Visualization Of Immune Responses Research Articles

In vivo visualization of immune responses during rejection of allogeneic tissue in the anterior eye chamber

Abstract The anterior chamber of the eye (ACE) was recently described as a novel imaging site in diabetes research by observing transplanted pancreatic islet activity in the eye of a living animal. The ACE technology is ideally suited for non-invasive and longitudinal in vivo imaging. We take advantage of the ACE window to observe immune responses, especially allorejection of islet cells by cytotoxic T lymphocytes. We transplant pancreatic islets from DBA/1J mice into C57BL/6 recipient knock-in mouse which endogenously express a fluorescent protein (GzmB-mTFP or Bonzo) in T cells. We follow the onset of the rejection after vascularization on islets, when immune cells start to infiltrate into ACE, until the end of the rejection process where the islets are cleaned up for roughly a month by repetitive two-photon microscopy. We find that T cells show less migration velocity on islets of foreign mice than on autologous tissue, indicating T cell activation and recognition on the islets. Interestingly, the temporal infiltration pattern of T cells during the rejection process is precisely regulated, showing enrichment of CD4+ T cells on the islets before arrival of CD8+ T cells in histological studies. We use the ACE platform as a more comprehensive environment to study immune response and focus on T cell killing. We aim to understand the mechanism and regulation of T cell-mediated killing in vivo and to further investigate the killing in gene-deficient mice (i.e. munc13-4 and perforin) that resemble to severe human immune disease.

Visualization of Immune Responses in the Cornea.

The eye has become a useful site for the investigation and understanding of local and systemic immune responses. The ease of access and transparency of the cornea permits direct visualization of ocular structures, blood vessels, and lymphatic vessels, allowing for the tracking of normal and pathological biological processes in real time. As a window to the immune system, we have used the eye to dissect the mechanisms of corneal inflammatory reactions that include innate and adaptive immune responses. We have identified that the ocular microenvironment regulates these immune responses by recruiting different populations of inflammatory cells to the cornea through local production of selected chemokines. Moreover, crosstalk between T cells and macrophages is a common and crucial step in the development of ocular immune responses to corneal alloantigens. This review summarizes the data generated by our group using intravital fluorescent confocal microscopy to capture the tempo, magnitude, and function of innate and adaptive corneal immune responses.

Abstract B25: Visualization of immune response to Hepatitis B vaccination by in vivo mouse imaging

Abstract By taking advantage of bioluminescence imaging to monitor luciferase expressing splenocytes from the luciferase transgenic mouse, we visulaized the enhancement of immune response against hepatitis B virus (HBV) vaccine with adjuvants. To visualize antigen in vivo, large hepatitis B virus antigen (L-HBsAg) was labeled with radioiodine (125I). B6 mice were intramuscularly vaccinated. The localization of 125I-L-HBsAg was monitored for 5 weeks using animal SPECT/CT. To monitor the immune response, the luciferase expressing splenocytes were injected intravenously into immunized B6 mice. Bioluminescence signals from splenocytes were measured by IVIS 100 system. Localization of inoculated L-HBsAg was successfully monitored using animal SPECT/CT. L-HBsAg was lasted for 5 weeks and diminished. In addition, the injected splenocytes were successfully visualized in immunized mice, homing to spleen within 30 minutes and were accumulated in lymph nodes within 5 hours. Accumulation of splenocytes at vaccination site was observed within 24 hours. Moreover, after 10 hours, mouse vaccinated with antigen and two adjuvants showed 4 times more accumulation of splenocytes at vaccination site compared to mouse vaccinated with antigen only. Six days later, mouse vaccinated with antigen and two adjuvants showed 1.7-4.73 fold increased luciferase intensity of splenocytes at spleen, lymph nodes and vaccination site compared to mouse vaccinated with antigen only. In conclusion, in vivo real-time bioluminescent monitoring of splenocytes homing and proliferation against vaccination successfully provides efficiency of adjuvants. Our imaging system can be used for evaluation of efficacy of vaccination by enhanced the proliferation and activation of splenocytes near the vaccination site. Note: This abstract was not presented at the conference. Citation Format: Ha Kim, Keejong Hong, Byung Cheol Ahn, Jung Sun Yum, Hyewon Youn. Visualization of immune response to Hepatitis B vaccination by in vivo mouse imaging. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B25.

Abstract A25: High-resolution visualization of immune response across the intact melanoma tumor

Abstract Tumor immunotherapy relies on improved anti-tumor activity of the immune cells. Efficacious immunotherapy often correlates with increased CD8+ T cell infiltration within the tumor, subsequently resulting into reduction in tumor growth and morphological remodeling. However, pan- melanoma visualization of immune cell infiltration and associated macro-scale alterations in tumor morphology and vasculature remains extremely challenging. This is largely owing to the dense pigmentation, thick extracellular matrix deposition and friable texture of the tumor, rendering it optically inaccessible using the routine histology and organ clarification techniques. Here, we describe a rapid and inexpensive optical clarification technique that enables examination of immune infiltration as well as vascular changes, both at tissue level as well as single cell-resolution within an intact tumor. The aforementioned technique, termed Tumor-optimized three-dimensional imaging of solvent cleared organs (T-DISCO), combines in vivo fluorescent labeling of the endogenous immune cells with organic solvents-based tissue clarification and eventual image acquisition using a standard confocal microscope. This technique when applied to the B16F10 melanoma model undergoing a multi-component immunotherapy, revealed changes within the tumor microenvironment, including vascular normalization and intra-tumoral trafficking of the T cells. Thus, T-DISCO represents a powerful tool to examine and investigate immune cell behavior deep within the intact tumor. Citation Format: Sudha Kumari, Kelly Moynihan, Darrell J. Irvine. High-resolution visualization of immune response across the intact melanoma tumor. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A25.

Understanding of Immune System by Visualization of Spatiotemporal Regulation of Immune Cells in the Entire Body

Immune system is high-dimensional integrated system distributed in the whole body. Many kinds of, total 10(11) of immune cells are regulated by receiving appropriate signals in appropriate places. We have been attempting to understand immune system by revealing spatiotemporal regulation of immune cells at the whole body level by "Visualization of immune response in vivo". Photoconvertible protein, "Kaede"-Tg mice allowed us to monitor cell-replacement and cell-movement in the whole body by marking cells with color of Kaede from green to red with exposure to violet light. It is applicable to small cell number populations in both lymphoid organs and also peripheral tissues under both normal and pathophysiological conditions. By using this system, we have demonstrated novel findings that "Naive CD4(+) T cell recirculation is an active process that they recirculate through lymphoid organs to seek limited niche for interacting with endogenous antigens and upregulate their function." and "Activated regulatory T cells emigrating from cutaneous immune response is responsible for termination of immune reponse." I will introduce these new tools of us and would like to discuss what is needed to understand immune system in the entire body.