Harnessing Favorable Dendritic Cells Features to Improve Antigen-Specific T Cell Expansion

Introduction: Allogeneic hematopoietic stem cell transplantation (aHSCT) relies mostly on its graft-versus-leukemia (GvL) activity, which is mediated by donor T cells, to eliminate cancer cells escaping the conditioning regimen. The GvL effect represents a superb illustration of the capacity of the immune system to recognize tumor antigens and to eliminate the cancer cells harboring these antigens. Dendritic cells (DCs) are the actors with the most important capacity to present tumor antigens to T cells so that they can exert their cytotoxicity toward specific cancer targets. They play a key role in vivo in patients, and can be harnessed ex vivo for the generation of adoptive T cell therapies as well as tumor vaccine strategies. Nevertheless, the long period of time required for DC generation from monocytes represents a most important factor limiting their clinical usage. While DCs are usually produced over 9 days (DC9), they can also be generated over shorter periods of time and within as few as 3 days (DC3). However, DC3 present different biologic features from DC9 and usually demonstrate limited maturation and priming activity. Goal: To enhance the clinical potential of DCs, we investigated a TLR-based strategy aiming to increase the ability of DC3 to mature and generate antigen-specific T cells. Methods: Dendritic cells were generated from monocytes, isolated from peripheral blood, and matured using the gold standard maturation cocktail (GM-CSF, IL-4, IL-6, IL-1β, TNFα and PGE2). We determined the effect of TLR-3 and TLR-7/8 agonists on DC3 maturation status, cytokine production, and T cell reactivity (DC3-TLR). DCs were pulsed with Wilm's Tumor 1 (WT1) antigen, a well-known tumor-associated antigen, and added to peripheral blood mononuclear cell (PBMC) cultures at 7-day intervals for a total of 21 days. Anti-WT1 T cell reactivity was measured through IFNγ, TNFα, IL-2, and CD107a expression upon WT1 peptide restimulation, after co-culture with WT1-pulsed DC9, DC3 or DC3-TLR. Results: Both DC3 and DC3-TLR were of smaller size than DC9 (p<0.001), and harbored reduced expression of CD80 (p<0.0001). DC3 and DC3-TLR also demonstrated superior antigen uptake and processing capacity compared to DC9 (p=0.004). In addition, we found that DC3-TLR showed increased expression of the co-stimulatory molecule CD80 (p=0.007), and enhanced production of pro-inflammatory cytokines (IL-1β, IL-6, IL-12, and TNFα) in comparison to untreated DC3 (all p<0.005). We then sought to investigate the antigen-specific priming activity of DC9, DC3 and DC3-TLR. Interestingly, addition of TLR agonists to DC3 significantly increased CD8+ T cell expansion. Those DC3-TLR-primed CD8+ T cells harbored an effector memory phenotype, with significantly lower expression of the exhaustion marker LAG3 compared to DC3-primed T cells (p=0.008). Importantly, upon restimulation with WT1 peptide, the frequency and absolute number of DC3-TLR-primed WT1-specific T cells expressing functional markers were significantly increased compared to DC9-primed cultures (all p<0.05). Further, DC3-TLR-primed T cells displayed strong cytotoxic activity toward WT1-expressing autologous blast cells. To further define the DC features favoring WT1-specific T cells expansion, DC9, DC3 and DC3-TLR underwent RNA sequencing. These different cell types harbored distinct gene signatures. Notably, several chemokine transcripts, and most importantly CCL3 and CCL4 transcripts were upregulated in DC3-TLR compared to both DC3 and DC9. CCL3 and CCL4 chemokine production was also increased in DC3-TLR over DC3. Importantly, blocking the CCL3- and CCL4-receptor CCR5 reduced WT1-specific T cells in DC3-TLR-primed cultures. The addition of CCL3 and CCL4 to DC3-primed cultures did not recapitulate the WT1-specific T cell expansion observed in DC3-TLR, suggesting that chemokine gradients are important for antigen-specific T cell expansion. Conclusions: We have identified distinct phenotypic and molecular DC features leading to improved antigen-specific T cell expansion. This TLR-based strategy resulted in enhanced CD80 expression, IL-12 production, and other proinflammatory cytokines and chemokines. Thus, DC3-TLR offer interesting characteristics for adoptive T cell therapies and vaccination strategies, enabling greater clinical applicability and potentially superior anti-cancer activity.

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