Activation of Regulatory T Cells with IL-2 and TNF-α to Promote Homing to the Gut and to Improve Acute GvHD Prevention
Authors: Mancusi Antonella , Bonato Samanta , Del Sordo Rachele , Piccinelli Sara , Hoxha Eni , Zei Tiziana , Iacucci Ostini Roberta , Falzetti Franca , Carotti Alessandra , Ruggeri Loredana , Giansanti Michele , Negrin Robert S. , Martelli Massimo Fabrizio , Velardi Andrea , Pierini Antonio 
Published: November 2019

INTRODUCTION
Adoptive immunotherapy with donor conventional T cells (Tcons) and CD4+CD25+FOXP3+regulatory T cells (Tregs) is used to enhance immune reconstitution in clinical trials of haploidentical transplantation. Donor Tregs are infused to prevent adverse alloreactions mediated by Tcons against normal tissues, which cause GvHD, in the absence of any post transplant immune suppression. This strategy proved to be effective in preventing leukemia relapse and chronic GvHD, allowing for ~70% event-free survival in patients with acute leukemia (Pierini et al. submitted). However, acute GvHD occurs in ~30% of the patients. We are investigating if ex-vivo stimulation of Tregs with a combination of low-dose IL-2 and TNF-α before adoptive transfer could improve acute GvHD prevention. Tregs express higher levels of the TNF receptor 2 (TNFR2) compared with Tcons and TNFR2 expression defines a Treg subpopulation with maximal suppressive capacity. Recent studies showed that activation of TNFR2 signaling in mouse Tregs reduced GvHD lethality in mouse models.
METHODS
Human CD4+or CD4+CD25+T cells were incubated for 60 hours with IL-2 (10 UI/ml) with or without TNF-α (20 ng/ml). Treg proliferation in the presence of allogeneic irradiated peripheral blood mononuclear cells (PBMC), and Treg-mediated suppression of Tcon proliferation were evaluated by CFSE assay.
Colon biopsies from patients with suspected GvHD were stained with anti-CD3 and anti-FOXP3 antibodies.
RESULTS
We developed a translatable in vitro system for improving human Treg function by adding a short term stimulation with low-dose IL-2 and TNF-α (IL-2/TNF) in the selection process. CD4+T cells were primed with IL-2/TNF before CD25 positive selection. When total CD4+T cells were stimulated with IL-2/TNF, CD25 expression was selectively upregulated on CD4+FOXP3+Tregs, but not on CD4+FOXP3- T cells. IL-2/TNF-primed Tregs maintained TNFR2 expression and expressed higher levels of FOXP3 and CD25 compared with Tregs stimulated with IL-2 alone (p<.05). They also had enhanced proliferation in response to allogeneic PBMC (p<.05) and suppressed Tcon proliferation more efficiently (p<.01). Whole transcript expression analyses showed IL-2/TNF priming increased the expression of genes associated with Treg activation, proliferation and function compared to IL-2 alone, such us OX-40, 4-1BB, CD69, CCR8, CD39 genes and components of the NF-κB signaling pathway.
We analyzed GvHD clinical signs of the last 20 patients who were diagnosed with grade II-IV acute GvHD after Treg/Tcon-based haploidentical transplantation at our institution. Nineteen presented with early-onset gut GvHD and 40% of those patients did not have any skin involvement. We hypothesized that preferential targeting to the gut was the consequence of defective homing of Tregs to the gut. Compared with Tcons, Tregs express higher levels of skin homing receptors (such as CCR4 and CLA-4), but lower levels of the gut homing receptor α4β7-integrin. We analyzed the infiltration of Tregs and Tcons in 31 colon biopsies from patients with suspected GvHD. The number of Tregs was higher in patients who were not diagnosed with acute GvHD compared with those who were (158.3/mm2 vs 86.3/mm2, p<.05). A similar trend was observed for the Treg:Tcon ratio (0.27 vs 0.15, p<.05). These results suggest that homing of Tregs to the gut plays an important role in the effectiveness of gut GvHD prevention. We also found that IL-2/TNF priming selectively upregulated α4β7-integrin expression on Tregs compared with freshly isolated cells (p<.01). Experiments in humanized mouse models are underway to ascertain if IL-2/TNF priming improves Treg homing to the gut and site-specific GvHD prevention.
CONCLUSION
The present study shows that activation of TNFR2 signaling in human Tregs enhances their proliferation and suppressive function. It also suggests that Treg suppressive function exerted in periphery could be critical for tissue-specific GvHD control. Moreover, IL-2/TNF-priming could promote homing of Tregs to the gut and their capability to inhibit gut GvHD. Thus, IL-2/TNF-priming may be critical to optimize the ongoing clinical trial of adoptive immunotherapy with Tregs for GvHD prevention. Future studies will ascertain if IL-2/TNF-priming of Tregs can also allow for the infusion of higher doses of Tcons to further enhance immune reconstitution and leukemia killing without increasing the risk of GvHD.

Disclosures
No relevant conflicts of interest to declare.