Social isolation in mice: behavior, immunity, and tumor growth.

The aim of this study was to investigate the behavioral, immunological, and neurological effects of long-term isolation in an animal model. Male C3H/eB mice wereraised in either social isolation or standard conditions for 6 weeks. At 10 weeks, each group was further divided into 3 sets. (A) Physical strength and behavior were evaluated with the grip strength, hot plate, staircase, and elevated plus-maze tests. Natural-killer cell activity and lymphocyte proliferation were measured. (B) Half the animals were subjected to electric shock with 3 reminders, and freezing time was evaluated at each reminder. Cortisone levels were evaluated after 16 weeks. (C)Mice were injected with 38 C-13 B lymphoma cells and followed for tumor size and survival. Strength evaluation yielded asignificantly lower body weight and grip strength in the socially isolated mice. Behavioral test results were similar in the two groups. The pattern of reactions to stress conditioning differed significantly, with the socially isolated mice showing an incline in freezing with each successive reminder, and the control mice showing a decline. The socially isolated mice had significantly attenuated tumor growth, with no significant difference in survival from control mice. There were no significant between-group differences in immunological parameters. In conclusion, social isolation serves as a model for chronic stress. It was associated with significant changes in stress conditioning reaction, resembling symptoms of post-traumatic stress disorder, and attenuated tumor development. No differences from controls were found in behavior tests, immune parameters, or survival after tumor cell inoculation.Lay summaryThis article explores biological and behavioral consequences of social isolation in a mice model. Our results show that social isolation leads to changes in the Hypothalamic-hypophyseal-adrenal axis, which in turn alter the response to stress. Additionally, social isolation was shown to impact tumor progression.

Stress conditioning in mice: alterations in immunity and tumor growth.

The neuroendocrine and autonomic nervous systems are known regulators of brain-immune interaction. However, the functional significance of this interaction under stress is not fully understood. We investigated the effect of a stress paradigm by applying electric foot shock followed by three reminders, on behavior, immune parameters, and lymphoma tumor growth. Male C3H mice were divided into two groups: Group 1-exposed to electric foot shock followed by three reminders, and Group 2-untreated (controls). Sets of mice underwent the elevated plus maze, staircase, and hot plate tests. After foot shock, natural killer (NK) cell activity, and lymphocyte proliferation were measured. In addition, sets of mice were either vaccinated twice with B-cell lymphoma 38C-13 immunoglobulin for determination of anti-idiotype (Id) antibodies in sera, or inoculated with tumor cells and monitored for tumor development and survival time. Mice exposed to electric foot shock followed by the three reminders had higher NK cell activity, levels of anti-Id antibodies, and a higher proliferation rate of splenocytes in response to mitogens, than the control mice. The exposed mice also showed attenuated tumor growth. Thus, the stress paradigm inhibited tumor development and lead to some immune changes that were not accompanied by behavioral changes.

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.

Current vaccination strategies for the treatment of B-cell lymphoma and multiple myeloma.

Therapeutic vaccines have been developed to induce immune responses capable of eradicating lymphoma and myeloma tumors. Most of these vaccines target the immunoglobulin idiotype (Id) as a tumor-specific antigen. Phase I/II clinical trials of Id vaccination in lymphoma demonstrated that some lymphoma patients could mount immune responses that were correlated with a favorable clinical outcome. These encouraging results initiated phase III trials of Id vaccination in lymphoma, the results of which have been recently released. Disappointingly, only one of three phase III studies achieved the primary end point of progression-free survival. Detailed analysis of these results is awaited to help identify factors that determine clinical efficacy of Id vaccination as reflected in the three trials. Unlike lymphoma, studies of Id vaccination in multiple myeloma have yielded far less evidence of clinical benefit. Although Id vaccines induce immune responses in myeloma patients, their efficacy is insufficient to provide a clear clinical benefit. Strategies to improve lymphoma and myeloma vaccines are currently tested with an emphasis on optimization of antigen delivery and presentation and modulation of the immune system toward enhancement of T-cell function. Despite many hurdles yet to overcome, it is hoped that newly developed strategies that augment both immune and clinical responses will allow effective vaccination resulting in tumor eradication.

Altered N-Linked Glycosylation in Follicular Lymphoma and Chronic Lymphocytic Leukemia: Involvement in Pathogenesis and Potential Therapeutic Targeting.

B-cell antigen receptor (BCR) expression is indispensable for survival of most B-cell malignancies. In follicular lymphoma (FL), N-linked glycosylation sites are introduced in the immunoglobulin (Ig) variable region genes. Oligosaccharides added to the acquired sites are unusually of the high-mannose type. These glycans interact with mannose-specific lectins, especially with dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN). Lectin binding to FL triggers persistent activating signals, suggesting that lectins within the tumor microenvironment promote cell survival and proliferation. Insertion of N-glycosylation sites in Ig variable region genes has been detected in other germinal center-associated lymphomas, specifically in subsets of diffuse large B-cell lymphomas and Burkitt's lymphomas, suggesting involvement of altered glycans in pathogenesis of these malignancies as well. Furthermore, the BCR in chronic lymphocytic leukemia (CLL) carries high-mannose oligosaccharides, albeit in the heavy chain constant rather than variable region. The high expression level of the unique glycoform, particularly in the more aggressive unmutated CLL subset, suggests a functional significance for this glycan in CLL. As lectin interaction with the BCR is critical for FL and probably for some other lymphomas, targeting this interaction is considered to be an interesting therapeutic strategy. Reagents for blockade of lectin-BCR interaction may include antibodies against high-mannose glycans and mannose-based oligosaccharide mimics or non-carbohydrate glycomimetics. Moreover, as this interaction triggers signaling pathways similar to those demonstrated for BCR engagement by antigen, BCR signal transduction inhibitors may emerge as effective therapeutics for lectin-driven malignancies.

Distinct membrane localization and kinase association of the two isoforms of CD58.

The adhesion molecule CD58 is involved in intercellular adhesion and in signal transduction. It is natively expressed in both a transmembrane form and a glycosylphosphatidylinositol (GPI)-anchored form, and hence provides a model for the study of two distinct membrane-anchored forms of the same protein in the same cell. We demonstrate here that the two isoforms of CD58 are localized in distinct membrane compartments. The GPI-anchored form localizes in lipid rafts, while the transmembrane form resides in nonraft domains. In addition to distinct membrane localization, the two isoforms of CD58 differ in their association with protein kinases. GPI-anchored CD58, residing in raft domains, is constitutively associated with protein kinases. However, cross-linking mediates a substantial increase in kinase activity which is predominantly associated with the transmembrane CD58 in nonraft membrane domains. The extensive inducible kinase activity, associated with transmembrane CD58, is demonstrated in wild-type cells as well as in GPI-deficient variant cells. Thus, although the transmembrane CD58 is excluded from rafts, it may trigger signaling independently of the GPI-linked isoform.

Determination of idiotype-specific T cells in idiotype-vaccinated mice.

Immunoglobulin idiotypes (Id) of malignant B lymphocytes and plasma cells are tumor-specific antigens that were extensively used in immunotherapy studies. The effector mechanisms, involved in resistance to tumor following Id vaccination, are a controversial issue. Since cell-mediated responses, rather than antibody responses, constitute powerful effectors against tumors, recent studies focused on the generation of Id-specific T cells. Traditional methods for assessment of cellular responses in murine models of Id vaccination are inadequate because of their low sensitivity and because they do not determine actual frequency of antigen-reactive T cells. Here, we use the highly sensitive enzyme-linked immunospot (ELISPOT) assay for enumeration of interferon gamma (IFN-gamma)-secreting cells in Id-vaccinated mice. Our experimental model consists of the murine B-lymphocyte tumor 38C-13, which expresses surface IgM, and the plasma cell tumor D2, which secretes IgM with idiotypic specificity identical to that of 38C-13. Vaccination of mice with purified 38C-13 IgM induced resistance to 38C-13 as well as to D2 tumor cells. Although immunized mice produced high levels of anti-Id antibodies that bound to 38C-13 cells, no binding of antibodies to D2 occurred, suggesting that cellular mechanisms mediated resistance to the plasma cell tumor. ELISPOT assays revealed that immunization induced a significant increase in the frequency of Id-specific IFN-gamma-secreting T cells. Depletion of T cell subsets demonstrated that both CD4(+) and CD8(+) T cells were involved in the response to Id. This is the first report on application of the ELISPOT assay for enumeration of Id-reactive T cells in a murine model of Id vaccination, providing a tool to study Id-specific T cell responses and to evaluate the efficacy of Id vaccines.

Distinct processing of the pre-B cell receptor and the B cell receptor.

It has been recently demonstrated that while oligosaccharide moieties of µ heavy chains in the B-cell receptor (BCR) are of the complex type as expected, those of the pre-BCR on the surface of pre-B cells contain oligosaccharide moieties of the high-mannose type only. This is unique, because high-mannose glycans are generally restricted to the endoplasmic reticulum and not presented on the surface of mammalian cells. In the present study, we examined the processing of the unusually glycosylated µ heavy chains in pre-B cells. We demonstrate that the pre-BCR reaches the cell surface by a non-conventional brefeldin A-sensitive monensin-insensitive transport pathway. Although pre-BCR complexes consist of µ heavy chains with high-mannose oligosaccharide moieties, they are stably expressed in the plasma membrane and demonstrate turnover rates similar to those of the BCR. Thus, rapid internalization cannot account for their low surface expression, as previously postulated. Rather, we demonstrate that the low pre-BCR abundance in the plasma membrane results, at least in part, from insufficient production of surrogate light chains, which appears to be a limiting factor in pre-BCR expression.

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.

All oligosaccharide moieties of the µ chains in the pre-BCR are of the high-mannose type.

Although it is well established that pre-BCR signaling governs proliferation and differentiation during B cell development, the components of the pre-BCR that are important for signaling are a matter of controversy. It has been suggested that signaling by the µ heavy chains of the pre-BCR induces survival and differentiation of pre-B cells, while the λ5 part of the pre-BCR is essential for proliferation and clonal expansion. However, the mechanism by which pre-BCR µ chains initiate differentiation signals is not clear. Using two variants of a murine B-lymphocyte cell line that differ only in surface expression of either BCR or pre-BCR, we demonstrated that surface µ chains in the pre-BCR are of the high-mannose type only, while those in the BCR are of the complex type. It is hypothesized that mannose-specific lectin-like molecules on accessory cells or in solution may function as the non-antigen ligand that triggers the pre-BCR.

Bispecific antibodies for cancer therapy.

Bispecific antibodies, in contrast to conventional monoclonal antibodies, can bind simultaneously two different antigens. Taking advantage of this virtue, they are mostly designed for immune effector cell redirection to tumors and for radionuclide pretargeting to tumors. Bispecific antibodies of the first generation were produced by chemical cross-linking or cell-fusion technologies. More recently, the application of genetic engineering technologies gave rise to numerous formats of bispecific antibody fragments and whole IgG molecules. Because bispecific antibodies enable therapeutic strategies that are not possible with conventional monoclonal antibodies, they attract strong interest. Several bispecific antibody formats have already shown clinical efficacy in cancer patients, catalyzing efforts to translate the imaginative bispecific antibody concepts into effective therapies.

Signal transduction by CD58: the transmembrane isoform transmits signals outside lipid rafts independently of the GPI-anchored isoform.

The adhesion molecule CD58 is natively expressed in both a glycosylphosphatidylinositol (GPI)-anchored form and a transmembrane form. We previously demonstrated that the two isoforms of CD58 are differentially distributed in the cell membrane. The GPI-linked form resides in lipid rafts while the transmembrane form resides outside lipid rafts. Following cross-linking a fraction of transmembrane CD58 redistributes to lipid rafts. It has also been demonstrated that ligand binding to CD58 induces biological functions such as cytokine production and immunoglobulin isotype switching, indicating that cell-cell interactions result in CD58-mediated signal transduction. However, the signaling pathways involved in these activation processes are poorly defined. Here we show for the first time that cross-linking of CD58 induces protein tyrosine phosphorylation of BLNK, Syk and PLCgamma, and activation of ERK and Akt/PKB. In addition, we studied how these signaling events relate to the distinct membrane localization of the two isoforms of CD58. We demonstrate that cross-linking of CD58 triggers signaling that is predominantly associated with transmembrane CD58 in nonraft microdomains. Moreover, signaling through transmembrane CD58 does not depend on coexpression of the GPI-linked isoform. Thus, despite the residence of its GPI-anchored isoform in lipid rafts and the translocation of a fraction of its transmembrane isoform to lipid rafts, CD58 signaling is triggered by the transmembrane isoform outside lipid rafts. These findings corroborate signaling outside lipid rafts, as opposed to the established notion that rafts function as essential platforms for signaling.

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.

Growth inhibition of myeloma cells by anti-idiotype antibodies in the absence of membrane-bound immunoglobulin.

Immunoglobulins are expressed as membrane-bound or secreted forms. Plasma cells produce little or no membrane immunoglobulin but secrete immunoglobulin molecules in large amounts. Immunoglobulin idiotypes of malignant B cells are tumor-specific antigens that may be targeted for immunotherapy. Thus, idiotype vaccination is being evaluated in clinical trials to control residual disease in multiple myeloma and non-Hodgkin's lymphoma. It is traditionally considered that anti-idiotype antibodies are not effective against plasma cell tumors, because the large amounts of immunoglobulin molecules secreted by the tumors block anti-idiotype antibodies, and because the absence of membrane immunoglobulin on the surface of these tumor cells renders them resistant to the effect of anti-idiotype antibodies. While the obstacle of abundant circulating idiotype may be obviated by reducing tumor burden to minimal residual disease, the absence of membrane immunoglobulin has been considered as a limiting factor that prevents tumor eradication by anti-idiotype antibodies. We demonstrate here that murine plasmacytoma cells can produce small amounts of membrane immunoglobulin M (IgM) heavy chains. However, the latter are precursor molecules that do not reach the cell surface. Although membrane-bound IgM is absent, the cells stain positively for surface IgM, reflecting molecules of the secreted form in the process of secretion. In spite of the relatively low levels of secreted immunoglobulin on the cell surface, anti-idiotype antibodies are effective in retardation of tumor growth in vivo. Thus, while there is no doubt that idiotype-specific cell-mediated responses are very important, myeloma patients in complete remission may additionally benefit from idiotype-specific humoral responses.

Environmental enrichment augments the efficacy of idiotype vaccination for B-cell lymphoma.

Environmental enrichment is known to positively influence the organism's 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.

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.

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 x 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 x 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.

Anti-idiotype x 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 x 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.