Harmful effects of the label around the viability, DNA-damage and apoptosis-necrosis induction, could be minimized while yielding a high contrast in in vivo PET images

Harmful effects of the label around the viability, DNA-damage and apoptosis-necrosis induction, could be minimized while yielding a high contrast in in vivo PET images. transferable to other immune cell populations. Keywords: PET imaging, mouse T cells, in vivo cell tracking, antibody-based cell labeling, airway DTHR Abstract T cells are key players in inflammation, autoimmune diseases, and immunotherapy. Thus, holistic and noninvasive in vivo characterizations of the temporal distribution and homing dynamics of lymphocytes in mammals are of special interest. Herein, we show that PET-based T-cell labeling facilitates quantitative, highly sensitive, and holistic monitoring of T-cell homing patterns in vivo. We developed a new T-cell receptor (TCR)-specific labeling approach for the intracellular labeling of mouse T cells. We found that continuous TCR plasma membrane turnover and the endocytosis of the specific 64Cu-monoclonal antibody (mAb)CTCR complex enables a stable labeling of T cells. The TCRCmAb complex was internalized within 24 Rabbit Polyclonal to ZFHX3 h, whereas antigen recognition was not impaired. Harmful effects of the label around the viability, DNA-damage and apoptosis-necrosis induction, could be minimized while yielding a high contrast in in vivo PET images. We were able to follow and quantify the specific homing of systemically applied 64Cu-labeled chicken ovalbumin (cOVA)-TCR transgenic T cells into the pulmonary and perithymic lymph nodes (LNs) of mice with cOVA-induced airway delayed-type hypersensitivity reaction (DTHR) but not into pulmonary and perithymic LNs of na?ve control mice or mice diseased from turkey or pheasant OVA-induced DTHR. Our protocol provides consequent advancements in the detection of small accumulations of immune cells in single LNs and specific homing to the sites of inflammation by PET using the internalization of TCR-specific mAbs as a specific label of Pectolinarigenin T cells. Thus, our labeling approach is applicable to other cells with constant membrane receptor turnover. Noninvasive in vivo imaging is an emerging method that enables the examination of T-cell migration kinetics, homing patterns, and the sites of T-cell proliferation and activation. These data are needed for a better understanding of the T-cell response during autoimmune diseases, allergies, infections, and cancer (1). Furthermore, this method reveals the basic mechanisms required for the understanding of T-cell-based immunotherapies and the development and optimization of personalized therapies Pectolinarigenin (2). CD4+ IFN-Cproducing T-helper cells (TH1) and IL-4Cproducing T-helper cells (TH2) are key players in organ-specific autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, bronchial asthma, and insulin-dependent diabetes mellitus (3). In addition, tumor-associated antigen-specific TH1 cells can inhibit tumor growth (2, 4C6). However, the exact mode of action, as well as the sites of in vivo homing, proliferation, and trafficking kinetics remain unknown for most immune cells; therefore, noninvasive imaging tools are required to visualize their location and trafficking patterns to enable organ-specific temporal quantification. For in vivo investigation of physiological lymphocyte trafficking, it is strictly required to establish a labeling method with little or no impairment to lymphocyte viability and functionality. One frequently used cell-labeling compound for PET is usually [64Cu]Pyruvaldehyde bis(N4-methylthiosemicarbazone) ([64Cu]PTSM), which has been applied to mouse lymphocytes, primate stem cells, and human dendritic cells (7C9). We recently established a [64Cu]PTSM-labeling protocol that minimizes the harmful effects on T-cell functions (10). Nevertheless, intracellular [64Cu]PTSM labeling continues to impair viability Pectolinarigenin and functionality as well as to induce apoptosis/necrosis and DNA damage in TH1 cells. The use of T-cell receptor (TCR)-specific radiolabeled monoclonal antibodies (mAbs) as a label might minimize the harmful effects of 64Cu on lymphocytes compared with [64Cu]PTSM labeling. Our study aimed to develop a new, highly specific intracellular labeling strategy for T cells that enables whole-body, noninvasive in vivo T-cell tracking. We targeted the antigen-specific TCR using radiolabeled mAbs. We labeled chicken-ovalbumin-TCR-transgenic TH1 cells (cOVA-TCRtg-TH1) with 64Cu-DOTACmodified cOVA-TCRCspecific mAbs in vitro and investigated the endocytosis-dependent intracellular accumulation of the mAbCTCR complex. We performed temporal and quantitative PET imaging of systemically transferred and labeled cOVA-TCRtg-TH1 cells in a mouse model of OVA-induced acute airway delayed-type hypersensitivity reaction Pectolinarigenin (DTHR) using chicken, turkey (t), and pheasant (ph) OVA to detect cOVA-specific T-cell homing. This.