Autologous tumor cell killing activity of tumor-associated lymphocytes in patients with head and neck carcinomas

In order to select the most cytotoxic effector cells for adoptive immunotherapy, lymphokine activated killer (LAK) cells, tumor infiltrating lymphocytes (TILs) and autologous mixed lymphocyte tumor cell culture (MLTC) cells derived from peripheral blood mononuclear cells (PBMC) in the same subject with head and neck carcinomas were prepared. The autologous tumor cell killing activity and cell surface phenotypes of each of the three effector cells were studied. MLTC cells cultured with interleukin-2 (IL-2) showed the strongest cytotoxic activity among these three different effector cells. Although TILs had suppressed killing activity immediately after isolation, after successive cultivations with IL-2, a cytotoxic activity against autologous tumor cells stronger than that of LAK cells appeared. Both IL-2 stimulated MLTC cells and TILs showed an enrichment of CD8 positive and CDU negative cells in a CD3 positive subpopulation.Abbreviations CD cluster differentiation - IL-2 interleukin-2 - LA lymphokine activated - LAK lymphokine activated killer - MLTC mixed lymphocyte tumor cell culture - NK natural killer - PBMC peripheral blood mononuclear cells - TILs tumor infiltrating lymphocytes     

The synergistic effects of interleukin 2 and interleukin 7 on the proliferation and autologous tumor cell lysis of tumor-associated lymphocytes

Interleukin-7 (IL-7) has an ability to stimulate the proliferation of pre-B cells. It has been shown that IL-7 can also activate T lymphocytes. We here demonstrate that IL-7 in combination with interleukin-2 (IL-2) can drive cell proliferation and enhance the autologous tumor cell lysis by peripheral blood mononuclear cells (PBMC) and autologous mixed lymphocyte tumor cell culture (MLTC)-derived effector cells (MLTC cells). These synergistic effects of IL-2 and IL-7 on the proliferation and the augmentation of autologous tumor cell lysis were found for both effector cells. These effects were inhibited by neutralizing antibodies to IL-2 or IL-7, and by a combination of both antibodies, significantly. In terms of phenotypical expression, CD3 positive cells comprised the vast majority of MLTC cells after culture in medium containing IL-2 and IL-7 with an increase of IL-2 receptor positive cells.Abbreviations CD cluster differentiation - IFN interferon - IL interleukin - JRU Japanese Reference Unit - LAK lymphokine activated killer - mAb monoclonal antibody - MLTC mixed lymphocyte tumor cell culture - PBMC peripheral blood mononuclear cells - TILs tumor infiltrating lymphocytes     

Lytic activity against primary AML cells is stimulated in vitro by an autologous whole cell vaccine expressing IL-2 and CD80

Despite being of the myeloid lineage, acute myeloid leukaemia (AML) blasts are of low immunogenicity, probably because they lack the costimulatory molecule CD80 and secrete immunosuppressive factors. We have previously shown that in vitro stimulation of autologous peripheral blood mononuclear cells (PBMCs) with primary AML cells modified to express CD80 and IL-2 promotes proliferation, secretion of Th1 cytokines and expansion of activated CD8⁺ T cells. In this study, we show that allogeneic effector cells (from a healthy donor or AML patients) when stimulated with IL-2/CD80 modified AML blasts were able to induce the lysis of unmodified AML blasts. Effector cells stimulated with IL-2/CD80AML blasts had higher lytic activity than cells stimulated with AML cells expressing CD80 or IL-2 alone. Similarly, AML patient PBMCs primed with autologous IL-2/CD80 AML cells had a higher frequency of IFN-γ secreting cells and show cytotoxicity against autologous, unmodified blasts. Crucially, the response appears to be leukaemia specific, since stimulated patient PBMCs show higher frequencies of IFN-γ secreting effector cells in response to AML blasts than to remission bone marrow cells from the same patients. Although studied in a small number of heterogeneous patient samples, the data are encouraging and support the continuing development of vaccination for poor prognosis AML patients with autologous cells genetically modified to express IL-2/CD80.     

Interleukin-4 augments the cytotoxic capacity of lymphocytes and monocytes in antibody-dependent cellular cytotoxicity

Summary Human peripheral blood mononuclear cells (lymphocytes and monocytes) (PBMC) were preincubated for 0–24 h with human recombinant interleukin-4 (IL-4) and used as effector cells in an 18 h antibody-dependent cellular cytotoxicity (ADCC) assay with mAb 17-1A (mouse IgG2A) against SW948 (a human colorectal carcinoma cell line). A statistically significant increase in the lytic capability was noted after 2–24 h of preactivation. IL-4 at 1 ng/ml induced the highest cell lysis while higher and lower concentrations were inferior or had no effect at all. Preactivation for 24 h induced a more effective lytic cell population than 2 h prestimulation: 63 LU (lytic units)/10⁶ cells vs 42 LU/10⁶ cells. Pretreatment with 1 ng/ml IL-4 for 2 h induced a statistically significant increase in the ADCC activity of PBMC (P <0.05), of monocytes (P <0.01) and E-rosette-negative cells (natural killer cells) (P <0.05) compared to non-activated cells. IL-4 did not induce lymphokine-activated killer activity of PBMC against SW948. The spontaneous cytotoxicity against K562 was, however, increased after stimulation with 1 ng/ml IL-4 for 2 h of E-rosette-negative non-adherent cells.     

Granulocyte-monocyte-colony-stimulating factor augments the cytotoxic capacity of lymphocytes and monocytes in antibody-dependent cellular cytotoxicity

Summary Human peripheral blood mononuclear cells (lymphocytes and monocytes) were preincubated for 0–24 h with human recombinant granulocyte-monocyte-colony-stimulating factor (GM-CSF) and used as effector cells in an 18 h antibody-dependent cellular cytotoxicity (ADCC) assay with SW948 (a human colorectal carcinoma cell line) as target cells and mAb 17-1A. A significant increase in the lytic capability was noted after 0.5–2 h of preactivation while longer preincubation times did not significantly increase the lytic potential. GM-CSF at 0.01 g/ml induced the best tumor cell lysis while higher concentrations were inhibitory. GM-CSF pretreatment induced a statistically significant increase in the lytic capacity of both monocytes and lymphocytes in ADCC as well as in the spontaneous cytotoxicity.     

Antibody-dependent cell-mediated cytotoxicity using a murine monoclonal antibody against human colorectal cancer in cancer patients

Summary Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by a murine monoclonal antibody against human colerectal carcinoma, antibody 19–9, with human effector cells was tested in 33 patients with various carcinomas, 16 patients with benign lesions, and 13 normal controls, using a 12-h ⁵¹Cr release assay using human colorectal cancer cells as targets. Peripheral blood mononuclear cells (PBM) from these groups of patients and normal controls achieved moderate levels of target cell lysis in the presence of the monoclonal antibody at the high effector to target cell ratio of 200:1. The ADCC activity of PBM in cancer patients was significantly higher than that in either normal persons or patients with benign lesions. Since the ADCC was shown to be mainly mediated by adherent monocytes in the PBM, ADCC activity of monocytes from cancer patients was compared to those from control groups at an effector to target cell ratio of 30:1. The results also showed that the lytic capacity of monocytes was significantly higher in cancer patients than that in the control populations.     

Artificial feeder cells expressing ligands for killer cell immunoglobulin-like receptors and CD94/NKG2A for expansion of functional primary natural killer cells with tolerance to self

Background aimsNatural killer (NK) cells are promising cells for immunotherapy of cancer, and there are ongoing efforts to improve their ex vivo expansion to clinically relevant numbers. This study focused on the development of a C1-, C2-, Bw4 killer cell immunoglobulin-like receptor (KIR) ligand and NKG2A ligand-containing feeder cell line for autonomous expansion of functional NK cells.MethodsPC3^PSCA-derived feeder cells expressing IL-2, 4-1BBL and membrane-bound IL-15-mutDAP12 (mIL-15d) fusion protein in combinations or alone were generated and used for expansion. Expanded NK cells were analyzed with respect to subpopulations, expression of NK cell receptors and immune checkpoint molecules as well as their cytotoxicity against K562 cells, cetuximab-marked tumor cells and autologous B cells.ResultsOnly combinatorial expression of IL-2 plus 4-1BBL or IL-2, 4-1BBL plus mIL-15d in feeder cells efficiently expanded NK cells and supported selective outgrowth of NK cells from peripheral blood mononuclear cell samples. Best expansion of NK cells was achieved using PC3^PSCA-IL-2-4-1BBL-mIL-15d feeder cells. Such expanded NK cells exhibited upregulation of natural cytotoxicity receptors, DNAM-1 and NKG2C and induced expression of high affinity IL-2 receptor, which were paralleled by attenuated KIR and increased expression of NKG2A and ILT2. In addition, elevated TIM-3 levels were noted and PD-1 and T cell immunoreceptor with Ig and ITIM domain (TIGIT) levels remained low. Expanded NK cells were highly cytolytic when encountering K562 cells and cetuximab-marked target cells but remained unresponsive to autologous B cells and target cells with protective levels of human leukocyte antigen.ConclusionsCollectively, the results demonstrate the feasibility of PC3^PSCA-IL-2-4-1BBL-mIL-15d feeder cells for robust expansion of NK cells, which remain tolerant to self and could be used in the future for adoptive cell therapy of cancer.     

Induction of cytotoxicity against autologous tumour cells by interleukin-12: evidence for intrinsic anti-tumor immune capacity in curatively resected gastrointestinal tumour patients

The aim of this study was to investigate the cell-mediated immune response in 14 patients undergoing curative resection for a gastrointestinal tumor by the induction of peripheral blood mononuclear cell (PBMC)-mediated immune activity against autologous tumour cells. PBMC were stimulated by interleukin-12 (IL-12; 100 IU/ml) and IL-2 (1,000 IU/ml) without contact with tumour cells for 36 h. Specific cytotoxic activity against autologous tumour cells (auTu), natural killer (NK)-sensitive cells (K562) and allogeneic tumour cells (RF48/HT29) was determined by fluorescence cytotoxicity assay. Additionally, inhibition experiments using the mononuclear antibodies (mAb) FMC16 and W6/32 against major histocompatibility complex I (MHC I) on autologous tumour cells were performed in order to determine the involvement of specific T lymphocytes. The cytotoxic activity of unstimulated PBMC did not differ between the three target cells. IL-12 caused a 3.2-fold increase in activity against auTu ( P=0.002). In contrast, after stimulation with IL-2, only a slight increase in activity was observed. After IL-12 stimulation, cytotoxic activity against auTu was 2.5- to 2.7-fold higher than the corresponding activity against K562/allogeneic tumour cells ( P=0.002/ P=0.006). After blocking of the MHC I complex on auTu by FMC 16 or W6/32 mAb, a 62.9%/74.4% reduction in the specific cytotoxicity of IL-12-stimulated PBMC was found. In summary, IL-12 induced an effective immune response against auTu, which was partly mediated by specific cytotoxic T lymphocytes (CTL). It was considered that de novo generation of this activity during 36 h incubation without antigen contact was hardly possible, but that the observed induction of effective anti-tumor cytotoxicity was rather based on the re-activation of a pre-existing immune potential from the tumour-host interaction. These findings indicate the existence of an autologous anti-tumor immune response following curative resection in patients undergoing surgery for solid tumours, which might influence the development of tumour recurrence from disseminated tumour cells. Making use of this capacity could constitute an attractive immunotherapeutical approach for curatively operated tumour patients.     

Paradoxical enhancement of interleukin-2-mediated cytotoxicity against K562 cells by addition of a low dose of methotrexate

Summary In vitro effects of methotrexate (MTX) on interleukin-2(IL-2)-mediated cytotoxicity of peripheral blood mononuclear cells (PBMC) were studied. PBMC were incubated with human recombinant IL-2 (25 U/ml) for 72 h; during the last 24 h, various concentrations (10 pM–1 µM) of MTX were added to the culture. Cytotoxicity against k562 cells was measured by a 4-h⁵¹Cr-release assay. The IL-2-mediated cytotoxicity was paradoxically increased at around a concentration (10 nM) MTX. Such a low concentration of MTX showed no anti-proliferative effect on cell growth. This enhancement with 10 nM MTX was shown only in an E-rosette⁺ (E⁺) population, but not in E-rosette^– (E^–). In addition, when E⁺ cells were treated with an anti-CD16 monoclonal antibody plus complement after incubation with IL-2 and MTX, MTX-induced enhancement was lost, suggesting that an E⁺CD16⁺ cell population was mainly involved in this augmentation. Positively sorted E⁺CD16⁺ cells showed similar enhancement of cytotoxicity after treatment with IL-2 plus MTX. On the other hand, MTX treatment did not show the phenotypical changes including of the E⁺CD16⁺ cells, indicating that this treatment did not affect the differentiation and proliferation of the specific cell subset. Our results indicate that a low dose of MTX could have a role in the regulation of immunological anti-cancer surveillance systems through the natural killer and lymphokine-activated cytotoxic cells.This work was supported in part by a Grant-in-Aid for Cancer Research (1–10) from the Ministry of Health and Welfare in Japan     

Comparison of the cytotoxic response against ovarian cancer by immune effector cells isolated and expanded from normal donors and ovarian cancer patients

Background/AimsThe aim of this study was to compare the cytotoxic response against ovarian cancer (OC) cells elicited by different immune effector cells in combination with the cytokines interleukin (IL)-2 and interferon (IFN) α-2b.MethodsOC cells were co-cultured with peripheral blood mononuclear cells (PBMC) from normal donors or OC patients and IL-2 or IFN α-2b alone or in combination, in order to determine the cytotoxicity. T cells were isolated from healthy donors to determine T cell cytotoxic activity. PBMC from healthy donors and OC patients were expanded in an IL-2/IL-7/IL-12 cocktail with and without anti-CD3 antibody, and the cytotoxic activity measured. Flow cytometry was performed on primary, selected and expanded cells to determine T, B, and natural killer- (NK) cell percentages.ResultsHealthy donor PBMC elicited a significant cytotoxic response (59%) compared with OC patient PBMC (7%). T cells enriched from normal donors elicited a significant cytotoxic response (18%) compared with controls lacking effector cells (1.4%); however, the cytotoxicity observed was significantly less compared with unselected PBMC. Expanded effector cells consisted primarily of T cells (98%) and the fold-expansion was significantly higher in the presence of anti-CD3 (19- versus 132-fold). No significant difference in the expansion (either fold-expansion or cell type) was observed between OC patients and healthy donors. Expanded cells from both healthy donors and OC patients elicited a significant cytotoxic response in the presence of IL-2 (19% and 22%) compared with controls.ConclusionsPBMC from OC patients do not elicit a significant cytotoxic response; however, ex vivo-expanded cells from OC patients are capable of cytotoxic killing similar to unexpanded T cells isolated from normal donors. These data provide the groundwork for further development of cellular therapy against OC.     

Generation of cytotoxic T lymphocytes from peripheral blood of leukaemic patients

Peripheral blood mononuclear cells from 13 patients with acute leukaemia were used to establish long-term interleukin-2-dependent cytotoxic T lymphocytes. Cells were grown in RPMI medium containing interleukin-2 (IL-2, 100 U/ml) and 2.5% conditioned medium prepared by activating normal lymphocytes with phytohaemagglutinin. Proliferation of IL-2-dependent CD3-positive lymphocytes was seen in 1 of 2 acute lymphocytic leukaemia cases (ALL), 1 of 4 acute myelogeneous leukaemia cases (AML) (M1) and 8 of 8 more differentiated AML. In 2 cases with detectable leukaemic cell markers (1 ALL and 1 AML) passageable cells were developed, that expressed normal T cell phenotypes (namely CD3, CD4, and CD8) at the expense of leukaemic cells. In 1 of 2 cases, long-term IL-2-cultured cells showed specific cytotoxic activity against autologous leukemic cells. The percentage killing against autologous and two allogeneic target cell lines at a 50/1 effector/target (E/T) ratio was 42%, 9% and 19% respectively. Similarly the cytotoxic activity of IL-2 activated from 4 different individuals against conventional tumour targets K562 and Daudi at a ratio of 50/1 was 29%–68% (median=55%) and 34%–78% (median=61%) respectively. It was also found that this killing potential of the activated cells was maintained for as long as culture was continued (median 23 days, range 17–75 days). The mechanism(s) of T cell proliferation at the expense of leukaemic blast cells in the case of a minority of leukaemic patients and the possible clinical therapeutic potential of these cells following in vitro IL-2 activation deserve further investigation.     

Lymphocytes infiltrating human ovarian tumors. I. Role of Leu-19 (NKH1)-positive recombinant IL-2-activated cultures of lymphocytes infiltrating human ovarian tumors

Tumor-infiltrating lymphocytes (TIL) were obtained from human ovarian tumors, expanded in the presence of IL-2 in culture and studied for cytotoxicity against fresh autologous and allogeneic ovarian carcinoma (CA) targets. TIL from ovarian tumors grew well in long term cultures, achieving from 8- to 682-fold expansion. TIL cultured with IL-2 were cytotoxic against both autologous and allogeneic fresh ovarian CA targets, and no specificity for autologous tumor could be demonstrated in any of the cultures. In all fresh TIL preparations, CD3+ lymphocytes were the major cell type and contained a high proportion (up to 51%) of activated (IL-2R+) cells as determined by two-color flow cytometry. Sorting of bulk TIL cultures followed by cytotoxicity assays identified the Leu-19+ cells, both CD3+ and CD3-, as effectors of cytotoxicity against autologous and allogeneic tumor cell targets. Cold target inhibition assays showed that allogeneic targets (both ovarian CA and a sarcoma) competed effectively with autologous ovarian CA targets for Leu-19+ effectors in TIL cultures. mAb to Leu-19 or Leu-2a did not block lysis of autologous targets by sorted effectors. OKT3 antibody augmented lysis of autologous targets by CD3+Leu-19- effectors only. These results show that non-MHC-restricted Leu-19+ effectors in cultures of TIL with 1000 U/ml of rIL-2 mediate lysis of autologous and allogeneic tumor cells. The CD3+Leu-19- cells, the main population in these cultures, do not mediate tumor lysis. To determine the phenotype of antitumor effectors in IL-2 cultures of TIL, cell sorting followed by functional assays are necessary.     

Natural killer (NK) and lymphokine-activated killer (LAK) cell functions from healthy dogs and 29 dogs with a variety of spontaneous neoplasms

To investigate natural killer (NK) and lymphokine-activated killer (LAK) cell functions from 10 healthy dogs and 29 dogs with a variety of spontaneous neoplasms, large granular lymphocytes (LGLs) from blood samples were separated by a 58.5% Percoll density gradient. LGLs were stimulated with a low dose of recombinant human interleukin 2 (rhIL-2) for 7 days. Cytotoxicity of effector cells against the susceptible CTAC cell line was measured before and after stimulation. Compared with those before stimulation, the percentage of LGLs after stimulation with rhIL-2 was found to be significantly increased (P<0.01) in both dogs with tumors and controls. However, the increase was significantly higher in control animals, indicating a defect in proliferation ability of NK cells in canine tumor patients. After stimulation with rhIL-2, lymphokine-activated killer (LAK) cell activity in dogs with tumors was significantly lower (P<0.01) when compared with controls. Reduced cytotoxicity of rhIL-2–activated NK cells in dogs with tumors seems to be attributable to the presence of a diminished proliferative capacity of NK cells and a decreased ability of LAK cells to lyse target cells. Further knowledge of the precise function of IL-2–activated NK cells in dogs with tumors may help to optimize new and therapeutically beneficial treatment strategies in canine and human cancer patients. Our findings suggest that the dog could also serve as a relevant large animal model for cancer immunotherapy with IL-2.     

The effect of IL-2 and IFN-beta on autologous tumor cell kill by Percoll separated LGL fractions

Low density fractions of Percoll density gradient centrifugation of peripheral mononuclear cells contained the majority of large granular lymphocytes (LGL). LGL were used for 5-hr 51Cr release cytotoxic assay against autologous tumor cells in 20 patients with hematological malignancies (9AML, 4ALL and 7NHL). Mean % cytotoxicity (% CTX) was 6.0%, and the addition of IFN-beta and IL-2 in the medium induced the significant increase of % CTX to 15.0% and 26.1%, respectively. When LGL cultured in medium containing IFN-beta and IL-2 were assessed for cytotoxicity daily for 8 days, the enhancement of % CTX by IFN-beta was declined in a few days, while the enhancement by IL-2 was sustained for more than 8 days. The pretreatment of LGL with anti Leu-11 (CD16) plus complement abrogated the enhancing effect by IFN-beta or IL-2, but not with anti Leu-1 (CD5) plus complement. When this treatment was done on day 8 of IL-2 cocultivation, anti Leu-11 plus complement suppressed cytotoxicity significantly, and anti Leu-1 plus complement also induced mild suppression. The phenotypic characteristics of cells revealed the significant increase of anti Leu-19+ cells in IL-2 stimulated day 8 cells. High density fractions of Percoll gradient contained mostly T lymphocytes and showed no cytotoxicity against autologous tumor cells. However, cocultivation with IL-2 for 8 days induced the cytotoxicity, associated with increased number of anti Leu-19+ cells. These results suggested that IL-2 induced cytotoxic activity against autologous tumor cells might be related to the increase of anti Leu-19+ cells.     

Synovial T cell hyporesponsiveness to myeloid dendritic cells is reversed by preventing PD-1/PD-L1 interactions

Introduction

The aim of this study was to investigate PD-1/PD-L1 involvement in the hyporesponsiveness of rheumatoid arthritis (RA) synovial fluid (SF) CD4 T cells upon stimulation by thymic stromal lymphopoietin (TSLP)–primed CD1c myeloid dendritic cells (mDCs).

Methods

Expression of PD-1 on naïve (Tn), central memory (Tcm) and effector memory (Tem) CD4 T cell subsets was assessed by flow cytometry. PD-L1 expression and its regulation upon TSLP stimulation of mDCs from peripheral blood (PB) and SF of RA patients were investigated by quantitative RT-PCR and flow cytometry. The involvement of PD-1/PD-L1 interactions in SF T cell hyporesponsiveness upon (TSLP-primed) mDC activation was determined by cell culture in the presence of PD-1 blocking antibodies, with or without interleukin 7 (IL-7) as a recognized suppressor of PD-1 expression.

Results

PD-1 expression was increased on CD4 T cells derived from SF compared with PB of RA patients. TSLP increased PD-L1 mRNA expression in both PB and SF mDCs. PD-L1 protein expression was increased on SF mDCs compared with PB mDCs and was associated with T cell hyporesponsiveness. Blockade of PD-1, as well as IL-7 stimulation, during cocultures of memory T cells and (TSLP-primed) mDCs from RA patients significantly recovered T cell proliferation.

Conclusion

SF T cell hyporesponsiveness upon (TSLP-primed) mDC stimulation in RA joints is partially dependent on PD-1/PD-L1 interactions, as PD-1 and PD-L1 are both highly expressed on SF T cells and mDCs, respectively, and inhibiting PD-1 availability restores T cell proliferation. The potential of IL-7 to robustly reverse this hyporesponsiveness suggests that such proinflammatory cytokines in RA joints strongly contribute to memory T cell activation.     

Preadministration of a T-suppressor factor enhances tumor immunity in DBA/2 mice

Summary Human peripheral blood mononuclear cells (PBM) activated with recombinant interleukin-2 (IL-2) generate potent lytic activity (LAK) against a variety of malignant cells. IL-2 alone is sufficient for LAK generation, but high concentrations are needed to generate optimal cytotoxicity. Our recent studies based on combinations of biological agents indicated that alternative activation pathways may exist. Synergy for LAK induction was investigated using IL-2 and tumor necrosis factor- (TNF). Single-cell suspensions of primary human lung carcinomas were prepared from seven established cell lines and 32 fresh tumor specimens. Not only were all cell lines sensitive to allogeneic LAK, but also all fresh tumors were sensitive to some degree to both autologous and allogeneic LAK lysis measured by a 4-h ⁵¹Cr-release assay. LAK-mediated cytotoxicity, induced with a combination of human recombinant IL-2 (Cetus, 100 U/ml) and TNF (Genentech, 500 U/ml), showed a mean fourfold increase (range 0.7–16.3) over IL-2 alone. No lytic activity was generated from PBM incubated with media or TNF alone. The sequence dependence of adding IL-2 and TNF in enhancing cytolytic activity was also studied. In vitro kinetics data revealed that the addition of TNF 2–6 h before the addition of IL-2 greatly increased LAK activity over that obtained from the simultaneous addition of the two cytokines. These results demonstrated (a) the synergy of IL-2 and TNF for generating LAK; (b) the lysis of fresh primary lung cancer cells by LAK; and (c) the sequence dependence of IL-2 and TNF for the induction of optimal LAK activity.This work was supported by NCI Grants RO2-CA45225 and CAO 9611-01, and by an award from the Prouss Foundation     

HLA-DR antigens induce proliferation and cytotoxicity of T cells against haptenated (TNP and FITC) self structures

Antisera directed against the heavy, the light, or reactive against the complex of both chains of HLA-DR antigens strongly inhibited proliferation of T cells induced by TNP- or FITC-labeled autologous cells when added at initiation of the cultures, but not 72 h later. T cells from cultures treated with the anti-DR sera were unresponsive to interleukin-2 (IL-2). Nonetheless, the anti-DR sera did not inhibit proliferation of T cells that had already acquired sensitivity to IL-2. The DR antibodies abrogated the synthesis of IL-2 induced by both TNP- and FITC-conjugated autologous cells. Treatment of TNP- and FITC-labeled autologous cell cultures with the four different types of anti-DR sera significantly inhibited the induction of cytotoxic T cells. However, DR antibodies added at the effector phase of cytotoxicity assays did not inhibit the cytotoxic activity. Effector T cells from cultures treated with the anti-DR sera were unresponsive to IL-2 and addition of IL-2 to these cultures did not restore the cytotoxic activity. In contrast, effector T cells from cultures performed in the absence of the anti-DR sera proliferated to IL-2 stimulation and addition of IL-2 to these cultures significantly increased the generation of killer cells specific for hapten-labeled self structures. From these results we concluded the following: (1) Both the heavy and the light chains of DR antigens participate actively in the activation of T cells by rendering resting T cells sensitive to IL-2 and by inducing production of the growth factor in TNP- and FITC-conjugated autologous cell cultures. (2) The heavy and light chains of the DR antigens play an essential role in the induction of cytotoxic T cells specific for hapten-labeled self structures, most likely by enabling cytotoxic T cells to respond to IL-2 and by inducing the IL-2 producer T cells to synthesize the growth factor.     

Role of CD16 (Fc receptor III) and interleukin-2 in the reactivation of natural killer cells after inhibitory target cell contact.

Contact with natural killer (NK)-resistant monolayer targets is an inhibitory signal to NK cells. In this study, we have analyzed the effect of such effector/target cell interactions on the CD16 (FcRIII) expression on lymphocytes and the role of CD16 and interleukin-2 (IL-2) in the reactivation of their cytolytic machinery. Coculturing peripheral blood mononuclear cells with NK-resistant monolayer cells did not change the percentage of CD 16-positive effector cells, although this treatment effectively inhibited their cytotoxicity against NK-sensitive targets. The inhibited effector cells partially regained their activity by incubating for 24 h in medium supplemented with 10% fetal calf serum (FCS), whereas human albumin-, newborn calf serum- or human AB serum-supplemented media had no reactivating effect. Monoclonal class IgG1, IgG2a and IgM anti-CD16 antibodies [Abs; 3G8, CLB-CD16 (CLB-FcR gr1) and Leu 11b], and normal rabbit IgG (NR-IgG) prevented the FCS-mediated reactivation of cytotoxicity, whereas nonreactive control Abs significantly enhanced it. The detection of the CD16 antigen by the monoclonal anti-CD16 Abs Leu 11a and Leu 11c was blocked by the above anti-CD16 Abs and NR-IgG, while the expression of other NK cell-associated surface molecules (CD2, CD56) remained unchanged. Mere blocking of CD16, using a short-term incubation with anti-CD16 Abs, had an insignificant effect on endogenous NK activity, suggesting that CD16 is involved in NK cell (re)activation rather than in the killing process itself. In the presence of IL-2, inactivated effector cells also regained their killing activity. The IL-2-induced reactivation was not inhibited by anti-CD16 Abs. The results suggest that FCS-derived factors and soluble nonreactive immunoglobulins enhance the NK activity of down-regulated effector cells via CD16, and that CD16 and IL-2 receptors represent alternative independent pathways of NK cell reactivation.     

Human recombinant interleukin-6 enhances antibody-dependent cellular cytotoxicity of human tumor cells mediated by human peripheral blood mononuclear cells

Summary The effects of human recombinant interleukin-6 (hrIL-6) on antibody-dependent cellular cytotoxicity (ADCC) activity mediated by human peripheral blood mononuclear cells (PMNC) were investigated. Human PMNC were preincubated for 24 h with various concentrations of hrIL-6 and were used as effector cells in a 4-h⁵¹Cr-release assay. The ability of hrIL-6 to augment ADCC was measured using anti-colorectal carcinoma mAbs D612, 17.1A and 31.1 (each directed against a distinct tumor antigen) and using three human colorectal carcinoma cell lines, LS-174T, WiDr and HT-29, as targets. A significant increase in ADCC activity was observed after PMNC were preincubated in 100–400 U/ml but not in lower concentrations of hrIL-6. Variations in activities of PMNC among donors were observed. Non-specific mAb showed no effect in augmenting ADCC activity. hrIL-6 treatment did not augment non-specific (non-mAb-mediated) cytotoxicity. The enhancement of ADCC activity was blocked by the addition of an antibody against hrIL-6 but not by an antibody to the IL-2 receptor (capable of blocking the induction of lymphokine-activated killer cell cytotoxicity by IL-2), suggesting that hrIL-6 augmentation of ADCC activity may not be mediated through IL-2. These results demonstrate that hrIL-6 augments ADCC activity of human PMNC using mAbs to human tumor antigens and human tumor cells as targets, suggesting a potential role for IL-6 in combination with anti-cancer antibodies for cancer immunotherapy.     

Suppressor cell regulation of the spontaneously induced in vitro secondary antibody response.

The cytotoxic effect of peripheral blood lymphocytes from patients with chronic active hepatitis (CAH) on Chang liver cells was studied using a chromium-51 release assay. Cytotoxic effector cell activity was unaffected or slightly enhanced after the removal of glass-adherent mononuclear cells, indicating that the major effector cell is not a classical monocyte. Lymphocytes from 12 of 18 patients with CAH were cytotoxic, and the cytotoxic cells were contained within T-cell-depleted fractions. Further studies revealed that these effector cells were K cells, not B cells. K cells were identified as Fs- and complement receptor-bearing cells without detectable surface immunoglobulin. Serial studies in CAH showed continuous cytotoxicity in contrast to transient cytotoxicity in acute viral hepatitis, suggesting that the cytotoxic activity of lymphocytes is responsible for persistence of the disease.