Alloresponses of Human T-Cells from Adult Peripheral Blood and Umbilical Cord Blood Are Differentially Impacted By Lenalidomide
Authors: Besley Caroline , Kotsiou Eleni , Petty Robert , Ajanthah Sangaralingam , Chelala Claude , Ghazaly Essam , Le Dieu Rifca , Davies Jeff , Gribben John 
Published: October 2019

Abstract
Immunomodulatory drugs (IMiDs), such as lenalidomide provide a tool to enhance both direct anti-tumor and graft-versus-tumor effects after allogeneic haematopoietic stem-cell transplantation (AHCT). However, early clinical experience with IMiDs after AHCT using adult peripheral blood (APB) as a stem cell source has been limited by induction of graft-versus-host disease. Characterization of the mechanisms by which IMIDs can modulate alloresponses of T-cells and identification of differential effects on T-cells from disparate cell sources could facilitate more effective use of these drugs in the setting of AHCT. We therefore used in vitro modelling, multi-parameter flow cytometry and gene expression analysis to delineate the impact of the widely used IMiD lenalidomide on quantitative and qualitative alloresponses mediated by T-cells derived from APB and umbilical cord blood (UCB).
We co-cultured carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled peripheral blood mononuclear cells (PBMC) from healthy adult donors or donated UCB units with irradiated allogeneic PBMC in the presence of 1μM lenalidomide or vehicle control. In this model, cellular and supernatant concentrations of lenalidomide (determined by mass spectrometry) were similar to in vivo levels achieved at doses used as maintenance after AHSCT in published studies. Functional alloresponses were quantified after 7-9 days of allogeneic co-culture by flow cytometry. In addition, co-culture responders were flow-sorted into alloproliferative or non-proliferative fractions and extracted RNA used for gene expression profiling.
We demonstrate that lenalidomide potentiates net alloproliferation of APB derived T-cells cells by selectively enhancing allospecific proliferation of CD8+ T-cells (median 58% (lenalidomide-treated) versus 43% (untreated), n=40, p<0.001). Although pre-treatment of allogeneic stimulators and responders had a modest potentiating effect, lenalidomide was required during allogeneic co-culture for maximal potentiation of CD8+ alloresponses of APB T-cells. CD8+ T-cells had enhanced effector memory differentiation, were enriched for polyfunctional effectors (capable of producing two or more pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-2) and/or expressing the lysosomal membrane-associated protein CD107a), and had a distinct gene expression profile with altered expression of key immunoregulatory genes and depletion of cellular ikaros levels.
In common with our findings in APB T-cells, lenalidomide also potentiated proliferation of alloreactive CD8+ T-cells from UCB (median 43% (lenalidomide-treated) vs 24% (untreated), n=17, p=0.0005) with similar polyfunctional effector memory differentiation, immunoregulatory gene expression changes and cellular ikaros depletion. Importantly, lenalidomide reduced allospecific proliferation of UCB CD4+ T-cells (median 58% (untreated) versus 41% (lenalidomide-treated), n=17, p<0.01) whereas alloproliferation of APB CD4+ T-cells was unaffected. The reduction in UCB CD4+ alloproliferation was accompanied by selectively expansion of CD4+CD25+FOXP3+ regulatory T-cells (median 7.6% (lenalidomide-treated) vs 5.2% (untreated), n=12, p 0.02), resulting in an overall reduction in net UCB T-cell alloproliferation after lenalidomide treatment.
Our findings show that lenalidomide has a qualitatively different impact on alloresponses of T-cells from different cell sources; alloresponses of APB T-cells are increased by lenalidomide via selective expansion of polyfunctional CD8+ effectors while lenalidomide limits alloresponses of UCB T-cells by reducing CD4+ effector expansion and increasing tolerogenic Treg. These findings have important implications for the future use of IMiDs in the setting of AHCT.

Disclosures
No relevant conflicts of interest to declare.