Clinical-grade regulatory T cells: Comparative analysis of large-scale expansion conditions.

Recent clinical trials have indicated the high potential of regulatory T cells (Tregs) in the prevention of acute and chronic graft-versus-host disease (GvHD) after hematopoietic stem cell transplantation, but immune interventions require large numbers of Tregs. With respect to their limited natural occurrence, development and optimization of protocols for large-scale expansion of clinical-grade Tregs are essential if considered for therapeutic use. We compared different clinical-grade large-scale expansion protocols for repetitive transfer of large numbers of Tregs in clinical trials for the prevention of acute and/or chronic GvHD. Donor Tregs were isolated using magnetic-activated cell sorting (MACS) technology with good manufacturing practice-compliant devices. CD8 and CD19 depletion followed by CD25 enrichment resulted in the isolation of CD4+CD25+CD127- Tregs with a mean purity of 77%. Cell populations were expanded ex vivo using X-Vivo 15 (±rapamycin), TexMACS (±rapamycin), and CellGro DC (±rapamycin) in the presence of interleukin-2. The highest rates of expansion of clinical-grade Tregs were observed for X-Vivo 15 and CellGro DC without rapamycin in compared with all other expansion media tested. The suppressive capacity of the expanded Treg population was maintained under all conditions investigated. Our data suggest that expansion with CellGro provides data comparable to those obtained with TexMACS or X-Vivo 15 with rapamycin, although all three conditions did not provide the same propagation rate as X-Vivo 15 alone. With respect to functionality, phenotype, and stability, CellGro DC medium represents a reasonable alternative for good manufacturing practice-compatible large-scale ex vivo expansion.

Granzyme A Is Required for Regulatory T-Cell Mediated Prevention of Gastrointestinal Graft-versus-Host Disease.

In our previous work we could identify defects in human regulatory T cells (Tregs) likely favoring the development of graft-versus-host disease (GvHD) following allogeneic stem cell transplantation (SCT). Treg transcriptome analyses comparing GvHD and immune tolerant patients uncovered regulated gene transcripts highly relevant for Treg cell function. Moreover, granzyme A (GZMA) also showed a significant lower expression at the protein level in Tregs of GvHD patients. GZMA induces cytolysis in a perforin-dependent, FAS-FASL independent manner and represents a cell-contact dependent mechanism for Tregs to control immune responses. We therefore analyzed the functional role of GZMA in a murine standard model for GvHD. For this purpose, adoptively transferred CD4+CD25+ Tregs from gzmA-/- mice were analyzed in comparison to their wild type counterparts for their capability to prevent murine GvHD. GzmA-/- Tregs home efficiently to secondary lymphoid organs and do not show phenotypic alterations with respect to activation and migration properties to inflammatory sites. Whereas gzmA-/- Tregs are highly suppressive in vitro, Tregs require GZMA to rescue hosts from murine GvHD, especially regarding gastrointestinal target organ damage. We herewith identify GZMA as critical effector molecule of human Treg function for gastrointestinal immune response in an experimental GvHD model.

Characterization of granulocyte colony stimulating factor for in vitro induction of regulatory T cells for cellular immune intervention in transplant medicine.

OBJECTIVES: The application of regulatory T cells in the field of solid-organ and hematopoietic stem cell transplantation is under investigation to develop novel cellular strategies for tolerance induction. Establishing in vitro procedures to induce and expand regulatory T cells seeks to overcome the limiting small number of this rare T cell population. The present study is based on growing evidence that granulocyte colony stimulating factor exerts immune regulatory function in the adaptive immune system and may induce regulatory T cells in vivo. MATERIALS AND METHODS: We analyzed the effect of recombinant granulocyte colony stimulating factor to directly convert CD4+CD25- T cells into regulatory T cells in vitro. Marker molecules were analyzed by quantitative reverse transcriptase-polymerase chain reaction and fluorescent-activated cell sorter analyses. Functional assays were performed to investigate the suppressive capacity of granulocyte colony stimulating factor stimulated T cells. RESULTS: Kinetic analyses of Foxp3 gene expression uncovered increased levels early after in vitro stimulation with granulocyte colony stimulating factor. However, protein analyses for the master transcription factor Foxp3 and other regulatory T cells revealed that granulocyte colony stimulating factor did not directly induce a regulatory T cell phenotype. Moreover, functional analyses demonstrated that granulocyte colony stimulating factor stimulation in vitro does not result in a suppressive, immune regulatory T cell population. CONCLUSIONS: Granulocyte colony stimulating factor does not induce regulatory T cells with a specific phenotype and suppressive potency in vitro. Therefore, granulocyte colony stimulating factor does not qualify for developing protocols aimed at higher regulatory T cell numbers for adoptive transfer strategies in solid organ and hematopoietic stem cell transplantation.

Reconstitution and phenotype of Tregs in CMV reactivating patients following allogeneic hematopoietic stem cell transplantation.

In experimental and clinical settings Tregs prevent graft-versus-host disease (GvHD) by inhibiting the proliferation and function of conventional T cells (Tconv). The suppressive potency of Tregs might also lead to the inhibition of protective antiviral T cell responses. As the control of CMV reactivation is important to improve the clinical outcome in allogeneic HSCT, we analyzed the Treg reconstitution in CMV reactivating patients with and without GvHD (n=47) in the first 6 months following transplantation. Most importantly, CMV reactivation does not correlate with the numerical reconstitution of CD4(+)CD25(high)CD127(-) Tregs. During CMV reactivation the proportion of Tregs within the CD4(+) T cell population decreased significantly independent of GvHD manifestation. A comprehensive FACS analysis was performed in order to characterize the phenotype of Tregs and Tconv cells in greater detail for activation, co-stimulation, proliferation, suppressive function and migratory capability. Interestingly, Tregs of patients with CMV reactivation showed a significantly higher CXCR3 expression. CD4(+) Tconv cells expressed significantly higher protein levels of the proliferation marker Ki67 correlating with a numerical increase of CD4(+) T cells. Our results indicate that Tregs are not inhibiting pathogen clearance by Tconv following HSCT, which is of high relevance for future Treg cell-based clinical trials in allogeneic HSCT.

Human regulatory T cells of G-CSF mobilized allogeneic stem cell donors qualify for clinical application.

Recent clinical studies demonstrate the high potency of regulatory T cells (Tregs) to control graft-versus-host disease in hematopoietic stem cell transplantation (SCT). However, the adoptive transfer of Tregs is limited by their low frequency in unstimulated donors and considerable concerns that G-CSF induced SC mobilization might have negative effects on the stability and function of Tregs. The isolation of Tregs from the G-CSF mobilized SC grafts would extend this novel strategy for tolerance induction to the unrelated setting and simplify global clinical application. We characterized CD4(+)CD25(high)CD127(-) Tregs from SC donors before and after G-CSF mobilization for their phenotype, function, and stability. After G-CSF application the Treg cell yield increased significantly. Donor Tregs retained their cytokine profile, phenotypic characteristics and in vitro expansion capacity after SC mobilization. Most importantly, in vivo G-CSF stimulated Tregs remained highly suppressive on the proliferation of effector T cells, also after in vitro expansion, and displayed a stable phenotype in epigenetic studies. The surface expression of CXCR3 is transiently reduced. However, donor-derived Tregs maintain their migratory properties after G-CSF stimulation. Therefore, the adoptive transfer of Tregs from G-CSF mobilized SC donors seems to be a feasible and safe strategy for clinical application in allogeneic SCT.

Isolation strategies of regulatory T cells for clinical trials: phenotype, function, stability, and expansion capacity.

Recent clinical results demonstrate the highly effective potency of regulatory T cells (Tregs) to control graft-versus-host disease (GvHD). In this presented study, we directly compared different Treg subpopulations in order to define the most promising Treg target cell population for cellular intervention studies with respect to their phenotype, functional properties, stability, and expansion capacity. Different Treg cell populations have been isolated from healthy donors and characterized by fluorescence activated cell sorting (FACS) analysis for their phenotypic marker and purity, functional properties by suppression assay, stability by Treg-specific demethylated region (TSDR) of the Foxp3 promoter and their in vitro expansion capacity. The direct comparison of the respective Treg target cell populations identified CD4(+)CD25(hi)CD127(-) and CD4(+)CD25(hi)ICOS(+) Tregs as the most promising Treg population for fresh cell infusions in clinical trials with respect to cell yield, phenotype, function, and stability. The CD4(+)CD25(hi) Tregs qualified as the best candidate for in vitro expansion combining a highly stable phenotype with strong suppressive potential and attractive cell yield after repetitive stimulation. The suppressive capacity of freshly isolated CD4(+)CD25(hi)CD45RA(+) and CD49d(-)CD127(-) Tregs is comparable to freshly isolated CD4(+)CD25(hi), but inferior to CD4(+)CD25(hi)CD127(-) and CD4(+)CD25(hi)ICOS(+) Tregs. In vitro expansion of CD4(+)CD25(hi)CD45RA(+) and CD49d(-)CD127(-) Tregs resulted in cell populations with less suppressive potency compared with CD4(+)CD25(hi) expanded Tregs correlating well with a higher TSDR demethylation level. In conclusion, future clinical trials should favor CD4(+)CD25(hi)CD127(-) and CD4(+)CD25(hi)ICOS(+) Tregs for direct Treg cell transfer, whereas CD4(+)CD25(hi) Tregs qualify as best candidate for in vitro expansion.

Human regulatory T cells in allogeneic stem cell transplantation.

GVHD is still one of the major complications after allogeneic stem cell transplantation. Whereas murine data have clearly shown the beneficial effects of regulatory T cells (Tregs) on the prevention of GVHD, data from the human system are rare. Here, we present a comparative dynamic analysis of CD4(+)CD25(hi)CD127(lo/-) Tregs from patients with and without GVHD analyzing the whole genome profile over the first 6 months after stem cell transplantation, representing the most sensitive time window for tolerance induction. The Treg transcriptome showed a high stability. However, the comparison of Treg transcriptomes from patients with and without GVHD uncovered regulated gene transcripts highly relevant for Treg cell function. The confirmative protein analyses demonstrated a significantly higher expression of granzyme A, CXCR3, and CCR5 in Tregs of immune tolerant patients. These results point to a reduced suppressive function of Tregs from GVHD patients with diminished migration capacity to the target organs.

Preconditioning therapy with lentiviral vector-programmed dendritic cells accelerates the homeostatic expansion of antigen-reactive human T cells in NOD.Rag1-/-.IL-2rγc-/- mice.

Dendritic cell (DC)-based immunization is a potent strategy to direct prompt and durable immune responses against viral reactivations after transplantations. Here, we show that overnight lentiviral vector (LV) gene transfer into human monocytes co-expressing granulocyte-macrophage colony stimulating factor and interleukin (IL)-4 induced self-differentiated DCs (SMART-DCs) with stable DC immunophenotype over weeks in culture and secreted several inflammatory cytokines. SMART-DCs injected subcutaneously in immunodeficient NOD.Rag1(-/-).IL2rγ(-/-) (NRG) mice 1 day after LV transduction were stable for a month in vivo. "Conventional" DCs (cDCs) and SMART-DCs were compared with regard to their potency to accelerate the expansion, biodistribution, and antigenic stimulation of autologous human T cells. Peripheral blood cells obtained from human cytomegalovirus (hCMV)-reactive donors and full-length hCMV pp65 antigenic protein or peptides were used. DCs loaded with pp65 were administered subcutaneously into NRG mice as a preconditioning treatment a week prior to intravenous infusion with T cells. Optical imaging analyses demonstrated that in mice preconditioned with SMART-DC-pp65, T cells were directly recruited to the immunization site and subsequently spread to the spleen and other organs. A dramatic expansion of both human CD8(+) and CD4(+) T cells could be observed within a few days after infusion, and this was associated with consistent measurable CD8(+) effector memory T-cell responses against different pp65 epitopes. Thus, this mouse model demonstrates the proof-of-principle for SMART-DCs to accelerate expansion of human lymphocytes, resulting in poly-functional and antigen-specific immune responses against hCMV-pp65.

Chemokine receptor CXCR5 supports solitary intestinal lymphoid tissue formation, B cell homing, and induction of intestinal IgA responses.

Solitary intestinal lymphoid tissue (SILT) comprises a spectrum of phenotypically diverse lymphoid aggregates interspersed throughout the small intestinal mucosa. Manifestations of SILT range from tiny lymphoid aggregates almost void of mature lymphocytes to large structures dominated by B cells. Large SILT phenotypically resemble a single Peyer's patch follicle, suggesting that SILT might contribute to intestinal humoral immune responses. In this study, we track the fate of individual SILT in vivo over time and analyze SILT formation and function in chemokine receptor CXCR5-deficient mice. We show that, in analogy to Peyer's patches, formation of SILT is invariantly determined during ontogeny and depends on CXCR5. Young CXCR5-deficient mice completely lack SILT, suggesting that CXCR5 is essential for SILT formation during regular postnatal development. However, microbiota and other external stimuli can induce the formation of aberrant SILT distinguished by impaired development of B cell follicles in CXCR5-deficient mice. Small intestinal transplantation and bone marrow transplantation reveal that defect follicle formation is due to impaired B cell homing. Moreover, oral immunization with cholera toxin or infection with noninvasive Salmonella fail to induce efficient humoral immune responses in CXCR5-deficient mice. Bone marrow transplantation of CXCR5-deficient recipients with wild-type bone marrow rescued B cell follicle formation in SILT but failed to restore full humoral immune responses. These results reveal an essential role of CXCR5 in Peyer's patch and SILT development and function and indicate that SILT do not fully compensate for the lack of Peyer's patches in T cell-dependent humoral immune responses.

Adaptation of solitary intestinal lymphoid tissue in response to microbiota and chemokine receptor CCR7 signaling.

Besides Peyer's patches, solitary intestinal lymphoid tissue (SILT) provides a structural platform to efficiently initiate immune responses in the murine small intestine. SILT consists of dynamic lymphoid aggregates that are heterogeneous in size and composition, ranging from small clusters of mostly lineage-negative cells known as cryptopatches to larger isolated lymphoid follicles rich in B cells. In this study, we report that in chemokine receptor CCR7-deficient mice SILT is enlarged, although unchanged in frequency and cellular composition compared with wild-type mice. This phenotype is conferred by bone marrow-derived cells and is independent of the presence of intestinal bacteria. Remarkably, particularly small-sized SILT predominates in germfree wild-type mice. Colonization of wild-type mice with commensal bacteria provokes an adjustment of the spectrum of SILT to that observed under specific pathogen-free conditions by the conversion of pre-existing lymphoid structures into larger-sized SILT. In conclusion, our findings establish that intestinal microbes influence the manifestation of gut-associated lymphoid tissues and identify CCR7 signaling as an endogeneous factor that controls this process.