Genetically engineered human islets protected from CD8-mediated autoimmune destruction in vivo.

Islet transplantation is a promising therapy for type 1 diabetes, but graft function and survival are compromised by recurrent islet autoimmunity. Immunoprotection of islets will be required to improve clinical outcome. We engineered human ß cells to express herpesvirus-encoded immune-evasion proteins, "immunevasins." The capacity of immunevasins to protect ß cells from autoreactive T-cell killing was evaluated in vitro and in vivo in humanized mice. Lentiviral vectors were used for efficient genetic modification of primary human ß cells without impairing their function. Using a novel ß-cell-specific reporter gene assay, we show that autoreactive cytotoxic CD8(+) T-cell clones isolated from patients with recent onset diabetes selectively destroyed human ß cells, and that coexpression of the human cytomegalovirus-encoded US2 protein and serine proteinase inhibitor 9 offers highly efficient protection in vitro. Moreover, coimplantation of these genetically modified pseudoislets with ß-cell-specific cytotoxic T cells into immunodeficient mice achieves preserved human insulin production and C-peptide secretion. Collectively, our data provide proof of concept that human ß cells can be efficiently genetically modified to provide protection from killing mediated by autoreactive T cells and retain their function in vitro and in vivo.

In situ recognition of autoantigen as an essential gatekeeper in autoimmune CD8+ T cell inflammation.

A current paradigm states that non-antigen-specific inflammatory cues attract noncognate, bystander T cell specificities to sites of infection and autoimmune inflammation. Here we show that cues emanating from a tissue undergoing spontaneous autoimmune inflammation cannot recruit naive or activated bystander T cell specificities in the absence of local expression of cognate antigen. We monitored the recruitment of CD8(+) T cells specific for the prevalent diabetogenic epitope islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)(206-214) in gene-targeted nonobese diabetic (NOD) mice expressing a T cell "invisible" IGRP(206-214) sequence. These mice developed islet inflammation and diabetes with normal incidence and kinetics, but their inflammatory lesions could recruit neither naive (endogenous or exogenous) nor ex vivo-activated IGRP(206-214)-reactive CD8(+) T cells. Conversely, IGRP(206-214)-reactive, but not nonautoreactive CD8(+) T cells rapidly homed to and accumulated in the inflamed islets of wild-type NOD mice. Our results indicate that CD8(+) T cell recruitment to a site of autoimmune inflammation results from an active process that is strictly dependent on local display of cognate pMHC and suggest that CD8(+) T cells contained in extralymphoid autoimmune lesions are largely autoreactive.

Preservation of recall immunity in anti-CD3-treated recent onset type 1 diabetes patients.

BACKGROUND: The safety of any immune modulating agent in type 1 diabetes mellitus (T1DM) involves its selectivity on autoimmunity and its preservation of recall and tumour immunity. METHODS: We performed lymphocyte proliferation tests on seven recent onset diabetic patients treated with anti-CD3 (Otelixizumab; ChAglyCD3) and five recent onset diabetic patients treated with placebo, on average 2 years after therapy. RESULTS: Proliferative responses towards common viral, bacterial and yeast antigens upon in vitro stimulation with a range of recall antigens in anti-CD3-treated T1DM patients were highly similar to those in placebo-treated T1DM patients. Similarly, T-cell responses towards autoantigens were equally low between the two groups, several years after diagnosis of T1DM. The proliferative response upon stimulation with the human suppressor protein p53 was invariably high in both anti-CD3- and placebo-treated patients, implying preserved anti-tumour immunity in anti-CD3 treatment. CONCLUSIONS: As long-term focus on side effects is key, we demonstrate in this sub-cohort of recent onset T1DM patients treated with Otelixizumab that recall immunity is preserved in spite of high-dose anti-CD3 treatment, adding to the safety of anti-CD3 treatment as an immune-modulatory agent in the treatment of T1DM.

Development and application of fully functional epitope-tagged forms of transforming growth factor-beta.

Administration of transforming growth factor-beta (TGF-beta) has been found to be of therapeutic benefit in various mouse disease models and has potential clinical usefulness. However, the ability to track the distribution of exogenously administered, recombinant forms of these proteins has been restricted by cross-reactivity with endogenous TGF-beta and related TGF-beta isoforms. We describe novel FLAG- and hemagglutinin (HA)-tagged versions of mature TGF-beta1 that retain full biological activity as demonstrated by their ability to inhibit the growth of Mv1Lu epithelial cells, and to induce phosphorylation of the TGF-beta signaling intermediate, smad 2. Intracellular FLAG- and HA-TGF-beta1 can be detected in transfected cells by confocal immunofluorescence microscopy. We also describe sandwich ELISAs designed to specifically detect epitope-tagged TGF-beta and demonstrate the utility of these tagged ligands as probes for TGF-beta receptor expression by flow cytometry. The design of these fully functional epitope-tagged TGF-beta proteins should facilitate studies such as the evaluation of in vivo peptide pharmacodynamics and trafficking of TGF-beta ligand-receptor complexes.

Peripheral blood hematopoietic stem and progenitor cell frequency is unchanged in patients with alpha-1-antitrypsin deficiency.

Granulocyte-colony-stimulating factor (G-CSF)-induced hematopoietic stem and progenitor cell (HSPC) mobilization is associated with the release of neutrophil-derived proteases. Previously, we have shown that alpha-1-antitrypsin (AAT) inhibits these proteases in mice, resulting in inhibition of HSPC mobilization. Here, we studied the relationship between AAT and HSPC in steady state and cytokine-induced mobilization in humans. Patients with alpha-1-antitrypsin deficiency (AATD) have an 85-90 % decrease of AAT in the peripheral blood (PB). We hypothesized that this leads to increased proteolytic activity in the bone marrow and increased steady-state PB HSPC numbers. Using flow cytometry and semi-solid cell culture, we found no significant difference in PB HSPC in AATD patients (n = 18) as compared to controls (n = 22). Healthy stem cell donors (n = 43) were mobilized with G-CSF for 5 days and the number of CD45(+)/CD34(+) HSPC were determined in PB. We found that, during mobilization, PB AAT levels increased significantly, positively correlating with PB CD45(+)/CD34(+) cells (r = 0.31, p = 0.005). In conclusion, although serum AAT levels and HSPC mobilization in healthy stem cell donors are positively correlated, AAT is not an indispensable protease-inhibitor in the constitutive circulation of HSPC. These findings suggest a model in which both protease-dependent and -independent pathways contribute to HSPC mobilization.

Local autoantigen expression as essential gatekeeper of memory T-cell recruitment to islet grafts in diabetic hosts.

It is generally believed that inflammatory cues can attract noncognate, "bystander" T-cell specificities to sites of inflammation. We have shown that recruitment of naive and in vitro activated autoreactive CD8⁺ T cells into endogenous islets requires local autoantigen expression. Here, we demonstrate that absence of an autoantigen in syngeneic extrapancreatic islet grafts in diabetic hosts renders the grafts "invisible" to cognate memory (and naive) T cells. We monitored the recruitment of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206₋214-reactive CD8⁺ T cells into IGRP206₋214-competent and IGRP206₋214-deficient islet grafts in diabetic wild-type or IGRP206₋214(-/-) nonobese diabetic hosts (harboring either naive and memory T cells or only naive IGRP206₋214-specific T-cells, respectively). All four host-donor combinations had development of recurrent diabetes within 2 weeks. Wild-type hosts recruited IGRP206₋214-specific T cells into IGRP206₋214(+/+) but not IGRP206₋214(-/-) grafts. In IGRP206₋214(-/-) hosts, there was no recruitment of IGRP206₋214-specific T cells, regardless of donor type. Graft-derived IGRP206₋214 activated naive IGRP206₋214-specific T cells, but graft destruction invariably predated their recruitment. These results indicate that recurrent diabetes is exclusively driven by autoreactive T cells primed during the primary autoimmune response, and demonstrate that local antigen expression is a sine qua non requirement for accumulation of memory T cells into islet grafts. These findings underscore the importance of tackling autoreactive T-cell memory after ß-cell replacement therapy.