Adoptive therapy with regulatory T (Treg) cells aiming at tipping the immune cell balance in favor of immune tolerance by dampening proinflammatory cells in a quantitative and qualitative fashion and treating autoimmune and alloimmune responses. In experimental systems, Treg have shown encouraging efficacies for the treatment of graft-versus-host and autoimmune diseases including type-1 diabetes.1,2 Those experimental successes, however, have not been translated clinically.
Overcoming the impediments affecting homeostasis and expansion when transferring Treg remains a major challenge. Quantitatively, transfer of antigen-specific Treg is particularly efficient when transferring fewer and specific cells, suppressing alloimmunity. From a qualitative perspective, Treg cell homeostasis requires both T cell receptor (TCR) stimulation and IL-2 support to express FoxP3, a process that is critical for sustaining suppressor functions and for preventing the conversion into proinflammatory cells. To achieve that Treg override the role of effector T cells, IL-2 can be administered in low doses; however, there is a serious toxicity risk of IL-2 therapy resulting into the so-called vascular leak syndrome as a consequence of damaging CD25+ endothelial cells and indirectly through the activation of NK cells. Therefore, IL-2 delivery at the correct place and time is crucial.
To address the quandary of IL-2 delivery, Eskandari et al3 recently developed a strategy linking the release of IL-2 in response to TCR signaling with the aim of balancing immune cell responses. This strategy is based on coating the surface of Treg with a nano-gel loaded with IL-2 by a redox-sensitive linker; under conditions that occur on the cell membrane upon TCR stimulation, the gel releases IL-2 to promote Treg amplification and persistence locally (Figure 1). This clever “backpack” approach promoted Treg selectively in tissues where TCR activation occurs. Such nano-gels releasing their cargo upon redox reaction were previously developed by the same group to deliver IL-15 to tumor-specific CD8+ T cells upon TCR signaling.4
Technically, the IL-2-Fc fusion protein is attached to a gel matrix through a redox-sensitive bis(N-hydroxysuccinimide) linker. The gel is further modified by an anti-CD45 antibody that acts as a noninternalizing surface anchor, facilitating the gel to load onto hematopoietic cells. The integral disulfide bond of the IL-2 crosslinker is cleaved by reducing capacities on the surface of Treg upon TCR activation, releasing IL-2 and capturing the IL-2 receptor CD25 to provide Treg survival signals.
In the graft and draining lymph nodes (representing antigen-rich locations), nano-gel loaded Treg encounter TCR activation, increasing their natural cell surface redox potential to release IL-2 while activating suppressor functions locally. Murine models from the Eskandari et al study3 provide experimental evidence for this spatial-temporal enhancement of Treg function that leads to increased skin allograft survival. Remarkably, and to the point, Treg activation was highest in tissues with peak alloantigen loads, indicating preferential on-target effects. In these allografts, nano-gel-coated Treg induced a gene expression signature associated with “infectious tolerance”; moreover, IL7 levels increased, helping to maintain intragraft Treg homeostasis. Correspondingly, receptors facilitating inflammatory immune cell adhesion and allograft rejection including E-selectin declined.
Although this strategy is an innovative and feasible solution to enhance and tightly control on-target function by Treg, some aspects need further consideration. For instance, how do murine models relate to clinical application? The authors reported on 10-fold increased Treg: CD8+ T cell ratio locally, indicating preferential Treg dominance that is not obtained upon systemic IL-2 treatment. This observation requires clinical testing as the Treg:CD8 T cell ratio is a strong predictor for therapeutic outcomes,5 in contrast to an absolute increase in Treg numbers in the graft or urine of kidney transplant recipients.
We also need to consider potential paracrine effects. Released IL-2 may activate neighboring effector T cells, in-turn, potentially displaying an increased redox potential on their surface, resulting in an amplified releasing of IL-2 from the nano-gel coat that may drive proinflammatory responses. Other suppressive cytokines such as IL-10 or TGF-β that do not stimulate inflammatory immune cells may be considered as an alternative backpack payload.
It remains also unclear how a long-lasting effect by nano-gel loaded Treg will be achieved? The supply of IL-2 by a nano-gel backpack is limited and it is therefore reasonable to assume that the release of IL-2 will decline over time. Moreover, the nano-gel will be diluted with every cell division. Both processes will act additively and therefore limit the therapeutic capacity in parallel to TCR activation. The situation may be balanced to some extent by repetitive applications of freshly “backpacked” Treg, but this remains a critical issue to solve.
Other “designer Treg” therapies are on the horizon. It has been shown that antigen-specific Treg redirected by a TCR or chimeric antigen receptor can efficiently dampen inflammatory responses in the allograft6; “backpacking” with an IL-2 nano-gel may support their activities. In contrast to “backpacked” cells, redirected T cells can be genetically engineered with a response element to release a transgenic cytokine “on demand” when the TCR or chimeric antigen receptor engages cognate antigen in targeted tissues.7 Upon antigen recognition, so-called TRUCK delivers the “payload” in a spatial-temporal fashion into the targeted tissue.
Beyond current technical limitations, the release of “backpacked” cytokines or other proteins on Treg represents an important step forward toward a more specific modulation of immune cell functions with relevance for transplantation.
1. Elias S, Rudensky AY. Therapeutic use of regulatory T cells for graft-versus-host disease. Br J Haematol. 2019; 187:25–38
2. Yu H, Paiva R, Flavell RA. Harnessing the power of regulatory T-cells to control autoimmune diabetes: overview and perspective. Immunology. 2018; 153:161–170
3. Eskandari SK, Sulkaj I, Melo MB, et al. Regulatory T cells engineered with TCR signaling-responsive IL-2 nanogels suppress alloimmunity in sites of antigen encounter. Sci Transl Med. 2020; 12:eaaw4744
4. Tang L, Zheng Y, Melo MB, et al. Enhancing T cell therapy through TCR-signaling-responsive nanoparticle drug delivery. Nat Biotechnol. 2018; 36:707–716
5. Preston CC, Maurer MJ, Oberg AL, et al. The ratios of CD8+ T cells to CD4+CD25+ FOXP3+ and FOXP3- T cells correlate with poor clinical outcome in human serous ovarian cancer. PLoS One. 2013; 8:e80063
6. Rosado-Sánchez I, Levings MK. Building a CAR-Treg: going from the basic to the luxury model. Cell Immunol. 2020; 358:104220
7. Chmielewski M, Hombach AA, Abken H. Of CARs and TRUCKs: chimeric antigen receptor (CAR) T cells engineered with an inducible cytokine to modulate the tumor stroma. Immunol Rev. 2014; 257:83–90