Expansion of human autologous cytotoxic T lymphocytes on fixed target tumor cells

Human tumor specific cytotoxic T lymphocytes (CTL) were expanded on formalin-fixed autologous target tumor cells derived from glioblastoma multiforme. Growth response of the CTL restimulated with the fixed target cells was larger than those with live target cells. The results suggest that formalin-fixed tumor cells will be stable sources of tumor antigen for efficient autologous CTL expansion and be useful for adoptive immunotherapy of tumors. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Assessment of the number of local cytotoxic T lymphocytes required for degradation of micrometer-size tumor spheroids

Adoptive immunotherapy with human cytotoxic T lymphocytes (CTL) is a promising cancer treatment. Previously we showed that human CTLs against various types of tumors can be efficiently produced by coculturing peripheral blood cells with target cells. The aims of this study were to simulate the interaction of CTLs and micrometer-size tumor tissues in vitro and to assess the required number of CTLs at local tumor sites for degradation of a tumor. Allogeneic CTLs against a human transitional cell carcinoma cell line and autologous CTLs against a renal cell carcinoma cell derived from a surgical specimen were generated. The cytotoxic activities of CTLs against tumor cells in monolayer culture and tumor spheroids formed in U-bottom 96-well culture plates were assessed. Both allogeneic and autologous CTLs showed greater destructive activity than lymphokine activated killer (LAK) cells against target tumor spheroids. CTLs inoculated at E/T ratios of 0.1 to 1 coexisted with the tumor spheroid for 5 to 6 days and then increased in number with apparently lethal activity against the tumor spheroid. In contrast to CTLs, the increase in LAK cell numbers was scarcely observed, and the proliferated LAK cells did not show cytotoxicity against the tumor spheroid. These observations suggest that, when a small number of CTLs reach a local tumor site, they can destroy micrometer-size tumors after considerable local proliferation. This revised version was published online in August 2006 with corrections to the Cover Date.     

A mathematical analysis of the interactions between immunogenic tumor cells and cytotoxic T lymphocytes.

Recent developments of biotechnology have enabled us to use immunotherapy against certain kinds of tumors in patients. However, it is reasonable to doubt if the immunotherapy can completely aid the rejection of tumors that have escaped from the immune system. In this paper, we propose a new mathematical model of tumor immunity by tumor-specific cytotoxic T lymphocytes (CTLs), since tumor-specific CTLs play an important role in tumor immunity. Using this model, we have mathematically investigated the interactions between immunogenic tumor cells (TCs) and tumor-specific CTLs and evaluated the availability of immunotherapies for tumors. The findings herein demonstrate that three kinds of dynamics of tumor immunity exist: i.e. (1) TCs continue to proliferate with CTLs; (2) TCs are rejected by CTLs; and (3) TCs equilibrate with CTLs, but with little possibility of the equilibrium. The findings also demonstrate that a sufficient increase in CTLs by immunotherapy can aid the rejection of TCs, but an insufficient increase in CTLs by immunotherapy causes only a transient regression of TCs. Clinically the findings mean that increasing tumor-specific CTLs, e.g., by vaccination or adoptive transfer of tumor-specific CTLs expanded ex vivo, can theoretically aid the rejection of TCs.     

Melanocyte‐specific,cytotoxic T cell responses in vitiligo: the effective variant of melanoma immunity?

Vitiligo is a relatively common progressive depigmentary condition that is believed to be due to the autoimmune‐mediated loss of epidermal melanocytes. An interesting aspect of vitiligo is its relation to melanoma: cytotoxic T lymphocytes directed to self‐antigens shared by normal melanocytes and melanoma cells are found in both conditions and might prove important in melanocyte destruction, yet the resulting immune reactions are completely different. From this standpoint, the selective destruction of pigment cells that occurs in cases of vitiligo is the therapeutic goal sought in melanoma research. In the present article, we will address these issues by reviewing current literature on the subject as well as by posing some speculations.     

Adoptive transfer of cytotoxic T lymphocytes induced by CD86-transfected tumor cells suppresses multi-organ metastases of C1300 neuroblastoma in mice

 In this study, we examined the therapeutic antitumor effect of cytotoxic T lymphocytes (CTL) generated against CD86-transfected mouse neuroblastoma C1300. We first generated the transfectant, CD86⁺C1300, expressing a high level of mouse CD86 on the cell surface. While CD86⁺C1300 cells were rejected in syngeneic A/J mice when inoculated subcutaneously, neither vaccination nor any therapeutic antitumor effect was obtained, implying that C1300 may be a poorly immunogenic tumor. However, in vitro stimulation of splenocytes from either C1300-bearing or CD86⁺C1300-rejecting mice with CD86⁺C1300 cells resulted in remarkable CTL activity against C1300 cells. The CTL activity induced by CD86⁺C1300 was mediated by T cell receptor/CD3 and CD8 and was further enhanced by the addition of interleukin-2. Intravenous inoculation of C1300 cells led to multiple organ metastases including the liver, lung, kidney, ovary, lymph node and bone marrow. To examine the therapeutic effect of CTL in this metastasis model, CTL induced by parental or CD86⁺C1300 cells were administrated into C1300-bearing mice. Adoptive transfer of CD86⁺C1300-induced CTL resulted in marked elimination of multi-organ metastases and prolonged survival in almost all mice, 70% of which survived indefinitely. These results indicate that adoptive transfer of CTL induced by CD86-transfected tumor cells in vitro would be effective and useful for tumor immunotherapy against poorly immunogenic tumors. Received: 18 November 1996 / Accepted: 3 March 1997     

Comparison of cytotoxic T lymphocyte responses against pancreatic cancer induced by dendritic cells transfected with total tumor RNA and fusion hybrided with tumor cell

Pancreatic cancer (PC) is a deadly human malignancy. Dendritic cell (DC)-based immunotherapy with whole tumor antigens demonstrates potential efficiency in cancer treatment. Tumor RNA and tumor fusion hybrid cells are sources of whole tumor antigens for preparing DC tumor vaccines. However, the efficacy of these sources in eliciting immune responses against PC has not yet to be directly compared. In the present study, patient-derived PC cells and DCs were fused (DC–tumor hybrids) and primary cultured PC cell-derived total RNA was electroporated into autologous DCs (DC–tumor RNA). The antitumor immune responses induced by DC–tumor hybrids and DC–tumor RNA were compared directly. The results showed that both RNA and hybrid methodologies could induce tumor-specific cytotoxic T lymphocyte (CTL) responses, but pulsing DCs with total tumor RNA could induce a higher frequency of activated CTLs and T-helper cells than fusing DCs with autologous tumor cells. In addition, DC–tumor RNA triggered stronger autologous tumor cell lysis than DC–tumor hybrids. It could be concluded that DCs pulsed with whole tumor RNA are superior to those fused with tumor cells in priming anti-PC CTL responses. Electroporation with total tumor RNA may be more suitable for DC-based PC vaccination.     

Transfer of Protection to Murine Typhoid Conferred by L-Form Salmonella typhimurium in Dependence of Cooperation between L Form-Adopted Macrophages and L Form-Induced Lyt-2+ T Cells

The effector cells responsible for protection to Salmonella typhimurium in C3H/HeJ mice, conferred by L-form S. typhimurium, were determined by cell transfer test. Nonfractionated spleen cells from 6-week immune mice but not from 24-week immune animals transferred anti-S. typhimurium immunity. Treatment with anti-macrophage antiserum and complement most effectively abolished protective capacity in 6-week immune cells, while anti-T cell monoclonal antibody plus complement reduced it to a lesser extent. However, adoptive protection was achieved only by transfer of immune macrophages along with Lyt-2⁺ T cells selected from 6-week immune spleen cells. These Lyt-2⁺ T cells were cytotoxic to Kupffer cells from C3H/HeJ mice which had been infected 48 hr previously and from the mice which had been immunized 1 week previously, but not to the cells from 6-week immune mice and from normal animals. Moreover, protective capacity in immune macrophages seemed to be correlated to the degree of colonization by the L forms, and the inability to transfer immunity of 24-week immune spleen cells may be due to the decrease in the L form-colonization. These results suggest that cooperation between the L form-colonized macrophages and L form-induced cytotoxic Lyt-2⁺ T cells contributes to anti-S. typhimurium immunity, and might imply the immunological difference between the 6-week immune phagocytes and the cells at an early stage of infection or immunization.     

The expression of CCL2 by T lymphocytes of mammary tumor bearers: role of tumor-derived factors

Tumor-associated chemokines, including CC chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2), are thought to play many roles in cancer progression. Here we demonstrate the novel finding that during growth of the D1-7,12-dimethylbenzanthracene-3 mammary tumor in BALB/c mice, there is a dramatic up-regulation of CCL2 in splenic T cells at both the mRNA and protein levels upon stimulation. Of particular relevance is the finding that tumor-infiltrating T cells also produce high levels of CCL2. While a variety of tumor cell lines have been found to produce CCL2, we found no detectable levels of CCL2 protein in supernatants of the cultured mammary tumor cells. Investigation of the mechanisms involved in CCL2 induction showed that treatment of splenic T cells with the tumor-derived factors GM-CSF and phosphatidyl serine (PS) resulted in increased CCL2 production. This increased production may be involved in the downregulation of IFN-gamma by the T cells of tumor-bearing mice previously reported in this model, as treatment of splenic T lymphocytes with CCL2 resulted in a decreased secretion of IFN-gamma by those cells.     

Expression of the inflammatory chemokines CCL2, CCL5 and CXCL2 and the receptors CCR1-3 and CXCR2 in T lymphocytes from mammary tumor-bearing mice

Chemokines and their receptors have been studied in several solid tumor models as mediators of inflammation. In turn, inflammation has been implicated in the promotion and progression of tumors, and as such, chemokines have been proposed as novel molecular targets for chemotherapy. While the expression of these molecules has been described in tumor cells, endothelial cells, macrophages and neutrophils, less attention has been paid to the expression profile of these molecules by T lymphocytes in the periphery or infiltrating the tumor. Using the D1-DMBA-3 murine mammary adenocarcinoma model, we aimed to better characterize the differential expression of chemokines and/or their receptors in the host and in the tumor microenvironment, and specifically, in the T cells of tumor-bearing mice compared to normal control animals. We found that T lymphocytes from tumor-bearing mice express the pro-inflammatory chemokines, CCL2, CCL5 and CXCL2, as well as the chemokine receptors, CCR1, CCR2, CCR3 and CXCR2.     

Spontaneous rejection of intradermally transplanted non-engineered tumor cells by neutrophils and macrophages from syngeneic strains of mice

It is not surprising that tumors arising spontaneously are rarely rejected by T cells, because in general they lack molecules to elicit a primary T-cell response. In fact, cytokine-engineered tumors can induce granulocyte infiltration leading to tumor rejection. In the present study, we i.d. injected seven kinds of non-engineered tumor cells into syngeneic strains of mice. Three of them (i.e. B16, KLN205, and 3LL cells) continued to grow, whereas four of them (i.e. Meth A, I-10, CL-S1, and FM3A cells) were spontaneously rejected after transient growth or without growth. In contrast to the i.d. injection of B16 cells into C57BL/6 mice, which induces infiltration of TAMs into the tumors, the i.d. injection of Meth A cells into BALB/c mice induced the invasion of cytotoxic inflammatory cells, but not of TAMs, into or around the tumors leading to an IFN-γ-dependent rejection. On day 5, the cytotoxic activity against the tumor cells reached a peak; and the effector cells were found to be neutrophils and macrophages. The i.d. Meth A or I-10 cell-immunized, but not non-immunized, mice rejected i.p.- or i.m.-transplanted Meth A or I-10 cells without growth, respectively. The main effector cells were CTLs; and there was no cross-sensitization between these two kinds of tumor cells, suggesting specific rejection of tumor cells by CTLs from i.d. immunized mice. These results indicate that infiltration of cytotoxic myeloid cells (i.e. neutrophils and macrophages, but not TAMs) into or around tumors is essential for their IFN-γ-dependent spontaneous rejection.     

Role of perforin, granzymes and the proliferative state of the target cells in apoptosis and necrosis mediated by bispecific-antibody-activated cytotoxic T cells

 Bispecific monoclonal antibodies (bi-mAb), directed against a tumor-associated antigen and the CD3 or CD28 antigen on T lymphocytes, induce activation of resting T lymphocytes and target-specific tumor cell lysis. We now show that both necrosis and apoptosis contribute to T-cell-mediated tumor cell destruction. Even though T cells up-regulate FAS/APO-1 expression upon bi-mAb stimulation, FAS/APO-1-mediated apoptosis does not contribute to bi-mAb-mediated destruction of Hodgkin’s cells. CD8⁺ lymphocytes were the most potent effectors of bi-mAb-mediated cytotoxicity and had the highest levels of mRNA coding for perforin and granzyme A and B. Ca²⁺-complexing agents, which abrogate perforin activity, led to decreased levels of necrosis, while inhibition of granzyme activity in effector or target cells had a similar effect on apoptosis. Granzyme-mediated apoptosis critically dependent on the proliferative state of the target cells, while perforin-induced necrosis was not cell-cycle-dependent. Our results underline the importance of the expression levels of perforin and granzymes in the effector T cells and of the proliferative state of the target cells in bi-mAb-mediated apoptosis and necrosis of tumor cells. Received: 5 December 1996 / Accepted: 16 January 1997     

The mouse chorionic gonadotropin beta-subunit-like (muCG beta l) molecule produced by tumor cells elicits the switch of T-cell immunity response from TH2 to TH1 in mice immunized with DNA vaccine based on rhesus monkey homologous CG beta (rmCG beta)

BACKGROUND: CG beta is expressed not only in placenta, but also in a wide range of tumors. To study DNA vaccine based on xenogeneic CG beta for cancer immuno-therapy, we investigated whether rhesus monkey CG beta (rmCG beta) DNA vaccine could induce protective T-cell responses and humoral responses in mouse. METHODS: We constructed a plasmid containing the rmCG beta coding sequence. Two cloned syngeneic SP2/0 myeloma cell lines that stably express muCG beta l (SP2/0-muCG beta l) and HN (SP2/0-HN) protein were established. Inoculation of these cell lines was made into mice that had been immunized with DNA vaccine. Specific IgG and IgG type were measured by ELISA and the cytokine expression was detected with RT-PCR. To measure the lymphocyte metabolic activity, the MTS assay was used. RESULTS: After injection of SP2/0-muCG beta l into mice that had been immunized with DNA vaccine, a significant increase in the IgG2a specific to the antigen (p < 0.05) and a decrease in the specific IgG1 (p < 0.05) were measured. The expression of T(H)1 but not T(H)2 cytokines, including IFN-gamma and IL-2, were detected in the splenocytes. However, injection of tumor cells expressing irrelevant or mock molecules into immunized mice could not induce these changes. The survival rate of vaccine-immunized mice injected with SP2/0-muCG beta l was as high as 58.3% after 55 days. CONCLUSIONS: The rmCG beta DNA vaccine has proved to be a potential strategy for protection against tumors with homologous molecules. The muCG beta l produced by tumors is able to elicit an immunity switch from T(H)2 to T(H)1 in vaccinated mice.     

Identification of hepatitis B virus-specific CTL epitopes presented by HLA-A33:03 in peripheral blood mononuclear cells from patients and transgenic mice

Cytotoxic T lymphocyte (CTL) epitopes in the HBV protein of hepatitis B virus (HBV) may play a key role in viral control and liver damage. The aim of this study was to identify and study the function of HLA-A^∗33:03-restricted CTL epitopes in HBV protein of the HBV genotypes B and C, which are epidemic in China. Sixteen HBV peptides were predicated by computational analysis, and synthesized peptides were examined for their affinity to HLA-A^∗33:03 using a stable cell line. After being analyzed by enzyme-linked immunospot and cytolytic activity assays, as well as the tetramers staining method using peripheral blood mononuclear cells isolated from HBV-infected patients, five peptides (Hbs245–253, HBs335–343, HBc119–127, HBc104–112, and HBp391–399) were chosen to further confirm their HLA_A^∗33:03 restriction in transgenic mice.     

Mechanism of target cell lysis by cytotoxic T lymphocytes (CTL) employing RSV-induced H-2 congenic and recombinant mouse tumor cells: demonstration of soluble mediators released from H-2-restricted CTL clones

The secretion and the specificity of cytotoxic mediators from H-2-restricted cytotoxic T lymphocytes (CTL) were examined using non-virus-producing target tumor cells induced by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) in B10 congenic and recombinant mice. By using rat concanavalin A supernatant, two H-2-restricted CTL clones were established from cytotoxic effector cells of B10.A(5R) mice primed with SR-RSV-induced syngeneic tumor Cell-free supernatants from the H-2-restricted CTL clones cocultured with syngeneic tumor cells had selectively high cytotoxic activity for syngeneic and H-2-compatible tumor cells, but not for H-2-incompatible tumor cells. YAC-1 cells, and B10.A(5R) blasts as defined in the 5-hr 51Cr-release assay. The cytotoxic activity was detected in the cell-free supernatants from the CTL clones cocultured with the CTL-sensitive syngeneic and H-2-compatible tumor cells, but not with the CTL-insensitive tumor cells and YAC-1 cells. The cytotoxic activity of the cell-free supernatant could be adsorbed by the syngeneic tumor cells, but not by YAC-1 and L(s) cells. Thus, the H-2-restricted CTL clones against SR-RSV-induced tumor cells were capable of releasing cytotoxic mediators by coculturing with syngeneic or H-2-compatible tumor cells, and the cytotoxic mediators showed a certain H-2-restricted manner in killing the target cells. These results suggest that the lysis of RSV-induced tumor cells by H-2-restricted CTL can at least in part be mediated by cytotoxic factors.     

Hepatic cellular immune responses in mice with "cure" and "non-cure" phenotype to Leishmania infantum infection: importance of CD8+ T cells and TGF-beta production

The objective of this study was to analyse hepatic cellular immune response of mice with "cure" and "non-cure" phenotypes to Leishmania infantum infection. During infection establishment, elevated TGF-beta levels and absence of a Th1 response may have contributed to parasite multiplication and to similar hepatic parasitic loads. Later in infection, an increase in the number and activation levels of CD8+ cells was observed simultaneously with parasite elimination, but only significant in "cure" strain. During this recovering phase, "non-cure" animals showed low Th2 cytokine levels, while TGF-beta production was higher than in "cure" mice. These results point out to a role for CD8+ T cells in liver acquired immune response and to TGF-beta regulation of "cure" and "non-cure" phenotype to L. infantum infection.     

Characterization of the cells that suppress the cytotoxic activity of T lymphocytes. II. Physical properties, specificity, and developmental kinetics of the inhibitory splenic non-T cell population.

The in vivo immune response to alloantigens (tumor allograft reaction and acute graft-versus-host reaction) generates a population of antigen-specific “killer” T cells (CTL) and a separate activity (INA) inhibiting target cell lysis in vitro. Using a simple mathematical model to quantitate each activity from ⁵¹Cr-release titration curves, we show that cells with inhibitory activity differ from CTL in several respects: INA resists destruction by RAMB + C, acts without specificity for the sensitizing alloantigen, and appears to act by a cell-contact mechanism which does not require that the inhibitory cells remain viable. Cells with INA produce noncompetitive inhibition, in contrast to the competitive inhibition produced by alloantigen. Velocity sedimentation separation indicates that INA is quite heterogenous; normal spleen contains low levels of INA, which is associated with small cells (s ? 3 mm/hr). Increased INA develops in a regular manner during an immune response and is associated with a larger inhibitor population (s > 4 mm/hr). Separation on plastic dishes and with carbonyl iron powder demonstrates that INA is not restricted to macrophages.