Derin B. Keskin

Cells Expressing Indoleamine 2,3-Dioxygenase Inhibit T Cell Responses

Pharmacological inhibition of indoleamine 2,3-dioxygenase (IDO) activity during murine gestation results in fetal allograft rejection and blocks the ability of murine CD8+ dendritic cells to suppress delayed-type hypersensitivity responses to tumor-associated peptide Ags. These observations suggest that cells expressing IDO inhibit T cell responses in vivo. To directly evaluate the hypothesis that cells expressing IDO inhibit T cell responses, we prepared IDO-transfected cell lines and transgenic mice overexpressing IDO and assessed allogeneic T cell responses in vitro and in vivo. T cells cocultured with IDO-transfected cells did not proliferate but expressed activation markers. The potency of allogeneic T cell responses was reduced significantly when mice were preimmunized with IDO-transfected cells. In addition, adoptive transfer of alloreactive donor T cells yielded reduced numbers of donor T cells when injected into IDO-transgenic recipient mice. These outcomes suggest that genetically enhanced IDO activity inhibited T cell proliferation in vitro and in vivo. Genetic manipulation of IDO activity may be of therapeutic utility in suppressing undesirable T cell responses.

A review of the current use of rituximab in autoimmune diseases.

Rituximab is a human/murine chimeric monoclonal antibody primarily used for treating non-Hodgkins B-cell lymphoma. Recently it has also been used in the treatment of several autoimmune diseases. A literature review was conducted to determine the efficacy of rituximab in the treatment of some of these autoimmune diseases. Multiple mechanisms proposed for the rituximab mediated B cell depletion are also discussed. The efficacy of rituximab is well-established and it is FDA approved for treatment of Rheumatoid arthritis. In this review, data on the use of rituximab is presented from 92 studies involving 1197 patients with the following diseases: systemic lupus erythematosus, idiopathic thrombocytopenic purpura, anti-neutrophil cytoplasmic antibody associated vasculitis, Graves disease, autoimmune hemolytic anemia, pemphigus vulgaris, hemophilia A, cold agglutinin disease, Sjogrens syndrome, graft vs. host disease, thrombotic thrombocytopenic purpura, cryoglobulinemia, IgM mediated neuropathy, multiple sclerosis, neuromyelitis optica, idiopathic membranous nephropathy, dermatomyositis, and opsoclonus myoclonus. The efficacy varies among different autoimmune diseases. The cumulative data would suggest that in the vast majority of studies in this review, RTX has a beneficial role in their treatment. While rituximab is very effective in the depletion of B cells, current research suggests it may also influence other cells of the immune system by re-establishing immune homeostasis and tolerance. The safety profile of RTX reveals that most reactions are infusion related. In patients with autoimmune diseases the incidence of serious and severe side effects is low. Systemic infection still remains a major concern and may result in death.

An immunogenic personal neoantigen vaccine for patients with melanoma

Effective anti-tumour immunity in humans has been associated with the presence of T cells directed at cancer neoantigens, a class of HLA-bound peptides that arise from tumour-specific mutations. They are highly immunogenic because they are not present in normal tissues and hence bypass central thymic tolerance. Although neoantigens were long-envisioned as optimal targets for an anti-tumour immune response, their systematic discovery and evaluation only became feasible with the recent availability of massively parallel sequencing for detection of all coding mutations within tumours, and of machine learning approaches to reliably predict those mutated peptides with high-affinity binding of autologous human leukocyte antigen (HLA) molecules. We hypothesized that vaccination with neoantigens can both expand pre-existing neoantigen-specific T-cell populations and induce a broader repertoire of new T-cell specificities in cancer patients, tipping the intra-tumoural balance in favour of enhanced tumour control. Here we demonstrate the feasibility, safety, and immunogenicity of a vaccine that targets up to 20 predicted personal tumour neoantigens. Vaccine-induced polyfunctional CD4+ and CD8+ T cells targeted 58 (60%) and 15 (16%) of the 97 unique neoantigens used across patients, respectively. These T cells discriminated mutated from wild-type antigens, and in some cases directly recognized autologous tumour. Of six vaccinated patients, four had no recurrence at 25 months after vaccination, while two with recurrent disease were subsequently treated with anti-PD-1 (anti-programmed cell death-1) therapy and experienced complete tumour regression, with expansion of the repertoire of neoantigen-specific T cells. These data provide a strong rationale for further development of this approach, alone and in combination with checkpoint blockade or other immunotherapies.

TGFβ promotes conversion of CD16+ peripheral blood NK cells into CD16− NK cells with similarities to decidual NK cells

During pregnancy the uterine decidua is populated by large numbers of natural killer (NK) cells with a phenotype CD56superbrightCD16−CD9+KIR+ distinct from both subsets of peripheral blood NK cells. Culture of highly purified CD16+CD9− peripheral blood NK cells in medium containing TGFβ1 resulted in a transition to CD16−CD9+ NK cells resembling decidual NK cells. Decidual stromal cells, when isolated and cultured in vitro, were found to produce TGFβ1. Incubation of peripheral blood NK cells with conditioned medium from decidual stromal cells mirrored the effects of TGFβ1. Similar changes may occur upon NK cell entry into the decidua or other tissues expressing substantial TGFβ. In addition, Lin−CD34+CD45+ hematopoietic stem/progenitor cells could be isolated from decidual tissue. These progenitors also produced NK cells when cultured in conditioned medium from decidual stromal cells supplemented with IL-15 and stem cell factor.

Systematic identification of personal tumor-specific neoantigens in chronic lymphocytic leukemia.

Genome sequencing has revealed a large number of shared and personal somatic mutations across human cancers. In principle, any genetic alteration affecting a protein-coding region has the potential to generate mutated peptides that are presented by surface HLA class I proteins that might be recognized by cytotoxic T cells. To test this possibility, we implemented a streamlined approach for the prediction and validation of such neoantigens derived from individual tumors and presented by patient-specific HLA alleles. We applied our computational pipeline to 91 chronic lymphocytic leukemias (CLLs) that underwent whole-exome sequencing (WES). We predicted ∼22 mutated HLA-binding peptides per leukemia (derived from ∼16 missense mutations) and experimentally confirmed HLA binding for ∼55% of such peptides. Two CLL patients that achieved long-term remission following allogeneic hematopoietic stem cell transplantation were monitored for CD8(+) T-cell responses against predicted or confirmed HLA-binding peptides. Long-lived cytotoxic T-cell responses were detected against peptides generated from personal tumor mutations in ALMS1, C6ORF89, and FNDC3B presented on tumor cells. Finally, we applied our computational pipeline to WES data (N = 2488 samples) across 13 different cancer types and estimated dozens to thousands of predicted neoantigens per individual tumor, suggesting that neoantigens are frequent in most tumors.

Induction of anti-tumor cytotoxic T cell responses through PLGA-nanoparticle mediated antigen delivery

Nanotechnology-based antigen delivery has been developing as a vaccine strategy due to its dose-sparing and prolonged antigen presentation features. In the current study, we examined the feasibility of nanoparticle (NP)-mediated delivery of antigenic peptides to efficiently induce cytotoxic T lymphocyte responses against tumor-associated self-antigens in C57BL/6 mouse models. The biodegradable poly(D,L-lactide-co-glycolide) nanoparticle (PLGA-NP) carrying murine melanoma antigenic peptides, hgp100(25-33) and TRP2(180-188), were prepared by double emulsion method. Efficient uptake of PLGA-NP by murine dendritic cells was shown in vitro and in vivo, using NP labeled with the fluorescent dye DiD. Intradermal injection of peptide-loaded PLGA-NP into mice induced antigen-specific T cell responses more strongly than the peptides mixed with Freunds adjuvant. More importantly, vaccination with PLGA-NP carrying both TRP2(180-188) and a toll-like receptor 4 agonist, monophosphoryl lipid A, significantly delayed growth of subcutaneously inoculated B16 melanoma cells in a prophylactic setting. Furthermore, the anti-tumor activity of NP-mediated peptide vaccination was significantly augmented by combined treatment with interferon-γ, which might prevent tumor escape through up-regulation of MHC class I expression on tumor cells. Our findings demonstrate the feasibility of NP-mediated antigen delivery for cancer immunotherapy, in particular when immune escape mechanisms of tumor cells are blocked simultaneously.

Developmental bias in cleavage-stage mouse blastomeres

BACKGROUND The cleavage-stage mouse embryo is composed of superficially equivalent blastomeres that will generate both the embryonic inner cell mass (ICM) and the supportive trophectoderm (TE). However, it remains unsettled whether the contribution of each blastomere to these two lineages can be accounted for by chance. Addressing the question of blastomere cell fate may be of practical importance, because preimplantation genetic diagnosis requires removal of blastomeres from the early human embryo. To determine whether blastomere allocation to the two earliest lineages is random, we developed and utilized a recombination-mediated, noninvasive combinatorial fluorescent labeling method for embryonic lineage tracing. RESULTS When we induced recombination at cleavage stages, we observed a statistically significant bias in the contribution of the resulting labeled clones to the trophectoderm or the inner cell mass in a subset of embryos. Surprisingly, we did not find a correlation between localization of clones in the embryonic and abembryonic hemispheres of the late blastocyst and their allocation to the TE and ICM, suggesting that TE-ICM bias arises separately from embryonic-abembryonic bias. Rainbow lineage tracing also allowed us to demonstrate that the bias observed in the blastocyst persists into postimplantation stages and therefore has relevance for subsequent development. CONCLUSIONS The Rainbow transgenic mice that we describe here have allowed us to detect lineage-dependent bias in early development. They should also enable assessment of the developmental equivalence of mammalian progenitor cells in a variety of tissues.

TRYPTOPHAN CATABOLISM AND T CELL RESPONSES

Cells expressing indoleamine 2,3 dioxygenase (IDO) play key roles in regulating adaptive immune responses orchestrated by T cells. In this report we discuss our working model, the tryptophan depletion hypothesis, to explain links between IDO expression and inhibition of T cell responses. We posit that IDO+ cells, particularly professional antigen presenting cells (APCs) promote T cell entry but block cell cycle progression due to tryptophan catabolism. We discuss experimental evidence supporting predictions from the tryptophan depletion hypothesis and the implications that this model has for understanding the origin of tolerant states that explain immunological paradoxes, such as fetal survival, tumor persistence and failure to eradicate pathogens like HIV that cause persistent infections.

Indoleamine 2,3-dioxygenase, immunosuppression and pregnancy

Pharmacologic inhibition of indoleamine 2,3-dioxygenase (IDO) activity during murine pregnancy results in maternal T-cell-mediated rejection of allogeneic but not syngeneic conceptuses. Increased risk of allogeneic pregnancy failure induced by exposure to IDO inhibitor is strongly correlated with maternal C3 deposition at the maternal-fetal interface. Here we review evidence that cells expressing IDO contribute to immunosuppression by inhibiting T-cell responses to tumor antigens and tissue allografts, as well as fetal tissues.

Mass Spectrometry Profiling of HLA-Associated Peptidomes in Mono-allelic Cells Enables More Accurate Epitope Prediction

SUMMARY Identification of human leukocyte antigen (HLA)‐bound peptides by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) is poised to provide a deep understanding of rules underlying antigen presentation. However, a key obstacle is the ambiguity that arises from the co‐expression of multiple HLA alleles. Here, we have implemented a scalable mono‐allelic strategy for profiling the HLA peptidome. By using cell lines expressing a single HLA allele, optimizing immunopurifications, and developing an application‐specific spectral search algorithm, we identified thousands of peptides bound to 16 different HLA class I alleles. These data enabled the discovery of subdominant binding motifs and an integrative analysis quantifying the contribution of factors critical to epitope presentation, such as protein cleavage and gene expression. We trained neural‐network prediction algorithms with our large dataset (>24,000 peptides) and outperformed algorithms trained on datasets of peptides with measured affinities. We thus demonstrate a strategy for systematically learning the rules of endogenous antigen presentation. Graphical Abstract Figure. No Caption available. Highlights24,000 HLA class I peptides were identified through a scalable MS‐based pipeline.Mono‐allelic data revealed binding motifs that were validated biochemically.Comprehensive analyses provide an updated portrait of antigen processing rules.Neural networks were trained for 16 alleles and outperform standard by 2‐fold. &NA; HLA class I binding prediction has traditionally been based on biochemical binding experiments. Abelin and colleagues present an LC‐MS/MS‐based workflow and analytical framework that greatly accelerates gains in prediction performance. Key advances include the discovery of sequence motifs and improved quantification of the roles of gene expression and proteasomal processing.