/A2 mice have been widely used to study human immunology because these mice serve as a host for human immune cells (13
). In addition, immune-deficient mice are being used to study various human diseases, including graft-versus-host disease after the injection of MHC-mismatched human PBMCs (29
) and also islet transplantation rejection by mismatching MHC class I molecules of donor PBMCs to recipient islet MHC class I molecules (12
Our goal is to study human autoimmunity, specifically type 1 diabetes, using a humanized mouse model by transferring HLA-A2 matched PBMCs from type 1 diabetic patients and NDDs into NOD-scid/γcnull/A2 mice. We observed proliferation and survival of T cells (CD3+, CD4+, and CD8+) and B cells for at least 6 weeks in these mice. Similar levels of T cells in the spleen and PLN were found in mice engrafted with PBMCs from type 1 diabetic patients and NDDs; however, more B cells were found in the spleen and PLN in mice that received PBMCs from type 1 diabetic patients. This demonstrates the ability of human immune cell subsets to engraft secondary lymphoid organs after transfer. Moreover, the higher levels of B cells in the PLN and spleen may reflect what occurs in the PLN and spleen of type 1 diabetic patients. NOD-scid mice that express a chimeric A2/Kb gene and were wild-type at the IL2r-γ chain locus were poorly engrafted (data not shown).
Through this model we have revealed an increased level of islet infiltration in mice that received type 1 diabetic PBMCs compared with NDD PBMCs. Previous studies using NOD-scid
mice and HLA-A1+
PBMCs from type 1 diabetic patients have demonstrated the presence of islet-reactive T cells in pancreatic tissue (31
), but these T-cell clones were not capable of intraislet infiltration. We have shown here that T and B cells preferentially infiltrate the islets of NOD-scid
/A2 mice that received type 1 diabetic PBMCs and that a significant number of CD8+
T cells from the islets and spleen are specific for IGRP, IA-2, IAP, and insulin (known diabetogenic epitopes). These cells were found at a higher frequency in the islets compared with the spleen, indicating the islets are a preferred site of autoreactive T-cell expansion and accumulation. Therefore, the extravasation and accumulation of human autoreactive islet-specific T cells from circulation into the islets suggest that PBMCs from type 1 diabetic patients are capable of infiltrating the target organs necessary for the induction of diabetes.
Circulating cytotoxic T lymphocytes isolated from HLA-A2+
type 1 diabetic patients have been shown to kill β-cells through recognition of a glucose-regulated preproinsulin epitope (18
). In our model, higher frequencies of diabetogenic epitope-specific CD8+
T cells from type 1 diabetic patients infiltrate the islets of NOD-scid
/A2 mice. Furthermore, these antigen specific cells are capable of producing IFN-γ after peptide stimulation.
Until now, no reported studies have demonstrated islet infiltration of NOD-scid
/A2 mice by antigen-specific T cells using HLA-A2–matched PBMCs from type 1 diabetic patients. This may be the result of poor engraftment in previous models that is often associated with natural killer cell activity or the lack of human costimulatory molecules such as human MHC molecules (32
). We identified an engraftment threshold and determined that 20–30 × 106
PBMCs is ideal for maximum engraftment in NOD-scid
/A2 mice. This signifies a size limitation for transferring human cells into mice, which is similar to previous findings using NOD-scid
Although many genetic loci contribute to the development of diabetes, initial genome scans attribute particular MHC class I and II alleles as major contributors to the development of autoreactive T-cell responses in both humans and NOD mice (35
). The process of developing diabetes is accelerated in NOD HLA-A2.1 MHC class I transgenic mice and is mediated by pathogenic A2-restricted T-cell responses (37
). In humans, two islet-associated epitopes, IA-2 and GAD65, have been identified as being recognized by HLA-A2–restricted CD8+
T cells, thus indicating that CD8+
T cells that are HLA-A2 restricted may contribute to β-cell death. Although it is not clear whether CD8+
T cells are involved during the early phases of type 1 diabetes, we do know that they are involved in the development of diabetes because diabetes does not develop in NOD mice lacking MHC class I. The importance of IGRP, IAPP, IA-2, and insulin-specific CD8+
T cells is still unknown in humans. However, GAD65 and IA-2 are major islet antigens targeted in human type 1 diabetes. The presence of an enriched population of epitope-specific CD8+
T cells in the islets after transfer may indicate the expansion of epitope-specific immunodominant T cells from patients with type 1 diabetes in these humanized mice.
Overt diabetes has not yet developed in any of the NOD-scid/γcnull/A2 mice that received PBMCs from HLA-A2+ type 1 diabetic patients, although one of the nine recipients of type 1 diabetes HLA-A2+ PBMCs did show elevated blood glucose levels up to 235 mg/dL. It is likely that in addition to the HLA-A2 molecule, this mouse model may be improved by the addition of human MHC class II molecules or other costimulatory molecules that would allow interaction of the donor cells with mouse cells in the spleen and lymph node and perhaps provide a better environment for engraftment in these tissues. In the current model, CD4+ T cells can interact with engrafted donor APCs via human class II only, whereas the CD8+ T cells can receive stimulation from both donor and recipient (NOD-scid/γcnull/A2 mouse) cells.
It is difficult to isolate large numbers of diabetogenic CD8+ T cells directly from the peripheral blood of patients with type 1 diabetes. Therefore, one of the advantages of this model is the 1,000-fold increase in epitope-specific CD8+ T cells observed in mice engrafted with type 1 diabetic PBMCs. Presumably, this will allow more studies of human diabetogenic T cells; for example, we can test the cross-reactivity of diabetogenic T cells to common pathogens. This model also allows us to identify, directly from type 1 diabetic patients, islet epitope-specific CD8+ T cells. The four epitopes tested here comprised 63–88% of the islet-infiltrating CD8+ T cells in three of six mice that received type 1 diabetic PBMCs. In the islets of the remaining mice engrafted with type 1 diabetic PBMCs, these epitopes were recognized by 19–27% of CD8+ T cell infiltrates. In the future, we plan to use larger pools of peptides derived from islet antigens, and this may lead to the identification of other islet-infiltrating T cells.
This model of autoimmunity may also be useful in predicting islet cell transplantation by analyzing the level of pre-existing diabetogenic T cells in candidate patients with type 1 diabetes. Therefore, with future developments of this model, researchers may investigate more directly the mechanisms underlying T cell responses. We can gain valuable insight into the pathogenesis of various autoimmune diseases and predict immunotherapeutic responses.