Transfer of long-lasting tumor immunity by immune T cells from MHC congenic mice: Migration,survival and tumor-protectivity of cytotoxic donor cells

Immunocompetent B10.D2 (H-2^d) mice are able to reject the highly malignant lymphoma ESb of DBA/2 (H-2^d) origin very effectively. Seven days after intravenous injection of the ESb tumor cells, B10.D2 mice developed a strong tumor-rejection response which was associated with the generation of anti-tumor T cells in their spleens with direct cytotoxic activity. Most of the cytotoxic potential was directed against the minor histocompatibility differences as demonstrated by the lysis of unrelated DBA/2 derived Eb tumor cells and normal DBA/2 but no B10.D2 derived ConA lymphoblasts. A previously performed clonal analysis, however, revealed a minority population of CTL clones which specifically recognized the ESb specific transplantation antigen (ESb-TATA). When transferred systemically into DBA/2 mice, the B10.D2 anti-ESb immune spleen cells could delay the outgrowth of s.c. transplanted ESb tumor cells. When the ESb tumor cells were experimentally distributed in a s.c. implanted sponge-matrix, the i.v. injected B10.D2 immune cells could confer complete protective immunity against the metastatic tumor, provided the recipients were pre-treated with 5 Gy to allow a better take of the allogeneic cells. The distribution of intravenously injected B 10D2 donor spleen cells was assessed in the recipients up to 50 days by cytotoxicity testing and assaying for the expression of the ₂ microglobulin allelic form b ( ₂m^b). These tests revealed a high propensity of donor cells to populate the spleen and lymph nodes of the DBA/2 recipients. Again this was particularly marked in sublethally irradiated mice where a long-lasting lymphoid chimerism was established.     

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.     

A novel tumour model system for the study of long-term protective immunity and immune T cell memory

We present a novel non-transgenic system to be used for studies on anti-tumour adoptive immunotherapy (ADI) and long-term T cell memory. Tumour-reactive donor immune cells against lacZ-transfected syngeneic tumour cells (ESbL-Gal) were generated from a naíve T cell repertoire in DBA/2 mice by a well-established priming/restimulation protocol, and transferred to tumour-inoculated athymic nu/nu mice. The donor immune cells efficiently mediated protective anti-tumour immunity involving both CD4(+) and CD8(+) T cells, and anti-metastatic effects were stronger in 4.5 Gy pre-irradiated than in non-irradiated tumour-inoculated hosts. Long-term persistence of beta-galactosidase (Gal)-specific T cells was shown ex vivo by tetramer staining of CD8(+) T cells specific for an immunodominant Gal epitope. Resistance of treated nu/nu mice against tumour rechallenge revealed the existence of long-term protective immune memory.     

Immunotherapeutic potential of whole tumour cells

Despite the identification of tumour antigens and their subsequent generation in subunit form for use as cancer vaccines, whole tumour cells remain a potent vehicle for generating anti-tumour immunity. This is because tumour cells express an array of target antigens for the immune system to react against, avoiding problems associated with major histocompatibility complex (MHC)-restricted epitope identification for individual patients. Furthermore, whole cells are relatively simple to propagate and are potentially efficient at contributing to the process of T cell priming. However, whole cells can also possess properties that allow for immune evasion, and so the question remains of how to enhance the immune response against tumour cells so that they are rejected. Scenarios where whole tumour cells may be utilised in immunotherapy include autologous tumour cell vaccines generated from resected primary tumour, allogeneic (MHC-disparate) cross-reactive tumour cell line vaccines, and immunotherapy of tumours in situ. Since tumour cells are considered poorly immunogenic, mainly because they express self-antigens in a non-stimulatory context, the environment of the tumour cells may have to be modified to become stimulatory by using immunological adjuvants. Recent studies have re-evaluated the relative roles of direct and cross-priming in generating anti-tumour immunity and have highlighted the need to circumvent immune evasion.     

Modification of tumor cells by a low dose of Newcastle disease virus

Summary Augmented tumor-specific T cell responses were observed against the high metastatic murine lymphoma variant ESb when using as immunogen ESb tumor cells that had been modified by infection with a low dose of Newcastle disease virus (NDV). Such virus-modified inactivated tumor cells (ESb-NDV) were potent tumor vaccines when applied postoperatively for active specific immunotherapy of ESb metastases. We demonstrate here that immune spleen cells from mice immunized with ESb-NDV contain enhanced immune capacity in both the CD4⁺, CD8^– and the CD4^–, CD8⁺ T cell compartments to mount a secondary-tumor-specific cytotoxic T cell response in comparison with immune cells from mice immunized with ESb. ESb-NDV immune CD4⁺, CD8^– helper T cells also produced more interleukin 2 after antigen stimulation than the corresponding ESb immune cells. There was no participation of either CD4⁺ or CD8⁺ virus-specific cells in the augmented response. The specificity of the T cells for the tumor-associated antigen remaind unchanged. Thus, there is the paradox that the virus-mediated augmentation of the tumor-specific T cell response in this system involves increased T helper activity but does not involve the recognition of viral epitopes as potential new helper determinants.Abbreviations CTL cytolytic T lymphocytes - IL-2 interleukin 2 - rIL-2 recombinant IL-2 - mAb monoclonal antibody - NDV Newcastle disease virus - SSC syngeneic spleen cell     

Tumor immunotherapy across MHC barriers using allogeneic T-cell precursors

We present a strategy for adoptive immunotherapy using T-lineage committed lymphoid precursor cells generated by Notch1-based culture. We found that allogeneic T-cell precursors can be transferred to irradiated individuals irrespective of major histocompatibility complex (MHC) disparities and give rise to host-MHC restricted and host-tolerant functional allogeneic T cells, improving survival in irradiated recipients as well as enhancing anti-tumor responses. T-cell precursors transduced to express a chimeric receptor targeting hCD19 resulted in significant additional anti-tumor activity, demonstrating the feasibility of genetic engineering of these cells. We conclude that ex vivo generated MHC-disparate T-cell precursors from any donor can be used universally for 'off-the-shelf' immunotherapy, and can be further enhanced by genetic engineering for targeted immunotherapy.     

Studies on the induction and expression of T cell-mediated immunity. IV. Non-overlapping populations of alloimmune cytotoxic lymphocytes with specificity for tumor-associated antigens and transplantation antigens.

Two non-overlapping populations of alloimmune cytotoxic T cells with specificity for tumor-associated antigens (TAA) and for histocompatibility antigens (H-2) were characterized by two independent methods. The heterogeneity of cytotoxic cells was demonstrated in spleen cells derived from BALB/c (H-2d) mice sensitized to EL-4 (H-2b) tumor and from C57BL/6 (H-2b) mice sensitized to G-35 (H-2d) tumor cells. Adsorption of immune lymphocytes on monolayers prepared with cells bearing the sensitizing H-2 antigens abrogated the in vitro cell-mediated cytotoxicity (CMC) directed against 51Cr-labeled normal target cells (spleen cells or ConA-activated spleen blasts), whereas significant cytolytic activity to the corresponding 51Cr-tumor cells was still retained. Likewise, in competitive inhibition assays, CMC to 51 Cr-tumor target cells was only partially inhibited by unlabeled normal cells, whereas CMC to 51Cr-normal target cells was completely abrogated. These results suggested that alloimmune cytotoxic lymphocytes are heterogeneous and can be subdivided into two independent populations of restricted specificity. Several experiments suggested that the effector cell population directed against TAA can no longer elicit a graft-vs-host (GVH) reaction in vivo. This was demonstrated by adoptive transfer into lethally-irradiated allogeneic recipients of cytotoxic or primed spleen cells fractionated on host target cell monolayers. Furthermore, these results demonstrated that both effector cells and memory cells possess high affinity binding receptors to corresponding H-2 antigens. The potential use of fractionated immune lymphocytes sensitized to tumor allografts in adoptive immunotherapy is discussed.     

Fate of H2-activated T lymphocytes in syngeneic hosts. III. Differentiation into long-lived recirculating memory cells.

Memory to H2 determinants was studied with an adoptive transfer system using a population of H2-activated blast T cells (T.TDL) obtained from thoracic duct lymph of irradiated F₁ hybrid mice injected with parental strain T cells. CBA T.TDL activated either to DBA/2 or C57BL determinants were transferred to syngeneic “B” mice. Thoracic duct lymphocytes (TDL) were obtained from the recipients 4–6 weeks later and tested for their capacity to produce (a) a graft-versus-host (GVH) reaction, (b) a mixed lymphocyte reaction (MLR) (measured by an in vivo technique) and (c) allograft rejection (suppression of the growth of allogeneic tumour cells in vivo). Control experiments involved testing the function of TDL obtained from “B” mice preinjected with TDL or no cells.TDL from “B” mice injected with TDL (passaged TDL) gave strong MLR and GVH reactions to both DBA/2 and C57BL determinants. Passaged T.TDL activated to C57BL antigens gave intermediate MLR and GVH reactions to the specific (C57BL) determinants but only very low responses to third-party (DBA/2) determinants; reciprocal results were obtained with passaged T.TDL activated to DBA/2 determinants. TDL from “B” mice given no cells failed to respond to either set of determinants.Since the responses by the passaged T.TDL did not exceed those by passaged TDL there was no evidence that adoptive transfer of T.TDL had conferred to the recipients a state of memory to either MLR or GVH determinants. Adoptive transfer did, however, lead to qualitative changes in the properties of T.TDL since, before transfer, they were unable to evoke GVH reactions or produce an MLR of normal kinetics.Passaged T.TDL were far superior to passaged TDL at suppressing the growth of allogeneic tumour cells. The protection was specific since protection against DBA/2 tumour cells was, cell for cell, 5–10 fold more effective with passaged T.TDL activated to DBA/2 determinants than with cells activated to C57BL determinants. No protection was observed with cells treated with anti-θ serum. The protective cells appeared to be precursors of effector cells rather than effector cells per se since they failed to lyse the tumour cells in vitro. These data suggest therefore that the descendants of T.TDL which survived after transfer to “B” mice were highly enriched in long-lived recirculating T lymphocytes reactive to determinants expressed by specific tumour allografts.     

NK cell-based cancer immunotherapy: from basic biology to clinical application

Natural killer(NK) cells, which recognize and kill target cells independent of antigen specificity and major histocompatibility complex(MHC) matching, play pivotal roles in immune defence against tumors. However, tumor cells often acquire the ability to escape NK cell-mediated immune surveillance. Thus, understanding mechanisms underlying regulation of NK cell phenotype and function within the tumor environment is instrumental for designing new approaches to improve the current cell-based immunotherapy. In this review, we elaborate the main biological features and molecular mechanisms of NK cells that pertain to regulation of NK cell-mediated anti-tumor activity. We further overview current clinical approaches regarding NK cell-based cancer therapy, including cytokine infusion, adoptive transfer of autologous or allogeneic NK cells, applications of chimeric antigen receptor(CAR)-expressing NK cells and adoptive transfer of memory-like NK cells. With these promising clinical outcomes and fuller understanding the basic questions raised in this review, we foresee that NK cell-based approaches may hold great potential for future cancer immunotherapy.     

Anti-θ Serum-indueed Suppression of the Cellular Transfer of Tumour-specific Immunity to a Syngeneic Plasma Cell Tumour

IT has been well documented that tumour-bearing mice can become resistant to their own tumours, especially with chemically induced fibrosarcomas^1–3 and the importance of cell-mediated immune responses rather than humoral antibody in the resistance to tumour transplants has been emphasized^3,4, although the exact mechanism of tumour cell destruction remains ill-defined. Studies in mice^5,6, using allogeneic tumour cells, have demonstrated that thymus-derived (T) lymphocytes are essential for the killing of tumour cells. In addition, using an in vitro method of immunization against histocompatibility antigens, tumour cell destruction either in vitro¹ or in vivo⁸ was shown to be due to T cells alone. In all of these latter studies, however, it is the strong H-2 histocompatibility antigens that are inducing the immune response and not the tumour-specific transplantation antigens (TSTA). We describe here a specific anti-TSTA response to a murine plasma cell tumour which can be transferred with lymphoid cells and which can be shown to involve the essential participation of T cells.     

Superiority of the ear pinna over a subcutaneous tumour inoculation site for induction of a Th1-type cytokine response

 This study examines whether a correlation may be found between Th1- or Th2-type cytokine responses and resistance or susceptibility to tumour growth. Cytokine profiles were investigated in a well-defined mouse tumour model in which the injection site and the genetic background determine the phenotype of either tumour resistance or tumour susceptibility. DBA/2-derived ESb lymphoma variant cells with high metastatic capacity were inoculated into syngeneic mice either s.c., where they grow and metastasize, or into the ear pinna (i.e.), where they do not grow because of induction of protective immunity. Alternatively, the tumour cells were injected s.c. or i.e. into allogeneic B10.D2 mice, which are resistant to the tumour although they are identical at the MHC locus. Between 1 and 10 days after tumour cell injection the spleen-derived mRNA was tested for cytokine gene expression or the spleen cells were analysed by FACScan for T cell activation. The strongest cytokine response was observed in i.e. inoculated B10.D2 mice. This was characterized by an early (days 2–3) peak of interferon γ (INF-γ), interleukin-2 (IL-2), IL-2 receptor α (IL-2Rα) and IL-4. The cytokine mRNA response of i.e. inoculated DBA/2 mice was quite similar except that no IFN-γ could be detected. In s.c. inoculated B10.D2 mice, the IL-2, IL-2Rα and IFN-γ responses were weaker than after i.e. injection while the IL-4 response was comparable. The most striking difference between these cytokine profiles from tumour-resistant mice and those of s.c. inoculated tumour-susceptible DBA/2 mice was a delay in the latter in the IL-2, IL-2Rα and IFN-γ responses and the observation that the IL-4 response was not down-regulated. The persisting IL-4 response could down-regulate a Th1-type response and thereby explain tumour susceptibility as a consequence of host conditioning. Received: 4 September 1997 / Accepted: 2 October 1997     

Failure of specific adoptive immunotherapy owing to survival and outgrowth of variant cells

Summary Adoptive immunotherapy, the transfer of spleen cells from immunized mice to mice with a small tumor, was usually curative for mice with the P815 mastocytoma provided that steps were taken to prevent the generation of tumor-induced suppressor cells in the recipient animal. However, failure of adoptive immunotherapy of the P815 tumor, resulting in regrowth of either the primary intradermal or a metastatic tumor, was observed in 10 out of 112 animals receiving graded doses of 7.5×10⁷ to 3.0×10⁸ immune spleen cells. Examination of the ten tumors in mice that failed to respond to therapy revealed that seven of them were significantly less susceptible than the original P815 tumor to rejection in vivo by transferred anti-P815-specific effector cells. In addition, nine of the ten therapy-failure tumors were also less susceptible than the original P815 tumor to lysis in vitro by P815-specific, but not DBA/2-specific, cytotoxic T lymphocytes. Sensitivity to lysis by tumor-specific cytotoxic T cells was not, however, strongly correlated with sensitivity to rejection in vivo by P815-specific effector spleen cells. Neither in vivo sensitivity to rejection, nor sensitivity to cytotoxic T cells, was correlated with alterations in class I major histocompatibility complex antigen expression. These results suggest that the survival and outgrowth of variant tumor cells was frequently the cause of failure of specific adoptive immunotherapy of the P815 tumor, and that selection for cells with a reduced sensitivity to killing by cytotoxic T cells was only one mechanism that might lead to an immunotherapeutic failure.This work was supported by a grant from RJR Nabisco Inc., a grant from the J. M. Foundation, and by USPHS grant CA-40597 awarded by the National Cancer Institute     

Fully MHC-disparate mixed hemopoietic chimeras show specific defects in the control of chronic viral infections

The establishment of mixed allogeneic chimerism can induce donor-specific transplantation tolerance across full MHC barriers. However, a theoretical disadvantage of this approach is the possibility that the state of mixed chimerism might negatively affect the recipient's immune competence to control pathogens. Previous studies using murine models have not supported this hypothesis, because they indicate that acute viral infections are cleared by chimeric animals with similar kinetics to that of unmanipulated controls. However, chronic or persistent viral infections often require a more complex and sustained response with cooperation between CD4 Th cells, CTL, and B cells for effective control. The current study indicates that profound defects become manifest in the control of chronic pathogenic infections in MHC-disparate mixed allogeneic chimeric mice. Furthermore, we show that ineffective priming of the donor-restricted CTL response leads to virus persistence, as well as severe T cell exhaustion. Our results further suggest that either T cell adoptive immunotherapy or selected MHC haplotype matching partially restore immune competence. These approaches may facilitate the translation of mixed chimerism therapeutic regimens.     

Complete remission of cancer in late-stage disease by radiation and transfer of allogeneic MHC-matched immune T cells: lessons from GvL studies in animals

Most immunotherapy studies in animal tumor models are performed in early stages of the disease. Reports on the studies of treatment in late stages of tumor growth and metastasis are much rarer. To guide future efforts for treatment in late-stage disease, a model of effective immune rejection of advanced metastasized cancer is reviewed and lessons therefrom are summarized. Already cachectic DBA/2 mice with a subcutaneously transplanted syngeneic tumor (ESb-MP lymphoma) of 1.5 cm diameter and with macroscopic liver and kidney metastases at 4 weeks could be successfully treated by a combination of sublethal (5 Gy) irradiation followed by a single transfer of 20 million anti-tumor immune spleen cells from tumor-resistant allogeneic MHC-B10.D2 mice. Following intravenous cell transfer, the primary tumors became encapsulated and were eventually rejected from the skin while visceral metastases gradually disappeared leaving behind only scar tissue. There was wound-healing at the site of the rejected primary tumor, and the animals survived long term without any tumor recurrence. The complete eradication of late-stage disease by adoptive cellular immunotherapy could be corroborated noninvasively by ³¹P-NMR spectroscopy of primary tumors and by ¹H-NMR microimaging of liver metastases. Conclusions from functional mechanistic studies in this model are summarized and clinical implications discussed.     

The existence of antibody-like activities against the weakly immunogenic minor histocompatibility antigens

BALB/c mice develop cytotoxic lymphocytes as well as produce specific antibodies against the minor histocompatibility antigens when injected with DBA/2 P815 cells. P815 cells grown in BALB/c mice have IgG antibodies on their surface as demonstrated by the binding of ¹²⁵I-labeled goat anti-mouse IgG and by complement-dependent cytotoxicity. Serum from BALB/c mice hyperimmunized with P815 cells blocked lymphocyte-mediated cytotoxicity by BALB/c immune peritoneal exudate cells. This blocking activity was removed by absorbing hyperimmune serum with DBA/2 spleen cells or P815 cells. This result suggests that specific antibodies were generated against the minor histocompatibility differences between BALB/c and DBA/2 mice. The experimental procedures described may be very useful in demonstrating minute quantities of antibody against minor histocompatibility antigens on tumor cells.     

CD4+ and CD8+ regulatory T cells generated ex vivo with IL-2 and TGF-beta suppress a stimulatory graft-versus-host disease with a lupus-like syndrome

Regulatory T cells generated ex vivo from conventional mouse T cells have been used to prevent and alter the course of a stimulatory graft-vs-host disease with a lupus-like syndrome. DBA/2 mouse T cells induce this syndrome when injected into (DBA/2 x C57BL/6) F(1) mice. Stimulating DBA/2 T cells with irradiated C57BL/6 in the presence of IL-2 and TGF-beta induced both CD4(+) and CD8(+) cells to develop potent suppressive activity and enhanced their survival. The IL-2 and TGF-beta-treated T cells lost their ability to induce graft-vs-host disease and, instead, prevented other parental T cells from inducing lymphoid hyperplasia, B cell activation, and an immune complex glomerulonephritis. Moreover, a single transfer of TGF-beta-conditioned T cells to animals that had already developed anti-dsDNA Abs decreased the titer, suppressed proteinuria, and doubled survival. This study raises the possibility that autologous regulatory T cells generated ex vivo have the potential to be used as an adoptive immunotherapy to induce allograft tolerance and to control autoimmunity.     

Ia Restriction Specificity of KLH-Specific T Cells from Allogeneic Bone Marrow Chimeras Is Influenced by Histocompatibility at the H-2 and Minor Histocompatibility Loci

Ia restriction specificity involved in T cell proliferative responses to keyhole limpet hemocyanin (KLH) has been analyzed using a variety of allogeneic bone marrow chimeras. The chimeric mice were prepared by reconstituting irradiated AKR, SJL, B10.BR and B10.A(4R) mice with bone marrow cells from B10 mice. When such chimeric mice had first been primed with KLH in complete Freund's adjuvant (CFA), T cells from H-2 incompatible fully allogeneic chimeras showed significantly higher responses to KLH in the presence of antigen-presenting cells (APC) of donor strain (B10) than APC of recipient strain. However, in H-2 subregion compatible chimeras, [B10→B10.A(4R)], which were matched at the H-2D locus and at minor histocompatible loci, the T cells could mount vigorous responses to KLH with antigen-presenting cells (APC) of either donor or recipient type. The same results were obtained as well with chimeras that had been thymectomized after full reconstitution of lymphoid tissues by donor-derived cells. A considerable proportion of KLH-specific T cell hybridomas established from [B10→B10.A(4R)] chimeras exhibited both I-A^b and I-A^k restriction specificities. The present findings indicate that the bias to donor Ia type of antigen specific T cells is determined by donor-derived APC present in the extrathymic environment but that cross-reactivity to the recipient Ia is influenced to some degree by histocompatibility between donor and recipient mice, even though the histocompatible H-2D locus and minor histocompatibility loci seem not to be directly involved in the I-A restricted responses studied herein.     

The use of cytotoxic T cells in the regulation of tumour growth in syngeneic mice

Summary Normal C57BL/6 (B6) spleen cells were cultured with syngeneic EL4 tumour cells, expanded in IL2-containing medium, and tested for anti-tumour activity in vitro and in vivo. The activated cells were highly cytotoxic for EL4 and to a lesser degree killed syngeneic B6 blasts and allogeneic (D2) P815 tumour cells. B6 or BDF1 mice that received these cultured cells by IP injection cleared ¹²⁵IUdR-labelled EL4 cells faster than untreated mice. However, this enhanced clearance was evident only 7–12 days after injection. Since the injected cells had a short half-life (<10% remaining after 48 h) the effect of these cells in vivo was most probably due to the activation of the host's immune system. Mice that received cultured cells survived significantly longer than untreated mice following a lethal dose of EL4 cells. Cultured cells were much more effective in prolonging survival when used in conjunction with cyclophosphamide (CY). In animals receiving either cultured cells with or without CY or CY alone tumour clearance was markedly enhanced 7–12 days after injection.When challenged with a small dose of EL4 tumour cells (1×10⁴ SC per mouse) three of ten B6 mice treated with B6 anti-EL4 cultured cells were able to survive indefinitely. The frequency of CTL precursors to EL4 from the spleen cells of these surviving animals was about five-fold higher than that of normal spleen cells. Furthermore, CTL derived from primed spleen cells were more specific for EL4 than those derived from normal spleen cells.Abbreviations B6 C57BL/6J - BDF1 (C57BL/6J×DBA/2J) F1 - ConA SN concanavalin A supernatant - CTL cytotoxic T lymphocytes - CTL-P cytotoxic T-lymphocyte precursors - CY cyclophosphamide - E/T effector-to-target ratio - IL2 interleukin 2 - IP intraperitoneal - IUdR iododeoxyuridine - IV intravenous - LPS lipopolysaccharide - MST mean survival time     

Tumor reactivity of immune T cells in short-term culture

 The adoptive transfer of immune T cells is capable of mediating the regression of established neoplasms in a variety of animal tumor models. The antitumor activity is invariably proportional to the number of cells transferred, thus methods to expand immune cell number while maintaining therapeutic efficacy have been extensively investigated. Here we demonstrate that a short-term culture of immune T cells can amplify the T cell number and enhance the therapeutic reactivity against established pulmonary tumor, while maintaining immunological specificity. In contrast, the therapeutic reactivity of immune T cells against established subcutaneous tumor is diminished by short-term culture. While cultured immune T cells are not cytotoxic in a 4-h Cr-release assay, they do specifically secrete interferon γ upon stimulation with tumor cells. T cells cultured after a single exposure to tumor are even more active against pulmonary tumor than T cells cultured from mice immunized repeatedly. This culture system can rapidly induce T cell proliferation and differentiation into mature effector cells, and the resulting cells demonstrate an enhanced ability to treat visceral metastases, but a decreased ability to treat subcutaneous tumor. Thus T cells cultured after a single exposure to tumor represent an ideal population of cells for use in human adoptive immunotherapy trials. Received: 18 July 1996 / Accepted: 27 September 1996     

The antitumor effect of tumor-draining lymph node cells activated by both anti-CD3 monoclonal antibody and activated B cells as costimulatory-signal-providing cells

To establish an efficient cell-culture system for adoptive immunotherapy, we attempted to use lipopolysacharide (LPS)-activated B cells (LPS blasts) as costimulatory-signal-providing cells in the in vitro induction of antitumor effector cells. Both normal and tumor-draining lymph node cells were efficiently activated by both anti-CD3 monoclonal antibody (mAb) and LPS blasts, and subsequently expanded by a low dose of interleukin-2 (IL-2; anti-CD3 mAb and LPS blasts/IL-2). The expanded cells were predominantly CD8⁺ T cells and showed a low level of tumor-specific cytotoxic T lymphocyte (CTL) activity. The adoptive transfer of B16-melanoma-draining lymph node cells expanded by anti-CD3 mAb and LPS blasts/IL-2 showed significant antitumor effect against the established metastases of B16 in combination with intraperitoneal injections of IL-2. This treatment cured all B16-bearing mice. In addition, these mice also showed tumorspecific protective immunity against B16 at the rechallenge. Considering that activated B cells express several kinds of costimulatory molecules, these findings thus indicate an efficacy of costimulation that is derived from activated B cells for the in vitro induction of tumor-specific CTL, in co-operation with anti-CD3 mAb. The culture system presented here may thus be therapeutically useful, providing potent effectors for adoptive immunotherapy against various types of cancer.