AC133 is one of a new panel of murine hybridoma lines producing monoclonal IgG antibodies (mAbs) to a novel stem cell glycoprotein antigen with a molecular weight of 120 kD. AC133 antigen is selectively expressed on CD34bright hematopoietic stem and progenitor cells (progenitors) derived from human fetal liver and bone marrow, and blood. It is not detectable on other blood cells, cultured human umbilical vein endothelial cells (HUVECs), fibroblast cell lines, or the myeloid leukemia cell line KG1a by standard flow cytometric procedures. All of the noncommitted CD34+ cell population, as well as the majority of CD34+ cells committed to the granulocytic/monocytic pathway, are stained with AC133 antibody. In vitro clonogenicity assays have demonstrated that the CD34+AC133+ double-positive population from adult bone marrow contains the majority of the CFU-GM, a proportion of the CFU-Mix, and a minor population of BFU-E. The CD34dim and AC133− population has been shown to contain the remaining progenitor cells. AC133-selected cells engraft successfully in a fetal sheep transplantation model, and human cells harvested from chimeric fetal sheep bone marrow have been shown to successfully engraft secondary recipients, providing evidence for the long-term repopulating potential of AC133+ cells. A cDNA coding for AC133 antigen has been isolated, which codes for a polypeptide consisting of 865 amino acids (aa) with a predicted size of 97 kD. This antigen is modeled as a 5-transmembrane molecule, a structure that is novel among known cell surface structures. AC133 antibody provides an alternative to CD34 for the selection and characterization of cells necessary for both short- and long-term engraftment, in transplant situations, for studies of ex vivo expansion strategies, and for gene therapy.

Accumulating evidence suggests that clinically efficacious cancer immunotherapies are driven by T cell reactivity against DNA mutation-derived neoantigens. However, among the large number of predicted neoantigens, only a minority is recognized by autologous patient T cells, and strategies to broaden neoantigen-specific T cell responses are therefore attractive. We found that naïve T cell repertoires of healthy blood donors provide a source of neoantigen-specific T cells, responding to 11 of 57 predicted human leukocyte antigen (HLA)-A*02:01-binding epitopes from three patients. Many of the T cell reactivities involved epitopes that in vivo were neglected by patient autologous tumor-infiltrating lymphocytes. Finally, T cells redirected with T cell receptors identified from donor-derived T cells efficiently recognized patient-derived melanoma cells harboring the relevant mutations, providing a rationale for the use of such "outsourced" immune responses in cancer immunotherapy.

ABSTRACT

AC133 is one of a new panel of murine hybridoma lines producing monoclonal IgG antibodies (mAbs) to a novel stem cell glycoprotein antigen with a molecular weight of 120 kD. AC133 antigen is selectively expressed on CD34bright hematopoietic stem and progenitor cells (progenitors) derived from human fetal liver and bone marrow, and blood. It is not detectable on other blood cells, cultured human umbilical vein endothelial cells (HUVECs), fibroblast cell lines, or the myeloid leukemia cell line KG1a by standard flow cytometric procedures. All of the noncommitted CD34+ cell population, as well as the majority of CD34+ cells committed to the granulocytic/monocytic pathway, are stained with AC133 antibody. In vitro clonogenicity assays have demonstrated that the CD34+AC133+ double-positive population from adult bone marrow contains the majority of the CFU-GM, a proportion of the CFU-Mix, and a minor population of BFU-E. The CD34dim and AC133− population has been shown to contain the remaining progenitor cells. AC133-selected cells engraft successfully in a fetal sheep transplantation model, and human cells harvested from chimeric fetal sheep bone marrow have been shown to successfully engraft secondary recipients, providing evidence for the long-term repopulating potential of AC133+ cells. A cDNA coding for AC133 antigen has been isolated, which codes for a polypeptide consisting of 865 amino acids (aa) with a predicted size of 97 kD. This antigen is modeled as a 5-transmembrane molecule, a structure that is novel among known cell surface structures. AC133 antibody provides an alternative to CD34 for the selection and characterization of cells necessary for both short- and long-term engraftment, in transplant situations, for studies of ex vivo expansion strategies, and for gene therapy.

Epstein-Barr virus (EBV) has long been suggested as a pathogen in multiple sclerosis (MS).Here, we used high-throughput sequencing to determine the diversity, compartmentalization, persistence, and EBV-reactivity of the T-cell receptor (TCR) repertoires in MS. TCR-β genes were sequenced in paired samples of cerebrospinal fluid (CSF) and blood from patients with MS and controls with other inflammatory neurological diseases. The TCR repertoires were highly diverse in both compartments and patient groups. Expanded T-cell clones, represented by TCR-β sequences >0.1%, were of different identity in CSF and blood of MS patients, and persisted for more than a year. Reference TCR-β libraries generated from peripheral blood T cells reactive against autologous EBV-transformed B cells were highly enriched for public EBV-specific sequences and were used to quantify EBVreactive TCR-β sequences in CSF. TCR-β sequences of EBV-reactive CD8 + T cells, including several public EBV-specific sequences, were intrathecally enriched in MS patients only, whereas those of EBV-reactive CD4 + T cells were also enriched in CSF of controls. These data provide evidence for a clonally diverse, yet compartmentalized and persistent, intrathecal T-cell response in MS. The presented strategy links TCR sequence to intrathecal T-cell specificity, demonstrating enrichment of EBV-reactive CD8 + T cells in MS. Keywords:Cerebrospinal fluid r Epstein-Barr virus r High-throughput sequencing r MS r TCR Additional supporting information may be found in the online version of this article at the publisher's web-site Correspondence: Dr. Andreas Lossius e-mail: andreas.lossius@rr-research.no * These authors contributed equally to this work as senior authors. Eur. J. Immunol. 2014. 44: 3439-3452 Introduction T cells are thought to play an essential role in the pathogenesis of multiple sclerosis (MS) [1]. The T cells access the central nervous system (CNS) via the cerebrospinal fluid (CSF) or cross the blood-brain barrier through the perivascular spaces, which communicate with the CSF [2]. The CSF is also contiguous with the extracellular fluid of the brain and is believed to reflect inflammation within the CNS better than blood [3]. Although moderately increased in MS [4], the low numbers of T cells in CSF have complicated the characterization of these cells. Thus, the clonal composition of T cells in the CSF and their relationship to T cells in the blood is poorly understood. Cloning and sequencing of individual T-cell receptors (TCRs) [5], spectratyping [6,7], or flow cytometric staining for TCR variable (V) β-chain families [8] has previously permitted mapping of only a narrow part of the intrathecal TCR repertoire. These studies have yielded partly conflicting results with respect to clonal diversity and TCRV β-chain usage, and the overlap between the T-cell populations in CSF and blood has not been quantified. Epstein-Barr virus (EBV) has consistently been associated with MS in epidemiological studies [9], and MS patients display a perturbed immune respo...

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IntroductionHuman cytomegalovirus (HCMV) infection represents a major clinical problem in immunocompromised persons, including transplant recipients and AIDS patients. Prevalence of HCMV seropositivity ranges between 50% and 90% in healthy adults; after primary infection, the virus establishes lifelong latency in the host. 1 Usually, CMV in immunocompromised patients is caused by the reactivation of latent virus. A number of studies have suggested that the virus may aggravate immunodeficiency by interfering with antigen presentation. [2][3][4][5][6][7] In these publications, dendritic cells (DCs) have been generated from monocytes or CD34 ϩ bone marrow progenitor cells after 5 to 12 days of cytokine-supplemented culture. Such monocyte-derived DCs (moDCs) or bone marrowderived Langerhans cells are highly potent stimulators of T-cell activation. Exposure of these DCs to HCMV leads to functional paralysis of the cells, causing impaired T-cell activation. [2][3][4][5][6][7] Several mechanisms for this immunosuppression have been suggested. First, major histocompatibility complex (MHC) class I and class II and costimulatory molecules are down-regulated, resulting in impaired antigen presentation and increased susceptibility to natural killer cell-mediated lysis. [2][3][4][5][6][7][8] Second, a virally induced, soluble immunosuppressive factor released by mature moDCs has been postulated by several groups and was recently identified as CD83. 5,7 Third, infection of immature moDCs is lytic and leads to cell death. 7 Studies of HCMV effects on moDCs and Langerhans cells have shed light on mechanisms for viral immune evasion. However, the results showing paralysis of antigen presentation also raise questions as to how HCMV can be so effectively controlled in the immunocompetent host. Most primary infections induce minimal symptoms, and there is no clear evidence for clinically relevant immunosuppression. 1 Reactivation of the latent virus is believed to happen frequently, 9,10 but it proceeds asymptomatically in a healthy host, indicating a successful immune response. Indeed, an impressively large part of the T-cell repertoire is HCMV specific in seropositive persons. Labeling with MHC class I tetramers in complex with HCMV peptides has shown that 1% to 4% of all CD8 ϩ T cells are specific for HCMV proteins. 11,12 In view of the low pathogenicity of HCMV in immunocompetent persons, paralysis of key antigen-presenting cells by the virus seems unlikely.Elegant studies in mice have elucidated mechanisms for successful immunity to murine CMV (MCMV). 13 After injection of virus into mice, a small subset of DCs, termed plasmacytoid dendritic cells (PDCs), produce high levels of IFN-␣, IL-12, and TNF-␣. The rapid innate response is followed by maturation of several welldefined DC subsets in the mouse spleen and initiation of a strong MCMV-directed T-cell response. Frequencies of infected DCs were generally low and were restricted to a subset expressing CD8␣. These results show a high degree of specialization between murine DCs ...