T cell F(ab')2 or Fab' conjugates, Gel affects the early steps in cellular intoxication more than RTA; Gel conjugates bind more avidly and accelerate the modulation of antigen. In contrast, when part of whole IgG conjugates, Gel does not affect the binding to or modulation of surface antigen compared with RTA, while it does increase conjugate cytotoxicity. These observations suggest that Gel may be delivered more efficiently into the cytosol than RTA. A divergent intracellular pathway for Gel is also supported by the inability of chemical potentiators, which strongly enhance RTA potency, to affect Gel potency. These properties of Gel might also be advantageous for immunoconjugates made with other MoAbs or receptor-binding molecules.     

Different Susceptibilities of Normal T Cells and T Cell Lines to Immunotoxins

In the context of ex vivo T cell elimination from bone marrow, the anti-T cell cytotoxic potential of immunotoxins (IT) prepared by conjugation of die monoclonal antibodies (MOAb) WT32 (CD3), T101 (CD5), and WT1 (CD7) to ricin A chaw was evaluated. The cytotoxicity of IT was based on protein synthesis inhibition in human T cell lines: GHI, CEM, HPB-ALL, and Jurkat, and appeared closely related to the antigen density and internalization rate of the IT. Normal unstimulated T cells appeared to he rather insensitive to IT not due to a low antigen density or decreased internalization. The cytotoxicity of IT to T cells could he enhanced considerably by NH₄Cl. Treatment of T cells with a cocktail of IT (10⁻⁸ m ) and 20 m m NH₄Cl resulted in a 5000-fold cytoreduction as measured by clonogenic assays of limiting T cell dilutions, whereas the haematopoietic progenitor cells remained unaltered. Stimulation of T cells with phytobaemag-glutinin (PHA) prior to incubation with IT considerably increased the sensitivity to IT treatment. Thus, normal T cells are less sensitive to anti-T cell IT than T cell lines and activated T cells. This suggests that a low protein synthesis is responsible for the resistance to IT. However, a high specific cytotoxicity of IT to normal T cells can be achieved in the presence of 20 m m ammonium chloride.     

Human T Lymphocyte Differentiation Antigens as Target for Immunotoxins or Complement-Mediated Cytotoxicity

Graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT) is initiated by immunocompetent T cells present in the graft. Selective elimination of distinct T-cell subsets or a sufficient, but not complete T-cell depletion, might abolish severe GVHD without graft rejection and loss of the anti-tumour potential. In this study we analysed the efficacy of different monoclonal antibodies (MoAb) WT32 (CD3), OKT4 (CD4), T101 (CD5), WT1 (CD7), and WT82 (CD8) with respect to their cytotoxicity to T cells either as immunotoxin (IT) or in combination with complement. The cytotoxic potential was assessed by protein synthesis inhibition and clonogenic assays. The ricin A conjugated MoAb exerted only a minor effect on blood or bone marrow T cells, although they were highly inhibitory to T-cell lines. However, in the presence of 20 mM ammonium chloride, IT directed against CD3, CD5, and CD7 were highly cytotoxic. IT directed against CD4 and CD8 were less effective, due to a low internalization. The complement-mediated cytotoxicity was efficient for all antigens used. The natural killer (NK) activity, as measured by cytotoxicity to K562, was hardly depressed by anti-CD3, anti-CD4, anti-CD5, and anti-CD8, but was eliminated by anti-CD7. All procedures used had only a minimal effect on haematopoietic progenitors as measured by CFU-GM and BFU-E assays. We concluded that, although the T-cell population can be eliminated with the combination of anti-CD3, anti-CD5, and anti-CD7 antibodies plus complement, IT with 20 mM NH4Cl appear to kill higher amounts of T cells. Selective elimination of CD4- and CD8-positive cells is effectively obtained by MoAb with complement.     

Cytolytic activity in T cell clones derived from human synovial rheumatoid membrane: inhibition by synovial fluid.

A panel of T cell clones was derived from the synovial membrane of a patient with rheumatoid arthritis (RA). We investigated whether T cell clones with cytolytic properties were present and whether T cell cytotoxicity was influenced by the presence of synovial fluid. These issues were studied using anti-CD3 and lectin-induced cytotoxicity assays. The majority of the T cell clones derived from the synovial membrane showed cytotoxic properties although non-cytotoxic clones were also found. Three clones (N11, N6 and N15) showed strong cytotoxicity (more than 40% lysis at an effector-to-target cell ratio of 10:1) whereas three clones (N16, N4 and N14) were non-cytotoxic (less than 20% lysis at an effector-to-target cell ratio of 10:1). The induction of cytotoxicity in the anti-CD3-driven system was shown to be dependent on the dose of anti-CD3 present. When synovial fluid was added to these assays a strong inhibition of cytotoxicity was found. This inhibition of cytotoxicity was found with synovial fluid samples of RA patients, as well as with non-RA synovial fluids. Both anti-CD3 and lectin-dependent cytotoxicity assays were strongly inhibited. In conclusion, T cell clones with cytotoxic activity can be isolated from rheumatoid synovial membrane. In the presence of synovial fluid these cytotoxic cells are inhibited to exert their cytotoxic function.     

Involvement of CD27/CD70 interactions in antigen-specific cytotoxic T-lymphocyte (CTL) activity by perforin-mediated cytotoxicity

CD27 molecules are shown to be essential in the regulation of the death, activation and differentiation of T and B cells. However, the influence of CD27 on cytotoxic T-cell function remains obscure. Autologous EBV transformed B-cell lines (LCL), which highly express CD27 ligand CD70, here stimulated T cells and induced the cytotoxic T-lymphocyte (CTL) activity via T-cell antigen receptors (TCR). The cytotoxicity against LCL was diminished when anti-CD70 blocking MoAb was added initially in the culture. Resting T cells killed more CD70-transfected P815 cells than wild type P815 cells in the presence of anti-CD3 MoAb as measured by a 4-h 51Cr release assay, and the cytotoxicity of both of the cell populations completely disappeared in the presence of concanamycin A (CMA). The expression of the perforin by the LCL-induced CTL in the presence of anti-CD70 blocking MoAb was diminished as compared with that without the blockage of CD27/CD70 interactions. The CTL induced by LCL did not kill Fas-transfected WR cells. CD27 signalling in the T cells did not affect Fas ligand (FasL) mRNA expression, LAK activity and IFN-gamma synthesis in humans. Our data demonstrate that CD27/CD70 interactions enhance the cytotoxicity of CTL in the induction phase through enhancement of killing activity induced via the perforin-dependent mechanism, but not via the Fas/FasL system.     

Natural cytotoxic cell activity in the snake Psammophis sibilans.

Thymocytes, splenocytes and peripheral blood mononuclear cells (PBMC) of the snake Psammophis sibilans consistently killed the human erythroleukemic cells K562 in a 4 h assay as judged by lactate dehydrogenase enzyme release. PBMC and splenocyte natural cytotoxicity (NC) increased proportionally with increase in the effector/target cell ratio. Spontaneous killer cell activity was consistently 2-3 times higher in peripheral blood (PB) than in spleen. On the other hand, thymocytes displayed low, yet detectable, NC. In an attempt to define the cell subpopulation responsible for natural killer (NK) activity, PBMC were depleted of macrophages or B lymphocytes before use in NK cell assays against K562 cells. Depletion of macrophages did not impair NK activity thus suggesting that macrophages do not mediate spontaneous lysis in the present 4 h assay. Conversely, removal of B lymphocytes by panning onto dishes coated with monoclonal antibody against snake Ig significantly reduced, but did not eliminate, PBMC spontaneous cytotoxicity. These data suggest that T, B and perhaps distinct NK cells participate in spontaneous lysis. This suggestion was confirmed by studies of NC in thymus, spleen and PB the year round. Strong NC was detected during spring and autumn when high numbers of leukocytes including T and B cells can be recovered from spleen and PB. Negligible spontaneous cytotoxicity was observed during early and mid-summer and in winter, periods of the year when snakes are thymus-less and contain few T and B cells in peripheral lymphoid organs. These findings, the first to document natural cytotoxic activity in snakes, were discussed in relation to the issue of NK cell identity in vertebrates.     

ADCC against human erythrocyte target cells: role of the anti-target cell antibodies in determining lymphocyte killer activity.

Studies were undertaken to investigate the role of anti-target cell antibodies in determining whether lymphocytes can mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro. Trinitrophenyl (TNP) modified Chang liver cells and human erythrocytes were employed as target cells and were coated with xenogeneic and allogeneic antibodies against TNP and natural cell surface antigens. Two cytotoxic effector cell populations were used: human peripheral blood mononuclear cells (PBMC) containing both lymphocytes and monocytes, and monocyte-depleted peripheral blood lymphocytes (PBL). With Chang targets, both PBMC and PBL mediated ADCC with xenogeneic anti-Chang and xenogeneic anti-TNP sera. With human erythrocyte targets, PBMC but not PBL mediated ADCC with human anti-blood group B serum, while both PBMC and PBL mediated ADCC with xenogeneic anti-TNP sera and also with a human anti-CD serum. These results demonstrate that the source of anti-target cell antibodies employed in ADCC reactions may determine whether or not lymphocytes are capable of mediating cytotoxicity.     

LAK-cell-mediated cytotoxicity against tumor cell targets used to monitor the stimulatory effect of interleukin-2: cytotoxicity, target recognition and phenotype of effector cells lysing the Daudi, T24 and K562 tumor cell lines.

The T24 transitional bladder carcinoma cell line, the Daudi Burkitt lymphoma cell line and the K562 erythroleukemia cell line have all been used as target cells in 51Cr release assays to measure the in vivo induced lymphokine-activated killer (LAK) cell cytotoxicity during interleukin-2 (IL-2) therapy of cancer patients. However, different relationships between the clinical response to IL-2 treatment and the LAK cytotoxicity have been reported using these three different target cells. The purpose of the present study was to evaluate whether the LAK cytotoxicities measured against these target cells represent similar effector-to-target-cell interactions, so similar conclusions may be drawn of 51Cr release assay results in which the cell lines are used as target cells. The cytotoxicity of peripheral blood mononuclear cells (PBMC) and PBMC depleted of different natural killer and T cell subsets was measured against the three targets. LAK cell recognition of targets was evaluated by cold target inhibition experiments, and the development of LAK-cell-mediated lysis with time was evaluated in 51Cr release assays of varying duration. This study shows that LAK-mediated lysis of T24 and Daudi cells was closely related and LAK cytotoxicity measured in 51Cr release assays against these two target cells may be measurement of similar effector-to-target cell interactions.     

Selective T cell subset depletion with anti-CD4 and anti-CD8 intact ricin immunotoxins.

In the present study we have comparatively analysed the specific activity of a panel of immunotoxins (ITs) prepared by coupling Ricin to several monoclonal antibodies (MoAbs) directed against the CD3, CD4 and CD8 T cell membrane molecules. Peripheral blood and bone marrow mononuclear cells (PBMC and BMMC) were incubated with the different ITs for 2 h, in the presence of 0.1 M lactose, washed and subsequently stimulated with either phytohemagglutinin (PHA) or an anti-CD3 MoAb. Our results indicate that the proliferative response of PBMC to both stimuli was specifically inhibited (greater than 95%) by either anti-CD3 IT or a combination of anti-CD4 and CD8 ITs, at concentrations comparable to those previously used for ex vivo T cell depletion (300 ng/ml). When used individually at the same dose, anti-CD8 and anti-CD4 ITs inhibited the PHA-induced PBMC proliferative response 40 and 70% respectively. When either anti-CD4 or anti-CD8 IT-treated cells were activated with PHA and cultured for 14 days in the presence of IL-2, less than 2% of the blasts expressed the corresponding antigens as assessed by flow cytometry analysis. Similar results were observed when BMMC were treated with the different ITs. In contrast, the growth of CFU-GM was minimally affected (0-25% inhibition). Our data indicate that ITs directed against T cell subsets are highly active and specific reagents that may be potentially useful for pre-transplant bone marrow purging.     

Impaired cytolytic activity in peripheral blood T cells from renal cell carcinoma patients

Classic T lymphocyte cytotoxicity is mediated through the T cell receptor (TCR). Defects in TCR signal transduction and cytolytic activity have been reported in tumor infiltrating T lymphocytes. We hypothesized that impaired cytotoxicity occurs in peripheral blood T cells from renal cell carcinoma (RCC) that can be reversed by exposure to rhIL-2. Peripheral blood mononuclear cells (PBMC) from 29 RCC patients and 29 healthy volunteers were isolated and cultured in the absence or presence of 10 IU/ml rhIL-2. A redirected cytotoxicity assay that requires TCR signal transduction was used with chromium-labeled P815 target cells, effector PBMC and anti-CD3 antibody. Target cell lysis was measured in "lytic units" (LU). Mean LU from RCC patients was lower than that of healthy volunteers (105.8 LU vs. 194.6 LU, P = 0.025). Exposure to rhIL-2 increased T cell-mediated lysis in both groups. Disruption of T cell cytotoxicity in RCC patients can be overcome by exposure to rhIL-2.     

Defective induction of T-cell help and natural killing following anti-CD3 stimulation of autoimmune lymphocytes

Anti-CD3 monoclonal antibody (MoAb) stimulates T cells in normal peripheral blood to proliferate and develop cytotoxic activity against NK-sensitive tumor cell lines. We now find that anti-CD3 MoAb also generates cytotoxic activity against a cell line (MEL-21) resistant to classical NK cell killing. After activation in vitro with anti-CD3 MoAb for 18 h, normal peripheral blood mononuclear cells (PBMNC) develop more HLA-DR-positive helper than suppressor T cells, manifest a functional helper effect as measured by increased IgG synthesis (P less than 0.01), as well as kill MEL-21 target cells. PBMNC from rheumatoid arthritis (RA) patients respond normally but mononuclear cells from rheumatoid arthritis synovial fluid (RASF) respond poorly. PBMNC from systemic lupus erythematosus (SLE) patients also respond poorly to anti-CD3 stimulation. Thus, the ability of anti-CD3 to stimulate IgG production and generate enhanced natural cytolytic activity are defective in both RASF and SLE lymphocytes.     

Organotypic culture of HPV-transformed keratinocytes: a model for testing lymphocyte infiltration of (pre)neoplastic lesions of the uterine cervix

 The aim of our study was to establish the relevance of an in vitro model for analysing the ability of human lymphocytes to infiltrate human papillomavirus (HPV)-associated (pre)neoplastic lesions of the uterine cervix. To mimic these lesions, we have used the organotypic raft culture of HPV-transformed keratinocytes (SiHa). The SiHa organotypic raft culture was co-cultured with resting or prestimulated (IL-2 or IL-2+anti-CD3 mAb) allogeneic peripheral blood mononuclear cells (PBMC) for 24 and 72 h. The majority of infiltrating cells were T lymphocytes. Occasional NK cells were also identified. The stimulation with IL-2+anti-CD3 mAb induced the highest number of infiltrating cells, with the maximum lymphocyte infiltration observed after 24 h of co-culture. The lymphocyte infiltration was associated with an increased number of apoptotic cells in the organotypic cultures. The ability of PBMC and purified T cell and NK cell populations to lyse HPV-transformed keratinocytes was also investigated on monolayer cultures. As expected in an allogenic model, the highest cytotoxicity was mediated by NK cells activated by IL-2 or IL-2+anti-CD3 mAb. The cytotoxic activity of T cells was weak but, interestingly, increased in the presence of phytohaemagglutinin A (PHA), assuming that T cells were able to kill HPV-infected keratinocytes when a bridge between T cells and keratinocytes was provided. In conclusion, the organotypic culture of HPV-transformed keratinocytes may provide an effective in vitro model for investigating novel T cell-based immunotherapy protocols for the treatment of HPV-associated lesions. Received: 1 September 1997 / Accepted: 9 December 1997     

Endothelial cell lysis induced by lymphokine-activated human peripheral blood mononuclear cells

In vitro exposure of human peripheral blood mononuclear cells (PBMC) to interleukin 2 results in the generation of lymphokine-activated killer (LAK) cells. Such LAK cells exhibit cytotoxicity against a spectrum of tumor target cell lines whereas they apparently do not affect normal tissues. In this report we show that PBMC that have been activated with T cell growth factor lyse trypsinized human umbilical cord venous endothelial cells as well as endothelial cell monolayers in a dose-dependent manner. Microscopic analysis showed that during the 4-h incubation period cell clumps containing detached endothelial cells and LAK cells were formed. When these clumps were evaluated with trypan blue the endothelial cells stained positive whereas LAK cells excluded the dye. No lysis occurred when fresh PBMC were added to target endothelial cells. The endothelial cell kill could not be blocked with an anti-LFA-1 antibody nor with intact OKT3 or F(ab')2 fragments of WT32. We conclude that lymphokine-activated PBMC exhibit cell-mediated endothelial cell detachment and lysis.     

Activation of human T cells with NK cell markers by staphylococcal enterotoxin A via IL-12 but not via IL-18

We have reported recently that mouse liver NK cells and NK1 x 1+ T cells were activated by bacterial superantigens via the IL-12 production from Kupffer cells. In the present study, we examined the effect of staphyloccoccal enterotoxin A (SEA) on human T cells with NK cell markers, CD56 or CD57 (NK-type T cells). After stimulating peripheral blood mononuclear cells (PBMC) with SEA, PBMC produced a large amount of IFN- and acquired a potent antitumour cytotoxicity. The in vitro depletion of either CD56+ TCR NK cells, CD56+ T cells or 57+ T cells from PBMC significantly inhibited the IFN- production from PBMC. When purified NK-type T cells, NK cells and regular T cells were cultured with monocytes and SEA they all produced IFN-, while the IFN- amounts produced by both NK-type T cells were greater than those produced by NK cells. NK cells as well as CD56+ T cells showed cytotoxicity against NK-sensitive K562 cells, whereas both NK-type T cells showed a more potent cytotoxicity against NK-resistant Raji cells than did NK cells. The IFN- production from each population as well as from whole PBMC was greatly inhibited by anti-IL-12 antibody but not by anti-IL-18 antibody. The antitumour cytotoxicity of whole PBMC was also significantly inhibited by anti-IL-12 antibody while the SEA-induced proliferation of PBMC was not affected by anti-IL-12 antibody. Furthermore, SEA-activated NK-type T cells as well as NK cells showed cytotoxicities against vascular endothelial cells. Our findings suggest that human NK-type T cells are thus involved in bacterial superantigen-induced immune response.     

Activation of human T lymphocytes. III. Triggering of bystander cytotoxicity in cytotoxic T cell clones by antibodies against the T3 antigen or by a calcium ionophore

The role of T cell differentiation antigens in antigen-specific and nonspecific cytotoxicity by human cytotoxic T lymphocyte (CTL) clones was investigated. In contrast to other reports, several monoclonal antibodies (mAb) against the T3 antigen only marginally blocked antigen-specific cytotoxicity at high concentrations but induced cytotoxicity against third party cells at concentrations from 10 to 0.001 micrograms/ml. Susceptibility to anti-T3-induced lysis was variable but was found with all target cells. Incubation of CTL with anti-T3 mAb even led to self-destruction of the CTL. The effect was independent of the presence of Fc receptors on the target cell and could be obtained with F(ab')2 fragments of the antibody as well. Only activated but not resting T cells could be induced to lyse by anti-T3. Furthermore, this type of bystander killing of target cells could also be induced by the Ca2+ ionophore A23187. Antibodies against the T8 differentiation antigen inhibited antigen-specific, oxidation-induced and anti-T3-induced cytotoxicity by T8+ CTL clones, whereas triggering by the ionophore A23187 was not inhibited. These results show that undirected killing can be triggered in CTL by activating a transducing molecule directly without involving the antigen receptor. Since this triggering of the lethal hit can still be inhibited by mAb against the T8 molecule, the T8 molecule probably has a regulatory role in a late phase of CTL triggering.     

Evidence for a regulatory role of the T8 (CD8) antigen in antigen-specific and anti-T3-(CD3)-induced lytic activity of allospecific cytotoxic T lymphocyte clones

The functional role of the T8 antigen of human T cells was studied by inhibition with anti-T8 monoclonal antibodies (mAb) of the cytotoxic action of T8+ cytotoxic T lymphocyte clones (CTL). All clones were allospecific and directed against HLA-B7. The ability of seven different anti-T8 mAb to inhibit the cytotoxicity of these alloreactive CTL clones corresponded with their avidity for a particular target cell. The lysis of cross-reactive antigen-bearing target cells was more readily blocked by anti-T8 mAb than lysis of the specific B7 target cell against which a clone was raised. The seven anti-T8 mAb showed a spectrum of CTL blocking ability ranging from strong blocking with all five CTL clones tested to weak inhibition of only two out of five clones. mAb inhibition of CTL reactivity and cold target inhibition studies with one of the five CTL clones indicate a post-binding role of the T8 molecule. Functional epitope mapping based on CTL blocking with the anti-T8 mAb resulted in the definition of one nonfunctional epitope on the T8 molecule which is only expressed on mature T lymphocytes and a cluster of closely related functional epitopes expressed on both thymocytes and mature T lymphocytes. Not only allospecific cytotoxicity, but also nonspecific cytotoxicity induced anti-T3 mAb in these allospecific clones was inhibited by anti-T8 mAb in the absence of HLA class I expression on the target cell (Daudi cell line). The hierarchy of blocking with anti-T8 mAb and the classification of functional epitopes on T8 in anti-T3-induced nonspecific cytotoxicity were similar to those obtained in blocking of allospecific reactivity of the CTL clones. This analogy points to an identical function of the T8 antigen in both allospecific and anti-T3-induced nonspecific cytotoxicity. If HLA class I molecules are the counter structures of the T8 antigen, then these results argue against an adhesion-like function of the T8 structure. The combined results show that the T8 molecule has a regulatory role in CTL activation. It is postulated that the T8 antigen might serve as a receptor that transduces a negative feedback signal for T cell activation which prevents T cell triggering by nonspecific interaction.     

Anomalous reactivity of anti-T cell sera on spleen cells from T-cell-deprived mice and on mitogen-stimulated nude mice spleen cells

Recent results have shown that various alloantisera which react in the expected fashion on lymphocyte populations can react anomalously against cultured tumor cell populations because of the presence of contaminating antibodies against murine leukemia virus (MLV) antigens. Since it is now known that activation of MLV can occur in certain types of dividing lymphocyte populations, anti-T cell sera were tested on lymphoid cell populations in which T cells were absent or greatly reduced in numbers, but where activation of MLV in the B cell population would be expected. Whereas normal, freshly harvested, nude mice spleen cells were unreactive, in vitro:stimulation of these cells with the B cell mitogen, lipopolysaccharide, led to a high degree of sensitivity to the cytotoxic effects of anti-T alloantisera or heteroantisera. Spleen cells from adult thymectomized, lethally-irradiated, bone marrow-reconstituted mice also showed unexpected reactivity with the anti-T cell sera. In both cases, reactivity was noted only if absorbed rabbit serum was used as a complement source in the cytotoxic assays. The anomalous anti-B cell activity of the anti-T cell sera could be removed by absorption with relatively small numbers of cells from Thy-negative cultured tumor cell lines, including fibrosarcomas, but not by absorption with thymocytes. Hence, activated or stimulated B cells may react strongly with allo-or hetero- anti-T cell sera under certain conditions, and this anomalous reactivity appears unrelated to the presence of the anti-T cell antibodies in the sera.     

Human CD8+ cytotoxic T cell responses to adenovirus capsid proteins

Adenoviruses (Ads) cause fatal disease in allogeneic stem cell transplant recipients, but there is no established therapy. Ad-specific CD8+ T cells were detected in PBMC from healthy adults at a mean frequency of 77 per 10(5) CD8+ T cells (range 8-260) by interferon-gamma ELISPOT and cytokine flow cytometry assays. CD8+ T cell lines from 7 of 7 donors exhibited MHC-class-I-restricted killing of targets expressing the capsid protein hexon. In contrast, cytotoxicity against the capsid proteins fiber and penton base was weaker or not detected. Two HLA-A2-restricted hexon epitopes and one HLA-B-restricted epitope were identified, all of which are adjacent to or overlap an HLA-DP4-restricted epitope in the highly conserved C-terminus. Thus, hexon is the immunodominant T cell target among capsid proteins and contains multiple C-terminal epitopes conserved among serotypes. These data support evaluation of donor lymphocyte infusions for treatment of Ad disease post-transplant.     

Dengue virus-induced human cytotoxic factor: production by peripheral blood leucocytes in vitro.

During dengue type 2 virus (DV) infection of mice a unique cytokine, the cytotoxic factor (CF), is produced which reproduces the pathological lesions seen in patients of dengue haemorrhagic fever (DHF). Recently we have observed a CF-like protein in the sera of DHF cases. The present study was undertaken to investigate whether DV can stimulate human peripheral blood mononuclear cells (PBMC) in vitro to produce human CF (hCF). Cultures prepared from PBMC or its enriched subpopulations were inoculated with 1000 LD50 of DV and controls with normal mouse brain suspension (NMB). Aliquots of cultures were harvested daily from 24 h to 96 h and their supernatant (CS) and cells were separated. CS were screened for viral titres and for the presence of hCF by cytotoxicity assay, inhibition ELISA, dot blot and Western blot tests using anti-mouse-CF antibodies. The RNA from the cells was screened in Northern blot and dot blot tests for the presence of mRNA for CF. It was observed that hCF appeared in the CS of DV-infected culture of PBMC and T-enriched cells at 48 h and was present until 96 h; no CF was detected in CS of B cells or monocyte cultures. The production of hCF was abrogated by pretreatment of the T cells with anti-CD4 antibodies but not with anti-CD8 antibodies, indicating that hCF was produced by CD4+ T cells. The Northern blot analysis using oligonucleotide probes prepared on the basis of amino-terminal sequence of mouse CF, showed presence of mRNA for hCF in PBMC and T cell cultures. DV replicated in PBMC cultures with peak titres at 48 h. The findings of the present study demonstrate that DV-induced hCF is produced by human T cells.     

Aggregation of MHC class I molecules on a CD8+ alphabeta T cell clone specifically inhibits non-antigen-specific lysis of target cells

MHC class I molecules are target molecules recognized by TCR or NK receptors encoded in the NK gene cluster or leukocyte receptor cluster. We show that aggregation of MHC class I molecules by specific monoclonal antibodies on cytotoxic T cells, inhibits the anti-CD94 redirected lysis of P815. This inhibition is not the consequence of apoptosis or anergy of the cytotoxic T lymphocytes. In contrast, aggregation of MHC class I molecules does not inhibit either the anti-CD3 redirected cytotoxicity or the CD94-triggered up-regulation of CD25 molecules of the same T cell clone. MHC class I ligand molecules expressed by antigen presenting cells and/or T lymphocytes could therefore be able to modulate nonspecific cytotoxicity upon interaction with MHC class I molecules expressed by effector cytotoxic T lymphocytes.