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.

A robust, good manufacturing practice-compliant, clinical-scale procedure to generate regulatory T cells from patients with amyotrophic lateral sclerosis for adoptive cell therapy.

Regulatory T cells (Tregs) play a fundamental role in the maintenance of self-tolerance and immune homeostasis. Defects in Treg function and/or frequencies have been reported in multiple disease models. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting upper and lower motor neurons. Compelling evidence supports a neuroprotective role for Tregs in this disease. Indeed, rapid progression in ALS patients is associated with decreased FoxP3 expression and Treg frequencies. Thus, we propose that strategies to restore Treg number and function may slow disease progression in ALS. In this study, we developed a robust, Good Manufacturing Practice (GMP)-compliant procedure to enrich and expand Tregs from ALS patients. Tregs isolated from these patients were phenotypically similar to those from healthy individuals but were impaired in their ability to suppress T-cell effector function. In vitro expansion of Tregs for 4 weeks in the presence of GMP-grade anti-CD3/CD28 beads, interleukin (IL)-2 and rapamcyin resulted in a 25- to 200-fold increase in their number and restored their immunoregulatory activity. Collectively, our data facilitate and support the implementation of clinical trials of adoptive therapy with ex vivo expanded and highly suppressive Tregs in patients with ALS.

Therapeutic strategies for the prevention and treatment of cytomegalovirus infection.

INTRODUCTION: CMV remains a significant cause of morbidity and mortality in immunosuppressed patients, particularly following allogeneic haematopoietic transplantation. This reflects the inability of depressed host immunity to contain viral replication, principally through the loss of T-cell function. There is a clear rationale for the restoration of CMV-specific immunity using adoptive T-cell immunotherapy. AREAS COVERED: This review analyses current treatment strategies for prophylaxis and preemptive treatment of CMV with a particular focus on patients following allogeneic haematopoietic transplantation. The main emphasis of this review is the role of adoptive T-cell therapy, particularly some of the newer direct selection technologies that allow the rapid generation of a GMP-compliant cellular product. Relevant studies were selected from PubMed. Search terms: allogeneic transplant, cytomegalovirus, multidrug-resistant virus, adoptive T-cell therapy. EXPERT OPINION: A number of early studies showed that T-cell therapies can be delivered safely and are efficacious. However, they relied on culture techniques that make wider application difficult. Newer direct selection techniques have allowed production of cellular products more rapidly, cheaply, and to GMP standards. Clinical trials will help define the role of these cellular products, which have the potential to alter our entire approach to the treatment of CMV infection.

Genetic modification of T cells.

Adoptively transferred T cells have shown activity in treating viral infections after hemopoietic transplantation and anti-tumor activity against some malignancies such as melanoma and lymphoma. Current research focuses on defining the optimum type of cell for transfer to improve persistence and genetically modifying infused T cells to augment function, overcome tumor evasion strategies and allow ablation should adverse effects occur.

High initial frequency of TCR-transgenic CD8 T cells alters inflammation and pathogen clearance without affecting memory T cell function.

In adoptive transfer experiments, the initial frequency of naïve TCR-transgenic T cells impacts CD8 T cell phenotype after acute infections. The exact reasons for the observed changes, however, are unclear and it is unknown whether alterations in phenotype translate into impaired memory T cell function as well. Here we perform in vivo comparisons of effector and memory CD8 T cells generated from high or low numbers of naïve precursors. We show that high numbers of adoptively transferred T cells exhibit effector functions that alter systemic inflammation and pathogen abundance in the initial days after infection. While these altered environmental conditions resulted in profound changes in primary effector and memory CD8 T cell phenotype, memory T cells derived from both high and low numbers of naïve precursors protected equally well against re-infection and generated secondary effector and memory T cells that were similar in numbers and phenotype. Our results confirm the necessity to use low numbers of naïve precursors to mimic endogenous immune responses but show at the same time that memory CD8 T cell function in adoptive transfers is independent of input numbers.

In vivo trafficking and survival of cytokine-induced killer cells resulting in minimal GVHD with retention of antitumor activity.

Cytokine-induced killer (CIK) cells are ex vivo-expanded T lymphocytes expressing both natural killer (NK)- and T-cell markers. CIK cells are cytotoxic against autologous and allogeneic tumors. We previously showed that adoptive transfer of allogeneic CIK cells in a murine model caused minimal graft-versus-host disease (GVHD). However, the precise mechanism of reduced GVHD is not fully understood. Therefore, we evaluated the trafficking and survival of luciferase-expressing CIK cells in an allogeneic bone marrow transplant model. The initial trafficking patterns of CIK cells were similar to conventional T cells that induced GVHD; however, CIK cells infiltrated GVHD target tissues much less and transiently. CIK cells accumulated and persisted in tumor sites, resulting in tumor eradication. We evaluated different properties of CIK cells compared with conventional T cells, demonstrating a slower division rate of CIK cells, higher susceptibility to apoptosis, persistent increased expression of interferon gamma (IFN-gamma), and reduced acquisition of homing molecules required for entry of cells into inflamed GVHD target organs that lack expression of NKG2D ligands recognized by CIK cells. Due to these properties, allogeneic CIK cells had reduced expansion and caused less tissue damage. We conclude that CIK cells have the potential to separate graft-versus-tumor effects from GVHD.

Presentation of proteins encapsulated in sterically stabilized liposomes by dendritic cells initiates CD8(+) T-cell responses in vivo.

Liposomes have been proposed as a vehicle to deliver proteins to antigen-presenting cells (APC), such as dendritic cells (DC), to stimulate strong T cell-mediated immune responses. Unfortunately, because of their instability in vivo and their rapid uptake by cells of the mononuclear phagocyte system on intravenous administration, most types of conventional liposomes lack clinical applicability. In contrast, sterically stabilized liposomes (SL) have increased in vivo stability. It is shown that both immature and mature DC take up SL into neutral or mildly acidic compartments distinct from endocytic vacuoles. These DC presented SL-encapsulated protein to both CD4(+) and CD8(+) T cells in vitro. Although CD4(+) T-cell responses were comparable to those induced by soluble protein, CD8(+) T-cell proliferation was up to 300-fold stronger when DC had been pulsed with SL-encapsulated ovalbumin. DC processed SL-encapsulated antigen through a TAP-dependent mechanism. Immunization of mice with SL-encapsulated ovalbumin led to antigen presentation by DC in vivo and stimulated greater CD8(+) T-cell responses than immunization with soluble protein or with conventional or positively charged liposomes carrying ovalbumin. Therefore, the application of SL-encapsulated antigens offers a novel effective, safe vaccine approach if a combination of CD8(+) and CD4(+) T-cell responses is desired (ie, in anti-viral or anti-tumor immunity).

The use of cytotoxic t cells for the prevention and treatment of epstein-barr virus induced lymphoma in transplant recipients.

Epstein-Barr Virus Lymphoproliferative Diseases (EBV-LPD) can affect patients receiving allogeneic hemopoietic stem cell or solid organ transplant. They are caused by impairment of EBV specific CD8+ cytotoxic T-lymphocyte (CTL) response due to the immunosuppression that follows these procedures. Despite different therapies LPD can still have a rapid and lethal course. A promising solution is the application of adoptive immunotherapy approaches such as ex vivo generated EBV-specific CTLs for prevention and therapy. Their infusion has been demonstrated to be safe and effective in allogeneic hemopoietic stem cell transplant recipients and their use after allogeneic solid organ transplant is also under evaluation.