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In vivo manipulation of Treg cells
mAb-primed host Tregs were adoptively transferred into BDF1 mice, less severe disease was observed.121
TNFRSF25 (DR3)/TNFSF15 (TL1A)
TNFRSF25 (DR3)/TNFSF15 (TL1A) targeting to manipulate Tregs in vivo. TNFRSF25 is constitutively expressed on Tregs and up-regulated upon activation on CD4+ and CD8+ T cells. Low levels are also present on NK, NKT cells and ILC2/3 subsets. Its natural ligand, TL1A, is primarily expressed on endothelial cells and upon activation on APC and T cells, including Tregs.122,123 TNFRSF25 co-stimulation promotes proliferation, effector function and survival, as well as apoptosis depending on signal strength.124,125
Recent studies, using either agonistic mAb (clone 4C12) or a TL1A-Ig fusion protein, have shown that triggering the TNFRSF25 pathway in the absence of antigen leads to a significant expansion of Tregs in vivo which is dependent on the TCR and IL-2.126,127 Notably, TNFRSF25 stimulation expands Tregs to a greater degree than CD25 stimulation (via IL-2) alone. Moreover, TNFRSF25 stimulation alone has the strongest in vivo effect on Treg expansion com- pared to other TNFRSF members including GITR, OX-40 or 4-1BB.126 Stimulating multiple receptors on Tregs can have synergistic effects, though the levels of expansion achieved depend on the molecular targets. Thus, targeting two TNF family members, TNFRSF25 (4C12) and OX-40 (OX-86)128,129 in the context of co-stimulation or vaccina- tion led to increased frequency of Tregs versus either alone. However, when both TNFRSF25 and CD25 are stimulat- ed,130 there is a marked elevation of the compartment, much greater than administration of TL1A-Ig or IL-2 alone or 4C12 together with OX-86. It is likely that the former is observed because sufficient IL-2 is provided to maintain the elevated Treg levels.
TNFRSF25-induced expansion, either alone or in combi- nation with IL-2, leads to upregulation of activation mark- ers on Tregs and enhanced suppressive activity.131,132 Our own studies have demonstrated that ICOS-1, Nrp-1, PD-1 and other molecules are more highly expressed after com- bined TNFRSF25 and CD25 in vivo stimulation compared with targeting the individual receptors.132 Tregs expanded in vivo via the TNFRSF25 are protective against allergic lung inflammation and EAE.126,127,133 Furthermore, they also prolonged graft survival in a mouse model of heterotopic allogeneic heart transplantation.134
TNFRSF25 (DR3)/TNFSF15 (TL1A) manipulation in pre- clinical allogeneic hematopoietic stem cell transplantation. As noted above, TNFRSF25 stimulation using 4C12 or TL1A- Ig selectively and dramatically promotes in vivo Treg expansion. Single administration of 4C12 resulted in a sig- nificant increase in splenic and lymph node Treg numbers with enhanced suppressive function.135 In a major MHC- mismatched GvHD model, recipients of 4C12-treated donor T cells showed a significant increase in overall sur- vival and diminished GvHD.130,135 Importantly, T cells from 4C12-treated donors exhibited preserved graft-versus- tumor (GvT) activity.135 Expansion of Tregs with 4C12 treatment induced phenotypic changes in Tregs, including higher expression of activation and maturation molecules.131 Mice treated with purified 4C12-Tregs (CD4+CD25+) showed diminished GvHD with improved survival, demonstrating that these Tregs possessed higher in vivo suppressive activity.131 Notably, administration of 4C12 into recipients with ongoing GvHD augmented
mortality and worsened the disease because upon alloantigen exposure TNFRSF25 stimulation induced effector T-cell proliferation and activation.131 However, prophylaxis of recipients with 4C12 induced host-derived Treg expansion with a reduction in GvHD severity.131 Critically, the success of this approach to prevent GvHD is dependent on the timing of TNFRSF25 stimulation and the status of donor T-cell activation.
Because of the success of IL-2 in pre-clinical and clinical GvHD studies and the expression of TNFRSF25 in the Treg population, we developed a strategy to transiently manipulate Tregs in vivo combining LD IL-2C with TL1A- Ig. This “two-pathway” approach markedly expands (5-7- fold) and selectively activates Tregs. Transplantation of TL1A-Ig/IL-2C Treg expanded donor spleen into recipi- ents resulted in amelioration of GvHD severity in both fully MHC-mismatched and MHC-matched aHSCT set- tings130,132 (Figure 2). In vivo Treg expansion was superior with TL1A-Ig/IL-2C treatment compared to 4C12 mAb administration and GvHD was more completely amelio- rated after transplant of two-pathway expanded donor Tregs. Furthermore, recipients of transplants using spleen cells from TL1A-Ig/IL-2 expanded donors demonstrated preserved GvL while GvHD was effectively diminished.130 In fact, because TL1A-Ig/IL-2 expanded Tregs expressed higher activation and functional molecules, very low num- bers of these expanded cells (corresponding to a ratio of 1:6 expanded Tregs/Tconv) very effectively suppress GvHD post aHSCT.132 Furthermore, TL1A-Ig/IL-2 expand- ed Treg therapy was shown to be as effective as post- transplant cyclophosphamide for GvHD prophylaxis while more rapid thymic reconstitution providing earlier recovery of recipient immune function.136 GvHD is pro- moted by alloreactive donor T cells and inflammation; therefore, we proposed to regulate both pathways. Donor Treg expansion was combined with EP11313 (a bromod- omain and extra-terminal bromodomain inhibitor, BETi) using short-term treatment in the recipient in a fully MHC-mismatched aHSCT. This strategy was found to significantly reduce early GvHD clinical scores including decreased ocular and skin involvement. Importantly, uti- lizing highly purified TL1A-Ig/IL-2 expanded donor Tregs in vivo, this second assessment of the combinatorial strat- egy further supported the notion that selective BETi can be used for GvHD treatment in combination with Treg adoptive therapy.137
CD28/CD80/86 (B7.1/2)
CD28/CD80/86 (B7.1/2) targeting to manipulate Tregs in vivo. CD28, a key co-stimulatory molecule, is expressed on all T cells and provides signals for activation and survival. Its ligands, CD80 and CD86, are expressed on APC. CD28 super-agonists (CD28SA) are mAbs which induce poly- clonal T-cell proliferation in the absence of TCR liga- tion.138,139 In rodents, in vivo application of CD28SA at low doses efficiently expands Tregs and partially inhibits expansion of autoreactive T cells. In addition, CD28SA expanded Tregs showed enhanced suppressive activity (increased IL-10 production) and migrated to inflamed tis- sues without causing cytokine release syndrome.139-142 Treg expansion by CD28SA is dependent on paracrine IL-2 from CD28SA-stimulated Tconv cells.142 Accordingly, CD28SA expanded Tregs are highly effective in the treat- ment/prevention of disease in rodent models of autoim- munity, like EAE,141 experimental autoimmune neuritis143
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