Alloanergization Method for Inducing Allospecific Hyporesponsiveness in PBMC Exposed to Allostimulation In Vitro in the Context of Costimulatory Molecule Blockade

  • Christine M. Barbon
  • Kenneth J. Janec
  • Rowan H. Kelner
  • James E. Norton
  • Eva C. GuinanEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1899)


Alloantigen-specific hyporesponsiveness can be induced in alloreactive T cells contained within the whole peripheral blood mononuclear cell (PBMC) population by stimulating these responder cells ex vivo with HLA-mismatched stimulator PBMC as the antigen presenting cell (APC) source, in the presence of a CD28 costimulation blocking agent. As a result of this approach, specific alloreactivity is markedly decreased (by 1–2 logs), but third-party alloresponses and in vitro responses relying on the activation of pathogen- and tumor-associated antigen T-cell functional activities are not globally impinged upon (Guinan et al. N Engl J Med 340(22):1704–1714, 1999, Davies et al. Transplantation 86(6):854–864, 2008, Davies et al. Cell Transplant 21(9):2047–61, 2012). This method has been used clinically to alloanergize bone marrow and PBMC allografts, creating ex vivo cell therapies for adoptive transfer to blood cancer patients at high risk of disease relapse whose best option was to receive haploidentical hematopoietic cell transplants. These early phase trials consisting of, or containing, alloanergized T-cell infusions show promise in reducing graft-versus-host disease (GvHD), providing more rapid immune reconstitution, and decreasing severe post-transplant infectious complications and disease relapse. Herein, we describe this straightforward technique for generating alloanergized PBMC as it is performed in the research lab setting using belatacept for CD28-mediated costimulatory blockade (CSB) and PBMC isolated by Ficoll Hypaque gradient centrifugation as responders and APC. We also describe methods for evaluating subsequent alloproliferation to first and third party stimulation as well as assessment of cell division, pathogen-specific immunity, or allosuppression. The technique has successfully been transferred to collaborating labs, largely owing to the flexibility of using fresh or frozen PBMC, the lack of a requirement for specially isolated APC populations, and the ability to scale up or scale down the cell numbers that are to be anergized.

Key words

Anergy Alloanergy Regulatory T cell (Treg) Alloantigen-specific response alloreactivity T-cell costimulation Mixed lymphocyte reaction (MLR) Immune tolerance Costimulatory molecule blockade 


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Christine M. Barbon
    • 1
  • Kenneth J. Janec
    • 2
  • Rowan H. Kelner
    • 2
  • James E. Norton
    • 2
  • Eva C. Guinan
    • 2
    • 3
    Email author
  1. 1.IO Bioscience, Oncology IMED Biotech UnitAstrazeneca, Gatehouse ParkWalthamUSA
  2. 2.Department of Radiation OncologyDana-Farber Cancer InstituteBostonUSA
  3. 3.Department of Radiation OncologyHarvard Medical SchoolBostonUSA

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