Recent Developments in Cellular Immunotherapy for HSCT-Associated Complications

Abstract

Allogeneic hematopoietic stem cell transplantation is associated with serious complications, and improvement of the overall clinical outcome of patients with hematological malignancies is necessary. During the last decades, posttransplant donor-derived adoptive cellular immunotherapeutic strategies have been progressively developed for the treatment of graft-versus-host disease (GvHD), infectious complications, and tumor relapses. To date, the common challenge of all these cell-based approaches is their implementation for clinical application. Establishing an appropriate manufacturing process, to guarantee safe and effective therapeutics with simultaneous consideration of economic requirements is one of the most critical hurdles. In this review, we will discuss the recent scientific findings, clinical experiences, and technological advances for cell processing toward the application of mesenchymal stromal cells as a therapy for treatment of severe GvHD, virus-specific T cells for targeting life-threating infections, and of chimeric antigen receptors-engineered T cells to treat relapsed leukemia.

Keywords: T cells; adoptive transfer; cell manufacture; chimeric antigen receptor; extracellular vesicles; immunomodulation; infection; mesenchymal stromal cells.

Figure 1

Transmission electron microscopy micrograph of whole-mounted extracellular vesicles-purified human MSCs. MSC-EVs exhibit a spheroid, cup-shaped morphology. Scale bar shows 100 nm. Photography courtesy of Monica Reis.

Figure 2

Overview of the bioactive molecules secreted by MSCs and their impact on cells of the innate and adaptive immune response. Some bioactive molecules are constitutively expressed by MSCs, while others are “licensed” by exposure to an inflammatory environment or upon TLR stimulation (241). Depending upon the bioactive secretion profile, MSCs can skew the differentiation of CD4⁺ T-helper cells into various T-cell subsets, each with distinct cytokine and gene expression profiles, can promote the generation of regulatory T cells (Tregs) and inhibit the proliferation of cytotoxic T cells (–244). MSCs can modulate the development of conventional and plasmacytoid DC (–247) while DCs generated in the presence of MSCs have functional properties typical of tolerogenic DCs (–250). Similarly, MSCs can polarize macrophages of the classical M1 pro-inflammatory phenotype to that of an alternative anti-inflammatory M2 phenotype (215), or directly induce this alternative phenotype by coculture (251). In contrast to other cell types, MSC modulation of B-cell function is poorly understood and the findings are contentious. Results from in vitro experiments show that while MSCs impair the proliferation and terminal differentiation of B cells (252) they have also been shown to stimulate antibody secretion (253). More recently, data have emerged which suggests that MSCs can promote the induction of regulatory B cells (Breg) (254). Neutrophils are an important mediator of the innate response and MSCs have been shown to enhance their survival through an IL-6-mediated mechanism, concomitant with the downregulation of reactive oxygen species, thereby conserving the pool of neutrophils primed to respond rapidly to infection (255). MSCs inhibit the proliferation and differentiation of monocytes to immature dendritic cells (DCs) (245). Natural Killer (NK) cells and MSCs have a reciprocal relationship; MSCs can inhibit the proliferation and cytotoxicity of resting NK cells and their cytokine production in vitro, while activated NK cells can be cytotoxic to MSCs (256). MSCs constitutively secrete Factor H which inhibits complement activation (257), conversely the complement activation products C3a and C5a released upon tissue damage are chemotactic factors for MSCs (258), recruiting them to sites of injury. Abbreviations: CCR, C-C chemokine; CD, cluster of differentiation; cDC, conventional dendritic cell; CTL, cytotoxic T-lymphocyte; GM-CSF, granulocyte-macrophage colony-stimulating factor; HGF, hepatocyte growth factor; HLA, human leukocyte antigen; IDO, indoleamine 2,3-dioxygenase; IFNγ, interferon-γ; Ig, immunoglobulin; IL, interleukin; MФ, macrophage; MHC, major histocompatibility complex; Mono, monocyte; Neutro, neutrophil; NF-κB, nuclear factor kappa B; PD-1, programed cell death protein-1; pDC, plasmacytoid dendritic cell; PGE₂, prostaglandin E₂; TGFβ, transforming growth factor β; Th, T-helper cell; TNFα, tumor necrosis factor α; tolDC, tolerogenic dendritic cell; TSG, TNF-α-stimulated protein.

Figure 3

Principle approach of adoptive T cell therapy for treatment of viral infections. Out of peripheral blood of the HSCT donor the virus-specific T cells are selected. The generated T cell product is infused into the patient suffering of viral complications after allogenic HSCT.

Figure 4

Methods for in vitro generation of a virus-specific T cell product. For the in vitro manufacture process blood is used as the cellular source, mostly derived from the stem cell donor. Selection of virus-specific T cell and thereby depletion of potentially alloreactive T cells from the blood can be achieved by different methods. (A) Activation and expansion: peripheral blood cells are incubated with viral antigen. Antigen-presenting cells (APC) phagocytose, process, and present the antigen as peptides on MHC molecules. Virus-specific T cells recognize their cognate viral antigenic peptide via the TCR, get activated, and later on start proliferating for several days. In many applications, additional repetitive antigen restimulations are performed to further increase the expansion and thereby the number and the purity of the virus-specific T cell population. Dependent on the viral antigen and APC used for the process, either CD4⁺ and/or CD8⁺ T cells are contained in the product. (B) MHC class I/peptide multimer technology: virus-specific T cells within peripheral blood become labeled by a MHC class I/peptide multimer reagent, which binds to the TCR of the viral peptide-specific T cells. After an additional labeling step with magnetic beads the CD8⁺ virus-specific T cells are magnetically enriched. (C) Cytokine-capture assay: peripheral blood cells are incubated with viral antigen, e.g., a peptide pool, for 4 h. APC present the peptides on MHC molecules to virus-specific T cells, which start producing IFN-γ. Cells are labeled with a catch matrix consisting of a CD45 antibody conjugated to an Anti-IFN-γ antibody. In this way, secreted IFN-γ is specifically captured on the cell surface of the activated virus-specific T cells. Subsequently, the cell-bound IFN-γ is detected with Anti-IFN-γ magnetic particles and the virus-specific T cells are magnetically enriched. Both CD4⁺ and CD8⁺ T cells are obtained by this method.

Figure 5

In vitro human skin explant assay as a model to investigate the potential of third-party CMV-specific T cells to elicit GvHR in an HLA-mismatched system. (A) CMV-specific T cells were isolated from blood of seropositive donors by IFN-γ secretion assay and expanded in vitro between 2 and 4 weeks with IL-2 and irradiated feeder cells. (B) CMV-specific T cell lines and unselected PBMCs from the same donor where exposed to HLA-mismatched PBMCs (recipient’s cells) in a mixed lymphocyte reaction for 7 days followed by incubation with recipient’s skin for further 3 days. Then skin biopsies were collected, fixed in formalin, and stained with hematoxylin and eosin. (C) The histopathological damage in the skin biopsies displays a readout of the allogeneic-HLA reactions caused by T cell activation. The images show that CMV-specific T cells do not cause GvHR (Grade I) as opposed to Unselected PBMCs (Grade II and III) from the same donor.

Figure 6

General workflow for adoptive therapy with CAR-modified T cells. Figure courtesy of Prof. Hinrich Abken.

Figure 7

Structure of different generations of CARs. Figure courtesy of Prof. Hinrich Abken.

Figure 8

In vitro manufacture process of a CAR-engineered T cell product.