Assessing the effectiveness of Interleukin-2 therapy in experimental type 1 diabetes.

AIM: Much focus of immunotherapy for type 1 diabetes (T1D) has been devoted on selectively boosting regulatory T (Treg) cells using low dose IL-2 due to their constitutive expression of IL-2Rα, CD25. However, several clinical trials using a low dose of IL-2 only showed a limited improvement of metabolic control. It can therefore be hypothesized that further decreasing IL-2 dosage may increase the selective responsiveness of Treg cells. METHODS: We induced experimental T1D using multiple low dose streptozotocin (STZ) injections and treated the mice with an ultra-low dose IL-2 (uIL-2, approximately 7-fold lower than low dose). Immune response was studied using multicolor flow cytometry. RESULTS: We found that uIL-2 did not protect STZ mice from developing hyperglycemia. It did neither increase Treg cell proportions, nor did it correct the phenotypic shift of Treg cells seen in T1D. It only partially decreased the proportion of IFN-γ+ T cells. Likewise, uIL-2 also did not protect the dysfunction of regulatory B (Breg) cells. Strikingly, when administered in combination with an anti-inflammatory cytokine IL-35, uIL-2 abrogated IL-35's protective effect. Low dose IL-2, on the other hand, protected half of the STZ mice from developing hyperglycemia. No difference was found in the Treg and Breg response, and it only tended to decrease CD80 expression in macrophages and dendritic cells. CONCLUSION: In conclusion, further decreasing IL-2 dosage may not be a suitable approach for T1D therapy, and the limited success suggests that an alternative low dose IL-2 therapy strategy or other immunotherapies should be considered.

Interleukin-35 Prevents Development of Autoimmune Diabetes Possibly by Maintaining the Phenotype of Regulatory B Cells.

The anti-inflammatory role of regulatory B cells (Breg cells) has been associated with IL-35 based on studies of experimental autoimmune uveitis and encephalitis. The role of Breg cells and IL-35+ Breg cells for type 1 diabetes (T1D) remains to be investigated. We studied PBMCs from T1D subjects and healthy controls (HC) and found lowered proportions of Breg cells and IL-35+ Breg cells in T1D. To elucidate the role of Breg cells, the lymphoid organs of two mouse models of T1D were examined. Lower proportions of Breg cells and IL-35+ Breg cells were found in the animal models of T1D compared with control mice. In addition, the systemic administration of recombinant mouse IL-35 prevented hyperglycemia after multiple low dose streptozotocin (MLDSTZ) injections and increased the proportions of Breg cells and IL-35+ Breg cells. A higher proportion of IFN-γ+ cells among Breg cells were found in the PBMCs of the T1D subjects. In the MLDSTZ mice, IL-35 administration decreased the proportions of IFN-γ+ cells among the Breg cells. Our data illustrate that Breg cells may play an important role in the development of T1D and that IL-35 treatment prevents the development of hyperglycemia by maintaining the phenotype of the Breg cells under an experimental T1D condition.

Kinetics of immune cell responses in the multiple low-dose streptozotocin mouse model of type 1 diabetes.

In type 1 diabetes (T1D), the insulin-producing ß cells are destructed by immune mechanisms. It has been hypothesized that the very first immune response in T1D onset comes from innate immune cells, which further activates the adaptive immune cells to attack the islets. Despite intensive research on characterization of islet-infiltrating immune cells, the kinetics of different immune cells in multiple low-dose streptozotocin (MLDSTZ)-induced T1D mouse model is still much unclear. Therefore, we investigated the proportions of innate immune cells such as neutrophils, dendritic cells (DCs), plasmacytoid dendritic cells (pDCs), macrophages, natural killer (NK) cells, and adaptive immune cells (T and B lymphocytes) in thymi, pancreatic-draining lymph nodes, and spleens of MLDSTZ mice on days 3, 7, 10, and 21 after the first injection of STZ by flow cytometry. The proportions of DCs and B cells were increased from day 3, while the proportions of B-1a lymphocytes and interferon-γ+ cells among NK cells were increased, but NK cells were decreased on day 10 in MLDSTZ-treated mice, illustrating that the initial immune response is induced by DCs and B cells. Later, the proportions of T helper 1 and cytotoxic T cells were increased from day 7, suggesting that the innate immune cells precede adaptive immune cell response in MLDSTZ mice. Altogether, our data demonstrate a possible sequence of events regarding the involvement of DCs, pDCs, NK cells, B-1a lymphocytes, B, and T cells at the early stage of T1D development.

Pro-tumoral immune cell alterations in wild type and Shb-deficient mice in response to 4T1 breast carcinomas.

To assess mechanisms responsible for breast carcinoma metastasis, 4T1 breast carcinomas were grown orthotopically in wild type or Shb knockout mice. Tumor growth, metastasis, vascular characteristics and immune cell properties were analyzed. Absence of Shb did not affect tumor growth although it increased lung metastasis. Shb knockout mouse tumors showed decreased redness and less developed vascular plexa located at the periphery of the tumors. No difference in overall tumor vascular density, leakage or pericyte coverage was noted between the genotypes although the average vessel size was smaller in the knockout. Tumors induced an increase of CD11b+ cells in spleen, lymph node, thymus, bone marrow and blood. Numbers of Shb knockout CD11b/CD8+ cells were decreased in lymph nodes and bone marrow of tumor bearing mice. Mice with tumors had reduced numbers of CD4+ lymphocytes in blood/lymphoid organs, whereas in most of these locations the proportion of CD4+ cells co-expressing FoxP3 was increased, suggesting a relative increase in Treg cells. This finding was reinforced by increased blood interleukin-35 (IL-35) in wild type tumor bearing mice. Shb knockout blood showed in addition an increased proportion of IL-35 expressing Treg cells, supporting the notion that absence of Shb further promotes tumor evasion from immune cell recognition. This could explain the increased number of lung metastases observed under these conditions. In conclusion, 4T1 tumors alter immune cell responses that promote tumor expansion, metastasis and escape from T cell recognition in an Shb dependent manner.