Induction of broad multifunctional CD8+ and CD4+ T cells by hepatitis B virus antigen-based synthetic long peptides ex vivo.

Introduction: Therapeutic vaccination based on synthetic long peptides (SLP®) containing both CD4+ and CD8+ T cell epitopes is a promising treatment strategy for chronic hepatitis B infection (cHBV). Methods: We designed SLPs for three HBV proteins, HBcAg and the non-secreted proteins polymerase and X, and investigated their ability to induce T cell responses ex vivo. A set of 17 SLPs was constructed based on viral protein conservation, functionality, predicted and validated binders for prevalent human leukocyte antigen (HLA) supertypes, validated HLA I epitopes, and chemical producibility. Results: All 17 SLPs were capable of inducing interferon gamma (IFNÉ£) production in samples from four or more donors that had resolved an HBV infection in the past (resolver). Further analysis of the best performing SLPs demonstrated activation of both CD8+ and CD4+ multi-functional T cells in one or more resolver and patient sample(s). When investigating which SLP could activate HBV-specific T cells, the responses could be traced back to different peptides for each patient or resolver. Discussion: This indicates that a large population of subjects with different HLA types can be covered by selecting a suitable mix of SLPs for therapeutic vaccine design. In conclusion, we designed a set of SLPs capable of inducing multifunctional CD8+ and CD4+ T cells ex vivo that create important components for a novel therapeutic vaccine to cure cHBV.

Enhanced HPV16 E6/E7+ tumor eradication via induction of tumor-specific T cells by therapeutic vaccination with virosomes presenting synthetic long peptides.

Therapeutic cancer vaccines trigger CD4 + and CD8 + T cell responses capable of established tumor eradication. Current platforms include DNA, mRNA and synthetic long peptide (SLP) vaccines, all aiming at robust T cell responses. SLPs linked to the Amplivant® adjuvant (Amplivant-SLP) have shown effective delivery to dendritic cells, resulting in improved immunogenicity in mice. We have now tested virosomes as a delivery vehicle for SLPs. Virosomes are nanoparticles made from influenza virus membranes and have been used as vaccines for a variety of antigens. Amplivant-SLP virosomes induced the expansion of more antigen-specific CD8 + T memory cells in ex vivo experiments with human PBMCs than Amplivant-SLP conjugates alone. The immune response could be further improved by including the adjuvants QS-21 and 3D-PHAD in the virosomal membrane. In these experiments, the SLPs were anchored in the membrane through the hydrophobic Amplivant adjuvant. In a therapeutic mouse model of HPV16 E6/E7+ cancer, mice were vaccinated with virosomes loaded with either Amplivant-conjugated SLPs or lipid-coupled SLPs. Vaccination with both types of virosomes significantly improved the control of tumor outgrowth, leading to elimination of the tumors in about half the animals for the best combinations of adjuvants and to their survival beyond 100 days.

Combination immunotherapy with synthetic long peptides and chemotherapy or PD-1 blocker for cancers caused by human papilloma virus type 16.

Therapeutic vaccination of premalignant conditions and of different stages of cancer can be accomplished with several platforms including DNA vaccines, RNA vaccines, synthetic long peptides (SLP), and recombinant viruses. We successfully used a therapeutic vaccine composed of SLP covering the complete sequence of the two oncogenic proteins E6 and E7 of human papillomavirus type 16 (HPV16) as monotherapy in patients with premalignant disease. However, combination treatment might be required in patients with (advanced) cancer because of the hostile cancer microenvironment for T cells in established HPV16+ cancer, often associated with systemic immunosuppression. In patients with late-stage recurrent or metastatic HPV16+ cancers, we have therefore combined treatment with the SLP vaccine, called ISA101b, with either standard-of-care chemotherapy or with immune checkpoint inhibition with anti-PD-1 monoclonal antibody. A strong vaccine-induced interferon gamma-producing T cell response to HPV16 E6/E7 was associated with significantly better survival. In a second phase 1/2 study, patients with recurrent or metastatic HPV16+ oropharyngeal cancer were treated with the combination of ISA101b and anti-PD-1 (nivolumab). In this trial, the clinical overall response rate (ORR) in 22 patients was 36%, twice the ORR in the nivolumab registration trial for this category of patients, and 2/22 patients had a complete clinical response that is ongoing after 4 1/2 years. Other promising strategies for late-stage cancer recipients are the infusion of expanded tumor-infiltrating lymphocytes or the infusion of T cell receptor transduced T cells, both directed against HPV16.

A unique immune signature in blood separates therapy-refractory from therapy-responsive acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) is an immune cell‒driven, potentially lethal complication of allogeneic hematopoietic stem cell transplantation affecting diverse organs, including the skin, liver, and gastrointestinal (GI) tract. We applied mass cytometry (CyTOF) to dissect circulating myeloid and lymphoid cells in children with severe (grade III-IV) aGVHD treated with immune suppressive drugs alone (first-line therapy) or in combination with mesenchymal stromal cells (MSCs; second-line therapy). These results were compared with CyTOF data generated in children who underwent transplantation with no aGVHD or age-matched healthy control participants. Onset of aGVHD was associated with the appearance of CD11b+CD163+ myeloid cells in the blood and accumulation in the skin and GI tract. Distinct T-cell populations, including TCRγδ+ cells, expressing activation markers and chemokine receptors guiding homing to the skin and GI tract were found in the same blood samples. CXCR3+ T cells released inflammation-promoting factors after overnight stimulation. These results indicate that lymphoid and myeloid compartments are triggered at aGVHD onset. Immunoglobulin M (IgM) presumably class switched, plasmablasts, and 2 distinct CD11b- dendritic cell subsets were other prominent immune populations found early during the course of aGVHD in patients refractory to both first- and second-line (MSC-based) therapy. In these nonresponding patients, effector and regulatory T cells with skin- or gut-homing receptors also remained proportionally high over time, whereas their frequencies declined in therapy responders. Our results underscore the additive value of high-dimensional immune cell profiling for clinical response evaluation, which may assist timely decision-making in the management of severe aGVHD.

The development of T cells from stem cells in mice and humans.

T cells develop from hematopoietic stem cells in the specialized microenvironment of the thymus. The main transcriptional players of T-cell differentiation such as Notch, Tcf-1, Gata3 and Bcl11b have been identified, but their role and regulation are not yet completely understood. In humans, functional experiments on T-cell development have traditionally been rather difficult to perform, but novel in vitro culture systems and in vivo xenograft models have allowed detailed studies on human T-cell development. Recent work has allowed the use of human severe combined immunodeficiency stem cells to unravel developmental checkpoints for human thymocyte development.

Overexpression of LMO2 causes aberrant human T-Cell development in vivo by three potentially distinct cellular mechanisms.

Overexpression of LMO2 is known to be one of the causes of T-cell acute lymphoblastic leukemia (T-ALL) development; however, the mechanisms behind its oncogenic activity are incompletely understood. LMO2-overexpressing transgenic mouse models suggest an accumulation of immature T-cell progenitors in the thymus as the main preleukemic event. The effects of LMO2 overexpression on human T-cell development in vivo are unknown. Here, we report studies of a humanized mouse model transplanted with LMO2-transduced human hematopoietic stem/progenitor cells. The effects of LMO2 overexpression were confined to the T-cell lineage; however, initially, multipotent cells were transduced. Three effects of LMO2 on human T-cell development were observed: (1) a block at the double-negative/immature single-positive stage, (2) an accumulation of CD4(+)CD8(+) double-positive CD3(-) cells, and (3) an altered CD8/CD4 ratio with enhanced peripheral T lymphocytes. Microarray analysis of sorted double-positive cells overexpressing LMO2 led to the identification of an LMO2 gene set that clustered with human T-ALL patient samples of the described "proliferative" cluster. In this article, we demonstrate previously unrecognized mechanisms by which LMO2 alters human T-cell development in vivo; these mechanisms correlate with human T-ALL leukemogenesis.

The functional relationship between hematopoietic stem cells and developing T lymphocytes.

In contrast to all other blood and immune cells, T lymphocytes do not develop in the bone marrow (BM), but in the specialized microenvironment provided by the thymus. Similar to the other lineages, however, all T cells arise from multipotent hematopoietic stem cells (HSCs) that reside in the BM. Not all HSCs give rise to T cells; but how many and what kind of developmental checkpoints are located along this intricate differentiation path is the subject of intense research. Traditionally, this process has been studied almost exclusively using mouse cells, but recent advances in immunodeficient mouse models, high-speed cell sorting, lentiviral transduction protocols, and deep sequencing techniques have allowed these questions to be addressed using human cells. Here we review the process of thymic seeding by BM-derived cells and T cell commitment in humans, discussing recent insights into the clonal composition of the thymus and the definition of developmental checkpoints, on the basis of insights from human severe combined immunodeficiency patients.

Identification of checkpoints in human T-cell development using severe combined immunodeficiency stem cells.

BACKGROUND: Severe combined immunodeficiency (SCID) represents congenital disorders characterized by a deficiency of T cells caused by arrested development in the thymus. Yet the nature of these developmental blocks has remained elusive because of the difficulty of taking thymic biopsy specimens from affected children. OBJECTIVE: We sought to identify the stages of arrest in human T-cell development caused by various major types of SCID. METHODS: We performed transplantation of SCID CD34(+) bone marrow stem/progenitor cells into an optimized NSG xenograft mouse model, followed by detailed phenotypic and molecular characterization using flow cytometry, immunoglobulin and T-cell receptor spectratyping, and deep sequencing of immunoglobulin heavy chain (IGH) and T-cell receptor δ (TRD) loci. RESULTS: Arrests in T-cell development caused by mutations in IL-7 receptor α (IL7RA) and IL-2 receptor γ (IL2RG) were observed at the most immature thymocytes much earlier than expected based on gene expression profiling of human thymocyte subsets and studies with corresponding mouse mutants. T-cell receptor rearrangements were functionally required at the CD4(-)CD8(-)CD7(+)CD5(+) stage given the developmental block and extent of rearrangements in mice transplanted with Artemis-SCID cells. The xenograft model used is not informative for adenosine deaminase-SCID, whereas hypomorphic mutations lead to less severe arrests in development. CONCLUSION: Transplanting CD34(+) stem cells from patients with SCID into a xenograft mouse model provides previously unattainable insight into human T-cell development and functionally identifies the arrest in thymic development caused by several SCID mutations.

The composition and differentiation potential of the duodenal intraepithelial innate lymphocyte compartment is altered in coeliac disease.

OBJECTIVE: Coeliac disease (CD), a gluten-induced enteropathy, alters the composition and function of duodenal intraepithelial T cells. The intestine also harbours four types of CD3-negative intraepithelial lymphocytes (IELs) with largely unknown function: CD56(-)CD127(-), CD56(-)CD127(+), CD56(+)CD127(-) and CD56(+)CD127(+). Here we aimed to gain insight into the potential function of these innate IELs in health and disease. DESIGN: We determined the phenotypes, relative abundance and differentiation potential of these innate IEL subsets in duodenal biopsies from controls and patients with CD or patients with refractory CD type II (RCDII). RESULTS: Hierarchical clustering analysis of the expression of 15 natural killer and T cell surface markers showed that innate IELs differed markedly from innate peripheral blood lymphocytes and divided innate IEL subsets into two main branches: a CD127(-) branch expressing high levels of interleukin (IL) 2/15Rß but no IL-21R, and a CD127(+) branch with the opposite phenotype. While CD was characterised by the contraction of all four innate IEL subsets, a selective expansion of CD56(-)CD127(-) and CD56(-)CD127(+) innate IEL was detected in RCDII. In vitro, in the presence of IL-15, CD56(-)CD127(-) IEL from controls and patients with CD, but not from patients with RCDII, differentiated into functional natural killer and T cells, the latter largely dependent on notch-signalling. Furthermore, compared with non-coeliac controls, CD56(-)CD127(-) IEL from patients with CD expressed more intracellular CD3ε and CD3γ and gave more pronounced T cell differentiation. CONCLUSIONS: Thus, we demonstrate previously unappreciated diversity and plasticity of the innate IEL compartment and its loss of differentiation potential in patients with RCDII.

Development of a diverse human T-cell repertoire despite stringent restriction of hematopoietic clonality in the thymus.

The fate and numbers of hematopoietic stem cells (HSC) and their progeny that seed the thymus constitute a fundamental question with important clinical implications. HSC transplantation is often complicated by limited T-cell reconstitution, especially when HSC from umbilical cord blood are used. Attempts to improve immune reconstitution have until now been unsuccessful, underscoring the need for better insight into thymic reconstitution. Here we made use of the NOD-SCID-IL-2Rγ(-/-) xenograft model and lentiviral cellular barcoding of human HSCs to study T-cell development in the thymus at a clonal level. Barcoded HSCs showed robust (>80% human chimerism) and reproducible myeloid and lymphoid engraftment, with T cells arising 12 wk after transplantation. A very limited number of HSC clones (<10) repopulated the xenografted thymus, with further restriction of the number of clones during subsequent development. Nevertheless, T-cell receptor rearrangements were polyclonal and showed a diverse repertoire, demonstrating that a multitude of T-lymphocyte clones can develop from a single HSC clone. Our data imply that intrathymic clonal fitness is important during T-cell development. As a consequence, immune incompetence after HSC transplantation is not related to the transplantation of limited numbers of HSC but to intrathymic events.

Sustained Engraftment of Cryopreserved Human Bone Marrow CD34(+) Cells in Young Adult NSG Mice.

Hematopoietic stem cells (HSCs) are defined by their ability to repopulate the bone marrow of myeloablative conditioned and/or (lethally) irradiated recipients. To study the repopulating potential of human HSCs, murine models have been developed that rely on the use of immunodeficient mice that allow engraftment of human cells. The NSG xenograft model has emerged as the current standard for this purpose allowing for engraftment and study of human T cells. Here, we describe adaptations to the original NSG xenograft model that can be readily implemented. These adaptations encompass use of adult mice instead of newborns and a short ex vivo culture. This protocol results in robust and reproducible high levels of lympho-myeloid engraftment. Immunization of recipient mice with relevant antigen resulted in specific antibody formation, showing that both T cells and B cells were functional. In addition, bone marrow cells from primary recipients exhibited repopulating ability following transplantation into secondary recipients. Similar results were obtained with cryopreserved human bone marrow samples, thus circumventing the need for fresh cells and allowing the use of patient derived bio-bank samples. Our findings have implications for use of this model in fundamental stem cell research, immunological studies in vivo and preclinical evaluations for HSC transplantation, expansion, and genetic modification.

Integrated transcript and genome analyses reveal NKX2-1 and MEF2C as potential oncogenes in T cell acute lymphoblastic leukemia.

To identify oncogenic pathways in T cell acute lymphoblastic leukemia (T-ALL), we combined expression profiling of 117 pediatric patient samples and detailed molecular-cytogenetic analyses including the Chromosome Conformation Capture on Chip (4C) method. Two T-ALL subtypes were identified that lacked rearrangements of known oncogenes. One subtype associated with cortical arrest, expression of cell cycle genes, and ectopic NKX2-1 or NKX2-2 expression for which rearrangements were identified. The second subtype associated with immature T cell development and high expression of the MEF2C transcription factor as consequence of rearrangements of MEF2C, transcription factors that target MEF2C, or MEF2C-associated cofactors. We propose NKX2-1, NKX2-2, and MEF2C as T-ALL oncogenes that are activated by various rearrangements.