Nurit Hollander

Environmental enrichment in mice decreases anxiety, attenuates stress responses and enhances natural killer cell activity.

The importance of environment in the regulation of brain, behaviour and physiology has long been recognized in biological, social and medical sciences. Animals maintained under enriched conditions have clearly been shown to have better learning abilities than those maintained under standard conditions. However, the effects of environmental enrichment (EE) on immunity and emotionality have been less documented and remain questionable. Therefore, we investigated the effect of EE on natural killer (NK) cell activity, psychological stress responses and behavioural parameters. Male C3H mice were housed either in enriched or standard conditions for 6 weeks. Behaviour was then examined by the grip‐strength test, staircase and elevated plus maze, and corticosterone levels and NK cell activity were measured. Furthermore, animals exposed to the stress paradigm, achieved by electric shock with reminders, were tested for freezing time in each reminder. Corticosterone levels were also measured. The EE mice showed decreased anxiety‐like behaviour and higher activity compared to standard mice, as revealed by a greater percentage of time spent in the open arms of the elevated plus maze, and a higher rate of climbing the staircase. A shorter freezing time in the stress paradigm and no corticosterone level reactivity were measured in EE mice. In addition, NK cell activity in spleens of EE mice was higher than that demonstrated in those of standard mice. Thus, EE has a beneficial effect on anxiety‐like behaviour, stress response and NK cell activity. The effect on NK cell activity is promising, due to the role of NK cells in host resistance.

Synergistic Effect of Dendritic Cell Vaccination and Anti-CD20 Antibody Treatment in the Therapy of Murine Lymphoma

Indolent B-cell lymphomas are characterized by repeated remissions and relapses with most patients eventually dying of the disease. Although combination treatments with chemotherapy and the anti-CD20 antibody rituximab improved duration of remissions and overall survival, the disease is essentially incurable. Thus, novel therapeutic approaches are needed. One such approach is active immunization with dendritic cells (DCs). Given that rituximab depletes patients of normal B cells, optimal vaccination strategies for rituximab-treated patients require induction of effector T cells. We have previously demonstrated in a murine model that idiotype (Id)-keyhole limpet hemocyanin-pulsed DCs induced Id-reactive CD8+ T cells and protection against tumor challenge in the absence of anti-Id antibodies. On the basis of these results, we investigated vaccination in a therapeutic model, in which mice carrying advanced tumors of the highly aggressive 38C-13 lymphoma were treated with chemotherapy and anti-CD20 antibodies combined with a DC-based vaccine. As a rule, cytoreduction by cyclophosphamide was required in each regimen of combination treatment, and vaccination with tumor cell-loaded DCs was more effective than vaccination with Id-keyhole limpet hemocyanin-loaded DCs. We demonstrated that under conditions of large primary tumors that had already spread to lymph nodes, when anti-CD20 antibody treatment showed minimal effect and DC vaccination had no effect, synergism between anti-CD20 antibodies and DC vaccines resulted in significant long-term survival that did not involve active antitumor antibody production. Combination treatments including tumor cell-loaded DC vaccines may therefore provide a strategy for enhancing the potency of therapy in rituximab-treated patients.

Dendritic cell-based therapeutic vaccination against myeloma: vaccine formulation determines efficacy against light chain myeloma.

Multiple myeloma is an incurable plasma cell malignancy. Immunotherapy in myeloma patients had limited success to date. We have previously demonstrated that dendritic cells (DCs) pulsed with autologous Ig Id induced Id-reactive CD8+ T cells and protection against a myeloma tumor challenge. In this work, we studied the therapeutic efficacy of chemotherapy combined with different formulations of DC-based vaccines in mice bearing large plasma cell tumors. The comparative study demonstrated that s.c. injection of DCs loaded with Id coupled to keyhole limpet hemocyanin, s.c. injection of DCs loaded with irradiated tumor cells, and intratumoral injection of naive DCs were similarly effective in mediating tumor regression and long-term survival. However, whereas the Id-keyhole limpet hemocyanin-DC vaccine was inefficient against myeloma cells that lost expression of the Ig H chain, intratumoral injection of naive DCs and s.c. injection of DCs loaded with irradiated tumor cells were highly effective against cells producing L chains only. This may be of particular importance for patients with L chain myeloma. Given that T cells respond primarily to peptides derived from H chain CDRs, attempts to treat L chain disease with myeloma protein-pulsed DCs may be futile. Vaccination with tumor cell-loaded DCs may, however, induce an effective antitumor response.

Anti-idiotype x anti-CD44 bispecific antibodies inhibit invasion of lymphoid organs by B cell lymphoma.

The demonstration that Abs to adhesion molecules can block tumor metastasis suggested their use for therapy. However, such Abs affect nonmalignant cells as well. To circumvent this adverse effect, we proposed the use of bispecific Abs that bind simultaneously to an adhesion receptor and to a tumor-specific Ag. Such bifunctional Abs bind more avidly to tumor cells that coexpress both target Ags than to normal cells. The Id of the surface Ig of malignant B lymphocytes is a tumor-specific Ag. Therefore, we produced bispecific Abs with specificity to the adhesion molecule, CD44, and to an idiotypic determinant of the murine B cell lymphoma, 38C-13. These anti-Id × anti-CD44 bispecific Abs blocked 38C-13 cell adhesion to hyaluronic acid, while not affecting adhesion of Id-negative cells. In vivo studies demonstrated that the bispecific Abs inhibited lymphoma cell dissemination to the lymph nodes, bone marrow, and spleen, and prolonged survival of tumor-bearing mice. Migration of 38C-13 cells to the lymphoid organs was inhibited by the bispecific Abs. Thus, the bispecific Ab-mediated reduction in metastasis resulted, at least in part, from reduced homing to these organs. In contrast to anti-CD44 monospecific Abs, the anti-Id × anti-CD44 bispecific Abs did not affect immune responses such as delayed-type hypersensitivity. Hence, bispecific Abs against adhesion molecules and tumor-specific Ags may selectively block tumor metastasis in a way which may leave at least part of the immune system intact.

Immunotherapy for B-Cell Lymphoma: Current Status and Prospective Advances

Therapy for non-Hodgkin’s lymphoma has progressed significantly over the last decades. However, the majority of patients remain incurable, and novel therapies are needed. Because immunotherapy ideally offers target selectivity, an ever increasing number of immunotherapies, both passive and active, are undergoing development. The champion of passive immunotherapy to date is the anti-CD20 monoclonal antibody rituximab that revolutionized the standard of care for lymphoma. The great success of rituximab catalyzed the development of new passive immunotherapy strategies that are currently undergoing clinical evaluation. These include improvement of rituximab efficacy, newer generation anti-CD20 antibodies, drug-conjugated and radio labeled anti-CD20 antibodies, monoclonal antibodies targeting non-CD20 lymphoma antigens, and bispecific antibodies. Active immunotherapy aims at inducing long-lasting antitumor immunity, thereby limiting the likelihood of relapse. Current clinical studies of active immunotherapy for lymphoma consist largely of vaccination and immune checkpoint blockade. A variety of protein- and cell-based vaccines are being tested in ongoing clinical studies. Recently completed phase III clinical trials of an idiotype protein vaccine suggest that the vaccine may have clinical activity in a subset of patients. Efforts to enhance the efficacy of active immunotherapy are ongoing with an emphasis on optimization of antigen delivery and presentation of vaccines and modulation of the immune system toward counteracting immunosuppression, using antibodies against immune regulatory checkpoints. This article discusses results of the various immunotherapy approaches applied to date for B-cell lymphoma and the ongoing trials to improve their effect.

Environmental Enrichment Augments the Efficacy of Idiotype Vaccination for B-cell Lymphoma

Environmental enrichment is known to positively influence the organisms psychologic and physiologic well-being. However, the effects of environmental enrichment on immune responses and cancer prognosis have not been clearly established and its impact on cancer therapy is unknown. Here, we report that environmental enrichment mediated a statistically significant improvement of the outcome of immunotherapy in an experimental model of B-cell lymphoma. When mice were immunized with an idiotype-vaccine, those maintained under enriched environmental conditions produced statistically significant higher levels of anti-idiotype antibodies and revealed more attenuated tumor growth than those housed in standard environments. Most strikingly, enriched tumor-bearing mice had statistically significant prolonged survival, with 44% of them disease-free compared with 0% in the standard rearing tumor-bearing mice. The possible mechanisms for the enhancement of immunotherapy by environmental enrichment are cognitive, physical activity, and psychologic. The demonstration of synergistic effect of cancer therapy and environmental enrichment on tumor rejection has important implication for cancer treatment.

Rejection of tumors of the B cell lineage by idiotype-vaccinated mice.

Abstract Idiotypic determinants of immunoglobulins of malignant B lymphocytes and plasma cells are tumor-specific antigens and have been used extensively in immunotherapy studies. The mechanisms involved in resistance to tumor challenge following idiotype vaccination are poorly understood. Although a predominant role has been attributed to anti-idiotype antibodies, both humoral and cellular immune responses are probably involved. Cell-mediated responses may be particularly effective against tumor cell variants that do not express the idiotype on the cell surface and are therefore resistant to anti-idiotype antibodies but continue to produce one of the original immunoglobulin polypeptides that may be processed and presented to T cells. In this report we describe two experimental models of idiotype vaccination in which antibodies are unlikely to play a role, and hence tumor immunity is attributed to cell-mediated responses. One model consists of the murine B lymphocyte tumor 38C-13 and its idiotype-negative variant DB2, which has lost the idiotypic specificity of the parental 38C-13 cell line through the production of a different light chain but expresses the original heavy chain. Vaccination of mice with the purified IgM of 38C-13 induced resistance to 38C-13 tumor cells as well as to the variant cells. Although immunized mice produced high levels of anti-idiotype antibodies that bound to 38C-13 cells, no binding of antibodies to DB2 cells occurred. The finding that idiotype-vaccinated mice were resistant to idiotype-negative DB2 cells suggested that cellular mechanisms are involved in mediating resistance. The second model consists of the two plasma cell line JLμs and JLμm, which produce IgM with an identical specificity. Whereas one of them (JLμs) secretes the IgM, the other one(JLμm) can neither secrete nor deposit it on the cell surface. Immunization against JLμs IgM followed by tumor challenge resulted in prolonged survival of both JLμs- and JLμm-challenged mice. Although sera of immunized mice contained high levels of anti-idiotype antibodies, they did not react with the plasmacytoma cells. Similarly to the results obtained in the 38C-13 experimental model, these results suggest that a non-antibody-mediated mechanism was involved in the resistance of mice to tumor growth.

Anti-idiotype × Anti-LFA-1 Bispecific Antibodies Inhibit Metastasis of B Cell Lymphoma

Abs to adhesion molecules can block tumor metastasis. However, they may also block the function of normal cells. To circumvent this adverse effect, we proposed the use of bispecific Abs that bind simultaneously to an adhesion receptor and to a tumor-specific Ag. Such Abs bind more avidly to tumor cells that coexpress both target Ags than to normal cells. The Id of the surface Ig of malignant B lymphocytes is a tumor-specific Ag. We therefore produced a bispecific Ab with specificity to the adhesion molecule LFA-1 and to the Id of the murine B cell lymphoma 38C-13. Here we demonstrate that this Ab blocked liver metastasis in mice carrying primary s.c. tumors and partially inhibited lymph node metastasis. Migration of 38C-13 cells to liver and lymph nodes was inhibited by the bispecific Ab, while migration to spleen was not affected. Hence, the bispecific Ab-mediated reduction in liver and lymph node metastasis resulted at least in part from reduced homing to these organs. In contrast to anti-LFA-1 monospecific Abs, the anti-Id × anti-LFA-1 bispecific Ab did not affect immune responses such as delayed-type hypersensitivity. Hence, bispecific Abs against adhesion molecules and against tumor-specific Ags may selectively block tumor metastasis in a way that may leave much of the immune system intact.

B-cell lymphoma and myeloma protection induced by idiotype vaccination with dendritic cells is mediated entirely by T cells in mice.

Immunoglobulin idiotypes (Id) of malignant B cells are tumor-specific antigens that may be targeted for immunotherapy. Id-directed immunotherapy by immunization with autologous Id has been initiated in clinical trials to control residual disease in B-cell lymphoma and multiple myeloma. The effector mechanisms responsible for destruction of B-cell tumors are a controversial issue. The authors show that vaccination with Id-pulsed dendritic cells (DCs) or with soluble Id-KLH in adjuvant induced immune responses that eliminated both B-cell lymphoma and myeloma in tumor-bearing mice; however, the two vaccination regimens resulted in distinct immune responses. Whereas soluble Id plus adjuvant induced high levels of anti-Id antibodies, the Id-pulsed DCs did not induce anti-Id or any antitumor antibodies. Immunization with Id-pulsed DCs induced a significant increase in the frequency of Id-reactive T cells. Depletion studies in DC-vaccinated mice showed that the predominant effector cells responsible for tumor rejection were of the CD8+ subset. The finding that DC-based Id vaccines elicit tumor protection, which is entirely based on cell-mediated effector mechanisms, is of particular importance for plasma cell tumors because these tumors do not express Id on the surface and hence do not bind anti-Id antibodies.

Enhanced therapeutic effect of B cell‐depleting anti‐CD20 antibodies upon combination with in‐situ dendritic cell vaccination in advanced lymphoma

The present standard of care for B cell non‐Hodgkins lymphoma includes the anti‐CD20 monoclonal antibody rituximab. Although combination treatments with chemotherapy and rituximab improved the duration of remissions and overall survival in indolent B cell lymphoma, the disease is essentially incurable. Thus, new therapeutic approaches are needed. One such approach is active immunization. Given that rituximab depletes both malignant and normal B cells, it is expected to impair humoral immune responses in vaccinated patients. Hence, optimal vaccination strategies for rituximab‐treated patients require induction of effector T cells, which can be achieved by dendritic cell (DC) vaccines. We have demonstrated in a mouse model that chemotherapy combined with DC vaccines was therapeutically effective. However, efficacy was related to tumour size at the onset of treatment, decreasing in correlation with increasing tumour burdens. We therefore examined whether, in spite of its low efficacy in advanced disease, DC vaccination may synergize with anti‐CD20 antibodies to enhance therapy. Lymphoma‐bearing mice were treated with cyclophosphamide, anti‐CD20 antibodies and an intratumoral DC vaccine. Results clearly demonstrated the enhanced therapeutic effect of this combination treatment. Thus, under conditions of disseminated disease, when either anti‐CD20 antibody treatment or vaccination showed insufficient efficacy, their combination resulted in synergism that mediated long‐term survival. We demonstrated further that the combination of antibody and vaccine induced T cell‐mediated anti‐tumour immune responses with long‐term memory. Combination treatments including tumour cell‐loaded DC vaccines may therefore provide a strategy for enhancing therapy in rituximab‐treated patients.