Related Experiment Video
Updated: Aug 8, 2026
Generation of Human Alloantigen-specific T Cells from Peripheral Blood
Published on: November 21, 2014
Differentiation of human alloreactive CD8(+) T cells in vitro
Rob J Rentenaar1, Jelle L G Vosters, Frank N J van Diepen
1Renal Transplant Unit, Department of Internal Medicine, Academic Medical Center, Amsterdam, The Netherlands. rrentenaar@yahoo.com
Insights
This study tracked CD8(+) T cell proliferation and differentiation in mixed lymphocyte cultures. While in vitro methods visualized alloantigen-specific CD8(+) T cells, they failed to detect activated cells in vivo after kidney transplantation.
Area of Science:
- Immunology
- Cellular immunology
- Transplantation immunology
Background:
- CD8(+) T cells play a crucial role in immune responses, including allograft rejection.
- Understanding the expansion and differentiation of alloantigen-reactive CD8(+) T cells is vital for managing transplant outcomes.
Purpose of the Study:
- To investigate the proliferation and differentiation kinetics of alloantigen-reactive CD8(+) T cells in vitro.
- To assess the utility of in vitro methods for detecting in vivo activated alloreactive CD8(+) T cells.
Main Methods:
- Mixed lymphocyte cultures (MLC) were used to stimulate T cells.
- Carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution was measured to assess T cell proliferation.
- Flow cytometry was employed to analyze T cell differentiation markers (CD45RA, CD27) and intracellular cytokine production (interferon-gamma).
Main Results:
- CD8(+) T cell proliferation was detectable by day 4 in MLC, with over 60% dividing by day 6.
- Differentiation was evidenced by decreased CD45RA and CD27 expression and acquisition of interferon-gamma production.
- In vitro methods successfully visualized expanded alloantigen-reactive CD8(+) T cells but failed to detect activated cells in vivo in kidney transplant recipients.
Conclusions:
- Short-term in vitro stimulation and cytokine measurement can visualize alloantigen-specific CD8(+) T cells.
- Current in vitro techniques may not be sensitive enough to detect circulating alloreactive CD8(+) T cells activated in vivo following allogeneic kidney transplantation.
Abstract:
Expansion and differentiation of alloantigen-reactive CD8(+) T cells in mixed lymphocyte cultures was followed by measurement of the loss of carboxyfluorescein diacetate succinimidyl ester (CFSE) fluorescence of responder cells. Proliferation of CD8(+) T cells became detectable on day 4 of culture and, 2 days later, > 60% of the CD8(+) T cells in culture were dividing alloreactive lymphocytes. In parallel with expansion, CD8(+) T-cell differentiation was initiated, as evidenced by an increase in the number of CD45RA(-) and CD27(-) T cells and acquisition of the ability to produce interferon-gamma after restimulation with the specific alloantigen. Finally, although short-term stimulation and measurement of intracellular cytokine production allowed visualization of alloreactive CD8(+) T cells expanded in vitro, this procedure did not detect circulating alloreactive CD8(+) T cells activated in vivo in recipients of allogeneic kidney grafts.
