Bispecific-monoclonal-antibody-directed lysis of ovarian carcinoma cells by activated human T lymphocytes

Summary Two different bispecific hybrid antibodies were established by fusing a hybridoma producing monoclonal antibody (mAb) against the pancarcinoma antigen KS1/4 with either of the two hybridomas OKT3 and 9.3, secreting antibodies reactive with the T cell determinants CD3 and CD28, respectively. The KS1/4 antibody reacts with a 40-kDa cell-surface glycoprotein antigen that is expressed on the surface of a variety of adenocarcinoma cells, including ovarian carcinoma. The ability of the bispecific antibodies 9.3KS1/4 and OKT3KS1/4 to direct peripheral blood mononuclear cells (PBMC) specifically against OVCAR-3 ovarian carcinoma target cells was measured in a 4-h⁵¹Cr-release assay. The bispecific antibodies were four to six times more potent in killing the OVCAR-3 target cells when compared to their parental antibodies either alone or in combination. A dose-dependent response was observed in the 10–10000 ng/ml range. The specificity of the targeting was demonstrated by the complete inhibition of cytotoxic activity following pre-incubation of tumor target cells with the parental mAb and by the lack of killing of KS1/4-negative target cell lines. An evaluation of the efficacy of PBMC from ovarian cancer patients as effector cells revealed that their specific cytotoxicity against OVCAR-3 cells was enhanced severalfold by bispecific antibodies as compared to parental antibodies. Furthermore, stimulation of PBMC with immobilized CD3 and interleukin-2 for 4 days resulted in an enhanced directed killing of human ovarian carcinoma cells by human T effector cells and the bispecific antibodies.     

Dendritic cells pulsed with gp96-peptide complexes derived from human hepatocellular carcinoma (HCC) induce specific cytotoxic T lymphocytes

Dendritic cells (DCs) are one of the most potent antigen-presenting cells (APCs) capable of activating immune responses. Different forms of tumor antigens have been used to load DCs to initiate tumor-specific immune responses. Heat shock proteins (HSPs) are considered natural adjuvants which have the ability to chaperone peptides associated with them presented efficiently by interaction with professional APCs through specific receptors. In the present study, we used HSP, gp96-peptide complexes, derived from human hepatocellular carcinoma (HCC) cells as antigens for pulsing DCs. We found that gp96-peptide complexes derived from HCC cells induced the maturation of DCs by enhancing expression of human leukocyte antigen class II, CD80, CD86, CD40, and CD83. The matured DCs stimulated a high level of autologous T cell proliferation and induced HCC specific cytotoxic T lymphocytes, which specifically killed HCC cells by a major histocompatability complex (MHC) class I restricted mechanism. These findings demonstrate that DCs pulsed with gp96-peptide complexes derived from HCC cells are effective in activating specific T cell responses against HCC cells.     

Study of HLA class I restriction and the directed antigens of cytotoxic T lymphocytes at the tumor sites of ovarian cancer

The molecular basis of T-cell-mediated recognition of ovarian cancer cells remains to be fully addressed. In this study we investigated HLA class I restriction and directed antigens of cytotoxic T lymphocytes (CTL) at the sites of ovarian cancer. Three HLA-class-I-restricted CTL lines were established from the tumor sites of ovarian cancer by culturing tumor-infiltrating lymphocytes or tumor-associated ascitic lymphocytes with interleukin-2: (1) HLA-A2402-restricted and ovarian-adenocarcinoma-specific CTL, (2) HLA-A2-restricted CTL recognizing histologically different cancers, and (3) HLA-B52-restricted and ovarian-cancer-specific CTL. HLA-A0201, HLA-A0206 and HLA-A0207 tumor cells were lysed by the HLA-A2-restricted CTL. HLA-B52 restriction of the third CTL line was confirmed by the transfection of HLA-B5201 cDNA into the tumor cells. The HLA-A2-restricted CTL recognized the SART-1, but not the MAGE-1 or MAGE-3 antigen. These results may facilitate a better understanding of the molecular basis of tumor-specific immunity at the tumor site of ovarian cancer. Received: 30 December 1998 / Accepted: 2 March 1999     

Recycled addition of CD4+ T cell-rich population for induction of human autologous cytotoxic T lymphocytes: A practically efficient method

When CD4⁺ T cell-rich population appears in theinitial trial in induction cultures of humanautologous cytotoxic T lymphocytes (CTL), the cultureresults frequently in no or weak killing activity andtherefore usually be discarded as an `unsuccessful'CTL induction culture. However, addition of theinitial CD4⁺ T cell-rich population enabledefficient induction of the autologous CTL in theensuing trials. The CTL thus generated exhibitedstronger killing activities against autologous braintumor cells and ovarian tumor cells than previouslyobserved. This simple recycling of the primed butinert CD4⁺ T cell-rich population for CTLinduction will promote clinical practice of adoptiveimmunotherapy of human tumors with autologous CTL.     

Detection of oligoclonal T lymphocytes in lymph nodes draining from advanced non-small-cell lung cancer

Despite the combined use of surgery and chemoradiotherapy, the poor prognosis of advanced non-smallcell lung cancer (NSCLC) requires the definition of new therapeutic approaches. The presence of T lymphocytes, with peculiar phenotypic, functional and molecular characteristics within the tumour, suggested the possible use of these cells, expanded in vitro, in protocols of adoptive immunotherapy. We have described how a population of oligoclonal T lymphocytes, derived from advanced NSCLC, can be expanded in vitro and has the capability of lysing autologous cancer cells. What is more important, we observed that patients with advanced NSCLC, treated with TIL expanded in vitro and recombinant interleukin-2, seemed to have a disease-free period longer than that of patients treated with conventional chemoradiotherapy. in an attempt to find new sources of specific lymphocytes for immunotherapy, we describe the analysis of the phenotypic, functional and molecular characteristics of T lymphocytes, derived from lymph nodes draining advanced NSCLC. In this paper we show that these cells, have restriction patterns of T cell receptor chain similar to those detectable in the population of infiltrating T lymphocytes. This finding suggests that T cells derived from draining lymph nodes of advanced NSCLC have peculiar characteristics and can be a suitable source of effector cells for protocols of adoptive immunotherapy in lung cancer treatment.     

Implications of HIV specific cytotoxic T lymphocytes in AIDS

The immune response to HIV in infected humans leads to the production of HIV specific cytotoxic T lymphocytes (CTL) which circulate in high frequencies. The presence of these CTL and their eventual protective activities have been studied by various laboratories, and correlations have been made with certain immunopathological manifestations of HIV infections. It seems probable that HIV-immune CTL participate in the induction of certain disorders by initiating inflammatory reactions in the lungs, central nervous system, and lymph nodes. Various virus antigens recognized by HIV-immune CTL on the surface of the infected cell have been identified, and the molecular definition of the epitopes recognized is well under way. Likewise, numerous HLA transplantation antigens that regulate HIV antigen recognition by CTL have been identified. These data are discussed in view of the development of an eventual vaccine and of functional immunotherapies. They are compared with results obtained in animal experimental systems.Deceased     

Increase in the ability of human cancer cells to induce cytotoxic T lymphocytes by ultraviolet irradiation

The roles of ultraviolet-B (UV) radiation in the immunogenicity of human cancer cells have not been fully studied. We have investigated the effects of UV radiation on metastatic melanoma and renal cell carcinoma cells with regard to MHC antigen expression and the ability to induce cytotoxic T lymphocyte (CTL) activity in peripheral blood mononuclear cells (PBMC) or tumor-infiltrating lymphocytes (TIL) against untreated autologous tumor cells. UV radiation respectively decreased or increased MHC class I expression of freshly isolated tumor cells or cultured tumor cells, and also decreased MHC class I expression of starved cultured tumor cells. It increased the ability of both freshly isolated and cultured tumor cells to induce CTL activity from PBMC against untreated autologous tumor cells. UV-irradiated subclones that were more susceptible to CTL lysis were more potent for CTL induction from TIL than either an untreated parental clone or a UV-irradiated subclone that was resistant to CTL lysis. In summary, UV radiation increased the ability of tumor cells to induce CTL activity without a corresponding effect on MHC antigen expression.This work was supported in part by a grant CA47891 from the National Cancer Institute, USA, a grant-in-aid of the comprehensive 10-years strategy for cancer control from ministry of a Health and Welfare, Japan, and the Ishibashi Research Fund, Japan     

The human cathelicidin, LL-37, induces granzyme-mediated apoptosis in cytotoxic T lymphocytes

LL-37 is a human cationic host defense peptide (antimicrobial peptide) belonging to the cathelicidin family of peptides. In this study, LL-37 was shown to kill stimulated CD8⁺ T cells (Cytotoxic T lymphocytes; CTLs) via apoptosis, while having no cytotoxic effect on non-stimulated CD8⁺ or CD4⁺ T cells or stimulated CD4⁺ T cells. Of interest, the CD8⁺ cells were much more sensitive to LL-37 than many other cell types. LL-37 exposure resulted in DNA fragmentation, chromatin condensation, and the release of both granzyme A and granzyme B from intracellular granules. The importance of granzyme family members in the apoptosis of CTLs following LL-37 treatment was analyzed by using C57BL/6 lymphocytes obtained from mice that were homozygous for null mutations in the granzyme B gene, the granzyme A gene, or both granzymes A and B. Granzymes A and B were both shown to play an important role in LL-37-induced apoptosis of CTLs. Further analysis revealed that apoptosis occurred primarily through granzyme A-mediated caspase-independent apoptosis. However, caspase-dependent cell death was also observed. This suggests that LL-37 induces apoptosis in CTLs via multiple different mechanisms, initiated by the LL-37-induced leakage of granzymes from cytolytic granules. Our results imply the existence of a novel mechanism of crosstalk between the inflammatory and adaptive immune systems. Cells such as neutrophils, at the site of a tumor for example, could influence the effector, activity of CTL through the secretion of LL-37.     

Immortalization of human T lymphocytes by oncogenes

Immortalized human T cell lines were established by cotransfecting c-Ha-ras and c-myc oncogenes to lymph node lymphocytes. The cell lines kept growing for 3 months after establishment without a decrease in growth rate. The cells did not require interleukin-2(IL-2) for their growth, but addition of IL-2 stimulated the growth of these cells. Flow cytometric analysis revealed that these cells were T cells expressing CD4 or CD8 antigens. A CD4 positive (CD4⁺) cell line produced IL-6, indicating that the cell line belongs to helper T cells. The CD8 positive (CD8⁺) cell line possessed cytotoxicity to tumor cells, indicating that the cell line were killer T cells. Both cell lines were able to proliferate in serum-free medium indefinitely.     

Cytokine biosynthesis by tumor-infiltrating T lymphocytes from human non-small-cell lung carcinoma

The purpose of this work was to assess the capacity of tumor-infiltrating leukocytes (TIL) from human non-small-cell lung carcinoma (NSCLC) specimens to synthesize type-1 and type-2 cytokines. Methods: TIL were isolated from tumors following digestion with collagenase/DNase and further enriched by ficoll-hypaque gradient centrifugation. Membrane phenotypes and intracellular cytokine protein expression of TIL were assessed by flow cytometry. Results: The majority of TIL expressed the CD3 antigen with a CD4:CD8 ratio of approximately 2:1. Other leukocytes such as macrophages (CD14), B lymphocytes (CD20), and natural killer (NK) cells (CD56) were also found to infiltrate the tumors, but in significantly lower numbers. Owing to the limited recovery of non-CD3⁺ leukocytes, our analysis of cytokine biosynthesis has focused on T lymphocytes. In the absence of activation, a small percentage of CD3⁺ TIL synthesized cytokines ( <4%). Following activation with anti-CD3+interleukin-2 (IL-2), CD3⁺ TIL synthesized predominantly a type-1 cytokine profile; however, the type-2 cytokines, IL-6 and IL-10, were also detected in a small percentage of infiltrating cells. Following activation with phorbol 12-myristate 13-acetate + ionomycin, CD3⁺ TIL also expressed more type-1 than type-2 cytokines and in significantly greater numbers of cells. The CD3⁺CD8⁺ component of the TIL synthesized only type-1 cytokines, whereas the CD3⁺CD4⁺ component synthesized both type-1 and type-2 cytokines. Conclusion: These results show that the majority of the TIL isolated from NSCLC specimens are T lymphocytes with the capacity to synthesize type-1 cytokines. Received: 24 March 1999 / Accepted: 9 September 1999     

Clonal analysis of in vivo activated CD8+ cytotoxic T lymphocytes from a melanoma patient responsive to active specific immunotherapy

To study in vivo activated cytolytic T cells, CD8⁺ T cells clones were isolated from a melanoma patient (HLA A2, A11) treated with active specific immunotherapy for 5 years. CD8⁺ T lymphocytes, purified by fluorescence-activated cell sorting, were cloned directly from the peripheral blood without antigen-presenting cells in the presence of irradiated autologous melanoma cells and recombinant interleukin-2 (IL-2) and IL-4. These conditions were inhibitory to de novo in vitro immunization. Of the 28 cytolytic CD8⁺ T cell clones, 21 lysed the autologous melanoma cell line (M7) but not the autologous lymphoblastoid cell line (LCL-7) nor the two melanoma cell lines, M1 (HLA A28) and M2 (HLA A28, A31), used to immunize the patient. The remaining 7 clones were also melanoma-specific, although their reactivities were broader, lysing several melanoma cell lines but not HLA-matched lymphoblastoid cells. Eight clones from the first group, ostensibly self-MHC-restricted, were expanded for further analysis. All expressed cluster determinants characteristic of mature, activated T cells, but not those of thymocytes, naive T cells, B cells or natural killer (NK) cells. They also expressed CD13, a myeloid marker. Of the 8 clones, 3 expressed both CD4 and CD8, but dual expression was not correlated with specificity of lysis. Two CD8⁺ and 2 CD4⁺ CD8⁺ clones were specific for the autologous melanoma cells, the other 4 were also reactive against other HLA-A2-positive melanomas. Cytotoxicity for both singly and doubly positive clones was restricted by HLA class I but not class II antigens. Analysis of the RNA expression of the T cell receptor (TCR) V and V gene segments revealed heterogeneous usage by the A2-restricted clones and, perhaps, also by the broadly melanoma-specific clones. Apparent TCR-restricted usage was noted for the self-MHC-restricted clones; 2 of the 4 expressed the V17/V7 dimer. Since the T cell clones were derived from separate precursors of circulating cytotoxic T lymphocytes (CTL), the V17/V7 TCR was well represented in the peripheral blood lymphocytes of this patient. In summary, we show that melanoma cells presented their own antigens to stimulate the proliferation of melanoma-reactive CD8⁺ CTL. CTL with a range of melanoma specificities and different TCR dimers were encountered in this patient, perhaps as a result of hyperimmunization. Restricted TCR gene usage was noted only for classical self-MHC-restricted CD8⁺ T cell clones, although lysis of the autologous melanoma cells was effected by a variety of TCR structures. Molecular definition of the TCR repertoire of well-characterized T cell clones in this and other patients should provide new insight into the human antitumor immune response.Supported by National Institutes of Health research grants CA 36233 and EY 9031, the Lucy Adams Memorial Fund and a grant from the Concern Foundation     

The induction of murine tumor infiltrating lymphocytes (TIL) by interleukin-2 or T cell growth factor

Mice were injected in the foot pad with either 5×10⁵ syngeneic plasmacytoma (MOPC104E) or fibrosarcoma cells (Meth A). Lymph nodes containing tumor cells were harvested 14 days later and cultured. In the presence of recombinant interleukin-2 (r-IL-2) predominantly tumor cells proliferated. Culture with T cell growth factor (TCGF) resulted in the growth of lymphoid cells. Concanavalin A (Con A) had only a modest effect on elimination of tumor cells in the culture. Tumor-infiltrating lymphocytes (TIL) prepared from the lymph nodes showed specific tumor-neutralizing activity when grown in the presence of TCGF. In vitro examination revealed that Meth A cells could not be lysed by TIL, while TIL from MOPC tumors showed tumor specific activity. This study may explain negative results in human trials with TIL induced by IL-2 alone.Abbreviations r recombinant - IL-2 interleukin-2 - TCGF T cell growth factor - TIL tumor infiltrating lymphocytes - Con A concanavalin A - HBSS Hanks' balanced salt solution     

Analysis of NK cells and chemokine receptors in tumor infiltrating CD4 T lymphocytes in human renal carcinomas

Recent data suggest that chemokines and chemokine receptors mediate leukocyte recruitment of all components of the antitumor response. This study aimed to phenotypically characterize the immune lymphocyte infiltrate in human renal cell carcinomas (RCCs) and at the invasive margin (tumor–host interface) and to define the association of these findings with established prognostic indicators. Tumor infiltrating lymphocytes (TILs) were obtained from 24 patients with RCC undergoing radical nephrectomy. Peripheral blood cells from 37 patients were also obtained before surgery. Our findings are consistent with the preferential recruitment of CD4+ Th1-polarized effector memory cells that express CXCR3/CCR5. These cells were the main component of TILs and expressed as CXCR3, CCR5, CD45RO, and CD95. Natural killer (NK) cells were found in significantly higher proportions in TILs of RCCs than in peripheral blood lymphocytes (PBLs) or in other tumors studied (colorectal and breast cancers), where these cells were found in small proportions. No differences in nuclear grade or other studied parameters were observed between the TILs and the lymphocytes present at the invasive margin, which showed a similar composition. However, differences were found according to the tumor stage. First, significantly fewer NK cells were observed in PBLs from metastatic patients. Second, a significantly lower proportion of CCR5/CXCR3/CD4+ cells and a higher proportion of CCR4/CD4+ cells were observed in metastatic patients, suggesting that preferential Th1-polarization may gradually change during the progression of renal cancer cells. Finally, the frequency of CD25/CD4+ cells was higher in metastatic patients. Although the sample of patients with metastasis was small, the overall results suggest a change in composition of the TILs that may potentially confer a selective advantage for tumor growth and may account for the suppression of an effective cytotoxic response.     

Comparative analysis of cytotoxic T lymphocyte response induced by dendritic cells loaded with hepatocellular carcinoma -derived RNA or cell lysate

The choice of the tumor antigen preparation used for dendritic cell (DC) loading is important for optimizing DC vaccines. In the present study, we compared DCs pulsed with hepatocellular carcinoma (HCC) total RNA or cell lysates for their capacity to activate T cells. We showed here that HCC total RNA pulsed-DCs induced effector T lymphocyte responses which showed higher killing ability to HCC cell lines, as well as higher frequency of IFN-γ producing of CD4+ and CD8+ T cells when compared with lysate pulsed-DCs. Both of RNA and lysate loading did not influence the changes of mature DC phenotype and the capacity of inducing T cell proliferation. However, HCC lysate loading significantly inhibited the production of inflammatory cytokines IL-12p70, IFN-γ and enhanced the secretion of anti-inflammatory cytokines IL-10 of mature DCs. Our results indicated that DCs loaded with HCC RNA are superior to that loaded with lysate in priming anti-HCC CTL response, suggesting that total RNA may be a better choice for DCs-based HCC immunotherapy.     

CD3- bone marrow cells augment the generation of cytotoxic T lymphocytes showing a preference for the X-chromosome linked gene product of stimulator cells

Responder cells, composed of both a limited number of nylon wool-passed lymph node (NW-LN) cells and an excess number of CD3+ cell-depleted bone marrow (CD3- BM) cells from the same strain of mice, were stimulated with allogeneic spleen cells in vitro. The CD3- BM cells augmented the generation of allogeneic major histocompatibility complex (MHC) class I specific cytotoxic T lymphocytes (CTL) from NW-LN cells. C3H/He (H-2k, C3H background) responder cells were stimulated with either B10.D2 (H-2d, B10 background) or BALB/c (H-2d, BALB background) spleen cells. In the former stimulation, the CTL induced lysed B10.D2 target cells more efficiently than the BALB/c cells. Furthermore, these CTL lysed more (B10.D2 x BALB/c) F1 male target cells than (BALB/c x B10.D2) F1 male. In the latter stimulation, the CTL lysed more BALB/c than B10.D2 cells, and more (BALB/c) x B10.D2) F1 male than (B10.D2 x BALB/c) F1 male. The reciprocal mixed lymphocyte cultures (MLC) were carried out, in which BALB/c responder cells were stimulated with either C3H/He or B10.BR (H-2k, B10 background) spleen cells. In the former stimulation, the CTL induced lysed more C3H/He or (C3H/He x B10.BR) F1 male target cells than B10.BR or (B10.BR x C3H/He) F1 male, and in the latter, the reciprocal results were obtained. These results suggested that the CTL induced had a preference for the X-chromosome linked gene products (Xlgp), besides the specificity for the allogeneic MHC class I, of the mice used as stimulator.     

Culture of tumour-infiltrating lymphocytes from melanoma and colon carcinoma: Removal of tumour cells does not affect tumour-specificity

The therapeutic potential of adoptive therapy using tumour-infiltrating lymphocytes (TIL) has been demonstrated in a number of clinical trials. However, freshly isolated tumour-infiltrating lymphocytes (TIL) are often impaired in their proliferative and cytotoxic responses, which limits their use in immunotherapy. Several hypotheses with regard to the poor effector function of TIL have been postulated, including the production of immunosuppressive factors by tumour cells. In a previous paper we reported the efficient expansion of immunoreactive TIL from a variety of solid tumours by stimulation with a combination of monoclonal antibodies (mAbs) against CD3 and CD28. In the present study we analysed whether this protocol would be improved by the removal of tumour cells at the start of the culture. We tested a highly immunogenic tumour, melanoma, and a poorly immunogenic tumour, colon carcinoma. Removal of tumour cells highly improved anti-CD3/CD28 stimulated expansion of TIL from colon carcinoma, resulting in a significantly higher percentage of potentially tumour-specific CD8-positive T-cells and a reduced CD4/CD8 ratio compared to expansion in the presence of tumour cells. In contrast, expansion and CD4/CD8 ratio of melanoma-derived TIL was not significantly influenced by the removal of autologous tumour cells. CD3/CD28-stimulated melanoma TIL cultured in the absence of tumour cells showed specific lysis of autologous tumour cells comparable to melanoma TIL cultured in high-dose IL2. However, no cytotoxicity could be detected in colon TIL irrespective of the culture conditions used. On the other hand, 3/8 colon carcinoma TIL cultures and 9/12 melanoma-derived TIL cultures showed IFN secretion upon stimulation with autologous tumour cells. We conclude that stimulation of TIL with a combination of mAbs to CD3 and CD28 in the absence of tumour cells induces efficient expansion of potentially tumour-specific cells from a highly and a poorly immunogenic tumour. Removal of tumour cells does not have a negative influence on the generation of tumour-specific T cells, while cell yield improves. Therefore, for large-scale cultures this protocol can efficiently induce the outgrowth of tumour-specific TIL, at the same time providing a useful source of autologous tumour cells that can be stored and used to direct or test antitumour specificity.     

Differential calcium response in HeLa and HeLa-Fas cells by cytotoxic T lymphocytes

We constructed a CD95 overexpressing HeLa cell line which was extremely sensitive towards CD95 mediated apoptosis. In these CD95 overexpressing cells, CD95 blocks the nuclear calcium signal induced by perforin positive and CD95 ligand positive killer cells. This phenomenon is highly relevant in states of inflammatory syndromes such as systemic inflammatory response syndrome (SIRS) and sepsis which are associated with a high probability to reactivate latent viruses due to a functional deficiency of cytotoxic effectors.