In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Séverine Bézie; Claire Usal; Carole Guillonneau

doi:10.3791/55510

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Method Article

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

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DOI:

10.3791/55510

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August 12th, 2017

Séverine Bézie¹^,² , Claire Usal¹^,² , Carole Guillonneau¹^,²

¹Centre de Recherche en Transplantation et Immunologie UMR 1064, INSERM, Université de Nantes, ²Institut de Transplantation Urologie Néphrologie (ITUN), CHU Nantes

Summary

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

Abstract

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

Introduction

Cardiac allograft in rat is a reliable organ transplant model to assess tolerance induction treatments, to decipher the mechanisms of tolerance induction and maintenance, and has the potential to induce functionally competent and dominant regulatory cells. The protocols below describe a fully mismatch heterotopic cardiac graft from a Lewis 1W donor rat (LEW.1W, RT1^u) into a Lewis 1A recipient rat (LEW.1A, RT1^a). In this graft combination, acute rejection occurs rapidly (in about 7 days) and can be easily assessed by graft beating measurement through palpation of the abdomen. Here we propose three protocols to induce tolerance to the cardiac allog....