Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

Aditi Upadhye; Melissa Marshall; James  C. Garmey; Timothy  P. Bender; Coleen McNamara

doi:10.3791/61003

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Method Article

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

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DOI:

10.3791/61003

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May 31st, 2020

Aditi Upadhye¹ , Melissa Marshall² , James C. Garmey² , Timothy P. Bender³ , Coleen McNamara²

¹Department of Microbiology, Immunology, Cancer Biology, University of Virginia, ²Cardiovascular Research Center, University of Virginia, ³Beirne B. Carter Center for Immunology Research, University of Virginia

Summary

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

Abstract

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Introduction

Recent studies have demonstrated considerable immune cell, and specifically B cell, phenotypic and functional heterogeneity depending on cell localization¹^,²^,³^,⁴^,⁵. B-1a cells are one such population with heterogeneous capacity to produce protective IgM antibodies; bone marrow B-1a cells secrete IgM constitutively and contribute significantly to plasma IgM titers⁶, while peritoneal B-1a cells have low-level IgM secretion at homeostasis and instead can be activated through innate toll....