Molecular interactions in human T-cell-mediated cytotoxicity to Epstein-Barr virus. I. Blocking of effector cell function by monoclonal antibody OKT3

The ability of different anti-human T-cell lymphocyte monoclonal antibodies to inhibit the effector function of the cytotoxic T-cell response against autologous Epstein-Barr virus (EBV)-infected B-cell targets has been tested. It was found that monoclonal antibody, OKT3, which reacts with most human T cells, blocks the effector cell function in the absence of complement, an effect that was dose dependent. When monoclonal antibody OKT3 was tested at a concentration of 1 μg/ml, inhibition of cytotoxicity ranged between 50 and 80%. The F(ab′)₂ fragment of OKT3 inhibited as well as the intact IgG molecule, indicating that the Fc portion of the antibody is not necessary for the cytotoxicity blocking. The Fab fragment of OKT3 had lower blocking activity per microgram of protein tested. Antibodies SC1, OKT11 (anti-pan T cell), OKT8 (anti-cytotoxic/suppressor subset), and L368 (anti-HLA) did not have any discernible blocking effects. However, antibodies SC1, OKT8, and L368 could abrogate the cytotoxic activity in the presence of complement. Blocking by OKT3 was not due to its being present on the cell surface in higher concentrations than the other monoclonal antibodies since cytofluorographic analysis demonstrated that the amount of OKT8 or L368 antibodies bound on the cells was greater than OKT3. In addition, blocking was not due to antigenic modulation since incubation with antibody OKT3-F(ab′)₂ was not associated with a significant decrease in the amount of its reactive antigen. Under the conditions tested OKT3 did not affect cell viability or cause agglutination.     

K562 killing by K, IL 2-responsive NK, and T cells involves different effector cell post-binding trigger mechanisms

The monoclonal antibody 13.3 specifically blocks the trigger process of the NK-K562 cytolytic sequence at a post-binding effector cell level. This antibody was used to define differences in the lytic trigger processes of NK and other mechanisms of K562 lysis. Monoclonal antibody 13.3 inhibited lysis of K562 target cells by freshly isolated peripheral blood lymphocytes (PBL) and purified large granular lymphocytes (LGL), but had no inhibitory effect on antibody-dependent cell-mediated cytotoxicity to K562 by these effectors. Lectin-dependent cellular cytotoxicity (LDCC) to this target cell was also unresponsive to 13.3. The 13.3-induced inhibition of NK-K562 lytic activity persisted when PBL were activated in culture with interleukin 2 (IL 2) for periods up to 48 hr. After 48 hr of culture, the degree of inhibition diminished progressively in medium containing fetal calf serum but not in medium containing autologous serum. This 13.3-unresponsive lytic activity in cultured PBL could be attributed to more than one cell type and was present in both the LGL and Fc gamma receptor-depleted T cell fraction. Thus, K562 lysis by freshly isolated human lymphocytes via NK, K, and LDCC mechanisms is characterized by heterogeneity of the post-binding effector cell trigger mechanism. K562 lysis by lymphocytes cultured with IL 2 is similarly heterogeneous.     

Higher cytolytic efficiency of an IgG2 alpha than of an IgG1 monoclonal antibody reacting with the same (or spatially close) determinant on a human high-molecular-weight melanoma-associated antigen

Serological and immunochemical assays showed that the monoclonal antibody (MoAb) 225.28S, an IgG_2α, and the MoAb 653.40S, an IgG₁, react with the same (or spatially close) antigenic determinant expressed on a set of molecules carrying a high-molecular-weight human melanoma-associated antigen. Neither monoclonal antibody mediates complement-dependent lysis of cultured melanoma cells, but both of them specifically mediate lysis of target cells in an antiglobulin cytotoxic assay and in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay. In the latter two assays the IgG_2α displays a higher lytic activity than the IgG₁. The differential lytic activity of the IgG_2α and IgG₁ monoclonal antibodies was detected also when the sensitivity of the ADCC assay was increased either by boosting the cytolytic activity of the effector cells or by enhancing the susceptibility to lysis of target cells.     

Two distinct cytotoxic T lymphocyte subpopulations in patients with Vogt-Koyanagi-Harada disease that recognize human melanoma cells

The functional properties of cytotoxic lymphocytes from patients with Vogt-Koyanagi-Harada disease ( VKH ) specific for human melanoma cells (P-36 melanoma cell line established from a patient with malignant melanoma) were investigated by using monoclonal antibodies specific for human T cell subsets. Peripheral blood lymphocytes (PBL) from patients with VKH showed significant cytotoxic activity against the P-36 (SK-MEL-28) human melanoma cell line, but not against a human cervical carcinoma of the uterus cell line (HeLa-S3 cell line) or against a mouse melanoma cell line (B-16 cell line) originating from a C57BL/6 strain mouse or against the EL-4 mouse lymphoma cell line from a C57BL/6 mouse. The cytotoxic activity of the patients' PBL against the P-36 melanoma cell line was markedly reduced by pretreatment of the PBL with monoclonal anti-human Leu-1 antibody plus rabbit complement, but it was reduced to much less extent by pretreatment with either monoclonal anti-human Leu-2a or Leu-3a antibody plus rabbit complement. The specific cytotoxic activity of the patients' PBL against the P-36 human melanoma cell line is, therefore, mediated by T cells bearing Leu-1+ Leu-2a+ or Leu-1+ Leu-3a+ antigens. Furthermore, the cytotoxic activity was shown to be blocked not only by anti-Leu-2a antibody specific to human cytotoxic/suppressor T cells but also unexpectedly by anti-Leu-3a antibody which has previously been considered to be specific to human inducer/helper T cells. The results of this study suggest that at least two distinct subpopulations of cytotoxic T cells specific for P-36 human melanoma cells are present in the peripheral blood of VKH patients. These cytotoxic T cells have different surface antigens, Leu-2a and Leu-3a.     

Activation of autoreactive cytolytic T lymphocyte clone against human melanoma by anti-T3 monoclonal antibody and autologous accessory cells

A cytotoxic T-lymphocyte (CTL) clone Tc1.8 was derived in a limiting dilution culture from a single cell that was derived from melanoma-involved lymph node lymphocytes activated in in vitro coculture against the autologous melanoma cells (VIP). The clone Tc1.8 (T3+, T8+, T4-, and Leu7-) expressed restricted cytolytic activity against only the autologous target VIP. As it aged in continuous culture containing interleukin 2, Tc1.8 lost cytolytic activity. The cytolytic function could be restored, however, with monoclonal antibody (MoAb) against T3 (OKT3) or with F(ab')2 fractions of OKT3, and upon restimulation with irradiated accessory cells. OKT3-mediated reinduction of cytotoxicity by the aged Tc1.8 could not be achieved if the T3 molecules were modulated from the effector cell surface following overnight incubation of Tc1.8 with saturating concentrations of OKT3 MoAb. Following reactivation with OKT3 Tc1.8 gained cytolytic function against NK targets in addition to VIP. Reactivation with F(ab')2 fractions of OKT3 and with autologous accessory cells, however, maintained its restricted antigen fidelity. The NK-like activity of Tc1.8 upon reactivation with OKT3 resulted from conjugate formation between the activated Tc1.8 and NK targets via the activating ligand itself. Thus, upon stimulation with anti-T3 MoAb and with autologous accessory cells, independently, the autoreactivity could be restored in an aged and inactive CTL clone.     

Cross-reactivity between human and murine lymphocyte antigens

The anti-H-2 alloantiserum D-32 [(BlO.A(2R) × C3H.SW) anti-C3H] is cytolytic to human lymphocytes. Fab₂ blocking assays, indirect immunoprecipitation and sequential immunoprecipitation experiments showed that the anti-H-2 alloantiserum D-32 recognizes antigenic determinants which are expressed on the heavy chain of subpopulations of HLA-A, B antigens. These determinants are different from those defining the serological polymorphism of the HLA-A, B, C system, are the same as or spatially close to those recognized by the anti-HLA-A, B monoclonal antibody Q6/64 and are expressed on rabbit, rat or guinea pig lymphocytes.     

Clonal and frequency analyses of tumor-infiltrating T lymphocytes from human solid tumors

A limiting dilution analysis (LDA) was used to assess the functional profiles of tumor-infiltrating lymphocytes (TIL) recovered from 15 human solid tumors. The microculture system applied in this study has been shown to allow virtually all normal peripheral blood T lymphocytes (PBL-T) to undergo clonal proliferation and was applied to obtain estimates of the frequency of both proliferating and cytolytic cells among the TIL population. A total of 624 microcultures proliferating in the presence of irradiated allogeneic spleen cells and interleukin 2 (IL 2) were expanded for clonal analysis. These TIL microcultures were assessed for surface antigen phenotype, IL 2 production (helper function) and for their cytolytic capabilities against the human erythroleukemic line K562 (natural killer (NK)-like activity) and P815, a mouse mastocytoma line, in the presence of phytohemagglutinin (PHA), i.e., lectin-dependent cell cytotoxicity (LDCC) which allows the detection of cytolytic activity irrespective of the antigenic specificity of the effector cells. Whenever feasible, cytolytic activity against autologous and allogeneic tumor cells was tested. LDA first demonstrated that the proliferative potential was decreased in T lymphocytes infiltrating human solid tumors (approximately 1 in 50 to 1 in 2 proliferating T lymphocyte precursors (PTL-P) in this series) as compared to normal PBL-T (1 in 2 to 1 in 1 PTL-P). The growth pattern in the titration cultures showed a remarkable agreement with the single-hit Poisson model implying that third party cells are unlikely to be involved in the reduced proliferative potential. Quantitative estimates of functional precursors showed that, in spite of reduced proliferative potential, cytolytic T lymphocyte precursors (CTL-P) against unknown antigens (LDCC-reactive) accounted for a considerable part of the microcultures in many cases. The precursor frequency of T lymphocytes with NK-like activity was usually low in situ (with the exception of glioma), whereas it was in the normal range in the patient's autologous PBL-T. In four evaluable cases, quantitative assessment showed that 1 in 200 to 1 in 1000 T lymphocytes from TIL was cytolytic against allogeneic tumor cells, which is in the range of alloreactive cytolytic T lymphocytes (CTL) generated in the mixed lymphocyte culture from normal PBL. Cytolytic activity against autologous target cells could not be quantitatively estimated but out of 88 clones from 4 patients, 3 clones originating from 2 glioma patients showed high lytic values against autologous tumor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of cellular immune response against autologous human melanoma. II. Mechanism of induction and specificity of suppression

The cytotoxic immune response in the peripheral blood lymphocytes (PBL) against an autologous malignant melanoma cell line, PJ-M, was found to be down-regulated in in vitro co-culture (IVC) selectively by unfractionated resident lymph node lymphocytes (derived from a lymph node infiltrated with the PJ-M melanoma cells) and T4+ as well as T8+ fractions of the resident lymph node-derived lymphocytes. In this study, the mechanism involved in, and the specificities of, cytotoxic immune response in this autologous system were examined at population and clonal levels. Resident lymph node lymphocytes were isolated from both involved and uninvolved lymph nodes from the same patient. Resident lymphocytes from both sources regulated the generation of cytotoxic immune response when both types of resident lymph node lymphocytes were further sensitized against the PJ-M cells in IVC and were expanded in interleukin 2 (IL 2). An IL 2-dependent homogeneous lymphocyte line (I-10:1) bearing the phenotype of a helper T cell (T4+) and a T4+ clone (I-10.3) of the I-10:1 line, established by limiting dilution culture, also down-regulated the generation of cytotoxic immune effector cells in the PBL in IVC against the PJ-M targets. The IL 2-dependent T4+ inducer line I-10:1 generated a functionally differentiated T8+ suppressor population(s) that, in turn, could abrogate cytotoxic response in fresh PBL in IVC against PJ-M cells. The inducer line I-10:1 and its subclone I-10.3 suppressed the generation of cytotoxic effector cells in the PBL in IVC selectively against the autologous PJ-M cells. Generation of cytotoxic allo-response in IVC was unaffected by the inducer lines. These results provide further evidence for the involvement of the regulatory network in cytotoxic immune response in an autologous human tumor system, and suggest a potential explanation for cytotoxic unresponsiveness against autologous melanoma cells.     

Effect of histamine and histamine antagonists on natural and antibody-dependent cellular cytotoxicity of human lymphocytes in vitro

The in vitro effect of histamine and its antagonists, cimetidine and clemastine fumarate, on natural killer (NK) and antibody-dependent cellular Cytotoxicity (ADCC) activities of human lymphocytes was investigated. The histamine 1 (H1) antagonist, clemastine fumarate, and the histamine 2 (H2) antagonist, cimetidine, but not histamine alone, inhibited the NK and ADCC activities of lymphocytes when added directly to the mixture of effector and target cells in a ⁵¹Cr-release assay. This inhibition was proportional to the concentration of drugs added and was observed at various effector to target ratios against several targets. H1 and H2 antagonists also inhibited NK activities of T cells as well as Percoll-separated, NK-enriched effector cells. The inhibition was significantly reversed by histamine. In target binding assays, clemastine fumarate and cimetidine also decreased the target binding capacity of effector lymphocytes. Further, PBL precultured with histamine (10^?3–10^?4M) for 24 hr showed a significant decrease in their NK and ADCC activities. In coculture experiments, PBL precultured with histamine suppressed the NK activity of normal autologous effector lymphocytes. PBL precultured with histamine showed an increased number of OKT8⁺ cells, as estimated using monoclonal antibodies. The suppression of Cytotoxicity was not due to either direct toxicity, steric hindrance, crowding, or cell death, but by functionally viable suppressor cells. An immunoregulatory role for histamine in NK and ADCC reactions is proposed.     

T-lymphocyte subset interactions in the cell-mediated immune response to Epstein-Barr virus

The T-lymphocyte subset interactions in the cell-mediated response to Epstein-Barr virus (EBV) were studied in an in vitro system in which the ability of T lymphocytes to inhibit outgrowth of autologous EBV-transformed B lymphocytes is assessed. Inhibition could be demonstrated within lymphocytes of both the T4 and T8 surface phenotypes. Outgrowth inhibition was observed more frequently when the effector T-cell population contained cells of both surface phenotypes. In blocking experiments the OKT3 antibody completely prevents development of outgrowth inhibitory activity; the OKT4 and OKT8 antibodies were less effective in interfering with outgrowth inhibitory function. Maximal blocking activity occurred when antibody addition occurred in the early phase of generation of suppressor function. Pharmacologically achievable concentrations of interferon-alpha restored outgrowth inhibitory activity after blocking with monoclonal antibody. EBV reactivation was easily demonstrable in a group of 10 renal allograft recipients who received OKT3 antibody for treatment of acute rejection. These studies suggest that the immunoregulatory control of proliferation of EBV-transformed B lymphocytes is complex, and involves a collaborative interaction of lymphocytes of both the T4 and T8 surface phenotypes.     

Lysis of autologous human melanoma cells by in vitro allosensitized peripheral blood lymphocytes

Summary Peripheral blood lymphocytes (PBL) of melanoma patients were sensitized in vitro with lymphocytes of a single donor or with a pool of lymphocytes of 5–20 different donors. After 6–7 days, the cytotoxic activity of the sensitized PBL was tested against cultured autologous tumor cells and lymphocytes in a ⁵¹Cr-release assay. Tumor lysis was observed in 13 of 16 cases in which patients' PBL (Pt-PBL) were stimulated by a pool of allogeneic lymphocytes and in five out of seven cases when single sensitization was performed. In no case was lysis of autologous normal lymphocytes or blasts seen. Cultivation of Pt-PBL with irradiated autologous tumor cells never led to the induction of lymphocytes cytotoxic to melanoma cells. Lysability by pool-activated autologous Pt-PBL of fresh cryopreserved tumor cells was compared to that of short-term cultured tumor cells, and no significant differences were observed. Cold-target inhibition experiments indicated that the cytotoxicity of Pt-PBL was tumor-restricted since only autologous melanoma cells but not lymphocytes were able to inhibit the reaction. These results indicate that activation of Pt-PBL is necessary in order to elicit or amplify their antitumor activity.     

T lymphocyte-mediated cytotoxicity against autologous EBV-genome-bearing B cells

We have stimulated human peripheral blood lymphocytes in vitro with autologous EBV-infected or noninfected B cells. A cytotoxic response was obtained only when virally infected cells were used. The activity of the effector cells was restricted by the major histocompatibility complex and was directed against EBV-genome-bearing targets. The highest cytolytic response was obtained when lymphocytes of individuals previously exposed to the virus (EBV-VCA positive) were used. Lymphocytes of noninfected donors (EBV-VCA negative) gave a low response; the relative frequency of their effector cells was at least 4-fold lower. Lymphocytes of newborns did not respond. The cytotoxic activity was mediated by T lymphocytes of the cytotoxic/suppressor subset, as determined by cytofluorographic analysis and antibody plus complement-mediated lysis, using monoclonal antibodies to human lymphocyte surface antigen.     

Possible role for cytotoxic lymphocytes in the pathogenesis of acute interstitial nephritis after recombinant interleukin-2 treatment for renal cell cancer

A patient with renal cell cancer developed acute renal failure due to biopsy-proven acute tubulo-interstitial nephritis (AIN) in the 6th week of continuous infusion of 9 × 10⁶ IU m^–2 day^–1 recombinant interleukin-2 (rIL-2). We investigated whether the AIN was the result of a cellular cytotoxic reaction induced by the rIL-2 treatment. The cytolytic activity of cryopreserved peripheral blood lymphocytes (PBL), isolated before and at the end of the rIL-2 treatment (at the time of AIN), was studied after 5 days of culture with or without rIL-2 or anti-CD28 and immobilized anti-CD3 antibodies. The PBL isolated before and at the end of the rIL-2 treatment showed cytolytic activity towards a number of allogeneic targets. However, only the PBL isolated at the end of the rIL-2 treatment showed, when stimulated with rIL-2 in vitro, significant cytolytic activity against an autologous renal cell line cultured from the AIN biopsy specimen and against an allogeneic renal cell cancer cell line. These PBL displayed no enhanced killing capacity towards autologous PBL and the melanoma cell line M14. These observations suggest that the AIN may be the result of a cytotoxic lymphocyte-mediated reaction induced by the rIL-2 treatment.     

Natural killer-like function of activated T lymphocytes: differential blocking effects of monoclonal antibodies specific for a 90-kDa clonotypic structure

A monoclonal antibody termed anti-NKTb has been generated following immunization of mice with cloned human cells (JT9) displaying natural killer (NK)-like activity. This antibody has the capacity to block cytotoxicity of the immunizing clone against several targets. In the present study, anti-NKTb was compared with a monoclonal antibody termed anti-NKTa that had previously been generated against JT9 cells and that had also been shown to block the NK-like function of these cells. The expression of a NKTb determinant, like that of NKTa, was found to be restricted to two NK active clones derived from the same individual, JT9 and JT10, both of which have the same mature T-cell phenotype (T3+, T8+, T11+). Comodulation, immunoprecipitation, and competitive binding experiments showed that both antibodies are directed to the same 90-kDa heterodimer associated with the T3 structure on the cell surface. However, cytotoxicity blocking studies suggested that NKTa and NKTb may represent functionally distinct epitopes of this 90-kDa molecule. Anti-NKTa uniformly blocked the cytotoxicity of both JT9 and JT10 cells when tested against 11 randomly selected target cell lines. In contrast, anti-NKTb totally blocked the cytotoxicity of these cloned cells against some targets (i.e., HPB-ALL, Nalm-1) but had very little effect when cytotoxicity was measured against other target cells (i.e., K562, U937, KG-1). This selective blocking effect, therefore, supports the notion that the heterodimer defined by the NKT antibodies is involved in the process of target cell recognition rather than in the cytolytic pathway of the cloned effector cells. Moreover, the unique functional effects of anti-NKTb suggest that additional levels of complexity exist in the specific recognition mechanisms of these clonal populations of NK active mature T lymphocytes.     

Cytotoxic T lymphocyte clones from peripheral blood and from tumor site detect intratumor heterogeneity of melanoma cells. Analysis of specificity and mechanisms of interaction

CTL clones isolated from PBL or from tumor-infiltrating lymphocytes (TIL) of a melanoma patient (pt665) were screened for specificity on a panel including autologous tumor cells from two distinct metastases (Me665/1, Me665/2), autologous EBV-transformed B cells and 15 allogeneic cell lines of different histology. Each clone displayed a peculiar cytolytic activity ranging from lysis of most targets (PBL clone 4C4) to preferential reactivity on the two autologous metastases (TIL clone 8B3). Blocking and modulation experiments, revealed that the lysis of autologous-Tu cells by TIL clone 8B3, but not by PBL clone 4C4, could be inhibited by mAb to HLA-class I and to CD3 Ag or by CD3 complex modulation. Clone 8B3 was tested also on a panel of 25 tumor clones from Me665/2, revealing that only 4 neoplastic clones were lysed (2/4, 2/14, 2/17, and 2/51). Cold target competition experiments indicated that the uncloned autologous melanomas and one tumor clone (2/17), but no two other tumor clones (2/10, 2/15), could compete with one another for lysis by 8B3. Determination of melanin content of tumor clones from Me665/2 revealed that the four neoplastic clones recognized by 8B3 possessed much lower melanin levels than all the other 20 clones not lysed by this effector.     

Generation of cytotoxic effector cells against human melanoma

Metastatic or tumor-draining lymph nodes from six of nine melanoma patients undergoing lymph node dissection for metastatic melanoma generated cytotoxic T cells against autologous melanoma when these lymph node cells were treated by in vitro sensitization and recombinant interleukin-2 (IL-2). During the initial lymphocyte culture (2–6 weeks), cross-reactivity with autologous tumor cells, K562 and Daudi cells was usually noted. Cold-target inhibition assay with K562 and Daudi showed K562/Daudi-associated antigens on melanoma cells. During the later phase of lymphocyte culture with repeated in vitro sensitization (over 6–10 weeks), cytotoxicity was noted against autologous and allogeneic melanoma cells but not against K562, Daudi cells or autologous fibroblasts. Repeated in vitro sensitization resulted in the selection of specific cytotoxic lymphocytes against melanoma. Cold-target inhibition assay with autologous and allogeneic melanoma cells revealed shared and individual antigens. Using blocking monoclonal antibodies, MHC-restricted killing was noted in the autologous system. Further, both the autologous and allogeneic systems could be mediated through adhesion molecules such as ICAM-1 and LFA-3 on melanoma cells and LFA-1 on T cells. This study suggests that a constellation of cytotoxic effector cells and melanoma-associated antigens may be pivotal in tumor killing. Thus, future adoptive immunotherapy should modulate and enhance this complex interaction.This work was supported by an Elsa, U. Pardee Foundation grant, the Arizona Chronic Disease Research Commission grant and partly by grant CA23074 from the National Institutes of Health, Bethesda, MD, 20892     

Antigen recognition by human T lymphocytes is linked to surface expression of the T3 molecular complex

Four distinct surface molecules on human T cells are defined by the monoclonal antibodies anti-T1, anti-T3 (anti-T3_A), anti-T11 and anti-T12. Following cell binding, anti-T3 (anti-T3_A) and anti-T1 induce independent modulation of their respective ligands, whereas anti-T11 and anti-T12 do not. To explore the biological consequences of this modulation, we used cloned populations of T4 and T8 cytotoxic T lymphocytes. Anti-T3 (anti-T3_A), but not anti-T1, inhibits cytotoxic T lymphocyte effector function by T4 and T8 clones as well as antigen-specific T cell recognition. The latter is not secondary to a generalized inhibitory effect since responsiveness to interleukin 2 is maintained. Moreover, after modulation, cytotoxic T lymphocytes recover cytolytic function in parallel with reexpression of surface T3 molecules. We provide evidence for a direct linkage between antigen recognition by T lymphocytes and surface expression of the T3 molecular complex.     

In vitro generation of human activated lymphocyte killer cells: separate precursors and modes of generation of NK-like cells and "anomalous" killer cells

The activation of human peripheral blood mononuclear cells (PBM) in culture leads to the generation of nonspecific killer cells. These cells, termed activated lymphocyte killer (ALK) cells, can kill fresh tumor cells and tumor cell lines, in addition to the natural killer (NK) cell sensitive target K562. ALK cells have features in common with both T and NK cells, but their nature and origin are unknown. In the present study, it is shown that ALK cells are in fact heterogeneous and can be generated from both large granular lymphocytes with the same phenotype as NK cells and from T cells. Cell populations enriched for NK cells, when cultured with lymphokines, rapidly acquired a T cell phenotype, enhanced cytolytic activity against K562, and the ability to lyse NK-insensitive target cells such as a melanoma cell line LiBr; these ALK cells were described as NK-like cells. On the other hand, of the cloned cells derived from PBM stimulated with irradiated B lymphoblasts and grown in lymphokines, the major proportion of cytolytic T cells (CTC) able to kill the specific stimulator lymphoblasts were also found to kill LiBr but not K562 cells. These ALK cells, which were derived from the same precursors as CTC, were designated anomalous killer (AK) cells. Consistent with this, the presence of the pan T monoclonal antibody UCHT1 from the beginning of mixed cell cultures inhibited the generation of CTC and of the AK-type of ALK cell, which killed melanoma cells, but not the NK type, which killed K562 targets. By contrast, at the effector cell level, the antibodies UCHT1 and OKT8 only blocked specific killing by CTC but did not block the killing of LiBr or of K562 targets by ALK cells. However, at the effector cell level there was additional evidence for the heterogeneity of ALK cells. Thus, monoclonal antibody 9.1C3, which blocks killing by freshly isolated NK cells, also blocked the killing of K562 targets by NK-like cells, but did not block B lymphoblast killing by CTC or melanoma cell killing by AK cells. It is concluded that after mixed lymphocyte culture, the majority of ALK cells measured by the killing of melanoma target cells arise from the same precursors and are under the same influences as classical CTC (AK cells), whereas cells killing K562 targets are derived from NK cells (NK-like cells). Once generated, the AK cells have a different mechanism of killing from both classical CTC and from NK and NK-like cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Blocked ricin-conjugated T cell immunotoxins: Effect of anti-CD6-blocked ricin on normal T cell function

Summary The biological properties of an immunotoxin composed of an anti-CD6 monoclonal antibody conjugated to whole ricin, which had been modified so that the galactose-binding sites of the B chain were blocked (blocked ricin), were examined. Treatment of peripheral blood lymphocytes with anti-CD6-blocked ricin for a 24-h period prevented T cell proliferation induced by phytohemagglutinin in a dose-dependent manner with concentrations causing 50% inhibition (IC₅₀) ranging from 5 pM to 30 pM. In contrast, treatment with either blocked ricin alone or with a control immunotoxin prepared with a B-cell-lineage-restricted monoclonal antibody gave IC₅₀ values of approximately 2 nM. Although shortening the duration of the anti-CD6-blocked ricin treatment to as little as 3 h had little significant effect on the observed inhibition, T cell viability experiments demonstrated that the magnitude of immunotoxin-induced killing after a given time period is significantly higher when the target cells become activated. Thus, from the initial concentration of cells treated with anti-CD6-blocked ricin placed in culture, 40%–45% viable cells remained after 2 days yet only 3%–9% remained if phorbol ester and Ca²⁺ ionophore were added; activation of T cells after mock treatment using blocked ricin plus nonconjugated anti-CD6 demonstrated that this effect was not the result of activation alone. The toxicity of anti-CD6-blocked ricin was also measured by inhibition of PHA-induced clonogenic growth of normal T cells. Continuous treatment of the cells using anti-CD6-blocked ricin at 0.1 nM resulted in a surviving fraction of about 3.5 × 10^–3; when immunotoxin treatment was for 24 h or less, the surviving fraction was only about 10^–1. As an indication of the unique specificity of anti-CD6-blocked ricin, immunotoxin pretreatment of potential responder cells prevented the generation of allogeneic cytolytic T lymphocytes in mixed lymphocyte cultures yet had little effect on the generation of interleukin-2-induced lymphokine-activated killer cell activity. We conclude that anti-CD6-blocked ricin demonstrates a cellular specificity and potency that make it a highly promising anti-T cell reagent.     

Human immunodeficiency virus-specific cytotoxic responses of seropositive individuals: distinct types of effector cells mediate killing of targets expressing gag and env proteins.

By using target cells that expressed isolated env, gag, p27nef, or p23vif molecules introduced by recombinant vaccinia viruses containing genes encoding these polypeptides, it was possible to identify env, gag, p27nef, and p23vif as cytolytic target antigens for freshly isolated blood cells from human immunodeficiency virus 1 (HIV-1) seropositive patients. Most of the patients tested (95%) manifested a specific cytotoxic activity against vaccinia virus-env-infected target cells. The env-specific cytotoxic activity was not restricted by the major histocompatibility complex and was not mediated by T lymphocytes, as shown by the absence of blocking effect with an anti-CD3 monoclonal antibody and by the inefficiency of CD3+, CD8+, or CD4+ and CD8+ depletion to reduce the cytotoxic activity against the env-expressing target cells. In the same conditions, the cytotoxic activity specific for gag was abrogated and gag major histocompatibility complex-restricted cytotoxic T lymphocytes were detected in 85% of the subjects tested. Therefore, in a HIV-1 seropositive subject, distinct types of effector cells mediate the lysis of target cells expressing gag and env proteins.