Modulation of Autoimmune T-Cell Memory by Stem Cell Educator Therapy: Phase 1/2 Clinical Trial

Abstract

Background: Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that causes a deficit of pancreatic islet β cells. The complexities of overcoming autoimmunity in T1D have contributed to the challenges the research community faces when devising successful treatments with conventional immune therapies. Overcoming autoimmune T cell memory represents one of the key hurdles.

Methods: In this open-label, phase 1/phase 2 study, Caucasian T1D patients (N = 15) received two treatments with the Stem Cell Educator (SCE) therapy, an approach that uses human multipotent cord blood-derived multipotent stem cells (CB-SCs). SCE therapy involves a closed-loop system that briefly treats the patient's lymphocytes with CB-SCs in vitro and returns the "educated" lymphocytes (but not the CB-SCs) into the patient's blood circulation. This study is registered with ClinicalTrials.gov, NCT01350219.

Findings: Clinical data demonstrated that SCE therapy was well tolerated in all subjects. The percentage of naïve CD4(+) T cells was significantly increased at 26 weeks and maintained through the final follow-up at 56 weeks. The percentage of CD4(+) central memory T cells (TCM) was markedly and constantly increased at 18 weeks. Both CD4(+) effector memory T cells (TEM) and CD8(+) TEM cells were considerably decreased at 18 weeks and 26 weeks respectively. Additional clinical data demonstrated the modulation of C-C chemokine receptor 7 (CCR7) expressions on naïve T, TCM, and TEM cells. Following two treatments with SCE therapy, islet β-cell function was improved and maintained in individuals with residual β-cell function, but not in those without residual β-cell function.

Interpretation: Current clinical data demonstrated the safety and efficacy of SCE therapy in immune modulation. SCE therapy provides lasting reversal of autoimmune memory that could improve islet β-cell function in Caucasian subjects.

Funding: Obra Social "La Caixa", Instituto de Salud Carlos III, Red de Investigación Renal, European Union FEDER Funds, Principado de Asturias, FICYT, and Hackensack University Medical Center Foundation.

Keywords: AIRE, autoimmune regulator; Autoimmunity; CB-SCs, human cord blood-derived multipotent stem cells; CCR7, C–C chemokine receptor 7; Cord blood stem cell; HLA, human leukocyte antigen; HbA1C, glycated hemoglobin; IL, interleukin; Immune modulation; M2, muscarinic acetylcholine receptor 2; MLR, mixed leukocyte reactions; MNC, mononuclear cells; Memory T cells; OGTT, oral glucose tolerance test; PBMC, peripheral blood mononuclear cells; R, responder; S, stimulator; SCE, Stem Cell Educator; T1D, type 1 diabetes; TCM, central memory T cells; TCR, T-cell receptor; TEM, effector memory T cells; TGF-β1, transforming growth factor-β1; Th, helper T cell; Tregs, regulatory T cells; Type 1 diabetes.

Figures

Fig. 1

Study flow chart.

Fig. 2

Diagram of SCE therapy for the treatment and follow-up studies. All the participants received two treatments with the SCE therapy. Human cord blood units were derived from healthy allogeneic donors. The preparation of CB-SC cultures SCE devices were cultured in serum-free culture medium and incubated at 37 °C, in 8% CO2. After 2–3 weeks, CB-SCs growing at 90% confluence were prepared for clinical trial. One Educator device was generated from one cord blood unit, and used for one subject at one treatment. Follow-up visits were scheduled 2, 8, 12, 18, 26, 40 and 56 weeks after treatment for clinical assessments and laboratory tests. Previous work demonstrated that participants receiving sham therapy failed to show changes in immune modulation (Zhao et al., 2012).

Fig. 3

Changes in immune markers in Caucasian T1D patients after SCE therapy. All subjects received two treatments with SCE therapy. After 3 months, all subjects received a 2nd treatment with SCE therapy. Follow-up visits were scheduled 2, 8, 12, 18, 26, 40 and 56 weeks after treatment for clinical assessments and laboratory tests. Patient lymphocytes were isolated from peripheral blood by Ficoll-Hypaque (γ = 1.077) for flow cytometry analyses in T1D patients at baseline and different time points after SCE therapy. Isotype-matched IgG served as control. (a) Immune cell quantification in peripheral blood. (b) Percentage of CD4+ and CD8+ T cells in peripheral blood. (c) Outline of the markers and approach for the characterization of different T-cell subpopulations. CD45RA and CCR7 were applied to characterize the naïve and memory T cells in the gated CD4+ (R2) T cells. Flow cytometry showed the baseline levels of T-cell populations (bottom left panel, orange) and those at 26 weeks post-treatment (bottom right panel, green) in the PBMCs of T1D patient. (d) Flow Analysis of naïve CD4+ and CD8+ T cells in peripheral blood, demonstrating an increase in the percentage of naïve CD4+ T cells at 26 weeks post treatment. (e) Flow Analysis of CD4+ TCM and CD8+ TCM cells in peripheral blood, demonstrating an increase in the percentage of CD4+ TCM cells at 18 weeks post treatment. (f) Flow Analysis of CD4+ TEM and CD8+ TEM cells in peripheral blood, demonstrating a decline in the percentage of CD4+ TEM and CD8+ TEM cells at 18 weeks and 26 weeks respectively post treatment. (g) Flow Analysis of CD4+ HLA-DR+ in peripheral blood, demonstrating a decline in their percentages at 26 weeks post treatment. (h) Flow Analysis of CD8+ HLA-DR+ T cells in peripheral blood, demonstrating a decline in their percentages at 26 weeks post treatment.

Fig. 4

Up-regulation of CCR7 expression on T cells in Caucasian T1D patients after SCE therapy. All subjects received two treatments with SCE therapy. After 3 months, all subjects received a 2nd treatment with SCE therapy. Follow-up visits were scheduled 2, 8, 12, 18, 26, 40 and 56 weeks after treatment for clinical assessments and laboratory tests. Patient lymphocytes were isolated from peripheral blood by Ficoll-Hypaque (γ = 1.077) for flow cytometry analyses in T1D patients at baseline and different time points after SCE therapy. Isotype-matched IgG served as control. The levels of CCR7 expression were analyzed by Kaluza Flow Cytometry Analysis Software and present as arbitrary unit (a.u.). (a) Up-regulation of CCR7 expression on Naïve CD4+ T cells. (b) Up-regulation of CCR7 expression on Naïve CD8+ T cells. (c) Up-regulation of CCR7 expression on CD4+ TCM cells. (d) Up-regulation of CCR7 expression on CD8+ TCM cells. (e) Modulation of CCR7 expression on CD4+ and CD8+ TEM cells. Data are shown as mean ± SD for all statistical analyses (a–e), paired Student's t test (a–e).

Fig. 5

Confirm the up-regulation of CCR7 expression on T cells by the ex vivo studies. (a) Phase contrast microscopy shows the formation of cell clusters with different sizes in the mixed leukocyte reactions (MLR), in absence (left panel) of CB-SCs, but disappeared in presence (right panel) of CB-SCs. (b and c) Cells from the mixed leukocyte reactions were collected for flow analysis after co-culture for 5 days. Responder cells (R) were co-cultured with allogeneic stimulator cells (S) in the presence of CB-SCs. The ratio of R:S was 1:2; the ratio of CB-SCs:R was 1:10. (b) Flow cytometry of CCR7 expression on the gated CD4+ T cells and CD8+ T cells. The untreated CD4+ lymphocytes showed two populations: one was positive for CCR7 expression; another was negative (or very dim) for CCR7 expression (Top left panel). The mean fluorescence intensities of both populations were increased after treatment with CB-SCs (bottom left panel). (c) Flow cytometry of CCR7 expression on Naïve CD4+ T cells, CD45RO+ CCR7+ TCM and CD45RO+ CCR7− TEM in the gated CD4+ T cells. The data showed the increase of the percentage of Naïve CD4+ T cells and CD4+ TCM in the presence of CB-SCs. The percentages of CD4+ TEM were decreased after treatment with CB-SCs.

Fig. 6

Effects of SCE therapy on β-cell function in Caucasian T1D subjects. All subjects received two treatments with SCE therapy (a–f). T1D subjects received two treatments with SCE therapy at the beginning and 3rd month respectively. Fasting (blue) and glucagon-stimulated C-peptide levels (brown) were examined at different time points according to the protocol. For glucagon-stimulated C-peptide production, glucagon (1 mg, i.v.) was administrated within 30 s, and six minutes later, plasma samples were collected for the C-peptide test by Ultrasensitive C-peptide ELISA kit. These data were from six T1D subjects with some residual islet β-cell function (Group A). (a–d) Recovered fasting and glucagon-stimulated C-peptide levels were retained in subject 1 through the final follow-up at 56 weeks post-treatments in subject 1–4 respectively. (e and f) show subjects 5 and 6 displayed some residual islet β-cell function beyond 10 years after diagnosis of T1D. After receiving SCE therapy, fasting C-peptide levels in Subject 5 initially decreased, but increased later at 40 weeks; fasting C-peptide levels in Subject 6 initially declined to 0.09 ng/ml at 26 weeks but improved to 0.21 ng/ml at 40 weeks. Their glucagon-stimulated C-peptide levels showed the similar tendencies as the fasting C-peptide levels.

Fig. 7

Proposed model for the molecular and cellular mechanisms underlying SCE therapy for the treatment of T1D. The up-regulation of CCR7 expression on CD4+ TCM, CD8+ TCM, CD4+ TEM, and CD8+ TEM cells after receiving SCE therapy (right panel) may lead to the evacuation of these infiltrated autoimmune cells (left panel) from insulitic lesions through the draining of lymphatic vessels in pancreatic islets (dashed line) of T1D subjects. This restoration of homeostasis in pancreatic islets may result in the regeneration of islet β cells via potential signaling pathways.