Paul von Hoegen

Modification of tumor cells by a low dose of Newcastle disease virus. III. Potentiation of tumor-specific cytolytic T cell activity via induction of interferon-alpha/beta.

Abstract To investigate possibilities of augmenting tumor-specific immune responses against the highly metastatic murine lymphoma ESb, we tested the effects of the interferon inducer newcastle disease virus (NDV) or of interferon- α β as costimulator in mixed lymphocyte-tumor cell cultures (MLTC) on the tumor-specific cytolytic T cell (CTL) response. Both approaches, namely stimulation of ESb immune spleen cells with NDV-modified stimulator cells or with ESb stimulator cells and exogenous IFN- α β , led to a selective potentiation of tumor-specific CTL activity. The potent activation of tumor-specific CTL precursor (CTLP) required the simultaneous presence of the specific ESb tumor antigen—possibly to mediate a signal via the corresponding T cell receptor—and costimulators—possibly to mediate second activation signals. Increased CTL activity required only very low amounts of NDV or IFN- α β . The generation of CTL activity in the MLTC cultures could be blocked by antisera to IFN- α β , not, however by control sera. Similar effects were observed in vivo , suggesting that IFN- α β not only caused an increase in CTL activity, but was essential for the generation of CTL activity. The reduction of the generation of CTL by antiserum to IFN- α β could be overcome by excess interferon, especially when using ESb-NDV as stimulator Cells.

Synergistic role of type I interferons in the induction of protective cytotoxic T lymphocytes

Abstract Differentiation of cytolytic T cells can be supported by type I and type II interferons (IFN). To characterize the role of type I interferons further we tested the role of recombinant IFN-α and IFN-β on the induction of a weak immune response, against a low immunogenic tumor, which has been shown to be increased by IFN. Both type I interferons IFN-α and IFN-β were able to support the differentiation of cytolytic T lymphocytes (CTL). In case of IFN-α no correlation with the antiviral activity could be seen by comparison of IFN-α 1 and IFN-α4. The maximal in vitro effects were achieved with very low concentrations in the range of 1–100 IU/ml. IFN-α showed the strongest effects, if added in the early phase of the mixed leukocyte culture, whereas IFN-β was most effective when given at the last day of the culture. In combination, both IFNs gave additional/synergistic effects, whereby addition of IFN-α at day 0 and IFN-β at day 4 led to maximal specific CTL responses. In vivo augmentation of the anti-tumor immune response by both types of IFNs supported the in vitro findings and also the synergistic effect of both types of IFNs could be demonstrated. Therefore we propose that IFN-α is relevant in the induction of CTL responses, i.e., the conversion of precursor T cells into mature cells and growth promotion whereby IFN-β might trigger the lytic machinery of the cells and promote differentiation. This synergistic efficacy is also operative in tumor rejection.

Tumour-specific CTL response requiring interactions of four different cell types and recognition of MHC class I and class II restricted tumour antigens

This study demonstrates that a syngeneic specific cytotoxic T lymphocyte (CTL) response to a class I major histocompatibility complex (MHC) positive tumour requires dual processing and recognition of tumour antigens. One type of antigen is processed and expressed in association with class I MHC at the surface of intact tumour cells. It is recognized by CD8 α,β TCR CTL in vitro and by protective immune T cells in vivo and thus functions as a tumour‐associated transplantation antigen (TATA). The other type of antigen is processed and expressed by distinct host APC in association with class II MHC. This is recognized by immune CD4 T cells which function as essential helper cells in the generation of the CD8 CTL response. These conclusions are supported by cell depletion and reconstitution experiments as well as by blocking experiments with monoclonal antibodies using the highly metastatic class II negative murine lymphoma ESb as a model system. The existence of two types of cognate T cell responses in a syngeneic anti‐tumour response was directly proved by the establishment of two types of tumour specific T cell lines which required as co‐stimulator either MHC class II positive APC or IL‐2. In suboptimal mixed lymphocyte tumour cell cultures either of these co‐stimulator functions was found to be limiting the overall anti‐tumour CTL response. The generation of the tumour specific CTL response could be blocked by monoclonal antibodies against all the molecules involved in the cognate interactions (i.e. class I MHC, CD8, class II MHC, CD4 and TCR) but not by anti‐CD2 or anti‐IgG. The strict requirement for helper cells and APC could be bypassed by the addition of recombinant IL‐2 but optimal triggering of CD8 CTL‐precursor required viable tumour stimulator cells. This well characterized in vitro assay may be useful (i) for monitoring the immune status of CD4 and CD8 immune T cells separately, for instance of tumour bearing and/or treated animals and (ii) for the development and testing of potent tumour cell vaccines with T cell stimulatory and/or co‐stimulatory activities.

New antigens presented on tumor cells can cause immune rejection without influencing the frequency of tumor-specific cytolytic T cells

The specific immune response against syngeneic tumors by T cells is dependent on the existence of tumor-associated transplantation antigens (TATA). In the case of the chemically induced DBA/2-derived lymphoma Eb and its highly metastatic variant ESb the immunogenicity of these antigens is not sufficient to prevent tumor growth. Therefore we tested in two systems the influence of additional antigens as possible helper determinants for the generation of tumor-specific immune responses. In the Eb tumor system additional antigens were induced by mutagenization. The frequency of cytotoxic T lymphocytes (CTL) in response to mutagenized Eb cells was higher than that in response to untreated Eb cells. Fine specificity analysis revealed there there was no increase in the CTL response against the original TATA, but an activation of additional CTL clones responding to mutagen-induced antigens. In the ESb tumor system we tested the effect of additional recognition of minor histocompatibility antigens on the frequency of TATA-specific CTL. Transplantation of ESb tumor cells into B10.D2 mice, which are H-2-identical but differ in minor antigens, results in strong tumor rejection responses. In a limiting dilution mixed-leukocyte-tumor microculture system it was found that the minor antigens are recognized at the clonal level as independent antigens. The overall frequency of anti-tumor CTL in ESb-immunized B10.D2 mice was about 1/3000. Among these, the frequency of TATA-specific CTL was 1/16,709 and thus not significantly different from that of syngeneic DBA/2 mice. Thus neither minor antigens nor mutagen-induced antigens acted in the Eb/ESb tumor system as helper determinants and did not increase the frequency of tumor-specific CTLs.

Deficiency in T cell responses of human fetal lymph node cells: A lack of accessory cells

Lymph node and spleen cells of human foetuses from the 18th to the 24th week of gestation were analysed with regard to their phenotypes and their functional capacities. Fetal mesenteric lymph nodes contain high percentages of CD45RA+ T cells and few B cells and monocytes, whereas the fetal spleen is comprised of equal numbers of T and B cells as well as monocytes/macrophages. Functional analysis of lymph node T cells revealed a lack of proliferative response to PHA or CD3 specific mAb, despite induction of expression of the activation marker CD69. Proliferation of LN cells and thymocytes was observed upon addition of exogenous IL‐2. An allogeneic EBV transformed tumour cell line, known to be an effective antigen presenting cell, could induce proliferation of LN cells without exogenous IL‐2 and fetal spleen cells could proliferate in response to all stimuli tested without additional IL‐2. Splenic non‐T cells could restore the proliferation of lymph node cells as efficiently as IL‐2 or the EBV transformed B cell line. Separated B cells were more effective than plastic adherent cells on a per cell basis. Naivity of the fetal immune system is therefore not only reflected by the expression of markers representative for naive lymphocytes but can also be due to the absence of potential accessory cells in the different lymphoid organs.

Specific eradication of micrometastases by transfer of tumour-immune T cells from major-histocompatibility-complex congenic mice

SummaryDBA/2 (H-2d) mice bearing a transplanted highly metastatic lymphoma (ESb) in a state of widely disseminated disease could be successfully treated by a combination of surgery (removal of the local tumour), irradiation (5 Gy) and adoptive immunotherapy. The immunotherapy was achieved by transfer of anti-ESb-immune spleen cells from B10.D2 mice, which express the same major histocompatibility complex (MHC) molecules as DBA/2. In contrast, anti-ESb-immune cells from MHC-disparate C57BL/6 mice did not confer protective immunity. The B10.D2 anti-ESb-immune T cells contain two types of cytolytic specificity as detected by limiting-dilution analysis: (1) clones with specificity for the ESb-tumour-associated transplantation antigen (TATA) (at low frequency), and (b) clones with specificity for minor DBA/2 histocompatibility (H) antigens (at high frequency). Immune B10.D2 cells raised against different tumour lines or against TATA− ESb tumour variants did not confer the 100% protection seen with immune cells against ESb TATA+ cells. Finally we demonstrate that the allogeneic immune cells are more potent in terms of protective immunity than corresponding syngeneic immune cells. The data suggest that the strong graft-versus-leukemia effect with immune T cells from allogeneic MHC-identical but not from MHC-disparate mice was due to T cells with MHC-restricted specificity for an ESb-associated TATA. A graft-versus-host reactivity that developed much later and could not be prevented was most likely due to T cells sensitized against normal minor H antigens of the host. Our results are of potential relevance for allogeneic bone marrow transplantation and adoptive immunotherapy protocols.