TY - JOUR
T1 - Assessment of lymphocyte migration in an ex vivo transmigration system
AU - Warren, Kristi J.
AU - Wyatt, Todd A.
N1 - Funding Information:
This work was funded by the American Lung Association (K.J.W.), the Memorial Eugene Kenney fund awarded to T.A.W. and K.J.W., generous start-up support from the University of Utah for K.J.W., and a Department of Veterans Affairs award to T.A.W. (VA I01BX0003635). T.A.W. is the recipient of a Research Career Scientist Award (IK6 BX003781) from the Department of Veterans Affairs. The authors wish to acknowledge editorial assistance from Ms. Lisa Chudomelka. The authors thank the UNMC Flow Cytometry core for their support in collecting the flow cytometry data generated for this manuscript.
Publisher Copyright:
© 2019 Journal of Visualized Experiments.
PY - 2019
Y1 - 2019
N2 - Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17-and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 µm pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.
AB - Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17-and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 µm pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.
KW - CCL17 (C-C Motif Chemokine 17)/TARC (thymus and activation related chemokine)
KW - CCL22 (C-C Motif Chemokine 22)/MDC (Macrophage-derived chemokine)
KW - CCR4 (CC Chemokine Receptor 4)
KW - CD4 T cell (CD4+ T helper cell)/Th2 cell
KW - ILC2: (Group 2 Innate Lymphoid Cell)
KW - Immunology and Infection
KW - Issue 151
KW - OVA (Chicken Egg Ovalbumin)
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U2 - 10.3791/60060
DO - 10.3791/60060
M3 - Article
C2 - 31589207
AN - SCOPUS:85072926997
SN - 1940-087X
VL - 2019
JO - Journal of Visualized Experiments
JF - Journal of Visualized Experiments
IS - 151
M1 - e60060
ER -