Tumor vaccine strategies after allogeneic T-cell depleted bone marrow transplantation

Vacinas tumorais pós-transplante alogênico de medula óssea depletado de células T

Artigo Especial / Special Article

Tumor vaccine strategies after allogeneic T-cell depleted bone marrow transplantation

James L.M. Ferrara

Allogeneic bone marrow transplantation is currently restricted to hematological malignancies because of a lack of anti-tumor activity against solid cancers. We have tested a novel treatment strategy to stimulate specific anti-tumor activity against a solid tumor after transplantation by vaccination with irradiated tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor. Using the B16 melanoma model, we found that vaccination elicited potent anti-tumor activity in recipients of syngeneic bone marrow transplantation in a time dependent fashion, and that immune reconstitution was critical for the development of anti-tumor activity. Vaccination did not stimulate anti-tumor immunity after allogeneic bone marrow transplantation because of the post-transplantation immunodeficiency associated with graft-versus-host disease. Remarkably, vaccination was effective in stimulating potent and long-lasting anti-tumor activity in recipients of T cell-depleted allogeneic bone marrow. Thus T cells derived from donor stem cells were able to recognize tumor antigens even though they remained tolerant to host histocompatibility antigens. Donor leukocyte infusion from a donor immunized with the recipient-derived B16 vaccines enhanced clinical activity of tumor vaccines without exacerbating graft-versus-host disease and CD4⁺ T cells are essential for this enhancement. These results demonstrate that vaccination of both donors and recipients can stimulate potent anti-tumor effects without the induction of graft-versus-host disease, and this strategy has important implications for the treatment of patients with solid malignancies.

Keywords: Granulocyte-macrophage colony-stimulating factor, bone marrow transplantation, graft-versus-host disease, graft-versus-tumor, cancer vaccination, T cell-depletion, donor leukocyte infusions

Introduction

Intensive chemo-radiotherapy alone mediates the anti-tumor effects of autologous bone marrow transplantation (BMT), but the conditioning regimen together with additional graft-versus-tumor (GVT) effects help to eliminate malignancy after allogeneic BMT (1, 2). However, relapse after BMT remains a major clinical problem, and since residual disease after BMT is frequently resistant to cytotoxic therapies, improved patient outcomes will likely require novel treatment approaches (3, 4).

Recently, a number of promising cancer vaccination strategies have been developed that significantly augment anti-tumor immunity in multiple rodent tumor systems (5, 6). Granulocyte-macrophage colony-stimulating factor (GM-CSF) based vaccines require the participation of both CD4 and CD8 positive T lymphocytes and likely involve improved tumor antigen presentation by host macrophages and dendritic cells (7). The principles delineated in these pre-clinical studies have proven relevant to patients with advanced renal cell carcinoma or malignant melanoma (8, 9).

Immune reconstitution following BMT is characterized by a recapitulation of lymphoid ontogeny and a lack of sustained transfer of clinically significant donor T and B cell immunity (10,11). Multiple quantitative and qualitative T and B cell defects have been described following both autologous and allogeneic BMT (10, 12), although with the passage of sufficient time most abnormalities resolve, except in the presence of chronic graft-versus-host disease (GVHD) which is associated with immunosuppression in both humans and mice (12-14). Despite the delay in immune reconstitution after BMT, some evidence suggests that vaccination may still be possible in this setting. Effective immunization with a live attenuated vaccine against measles, mumps, and rubella has been reported two years after BMT (15).

To determine the relationship between immunologic reconstitution and responsiveness to vaccination, we performed a time-course analysis of vaccination after syngeneic BMT, in a well defined animal model (16). Mice were challenged with live B16 cells one week following immunization. As expected, tumor challenge was uniformly lethal in control animals vaccinated with irradiated, wild type B16 cells; the kinetics of tumor development was similar between transplant recipients and naive mice. By contrast, vaccination with GM-CSF secreting B16 cells resulted in substantial anti-tumor immunity at both 4 and 6 weeks after BMT. Immunophenotyping of splenocytes revealed that numbers of CD4⁺, CD8⁺, and B220⁺ cells at 6 weeks after BMT were significantly greater than at 4 weeks but were comparable to 8 weeks after BMT. These results suggested that immune reconstitution of T cells was critical for the generation of anti-tumor immunity post-BMT.

Vaccine efficacy was then assessed after allogeneic BMT. Vaccination resulted in substantial anti-tumor immunity in both non-transplanted animals and in recipients of syngeneic BMT. By contrast, 0% of the vaccinated recipients of allogeneic BMT survived the tumor challenge. In addition, vaccination failed to alter the kinetics of tumor development in recipients of allogeneic BMT, demonstrating a lack of primary anti-tumor activity. Similar results were obtained in a second BMT strain combination, in which donor and recipient differed only in MiHA.

GVHD is known to cause significant delays in immunologic reconstitution after BMT (12-14), and we hypothesized that poor immunologic reconstitution in the context of GVHD impaired anti-tumor activity. Immunophenotyping of splenocytes 7 week post-BMT revealed severely reduced T and B lymphocyte numbers in recipients of allogeneic BMT with significant GVHD as previously described (17,18), whereas numbers of CD4⁺ T cells, NK cells, B cells, and myeloid cells, but not CD8⁺ cells, were normal 7 weeks after syngeneic BMT.

T cell depletion (TCD) of the donor inoculum is able to prevent the immunosuppression associated with GVHD after allogeneic BMT but it also impairs immune reconstitution in clinical BMT (19). We therefore asked whether TCD of semi-allogeneic BM could also provide for sufficient immune reconstitution to provide anti-tumor immunity in this allogeneic BMT model. Vaccination stimulated the development of striking anti-tumor activity, equivalent to that observed in non-transplanted vaccinated animals activity (16). Similar effects were found after BMT across MiHA differences, where vaccination after TCD BMT also resulted in substantial levels of anti-tumor activity.

The effect of vaccination on tumor specific T cell responses was analyzed in vitro one week after vaccination. Allogeneic TCD BMT recipients showed normal numbers of all cell phenotypes by six weeks after BMT except CD8⁺ cells. T cell proliferation to B16 stimulators in these animals was restored to normal levels. Cytokine responses in vaccinated TCD BMT recipients were never less than responses after syngeneic BMT and often equivalent to that seen in vaccinated naïve animals. The development of proliferation and cytokine production to B16 in vitro correlated closely with the efficacy of the vaccine and tumor destruction in vivo. Comparable results were obtained in a second donor-recipient strain combination.

To determine the effect of vaccination on GVHD severity, we monitored the survival and clinical GVHD score (range 0-10) of immunized allogeneic BMT recipients, as previously described (20). GVHD was severe in the haploidentical BMT model, with 36% mortality from GVHD by the time of vaccination. Clinical scores of GVHD severity in surviving allogeneic animals were elevated by four weeks after allogeneic BMT but they were mild or absent in recipients of syngeneic or TCD BMT. Importantly, vaccination did not exacerbate GVHD in any group, and in particular it did not cause increased skin disease or depigmentation, as has been reported in other strategies to eliminate B16 tumors (21). Similar results were observed in the second BMT model across MiHA differences, where GVHD was relatively mild and only 15% of the animals died by the time of vaccination.

In light of this absence of GVHD following vaccination, we evaluated T cell responses to host antigens in vitro in recipients of allogeneic TCD BMT. Vaccination produced equivalent cytotoxic responses to B16 tumors after allogeneic TCD BMT and syngeneic BMT. As expected, T cells from vaccinated mice lysed allogeneic ConA blasts but did not lyse syngeneic ConA blasts. While unvaccinated donor mice possessed little detectable cytotoxicity against allogeneic B16 cells, vaccination significantly enhanced this cytotoxicity, similar to observations in immunized melanoma patients (9). Vaccination did not augment cytolytic activity against B6 ConA blasts, confirming tolerance to host antigens in vitro. Thus, GM-CSF based tumor cell vaccines were able to stimulate effective anti-tumor immunity, and did not elicit immune responses to host alloantigens either in vitro or in vivo.

To determine if vaccination stimulated the development of long-lasting anti-tumor immunity, we challenged mice that had rejected an initial tumor inoculum of 10⁶ wild type B16 cells at 5 months after immunization. 70% of syngeneic BMT and 100% of allogeneic TCD BMT recipients eliminated the second tumor challenge, demonstrating the induction of immunologic memory by this vaccination strategy (16).

We next examined whether immunization to donor leukocyte infusion (DLI) donors with recipient-derived tumor vaccines could induce a tumor-specific response in vitro. One week after vaccination of allogeneic donor mice with irradiated GM-CSF secreting B16 cells, T cell proliferative and cytokine responses were measured in vitro. After vaccination, T cells vigorously proliferated and produced large amounts of both Th1 and Th2 cytokines (IFN-g, IL-2, GM-CSF, IL-4, IL-5 and IL-10) to B16 stimulators, confirming that vaccination with recipient-derived tumor vaccines preferentially stimulates tumor-specific T cell responses (22).

We next examined the effects of DLI on GVHD and GVT activity after allogeneic TCD BMT. Following TCD BMT, recipients were vaccinated and also injected with low-dose DLI. One week later, all recipients were challenged with 1 x 10⁶ live wild type B16 cells, which were uniformly lethal to unimmunized semiallogeneic mice (16). Vaccination to recipients alone resulted in 22% tumor-free survival (TFS). Non-immune DLI given concurrently with vaccination did not impact the efficacy of the vaccine or exacerbate GVHD, but DLI from immunized donors significantly improved the efficacy of vaccination. In contrast, DLI alone did not confer any significant anti-tumor immunity, regardless of immunization status of the donor (data not shown). Importantly, immunized DLI did not cause clinical GVHD as assessed by the clinical GVHD score. These results demonstrate that immunized DLI increases the efficacy of the tumor vaccine given to recipients without breaking tolerance to host antigens.

We then investigated which cell components in the DLI inoculum are responsible for enhanced vaccine efficacy. CD4⁺ T cells, CD8⁺ T cells, and B220⁺ B cells were positively selected by AutoMACS from spleens of immunized DLI donors. DLI of either CD4⁺ T cells, CD8⁺ T cells or B cells were injected in BMT recipients concurrently with vaccination, followed by tumor challenge 1 week later. Vaccination without DLI produced 11% TFS and the addition of immunized T-DLI again significantly increased the efficacy of recipient vaccination to 50%. This effect of immunized T-DLI was due solely to the presence of CD4⁺ T cells in the DLI inoculum: the TFS of recipients who received immunized CD4-DLI concurrently with recipient vaccination was 57%. By contrast, immunized CD8-DLI and B-DLI did not increase the efficacy of recipient vaccination (TFS 0% and 7%, respectively). Lastly, immunized CD4-DLI given at the time of recipient vaccination did not exacerbate GVHD as assessed by clinical scores and by histopathologic scores of the liver 4 weeks after DLI.

Remarkably, our vaccination strategy was extremely effective after allogeneic BMT when the donor inoculum was depleted of T cells to prevent GVHD and resulted in mixed chimerism. This efficacy was manifest both in terms of tumor protection and the development of T cell responses specific for B16 melanoma antigens. The induction of tumor-specific cytokine production, proliferation, and cytotoxicity following vaccination was closely associated with efficacy of vaccination evident after both allogeneic TCD BMT and syngeneic BMT. Reconstitution to normal levels of CD4⁺ T cells (but not CD8⁺ T cells) was observed by 6 weeks after TCD BMT as well as syngeneic BMT. These findings demonstrate that TCD that prevents the development of GVHD allows sufficient reconstitution of T cells from donor stem cells to restore the efficacy of vaccination. In this case, a functional thymus is critical for repopulation of the periphery with competent T cells since expansion of donor T cells is not an option after TCD BMT. Unfortunately, such rapid reconstitution is unlikely to occur in adult humans, where the age-related reductions in thymic regenerative capacity often result in incomplete restoration of T cell homeostasis after TCD BMT (23). Novel approaches to stimulate immune reconstitution will be required in older patients with poor thymic function.

Interestingly, the protective anti-tumor immunity induced by GM-CSF vaccination was long-lasting and displayed immunologic memory, evidenced by the ability of vaccinated mice to reject a tumor challenge five months later. Clinical studies of BMT patients show a loss of donor-derived immunity (11,24,25), suggesting the need for antigenic stimulation to an immune system that is newly generated from donor bone marrow cells; hence the recommendation of post-BMT vaccination against infectious agents (26).

In additional studies, we determined that administration of DLI at the time of recipient vaccination could generate additional anti-tumor activity in BMT recipients. DLI from immunized donors alone induced neither GVHD nor GVT activity in this system, although it has been shown that tumor-specific T cell immunity in donors could be transferred to BMT recipients (24,27,28). In our study, however, DLI from immunized donors given at the time of vaccination greatly enhanced its efficacy. Immunization of both donors and recipients was essential for an optimal effect, suggesting that the improved anti-tumor immunity involves adoptive transfer of activated/effector cells, as well as restimulation of these effectors cells by vaccination. We also found that CD4⁺, rather than CD8⁺ T cells in the DLI inoculum are primarily responsible for this enhanced effect. Although much attention has been given to the role of CD8⁺ cytotoxic T cells in anti-tumor immune responses, several lines of evidence suggest an important role for CD4⁺ T cells in mediating systemic anti-tumor effector functions (29-31). However, the split between CD4 and CD8 may be unique to this model, and it remains to be determined if this response can be generalized to other tumor/transplant systems. In humans, these results are consistent with clinical data showing that low numbers of CD4⁺ DLI can induce GVT activity against chronic myelogenous leukemia and multiple myeloma (32).

Our experiments clearly show that vaccination of recipients with GM-CSF secreting tumor cells after TCD BMT generates anti-tumor activity that is separable from GVHD. Immunization of recipients rather than donors may have several advantages; vaccinations can [1] be administered after the acquisition of tolerance to host antigens by donor cells; [2] stimulate the newly developing immune system, resulting in long-lasting immunity; and [3] avoid unnecessary exposure of healthy donors to tumor cells and foreign proteins such as alloantigens.

The efficacy of DLI from vaccinated donors suggests that primary immune responses to B16 tumors are not directed at host alloantigens. Therefore the anti-tumor activity induced by this vaccine might be exploited after allogeneic BMT, although the ability to work in other tumor models needs to be determined. Since TCD BMT is associated with a marked reduction in the frequency and intensity of GVHD as well as reduced anti-tumor activity (33), the ability of the combined strategy to increase anti-tumor immunity without GVHD has important clinical implications.

Vacinas tumorais pós-transplante alogênico de medula óssea depletado de células T

Resumo

O transplante alogênico de medula óssea atualmente é indicado para as doenças hematológicas devido à falta de atividade anti-tumoral contra os tumores sólidos. Nós testamos uma nova estratégia de tratamento para estimular a atividade específica anti-tumoral contra tumores sólidos após transplantação, por meio de vacinação com células tumorais irradiadas preparadas para secretar fatores estimuladores de colônia macrofágica-granulocítica. Usando o modelo de melanoma B16, verificamos que a vacinação induz uma potente atividade anti-tumoral em pacientes transplantados com medula óssea singênica e que a reconstituição imune foi importante para o desenvolvimento da atividade anti-tumoral. A vacinação não estimulou a imunidade anti-tumoral após o transplante de medula óssea alogênico devido à imunodeficiência pós-transplante associada com a doença do enxerto contra o hospedeiro. A vacinação foi notadamente efetiva na estimulação potente e na atividade anti-tumoral a longo prazo em receptores de medula óssea depletada de células T. Assim, as células T derivadas das células tronco do doador foram eficazes no reconhecimento de antígenos tumorais mesmo quando permaneceram tolerantes aos antígenos de histocompatibilidade do hospedeiro. A infusão de leucócitos do doador imunizado com vacina B16 derivada do receptor aumentou a atividade clínica das vacinas tumorais sem agravar a doença do enxerto contra o hospedeiro e as células CD4+ foram essenciais para este aumento. Estes resultados demonstram que a vacinação de doadores e pacientes pode estimular os efeitos anti-tumorais sem a indução da doença do enxerto contra o hospedeiro e esta estratégia tem importantes implicações no tratamento de pacientes com tumores sólidos.

Palavras-chave: Fator estimulador de colônia macrofágica-granulocítica, transplante de medula óssea, Doença Enxerto Contra o Hospedeiro, enxerto contra tumor, vacina, depleção de células T, suspensão de linfócitos de doadores

Recebido: 10/06/2002

Aceito: 15/06/2002

Departments of Internal Medicine and Pediatrics, University of Michigan Cancer Center, Ann Arbor, MI, USA

Correspondence to: James L. M. Ferrara

University of Michigan Cancer Center

1500 E. Medical Center Drive. Ann Arbor, MI 48109-0942

Phone: [phone]. Email: [e-mail]

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Publication Dates

• Publication in this collection
  14 Jan 2003
• Date of issue
  2002