Tatiana D. Zorina

Bone Marrow‐Derived Dendritic Cells Serve as Potent Adjuvants for Peptide‐Based Antitumor Vaccines

Dendritic cells (DCs) are considered the most effective antigen‐presenting cells (APCs) for primary immune responses. Since presentation of antigens to the immune system by appropriate professional APCs is critical to elicit a strong immune reaction and DCs seem to be quantitatively and functionally defective in the tumor host, DCs hold great promise to improve cancer vaccines. Even though they are found in lymphoid organs, skin and mucosa, the difficulty of generating large numbers of DCs has been a major limitation for their use in vaccine studies. A simple method for obtaining DCs from mouse bone marrow cells cultured in the presence of GM‐CSF + interleukin 4 is now available. In four different tumor models, mice injected with DCs grown in GM‐CSF plus interleukin 4 and prepulsed with a cytotoxic T lymphocyte‐recognized tumor peptide epitope developed a specific cytotoxic T lymphocyte response and were protected against a subsequent tumor challenge with tumor cells expressing the relevant tumor antigen. Moreover, treatment of day 5‐14 tumors with peptide‐pulsed DCs resulted in sustained tumor regression in five different tumor models. These results suggest that presentation of tumor antigens to the immune system by professional APCs is a promising method to circumvent tumor‐mediated immunosuppression and is the basis for ongoing clinical trials of cancer immunotherapy with tumor peptide‐pulsed DCs.

Recovery of the Endogenous β Cell Function in the NOD Model of Autoimmune Diabetes

In light of accumulating evidence that the endocrine pancreas has regenerative properties and that hematopoietic chimerism can abrogate destruction of β cells in autoimmune diabetes, we addressed the question of whether recovery of physiologically adequate endogenous insulin regulation could be achieved in the nonobese diabetic (NOD) mice rendered allogeneic chimerae. Allogeneic bone marrow (BM) was transplanted into NOD mice at the preclinical and overtly clinical stages of the disease using lethal and nonlethal doses of radiation for recipient conditioning. Islets of Langerhans, syngeneic to the BM donors, were transplanted under kidney capsules of the overtly diabetic animals to sustain euglycemia for the time span required for recovery of the endogenous pancreas. Nephrectomies of the graft‐bearing organs were performed 14 weeks later to confirm the restoration of endogenous insulin regulation. Reparative processes in the pancreata were assessed histologically and immunohistochemically. The level of chimerism in NOD recipients was evaluated by flow cytometric analysis. We have shown that as low as 1% of initial allogeneic chimerism can reverse the diabetogenic processes in islets of Langerhans in prediabetic NOD mice, and that restoration of endogenous β cell function to physiologically sufficient levels is achievable even if the allogeneic BM transplantation is performed after the clinical onset of diabetes. If the same pattern of islet regeneration were shown in humans, induction of an autoimmunity‐free status by establishment of a low level of chimerism, or other alternative means, might become a new therapy for type 1 diabetes.

Transplanted Hematopoietic Cells Seed in Clusters in Recipient Bone Marrow In Vivo

The process of hematopoietic stem and progenitor cell (HSPC) seeding in recipient bone marrow (BM) early after transplantation is not fully characterized. In vivo tracking of HSPCs, labeled with PKH dyes, through an optical window surgically implanted on the mouse femur revealed that transplanted cells cluster in the recipient BM. Within the first day after intravenous injection, 86 ± 6% of the cells seeded in clusters (p < 0.001 versus scattered cells) in the endosteal surfaces of the epiphyses. The primary clusters were formed by concomitant seeding of 6‐10 cells over an area of ∼70 μm, and secondarily injected cells did not join the already existing clusters but formed new clusters. Major antigen‐disparate HSPCs participated in formation of the primary clusters, and T lymphocytes were also incorporated. After 4 to 5 days, some cellular clusters were observed in the more central regions of the BM, where the brightness of PKH fluorescence decreased, indicating cellular division. These later clusters were classified as secondary, assuming that the mechanisms of migration in the BM might be different from those of primary seeding. Some clusters remained in the periphery of the BM and retained bright fluorescence, indicating cellular quiescence. The number of brightly fluorescent cells in the clusters decreased exponentially to two to three cells after 24 days (p < 0.001). The data suggest that the hematopoietic niche is a functional unit of the BM stromal microenvironment that hosts seeding of a number of transplanted cells, which form a cluster. This may be the site where auxiliary non‐HSPC cells, such as T lymphocytes, act in support of HSPC engraftment.

Distinct characteristics and features of allogeneic chimerism in the NOD mouse model of autoimmune diabetes.

The adaptation of allogeneic chimerism in treatment of autoimmune diabetes has been shown as a promising approach in numerous studies in both experimental and clinical settings. Establishment of hemopoietic chimerism in NOD mice is the most adequate animal model to study mechanisms involved in the multiple aspects of the curative effects of chimerism in autoimmunity-prone individuals. However, there are some discrepancies in the current literature for parameters and criteria used to characterize chimerism in the NOD model. This study was aimed to standardize the criteria for the different pathological stages of diabetogenesis in chimeric versus unmanipulated NOD mice. We report two well-defined scoring systems and a new Index N for the assessment of the pathological characteristics of diabetogenesis and GVHD in chimeric NOD mice. Also, we have demonstrated that, in the NOD model, recipient conditioning resulting in as low as 1% of chimerism is sufficient to promote engraftment of the BM donor-specific islets of Langerhans.

Regulation of dendritic cell expansion in aged athymic nude mice by FLT3 ligand.

This report describes age-related alterations of dendritic cells (DC) distribution in nude athymic mice in vivo and reversal of certain age-dependent defects by an in vivo administration of hematopoietic growth factor FLT3 ligand (FLT3L). There are decreased percentages of CD11c(+) DC in the bone marrow and spleen and a reduced expression of MHC class II and CD86 molecules on DC in old nude mice. The decreased levels of CD11c(+) DC were due to the CD8alpha(-) DC subset. The distribution of CD11c(+) CD8alpha(+) DC in the lymphoid tissues was not different in young and old mice. The effect of in vivo administration of FLT3L on the generation and distribution of DC in the lymphoid tissues in young and old nude mice was also evaluated. Although, FLT3L had a higher inductive potential on the expansion of DC from the bone marrow in the elderly mice, the total level of CD11c(+) DC in the young animals was still significantly higher as compared to the old animals. Interestingly, FLT3L induced a pronounced redistribution and accumulation of MHC class II(+) DC in the lymphoid tissues in old mice, markedly increased the accumulation of CD8alpha(-) DC in the bone marrow in both young and old nude mice, and elevated both CD8alpha(-) and CD8alpha(+) DC in the spleen in young mice. However, only the level of CD8alpha(+) DC was up regulated in the spleen in old athymic mice after FLT3L-based therapy. In summary, abnormalities in DC generation and distribution in old athymic mice could be, in part, circumvented by the in vivo administration of FLT3L.

Treg cells in pancreatic lymph nodes: the possible role in diabetogenesis and β cell regeneration in a T1D model

Previously, we established a model in which physiologically adequate function of the autologous β cells was recovered in non-obese diabetic (NOD) mice after the onset of hyperglycemia by rendering them hemopoietic chimera. These mice were termed antea-diabetic. In the current study, we addressed the role of T regulatory (Treg) cells in the mechanisms mediating the restoration of euglycemia in the antea-diabetic NOD model. The data generated in this study demonstrated that the numbers of Treg cells were decreased in unmanipulated NOD mice, with the most profound deficiency detected in the pancreatic lymph nodes (PLNs). The impaired retention of the Treg cells in the PLNs correlated with the locally compromised profile of the chemokines involved in their trafficking, with the most prominent decrease observed in SDF-1. The amelioration of autoimmunity and restoration of euglycemia observed in the antea-diabetic mice was associated with restoration of the Treg cell population in the PLNs. These data indicate that the function of the SDF-1/CXCR4 axis and the retention of Treg cells in the PLNs have a potential role in diabetogenesis and in the amelioration of autoimmunity and β cell regeneration in the antea-diabetic model. We have demonstrated in the antea-diabetic mouse model that lifelong recovery of the β cells has a strong correlation with normalization of the Treg cell population in the PLNs. This finding offers new opportunities for testing the immunomodulatory regimens that promote accumulation of Treg cells in the PLNs as a therapeutic approach for type 1 diabetes (T1D).

Development of Dendritic Cell-Based Genetic Vaccines for Cancer

Cytotoxic T lymphocytes (CTL) are an important component of the host’s immune response to cancer1,2. A number of genes encoding tumor-associated antigens (TAA) and their peptide products which are recognized by CTL in the context of major histocompatibility complex (MHC) class I molecules have recently been identified3,4. Our group has focused on the translation of these new insights into the development and application of novel immunotherapies.

Infectious Diseases in Cancer Patients: An Overview

The predisposition of cancer patients to infectious diseases which contribute to the gravity of their prognosis is well documented. The current success in therapy of both malignancies and infections is unprecedented. However, the overall co-morbidity of these conditions is still a major problem in management of these patients. Paradoxically, to some degree the problem of containing infectious complications is directly associated with the vigor of the anti-cancer therapeutic regimens. The objective of this chapter is to provide an up to date overview of our understanding of the infectious complications in cancer patients based on the type of infection and immune responses.