Natural Polymorphisms in Tap2 Influence Negative Selection and CD4 : CD8 Lineage Commitment in the Rat

Contains fulltext : 136368.pdf (publisher's version ) (Open Access)Genetic variation in the major histocompatibility complex (MHC) affects CD4ratioCD8 lineage commitment and MHC expression. However, the contribution of specific genes in this gene-dense region has not yet been resolved. Nor has it been established whether the same genes regulate MHC expression and T cell selection. Here, we assessed the impact of natural genetic variation on MHC expression and CD4ratioCD8 lineage commitment using two genetic models in the rat. First, we mapped Quantitative Trait Loci (QTLs) associated with variation in MHC class I and II protein expression and the CD4ratioCD8 T cell ratio in outbred Heterogeneous Stock rats. We identified 10 QTLs across the genome and found that QTLs for the individual traits colocalized within a region spanning the MHC. To identify the genes underlying these overlapping QTLs, we generated a large panel of MHC-recombinant congenic strains, and refined the QTLs to two adjacent intervals of approximately 0.25 Mb in the MHC-I and II regions, respectively. An interaction between these intervals affected MHC class I expression as well as negative selection and lineage commitment of CD8 single-positive (SP) thymocytes. We mapped this effect to the transporter associated with antigen processing 2 (Tap2) in the MHC-II region and the classical MHC class I gene(s) (RT1-A) in the MHC-I region. This interaction was revealed by a recombination between RT1-A and Tap2, which occurred in 0.2% of the rats. Variants of Tap2 have previously been shown to influence the antigenicity of MHC class I molecules by altering the MHC class I ligandome. Our results show that a restricted peptide repertoire on MHC class I molecules leads to reduced negative selection of CD8SP cells. To our knowledge, this is the first study showing how a recombination between natural alleles of genes in the MHC influences lineage commitment of T cells

Association of MicroRNA-618 Expression With Altered Frequency and Activation of Plasmacytoid Dendritic Cells in Patients With Systemic Sclerosis

Objective. Plasmacytoid dendritic cells (PDCs) are a critical source of type I interferons (IFNs) that can contribute to the onset and maintenance of autoimmunity. Molecular mechanisms leading to PDC dysregulation and a persistent type I IFN signature are largely unexplored, especially in patients with systemic sclerosis (SSc), a disease in which PDCs infiltrate fibrotic skin lesions and produce higher levels of IFN alpha than those in healthy controls. This study was undertaken to investigate potential microRNA (miRNA)-mediated epigenetic mechanisms underlying PDC dysregulation and type I IFN production in SSc.Methods. We performed miRNA expression profiling and validation in highly purified PDCs obtained from the peripheral blood of 3 independent cohorts of healthy controls and SSc patients. Possible functions of miRNA-618 (miR-618) on PDC biology were identified by overexpression in healthy PDCs.Results. Expression of miR-618 was up-regulated in PDCs from SSc patients, including those with early disease who did not present with skin fibrosis. IFN regulatory factor 8, a crucial transcription factor for PDC development and activation, was identified as a target of miR-618. Overexpression of miR-618 reduced the development of PDCs from CD34+ cells in vitro and enhanced their ability to secrete IFN alpha, mimicking the PDC phenotype observed in SSc patients.Conclusion. Up-regulation of miR-618 suppresses the development of PDCs and increases their ability to secrete IFN alpha, potentially contributing to the type I IFN signature observed in SSc patients. Considering the importance of PDCs in the pathogenesis of SSc and other diseases characterized by a type I IFN signature, miR-618 potentially represents an important epigenetic target to regulate immune system homeostasis in these conditions

PD-L1 microSPECT/CT Imaging for Longitudinal Monitoring of PD-L1 Expression in Syngeneic and Humanized Mouse Models for Cancer

Antibodies that block the interaction between programmed death ligand 1 (PD-L1) and PD-1 have shown impressive responses in subgroups of patients with cancer. PD-L1 expression in tumors seems to be a prerequisite for treatment response. However, PD-L1 is heterogeneously expressed within tumor lesions and may change upon disease progression and treatment. Imaging of PD-L1 could aid in patient selection. Previously, we showed the feasibility to image PD-L1(+) tumors in immunodeficient mice. However, PD-L1 is also expressed on immune cell subsets. Therefore, the aim of this study was to assess the potential of PD-L1 micro single-photon emission tomography/computed tomography (microSPECT/CT) using radiolabeled PD-L1 antibodies to (i) measure PD-L1 expression in two immunocompetent tumor models (syngeneic mice and humanized mice harboring PD-L1 expressing immune cells) and (ii) monitor therapy-induced changes in tumor PD-L1 expression. We showed that radiolabeled PD-L1 antibodies accumulated preferentially in PD-L1(+) tumors, despite considerable uptake in certain normal lymphoid tissues (spleen and lymph nodes) and nonlymphoid tissues (duodenum and brown fat). PD-L1 microSPECT/CT imaging could also distinguish between high and low PD-L1-expressing tumors. The presence of PD-L1(+) immune cells did not compromise tumor uptake of the human PD-L1 antibodies in humanized mice, and we demonstrated that radiotherapy-induced upregulation of PD-L1 expression in murine tumors could be monitored with microSPECT/CT imaging. Together, these data demonstrate that PD-L1 microSPECT/CT is a sensitive technique to detect variations in tumor PD-L1 expression, and in the future, this technique may enable patient selection for PD-1/PD-L1-targeted therapy

MCLA-117, a CLEC12AxCD3 bispecific antibody targeting a leukaemic stem cell antigen, induces T cell-mediated AML blast lysis

Objective: We report the characterization of MCLA-117, a novel T cell-redirecting antibody for acute myeloid leukaemia (AML) treatment targeting CD3 on T cells and CLEC12A on leukaemic cells. In AML, CLEC12A is expressed on blasts and leukaemic stem cells. Methods: The functional capacity of MCLA-117 to redirect resting T cells to eradicate CLEC12APOS tumor cells was studied using human samples, including primary AML samples. Results: Within the normal hematopoietic compartment, MCLA-117 binds to cells expressing CD3 and CLEC12A but not to early myeloid progenitors or hematopoietic stem cells. MCLA-117 induces T cell activation (EC50 = 44 ng/mL), T cell proliferation, mild pro-inflammatory cytokine release, and redirects T cells to lyse CLEC12APOS target cells (EC50 = 68 ng/mL). MCLA-117-induced targeting of normal CD34POS cells co-cultured with T cells spares erythrocyte and megakaryocyte differentiation as well as preserves mono-myelocytic lineage development. In primary AML patient samples with autologous T cells, MCLA-117 robustly induced AML blast killing (23–98%) at low effector-to-target ratios (1:3–1:97). Conclusion: These findings demonstrate that MCLA-117 efficiently redirects T cells to kill tumour cells while sparing the potential of the bone marrow to develop the full hematological compartment and support further clinical evaluation as a potentially potent treatment option for AML

Natural Polymorphisms in Tap2 Influence Negative Selection and CD4:CD8 Lineage Commitment in the Rat

Genetic variation in the major histocompatibility complex (MHC) affects CD4∶CD8 lineage commitment and MHC expression. However, the contribution of specific genes in this gene-dense region has not yet been resolved. Nor has it been established whether the same genes regulate MHC expression and T cell selection. Here, we assessed the impact of natural genetic variation on MHC expression and CD4∶CD8 lineage commitment using two genetic models in the rat. First, we mapped Quantitative Trait Loci (QTLs) associated with variation in MHC class I and II protein expression and the CD4∶CD8 T cell ratio in outbred Heterogeneous Stock rats. We identified 10 QTLs across the genome and found that QTLs for the individual traits colocalized within a region spanning the MHC. To identify the genes underlying these overlapping QTLs, we generated a large panel of MHC-recombinant congenic strains, and refined the QTLs to two adjacent intervals of ∼0.25 Mb in the MHC-I and II regions, respectively. An interaction between these intervals affected MHC class I expression as well as negative selection and lineage commitment of CD8 single-positive (SP) thymocytes. We mapped this effect to the transporter associated with antigen processing 2 (Tap2) in the MHC-II region and the classical MHC class I gene(s) (RT1-A) in the MHC-I region. This interaction was revealed by a recombination between RT1-A and Tap2, which occurred in 0.2% of the rats. Variants of Tap2 have previously been shown to influence the antigenicity of MHC class I molecules by altering the MHC class I ligandome. Our results show that a restricted peptide repertoire on MHC class I molecules leads to reduced negative selection of CD8SP cells. To our knowledge, this is the first study showing how a recombination between natural alleles of genes in the MHC influences lineage commitment of T cells

Regulation of class I expression by classical and inverse cim.

<p>(A) CD68+ cells were stained on the cell surface with OX18 (anti-class Ia and Ib). Histograms show representative samples from DA, DA.1H and DA.1H derived strains with different alleles of <i>RT1-A</i> and <i>Tap2</i> as stated on top. Data from all individuals are shown in scatterplot (far right); * significant compared to DA.1H (1H); ** significant compared to DA. (B) CD68+ cells stained with a class Ia specific antibody (F16-4-4). (C) T cells from animals shown in (A) stained intracellularly with OX18 (scatterplot) and F16-4-4 (histogram). (D–E) Subsets of leukocytes from DA and DA.1IR85 spleen stained extracellularly (D) and intracellularly (E) with OX18. Data are representative of 6 individuals per group. (F) Surface expression of MHC class I (OX18) on CD68+ cells from DA and DA.1U congenic strains. (G) CD68+ cells (same as in F) stained with F16-4-4. Vertical lines in scatterplots show mean values. Representative results of at least two independent experiments are shown.</p

Class-I modification reduces negative selection of CD8 cells.

<p>(A) The QTLs in <i>Tcs1</i> and <i>Tcs2</i> did not affect the total number of double negative cells (DN; CD4−, CD8b−). B cells (CD45RA+) were excluded from the DN gate. (B) DN cell maturation is defined by CD45RC and CD2 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004151#pgen.1004151.s010" target="_blank">Fig. S10</a>). DN cells in DA.1FR9 showed a lower frequency of early thymic precursors (ETP) compared to DA. Counter plot shows gating strategy with numbers indicating percent (%) of parent population (stated above plot). (C) Counter plot shows CD45RC−, CD2^hi DN cells from DA.1UR83, and scatter plots the frequency of TCRβ− and TCRβ+ (DN4 in mouse) cells in DA and DA.1UR83. (D) Thymi from <i>Tcs2</i>-congenic strains with TAP-B (HR10 and UR10) contained fewer double positive (DP) cells but more cells (in %) with high TCR expression. (E) TAP-B strains (HR10 and UR10) showed higher frequencies (histograms) and total numbers (scatter plots) of CD8 single positive (SP) cells with high TCR expression. Numbers (%) in histograms represent mean-values ±SD of cells with high TCR expression (gated, <i>n</i> = 5). (F) Frequencies (histograms; <i>n</i> = 5) and total numbers (scatter plot) of CD8SP cells with high TCR expression in DA.1H (RT1-A^h, TAP-B) and in strains with low levels of surface MHC class I (HR83 [RT1-A^h, TAP-A] and IR85 [RT1-Aⁿ, TAP-A]). (G) Virtually all CD4SP cells express high levels of TCR. Histogram shows expression of TCR on CD4SP thymocytes in DA (<i>n</i> = 5). Scatterplot shows total number of CD4SP cells per thymus in <i>Tcs2</i>-congenic strains.</p