Histocompatibility Barriers Research Articles

Immune Editing: Overcoming Immune Barriers in Stem Cell Transplantation.

Human pluripotent stem cells have the potential to revolutionize the treatment of inborn and degenerative diseases, including aging and autoimmunity. A major barrier to their wider adoption in cell therapies is immune rejection. Genome editing allows for tinkering of the human genome in stem and progenitor cells and raises the prospect for overcoming the immune barriers to transplantation. Initial attempts have focused primarily on the major histocompatibility barrier that is formed by the human leukocyte antigens (HLA). More recently, immune checkpoint inhibitors, such as PD-L1, CD47, or HLA-G, are being explored both, in the presence or absence of HLA, to mitigate immune rejection by the various cellular components of the immune system. In this review, we discuss progress in surmounting immune barriers to cell transplantation, with a particular focus on genetic engineering of human pluripotent stem and progenitor cells and the therapeutic cell types derived from them.

Cross presentation: more exotic pathwaysEpigenetic repression of MHC class I antigen presentation enables immune evasion in cancer

Loss of MHC class I antigen presentation in cancer cells is a frequent cause of primary and acquired resistance to immunotherapy. Using a genome wide CRISPR/Cas9 screen we identify a highly conserved epigenetic repressive complex that mediates the coordinated transcriptional silencing of the MHC class I antigen processing and presentation pathway in a range of cancers, promoting immune evasion. Disruption of the complex restores tumour MHC class I antigen presentation allowing effective targeting by CD8+ T-cells. This mechanism of MHC class I repression is evolutionarily conserved and allows tumours to overcome histocompatibility barriers and grow as an allogeneic transplant in immunocompetent recipients in a mouse model of lung cancer and the transmissible Tasmanian Devil Facial Tumour 1 (DFT1). I will discuss the molecular mechanisms governing MHC-I repression both during normal development and in cancer and highlight how cancer cells co-opt an evolutionarily conserved, lineage specific function of an epigenetic complex to evade immune surveillance.

Alloreactive Immune Response Associated to Human Mesenchymal Stromal Cells Treatment: A Systematic Review.

The well-known immunomodulatory and regenerative properties of mesenchymal stromal cells (MSCs) are the reason why they are being used for the treatment of many diseases. Because they are considered hypoimmunogenic, MSCs treatments are performed without considering histocompatibility barriers and without anticipating possible immune rejections. However, recent preclinical studies describe the generation of alloantibodies and the immune rejection of MSCs. This has led to an increasing number of clinical trials evaluating the immunological profile of patients after treatment with MSCs. The objective of this systematic review was to evaluate the generation of donor specific antibodies (DSA) after allogeneic MSC (allo-MSC) therapy and the impact on safety or tolerability. Data from 555 patients were included in the systematic review, 356 were treated with allo-MSC and the rest were treated with placebo or control drugs. A mean of 11.51% of allo-MSC-treated patients developed DSA. Specifically, 14.95% of these patients developed DSA and 6.33% of them developed cPRA. Neither the production of DSA after treatment nor the presence of DSA at baseline (presensitization) were correlated with safety and/or tolerability of the treatment. The number of doses administrated and human leucocyte antigen (HLA) mismatches between donor and recipient did not affect the production of DSA. The safety of allo-MSC therapy has been proved in all the studies and the generation of alloantibodies might not have clinical relevance. However, there are very few studies in the area. More studies with adequate designs are needed to confirm these results.

The immunopeptidomes of two transmissible cancers and their host have a common, dominant peptide motif.

SummaryTransmissible cancers are malignant cells that can spread between individuals of a population, akin to both a parasite and a mobile graft. The survival of the Tasmanian devil, the largest remaining marsupial carnivore, is threatened by the remarkable emergence of two independent lineages of transmissible cancer, devil facial tumour (DFT) 1 and devil facial tumour 2 (DFT2). To aid the development of a vaccine and to interrogate how histocompatibility barriers can be overcome, we analysed the peptides bound to major histocompatibility complex class I (MHC‐I) molecules from Tasmanian devil cells and representative cell lines of each transmissible cancer. Here, we show that DFT1 + IFN‐γ and DFT2 cell lines express a restricted repertoire of MHC‐I allotypes compared with fibroblast cells, potentially reducing the breadth of peptide presentation. Comparison of the peptidomes from DFT1 + IFNγ, DFT2 and host fibroblast cells demonstrates a dominant motif, despite differences in MHC‐I allotypes between the cell lines, with preference for a hydrophobic leucine residue at position 3 and position Ω of peptides. DFT1 and DFT2 both present peptides derived from neural proteins, which reflects a shared cellular origin that could be exploited for vaccine design. These results suggest that polymorphisms in MHC‐I molecules between tumours and host can be ‘hidden’ by a common peptide motif, providing the potential for permissive passage of infectious cells and demonstrating complexity in mammalian histocompatibility barriers.

Inflammatory Mediators in Glioma Microenvironment Play a Dual Role in Gliomagenesis and Mesenchymal Stem Cell Homing: Implication for Cellular Therapy.

Glioblastoma is the most aggressive malignant primary brain tumor, with a dismal prognosis and a devastating overall survival. Despite aggressive surgical resection and adjuvant treatment, average survival remains approximately 14.6 months. The brain tumor microenvironment is heterogeneous, comprising multiple populations of tumor, stromal, and immune cells. Tumor cells evade the immune system by suppressing several immune functions to enable survival. Gliomas release immunosuppressive and tumor-supportive soluble factors into the microenvironment, leading to accelerated cancer proliferation, invasion, and immune escape. Mesenchymal stem cells (MSCs) isolated from bone marrow, adipose tissue, or umbilical cord are a promising tool for cell-based therapies. One crucial mechanism mediating the therapeutic outcomes often seen in MSC application is their tropism to sites of injury. Furthermore, MSCs interact with host immune cells to regulate the inflammatory response, and data points to the possibility of using MSCs to achieve immunomodulation in solid tumors. Interleukin 1β, interleukin 6, tumor necrosis factor α, transforming growth factor β, and stromal cell–derived factor 1 are notably up-regulated in glioblastoma and dually promote immune and MSC trafficking. Mesenchymal stem cells have widely been regarded as hypoimmunogenic, enabling this cell-based administration across major histocompatibility barriers. In this review, we will highlight (1) the bidirectional communication of glioma cells and tumor-associated immune cells, (2) the inflammatory mediators enabling leukocytes and transplantable MSC migration, and (3) review preclinical and human clinical trials using MSCs as delivery vehicles. Mesenchymal stem cells possess innate abilities to migrate great distances, cross the blood-brain barrier, and communicate with surrounding cells, all of which make them desirable “Trojan horses” for brain cancer therapy.

How Adoptive Immunotherapy with Conventional T and Regulatory T Cells Exerts a Gvl Effect without GvHD, after Haploidentical Hematopoietic Transplantation

▪Post-transplant relapse is still a major cause of treatment failure in high-risk acute leukemia (AL) patients. Attempts to manipulate donor T cell alloreactivity to spare normal tissues while killing leukemic cells have been largely unsuccessful. In the search for strategies to separate the GvL effect from GVHD, we investigated the role of a thymic-derived CD4+CD25+ FoxP3+ regulatory T-cell subpopulation (Tregs) that physiologically helps maintain immunological self-tolerance and immune homeostasis. Evidence from murine bone marrow (BM) transplantation across major histocompatibility barriers showed co-infusion of conventional T lymphocytes (Tcons) with Tregs suppressed lethal GVHD without impairing Tcon activity against malignant diseases. In 69 high-risk AL patients who had received an HLA haploidentical T cell-depleted hematopoietic transplant and no post-transplant immunosuppressive GvHD prophylaxis, adoptive immunotherapy with donor Tregs (2 × 106/kg) and Tcons (1 × 106/kg) protected patients from GvHD (Di Ianni et al., Blood 2011) and largely prevented post-transplant leukemia relapse. In fact, only 5% of patients relapsed (Martelli et al., Blood 2014). However, also because such patients had advanced stage disease, TRM was still in the range of that of T cell-depleted haplo transplants, (i.e. 40%, Aversa et al., JCO 2005). A current cohort of AL patients was conditioned with total body irradiation (TBI) (8Gy as single dose TBI or 13.5 Gy as fractionated TBI), cyclophosphamide (30mg/kg), thiotepa (8mg/kg) and fludarabine (200mg/m2). To date, 24 high-risk AL patients (7 ALL, 17 AML) have been enrolled. Twenty-two of the 24 patients engrafted, one AML patient relapsed, 4/22 developed aGvHD (4/4 are alive) and one developed cGvHD, TRM was exceptionally low (Fig. 1). At a median follow up of 2 years, 19 patients are alive. Consequently, probabilities of leukemia-free and leukemia/cGVHD-free survivals are good (Fig. 1). Hypotheses have been put forward to explain the ability of Tregs to prevent of GvHD while preserving the GvL effect mediated by Tcons. However, the mechanism is still unknown. In humans, naïve CD45RA+ Tregs express CXCR4 BM homing receptor and preferentially localize to the BM while memory CD45RO+ Tregs display lower CXCR4 expression and home to the periphery (Booth et al., J Immunol. 2010). Since Tregs that are recovered from peripheral blood and are used for adoptive immunotherapy are CD45RO+ and largely CxCR4 negative, we hypothesized that GvL without GvHD might be due to unopposed Tcon alloreactivity in the BM combined with regulated T cell alloreactivity at the periphery. In order to verify such hypothesis, we infused NSG mice with human leukemia and human Tregs and Tcons. Mice that received leukemia and haploidentical Tcons (without Tregs) cleared leukemia but died of GvHD. T cells harvested from their BM, spleen and liver were predominantly CD8+ and displayed alloreactivity against leukemia. Mice that received leukemia and Tcons plus Tregs were rescued from leukemia and survived without GvHD. T cells harvested from spleen and liver were composed of CD8+ T cells (40%) and CD4+T cells (60%). Purified CD4+ T cells had retained their regulatory function as they inhibited mixed lymphocyte reaction. Purified CD8+ T cells displayed no alloreactivity against leukemia. In contrast, T cells harvested from BM were still predominantly CD8+ and still displayed alloreactivity against leukemia suggesting Tcons had retained their alloantigen recognition. Finally, in mice treated with Tregs alone, T cells were recovered only in the spleen and liver, they displayed a CD4+ phenotype and inhibited mixed lymphocyte reaction. No T cells were found in the BM. In contrast, when we infused human purified CD45RA+ naïve Tregs (that display the CXCR4 BM homing receptor), mice died of leukemia progression despite the co-infusion of large doses of Tcons. In the BM we isolated CD8+ T cells that displayed no ability to kill leukemia and CD4+ T cells that displayed regulatory function. Thus, naïve Tregs homed to the BM and blocked the GvL effect of T cons. When CXCR4 was blocked with an anti-CXCR4 antibody, naïve Tregs could not home to the BM and T cons killed leukemia. In conclusion, Treg-Tcon adoptive immunotherapy confines the graft-versus-host alloreaction to the hematopoietic system and, consequently, allows a GvL effect without GvHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Lessons learnt from the Tasmanian devil facial tumour regarding immune function in cancer.

Genetic and genomic technologies have facilitated a greater understanding of the Tasmanian devil immune system and the origins, evolution and spread of devil facial tumour disease (DFTD). DFTD is a contagious cancer that has caused significant declines in devil populations across Tasmania. Immune responses to DFTD are rarely detected, allowing the cancer to pass between individuals and proliferate unimpeded. Early immunosenscence in devils appears to decrease anti-tumour immunity in older animals compared to younger animals, which may increase susceptibility to DFTD and explain high DFTD prevalence in this age group. Devils also have extremely low major histocompatibility complex (MHC) diversity, and multiple alleles are shared with the tumour, lowering histocompatibility barriers which may have contributed to DFTD evolution. DFTD actively evades immune attack by down-regulating cell-surface MHC I molecules, making it effectively invisible to the immune system. Altered MHC I profiles should activate natural killer (NK) cell anti-tumour responses, but these are absent in DFTD infection. Recent immunisation and immunotherapy using modified DFTD cells has induced an anti-DFTD immune response and regression of DFTD in some devils. Knowledge gained from immune responses to a transmissible cancer in devils will ultimately reveal useful insights into immunity to cancer in humans and other species.

Producing proT cells to promote immunotherapies.

T lymphocytes are critical mediators of the adaptive immune system and they can be harnessed as therapeutic agents against pathogens and in cancer immunotherapy. T cells can be isolated and expanded from patients and potentially generated in vitro using clinically relevant systems. An ultimate goal for T-cell immunotherapy is to establish a safe, universal effector cell type capable of transcending allogeneic and histocompatibility barriers. To this end, human pluripotent stem cells offer an advantage in generating a boundless supply of T cells that can be readily genetically engineered. Here, we review emerging T-cell therapeutics, including tumor-infiltrating lymphocytes, chimeric antigen receptors and progenitor T cells (proT cells) as well as feeder cell-free in vitro systems for their generation. Furthermore, we explore their potential for adoption in the clinic and highlight the challenges that must be addressed to increase the therapeutic success of a universal immunotherapy.

Animal Models for Preclinical Development of Allogeneic Hematopoietic Cell Transplantation.

Since its inception in the 1950s, hematopoietic cell transplantation (HCT) has become a highly effective clinical treatment for malignant and nonmalignant hematological disorders. This milestone in cancer therapy was only possible through decades of intensive research using murine and canine animal models that overcame what appeared in the early days to be insurmountable obstacles. Conditioning protocols for tumor ablation and immunosuppression of the recipient using irradiation and chemotherapeutic drugs were developed in mouse and dog models as well as postgrafting immunosuppression methods essential for dependable donor cell engraftment. The random-bred canine was particularly important in defining the role of histocompatibility barriers and the development of the nonmyeloablative transplantation procedure, making HCT available to elderly patients with comorbidities. Two complications limit the success of HCT: disease relapse and graft versus host disease. Studies in both mice and dogs have made significant progress toward reducing and to some degree eliminating patient morbidity and mortality associated with both disease relapse and graft versus host disease. However, more investigation is needed to make HCT more effective, safer, and available as a treatment modality for other non-life-threatening diseases such as autoimmune disorders. Here, we focus our review on the contributions made by both the murine and canine models for the successful past and future development of HCT.

A Clinically Relevant Protocol Induces Tolerance to a Vascularized Composite Allograft Across Major Histocompatibility Barrier In A Large Animal Model

A Clinically Relevant Protocol Induces Tolerance to a Vascularized Composite Allograft Across Major Histocompatibility Barrier In A Large Animal Model

Abstract 609: Overcoming host histocompatibility barrier to create a renewable source of off-the-shelf effector lymphocytes for adoptive immunotherapy

Abstract Encouraging clinical outcomes in autologous cellular immunotherapy have garnered hope and excitement. However, limitations of patient-derived cancer immunotherapies remain to be addressed to deliver reliable and efficacious therapies with broader applicability. Induced pluripotent stem cells (iPSCs) are a unique, renewable source for the continuous generation of cellular therapeutics and represent a highly promising approach for overcoming many of the limitations of autologous therapy. To advance the promise of iPSC technology as an “off-the-shelf” (OTS) source of cellular therapeutics, several considerations need to be addressed. Ensuring the persistence of allogeneic OTS therapies after adoptive cell transfer across histocompatibility barriers is a key requirement. Establishing a master cell line from genetically engineered clonal iPSC lines with the capacity to continuously generate homogenous populations of highly functional effector cells will also be necessary. Here we demonstrate a comprehensive approach for the generation of immune tolerant effector cells derived from a genetically engineered iPSC master cell line. We successfully combined deletion of classical human leukocyte antigen molecules with expression of immunosuppressive proteins to generate clonal iPSC lines with the ability to escape immune rejection. Utilizing in vitro quantitative live cell analysis we show that OTS-iPSCs elicit a significantly decreased cytotoxic response from both peripheral blood (PB)-NK cells (47.9 vs. 91.4% survival at 3:1 E:T ratio) and PB-T cells (>2.7-fold greater number of OTS-iPSC derived cells remaining at 88 hrs). Additionally, mixed lymphocyte reactions employing unfractionated PB mononuclear cells resulted in significantly decreased activation and proliferation of CD8+ T cells (63.4 vs. 29.6%), CD4+ T cells (70.9 vs. 17.3%) and NK cells (46.8 vs. 11.6%). In preclinical mouse models we demonstrate that OTS-iPSCs exhibit improved persistence in vivo. Bilateral engraftments were established in non-conditioned, fully immune-competent recipient mice using luciferized wildtype and OTS-iPSCs. Daily bioluminescence imaging revealed a significant increase in persistence of OTS-iPSCs during the 48-196 hour post injection window (>5.5 fold greater luminescence at 96 hrs). Using our potent chemically-defined stage-specific monolayer hematopoietic differentiation platform, we demonstrate that OTS-iPSC derived CD34 expressing hematopoietic cells are reproducibly scaled and readily give rise to functional lymphocytes carrying the engineered targeted modality in a homogenous manner (95 +/- 5%). The outlined preclinical data illustrate that iPSCs are an ideal renewable source for OTS hematopoietic cell-based immunotherapies and represent a potentially exponential advancement in adoptive immunotherapy. Citation Format: Raedun L. Clarke, Matthieu Bauer, Ryan Bjordahl, Jeffrey Sasaki, Brian Groff, Svetlana Gaidarova, Tom Tong Lee, Weijie Lan, Michelle Burrascano, Ramzey Abujarour, Greg Bonello, Megan Robinson, Stewart Abbot, Scott Wolchko, Daniel Shoemaker, Bob Valamehr. Overcoming host histocompatibility barrier to create a renewable source of off-the-shelf effector lymphocytes for adoptive immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 609. doi:10.1158/1538-7445.AM2017-609

Abstract 3755: Renewable and genetically engineered natural killer cells for off-the-shelf adoptive cellular immunotherapy

Abstract The unique attributes of a combinatorial tumor recognition system, diminished off-tumor cytotoxicity, and multifaceted effector function make natural killer (NK) cells a prime candidate for a universal approach to cancer immunotherapy. In addition, NK cells are the principal mediator of antibody-directed cellular cytotoxicity (ADCC). However, NK cell function is often impaired in the setting of cancer, reducing the effectiveness of the endogenous immune system and the therapeutic efficacy of monoclonal antibodies. To address the need for advanced and combinatorial cancer therapies, we developed a unique and effective strategy to create a renewable source of engineered “off-the-shelf” NK cells with augmented function, including enhanced ADCC and persistence. Key challenges associated with genetic editing, limited expansion, persistence and variability of peripheral blood (PB)-derived NK cells were overcome by utilizing our induced pluripotent stem cell (iPSC) technology as the unlimited starting material for the reproducible and consistent derivation of engineered NK cells. Through targeted transgene integration, we produced a clonal iPSC master cell line to continuously produce NK cells engineered to uniformly express a high affinity, non-cleavable version of CD16 (hnCD16-NK). In directed differentiation, the hnCD16-NK cells displayed homogeneous expression of CD16 (>95%) and a mature CD56+ NK cell phenotype, as exhibited by expression of KIR, NCRs, DNAM-1, and NKG2D. In contrast to endogenous CD16 expression, the engineered hnCD16 molecule was shown to be cleavage resistant upon NK cell activation (>95% CD16+ hnCD16-NK vs. <10% CD16+ PB-derived NK cell, upon target cell-mediated activation), and demonstrated enhanced antibody binding compared to PB-derived NK cells expressing the low-affinity variant. In addition to increased expression of the cytolytic molecules perforin and granzyme B and enhanced direct cytotoxicity against tumor targets, hnCD16-NK cells displayed superior ADCC capacity and cytokine production in response to CD16 stimulation. Importantly, manufacture of hnCD16-NK cells was proven to be highly scalable, delivering up to 107 fold expansion over a 35 day period. The maintained proliferative capacity can be in part associated with longer telomere length seen in hnCD16-NK cells. Furthermore, deletion of classical human leukocyte antigen molecules and ectopic expression of immunosuppressive proteins engineered at the iPSC level provided the ability of hnCD16-NK cells to potentially overcome the host histocompatibility barrier and to improve persistence in the allogeneic setting. In conclusion, the preclinical data presented herein highlight the therapeutic value of hnCD16-iNK cells as an ideal ADCC-mediated “off-the-shelf” NK cell-based immunotherapeutic product with augmented persistence, anti-tumor capacity, manufacturing reliability and preclinical efficacy. Citation Format: Ryan Bjordahl, Frank Cichocki, Raedun Clarke, Svetlana Gaidarova, Brian Groff, Paul Rogers, Stacey Moreno, Ramzey Abujarour, Greg Bonello, Tom Lee, Weijie Lan, Matthieu Bauer, Dave Robbins, Betsy Rezner, Sarah Cooley, Bruce Walcheck, Stewart Abbot, Bruce Blazar, Scott Wolchko, Daniel Shoemaker, Jeffrey S. Miller, Bahram Valamehr. Renewable and genetically engineered natural killer cells for off-the-shelf adoptive cellular immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3755. doi:10.1158/1538-7445.AM2017-3755

Generation of Hypoimmunogenic Induced Pluripotent Stem Cells for Allogeneic Cell and Tissue Transplantation

Background: The most urgent problem facing transplantation today is the lack of suitable donor organs and tissues. One possible alternative may be cell transplantation with the aim to replace, repair, or enhance the biological function of the diseased organ. Induced pluripotent stem cells are promising candidates for cell-based therapies. Transplanted autologous iPS cell do not cross histocompatibility barriers since such cells are genetically identical to the transplant recipient. However, for acute diseases such as myocardial infarction, treatment options must be readily available, and generating autologous iPSCs for treatment purposes would take too long. Therefore, identifying alternative methods for preventing rejection of iPSCs would be of great interest. In this study, we generated MHC I and MHCII -deficient mouse iPSCs (miPSCs) via disruption of beta-2-microglobulin and C2TA. The antigenicity was investigated in in vivo and in vitro assays. Methods: IPSCs were generated from C57BL/6 mouse fibroblasts by non-viral minicircle transfection of the transcription factors c-Myc, Sox-2, Klf4 and Oct4. The CRIPSR/Cas9 technology was used to generate β2m and C2TA knockout miPSCs (β2m-C2TA−/− miPSCs). Unmodified and β2m-C2TA −/− miPSCs as well as their derivates were injected in syngeneic C57BL/6 mice and in allogeneic BALB/c mice. Cellular response was quantified and bioluminescence imaging was performed to assess cell survival. Results: FACS analysis showed a lack of MHC I and MHC II expression on the β2m-C2TA−/− iPSCs but a normal expression in unmodified iPSCs. After allogeneic transplantation into BALB/c mice, β2m-C2TA−/− miPSCs generated a significantly weaker response in both IFNγ (p<0.01) and IL-4 (p<0.01) ELISPOT assays. Teratoma formation occurred in C57BL/6 mice receiving unmodified iPSCs (100%) and BALB/c mice receiving β2m-C2TA−/− iPSCs (100%), whereas BALB/c mice receiving unmodified iPSCs did not form any teratoma (0%). Indeed, also miPSC derivates, such as cardiomyocytes, endothelial and islet cells showed long-term survival after allogeneic transplantation. Conclusion: Our results clearly demonstrate the hypoimmunogenicity of β2m-C2TA−/− iPSCs after allogeneic transplantation. Thus, MHC I MHC II deficient iPSCs might serve an unlimited cell source for the generation of universally compatible “off-the-shelf” cells grafts or tissues in future clinical applications. Christiane Pahrmann.

Cancer as a contagious disease.

Contagious cancers are malignant cells that are physically transferred between individuals as a natural allograft, forming new clonal tumours. These cancers are highly unusual, but have emerged in 2 mammalian species, the dog and the Tasmanian devil, as well as 4 species of bivalve. The transfer of malignant cells in mammals should initiate a robust immune response and although invertebrates have a less complex immune system, these species still have mechanisms that should prevent engraftment and protect against cellular parasitism. Here the naturally occurring contagious cancers are reviewed to determine what features are important and necessary for the emergence and spread of these types of cancer, with a focus on the mammalian contagious cancers and how they successfully cross histocompatibility barriers.

Brief treatment with a highly selective immunoproteasome inhibitor promotes long-term cardiac allograft acceptance in mice

Constitutive proteasomes (c-20S) are ubiquitously expressed cellular proteases that degrade polyubiquitinated proteins and regulate cell functions. An isoform of proteasome, the immunoproteasome (i-20S), is highly expressed in human T cells, dendritic cells (DCs), and B cells, suggesting that it could be a potential target for inflammatory diseases, including those involving autoimmunity and alloimmunity. Here, we describe DPLG3, a rationally designed, noncovalent inhibitor of the immunoproteasome chymotryptic subunit β5i that has thousands-fold selectivity over constitutive β5c. DPLG3 suppressed cytokine release from blood mononuclear cells and the activation of DCs and T cells, diminished accumulation of effector T cells, promoted expression of exhaustion and coinhibitory markers on T cells, and synergized with CTLA4-Ig to promote long-term acceptance of cardiac allografts across a major histocompatibility barrier. These findings demonstrate the potential value of using brief posttransplant immunoproteasome inhibition to entrain a long-term response favorable to allograft survival as part of an immunomodulatory regimen that is neither broadly immunosuppressive nor toxic.

Genetic Engineering of Pluripotent Cells for the Continuous Derivation of Off-the-Shelf Effector Lymphocytes with Enhanced Therapeutic Persistence By Overcoming the Host Histocompatibility Barrier

Encouraging clinical outcomes in autologous cellular immunotherapy have garnered hope and excitement. However, considerable challenges and limitations of patient-derived cancer immunotherapies remain and need to be addressed in order to consistently deliver reliable and efficacious therapies with broadened applicability. Human induced pluripotent stem cells (hiPSCs) are a unique, renewable source for the continuous generation of cellular therapeutics for the treatment of hematological and non-hematological malignancies, and represent a highly promising approach for overcoming many of the limitations of autologous therapy. To advance the promise of hiPSC technology as an "off-the-shelf" source of cellular therapeutics, several considerations need to be addressed. Enabling cell transfer across histocompatibility barriers to permit persistence and therapeutic efficacy in an allogeneic setting is a key requirement. In addition to improving persistence, the ability to overcome histocompatibility barriers may facilitate multi-dosing regimens which may be a requirement in more advanced and complicated disease settings.Genetic incompatibilities between donor and recipient among the classical human leukocyte antigen (HLA) molecules is the leading cause of alloresponse by the host immune system and is currently mitigated by immunosuppressive strategies. Unfortunately, this treatment strategy is not only a stressful event for the patient but also damages the endogenous immune system, compromising the patient’s ability to continue to fight the disease and opportunistic infections. Genetic editing of the HLA genes to generate histocompatible universal cell products is a viable opportunity that is currently being investigated. In addition to selective editing of unique genes to avoid a T cell mediated alloresponse, additional considerations such as natural killer (NK) cell-mediated rejection will need to be addressed. We have previously demonstrated that our proprietary reprogramming platform supports efficient and rapid derivation of clonal hiPSC lines with properties indicative of the naïve state of pluripotency. In addition to maintaining a homogeneous renewable population of hiPSCs, our platform is amenable to precise multi-gene and multi-loci targeted engineering at the single cell level, in both nuclease -dependent and -independent strategies. Furthermore, we have shown through small molecule-guided differentiation protocols, these highly-stable pluripotent cell lines can be banked and repeatedly tapped to consistently produce homogenous populations of immune cells with enhanced effector properties.Here we demonstrate a multi-faceted and comprehensive approach for the generation of immune tolerant hiPSCs and hiPSC-derived immune effector cells. We successfully combined deletion of classical HLA molecules with enforced expression of robust immunosuppressive proteins, including non-classical HLA molecules, to generate clonal hiPSC lines with the ability to escape immune rejection for "off-the-shelf" (OTS-hiPSCs) cellular immunotherapy. Utilizing in vitro real-time quantitative live cell analysis we determined that OTS-hiPSCs elicit a significantly decreased cytotoxic response from both activated peripheral blood (PB)-NK cells and primed PB-T cells compared to wildtype controls. Furthermore we demonstrate that OTS-hiPSCs exhibit improved persistence in xenograft studies in vivo. Bilateral teratomas were formed in a non-conditioned, fully immune-competent recipient mice using luciferized wildtype and OTS-hiPSCs. Daily bioluminescence imaging over a period of 7 days revealed a significant increase (>50 fold difference) in persistence of OTS-hiPSCs compared to wildtype hiPSCs during the 60-144 hour post injection window. Lastly we demonstrate that OTS-hiPSCs can successfully differentiate into functional effector lymphocytes using our potent chemically-defined monolayer hematopoietic differentiation platform. Our current studies focus on the functional characterization of OTS-hiPSC-derived effector lymphocytes in humanized mouse models and generating increased potency of OTS-hiPSC-derived effector lymphocytes through precise genetic engineering of antigen targeting and costimulatory proteins to create and optimized source of "off-the-shelf" cell-based immunotherapies. DisclosuresBauer:Fate Therapeutics: Employment. Clarke:Fate Therapeutics: Employment. Sasaki:Fate Therapeutics: Employment. Groff:Fate Therapeutics: Employment. Lee:Fate Therapeutics: Employment. Lan:Fate Therapeutics: Employment. Abujarour:Fate Therapeutics: Employment. Bonello:Fate Therapeutics: Employment. Burrascano:Fate Therapeutics: Employment. Robinson:Fate Therapeutics: Employment. Bjordahl:Fate Therapeutics, Inc: Employment. Gaidarova:Fate Therapeutics: Employment. Abbot:Fate Therapeutics: Employment. Wolchko:Fate Therapeutics: Employment. Shoemaker:Fate Therapeutics: Employment, Equity Ownership. Valamehr:Fate Therapeutics, Inc: Employment.

Third Party Cytokine-Induced Killer Cells Eliminate Highly Activated T-Cells, Leading to Protect from Murine Lethal Graft-Versus-Host Disease

Protecting from lethal graft-versus-host disease (GVHD) while preserving graft-versus-tumor effects by adoptive immunotherapy would be an ideal goal in allogeneic hematopoietic stem cell transplantation (HSCT), especially using third party derived cells, because the use of third party cells would open a huge source of availability and feasibility across major histocompatibility barriers. We have demonstrated that third party cytokine-induced killer (CIK) cells protected from murine lethal GVHD with faster hematological recovery, which are ex vivo-expanded T lymphocytes expressing both natural killer and T-cell markers with strong cytotoxic activity both against autologous and allogeneic tumor cells. However the mechanism responsible for ameliorating acute GVHD by third party CIK cells are still unknown.First, lethally irradiated Balb/c (H-2d) mice were given C3H (H-2k) bone marrow cells with C3H whole splenocytes to induce lethal GVHD with/without third party (C57/BL6 (H-2b)) CIK cells or third party whole splenocytes on day 0 or day4. Mice receiving third party CIK cells showed much less GVHD and significant survival benefit compared those with third party splenocytes as shown below (p<0.05). On day7 after BMT, the percentage of CD69 on CD8 T-cells in peripheral blood (PB) in the mice receiving third party CIK cells was significantly lower than those in GVHD control mice as shown below (p<0.05), meanwhile donor derived not-activated T-cells were preserved in both CD4 and CD8 T-cells. From these results, we hypothesized that third party CIK cells prevent acute GVHD by selectively depleting highly proliferative alloreactive T-cells stimulated early after transplant, while sparing naïve T-cells. To test this, we performed mixed lymphocyte reaction (MLR) by adding third party CIK cells or third party splenocytes (Stimulator: Balb/c splenocytes, Responder: C3H splenocytes, third party cells: C57/BL6). The percentage of CD69 on responder derived CD8 T-cells in the culture mixture with third party CIK cells was much less compared to that with third party splenocytes as shown below (p<0.05). We have previously shown that allogeneic CIK cells had cytotoxicity against cultured dendritic cells (DCs) by 51Cr release assay. Therefore reduced alloreaction by third party CIK cells might be due in part to the elimination of antigen presenting cells such as DCs. Next, to demonstrate that third party CIK cells could eliminate responder cells directly and then reduce the alloreaction, we performed MLR like experiments using PHA to activate T-cells strongly instead of antigen presenting cells as a stimulator. As shown below, we have demonstrated that the percentage of highly activated T-cells (CD8+CD69+) in third party CIK added plates was significantly lower compared to that in third party splenocytes added plates, indicating that third party CIK cells could have direct effects against highly activated T-cells.To further clarify the mechanisms of how third party CIK cells reduce the highly activated T-cells, we performed MLR as mentioned above with/without the neutralizing antibody against NKG2D, which is an activator receptor expressed on NK cells. Although CIK cells have cytotoxicity against tumor cells via NKG2D signaling pathway in part, adding NKG2D antibody did not show a statistical significant difference in MLR assays. It suggested that third party CIK cells could suppress alloreactive T-cells by several pathways other than NKG2D signaling pathway.Finally, we identified the in vivo kinetics of third party CIK cells after BMT. Third party derived CIK cells could be identified as a rare subpopulation in recipient peripheral blood for some days after CIK infusion, but all of them could not be detected anymore by day 7 after infusion. Whereas third party whole splenocytes expanded in recipient peripheral blood and mice received third party splenocytes with donor BM and splenocytes died until day7 because of GVHD induced by third party splenocytes.In conclusion, infusion of third party CIK cells has strong potential to prevent murine lethal GVHD probably due to selectively depleting highly proliferative alloreactive T-cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Thymocytes self-renewal: a major hope or a major threat?

Thymus transplants were never used to correct T-cell intrinsic deficiencies, as it is generally believed that thymocytes have short intrinsic lifespans. This notion is based on multiple thymus transplantation experiments, where it was shown that thymus-resident cells were rapidly replaced by progenitors migrating from the bone marrow (BM). This substitution occurs even when bone marrow precursors are unable to generate T cells, as in Rag1/2(-) or severe combined immunodeficiency (SCID)-deficient mice. In contrast, two groups reported that neonatal thymi transplanted into mice that cannot respond to IL-7 harbor populations with extensive capacity to self-renew, which maintain continuous thymocyte generation for several months after surgery. The consequences of this self-renewal capacity differed in these two laboratories. We found that these thymus transplants rapidly reconstitute the full diversity of peripheral T-cell repertoires 1month after surgery, the earliest time point studied. Moreover, transplantation experiments performed across major histocompatibility barriers show that allogeneic-transplanted thymi are not rejected, and allogeneic cells do not induce graft-versus-host disease, both syngeneic and allogeneic transplants inducing rapid protection from infection. These results indicate a potential use of neonatal thymus transplants to correct T-cell intrinsic deficiencies. The other group observed that continuous thymocyte renewal from BM precursors was fundamental to prevent tumor development. In the absence of this input, thymocytes from the transplanted thymus generated tumors with all the characteristics of T-cell acute lymphoblastic leukemia (T-ALL). Moreover, they suggested that the absence of BM competition was responsible for the T-ALLs developing in X-linked severe combined immunodeficiency (SCID)-X1 patients, deficient in the expression of IL2-Rγc . These patients were treated with autologous CD34(+) cells transfected with virus vectors expressing γc in the absence of myeloablation. We here review the potential therapeutic impact of thymus transplantation and compare the results of these two laboratories aiming to find an answer to the 'Dr Jekill versus Mr. Hyde' status of thymus transplantation experiments.

Donor Leukocyte Infusion Mediated Graft-Versus-Host Responses in a Pre-Clinical Swine Model of Haploidentical Hematopoietic Cell Transplantation

The donor-anti-host cellular reactivity associated with allogeneic hematopoietic cell transplantation (HCT) is a powerful anti-leukemia treatment. However, graft-versus-host disease (GVHD) continues to limit HCT, especially across major histocompatibility (MHC) barriers. We previously described successful hematopoietic stem cell engraftment in MHC-mismatched swine without GVHD using novel reduced intensity conditioning (RIC) regimens consisting of partial, transient recipient T-cell depletion (with or without thymic or low-dose total body irradiation), and a short course of cyclosporine A (CyA). Here, we report that stable chimeric animals generated with these protocols are strongly resistant to donor leukocyte infusion (DLI) mediated GVH effects (n=17). Delayed DLI in tolerant chimeras at a dose consisting of 10-50X106 T cells /kg (Tc/kg) failed to induce conversion to full donor chimerism or cause GVHD. Sensitizing the donor with recipient skin prior to collecting donor cells for DLI did not have any demonstrable effect on DLI outcome (n=2). This resistance to DLI mediated GVH reactivity could be overcome in some cases (n=9) by either 1) increasing the dose of DLI, 2) removing chimeric host peripheral blood cell populations through extensive leukapheresis prior to DLI or 3) delivering lymphocytes fully mismatched to the host but not to the original HCT donor within the chimera. However, conversion to full donor chimerism in some recipients was associated with GVHD. Our studies suggest that delivery of non-manipulated HCT grafts under reduced intensity conditioning can induce a potent, GVHD free immune tolerant state that is strongly resistant to DLI. DLI induced conversion to full donor chimerism is often associated with GVHD despite full immune reconstitution of the host and lack of known inflammatory stimuli.

Freeze and Thaw of CD4+CD25+Foxp3+ Regulatory T Cells Results in Loss of CD62L Expression and a Reduced Capacity to Protect against Graft-versus-Host Disease.

The adoptive transfer of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in murine models of allogeneic hematopoietic cell transplantation (HCT) has been shown to protect recipient mice from lethal acute graft-versus-host disease (GVHD) and this approach is being actively investigated in human clinical trials. Here, we examined the effects of cryopreservation on Tregs. We found that freeze and thaw of murine and human Tregs is associated with reduced expression of L-selectin (CD62L), which was previously established to be an important factor that contributes to the in vivo protective effects of Tregs. Frozen and thawed murine Tregs showed a reduced capacity to bind to the CD62L binding partner MADCAM1 in vitro as well as an impaired homing to secondary lymphoid organs in vivo. Upon adoptive transfer frozen and thawed Tregs failed to protect against lethal GVHD compared with fresh Tregs in a murine model of allogeneic HCT across major histocompatibility barriers. In summary, the direct administration of adoptively transferred frozen and thawed Tregs adversely affects their immunosuppressive potential which is an important factor to consider in the clinical implementation of Treg immunotherapies.