High-dose IL2 immunotherapy can induce long-lasting cancer regression but is toxic and insufficiently efficacious. Improvements are obtained with IL2/anti-IL2 complexes (IL2Cx), which redirect IL2 action to CD8+ T and natural killer (NK) cells. Here, we evaluated the efficacy of combining IL2Cx with blockade of inhibitory immune pathways. In an autochthonous lung adenocarcinoma model, we show that the IL2Cx/anti-PD-1 combination increases CD8+ T-cell infiltration of the lung and controls tumor growth. In the B16-OVA model, which is resistant to checkpoint inhibition, combination of IL2Cx with PD-1 or CTLA-4 pathway blockade reverses that resistance. Both combinations work by reinvigorating exhausted intratumoral CD8+ T cells and by increasing the breadth of tumor-specific T-cell responses. However, only the IL2Cx/anti-CTLA-4 combination is able to rescue NK cell antitumor function by modulating intratumoral regulatory T cells. Overall, association of IL2Cx with PD-1 or CTLA-4 pathway blockade acts by different cellular mechanisms, paving the way for the rational design of combinatorial antitumor therapies.
©2019 American Association for Cancer Research.

Treatment of patients bearing human papillomavirus (HPV)-related cancers with synthetic long-peptide (SLP) therapeutic vaccines has shown promising results in clinical trials against premalignant lesions, whereas responses against later stage carcinomas have remained elusive. We show that conjugation of a well-documented HPV-E7 SLP to ultra-small polymeric nanoparticles (NP) enhances the antitumor efficacy of therapeutic vaccination in different mouse models of HPV+ cancers. Immunization of TC-1 tumor-bearing mice with a single dose of NP-conjugated E7LP (NP-E7LP) generated a larger pool of E7-specific CD8+ T cells with increased effector functions than unconjugated free E7LP. At the tumor site, NP-E7LP prompted a robust infiltration of CD8+ T cells that was not accompanied by concomitant accumulation of regulatory T cells (Tregs), resulting in a higher CD8+ T-cell to Treg ratio. Consequently, the amplified immune response elicited by the NP-E7LP formulation led to increased regression of large, well-established tumors, resulting in a significant percentage of complete responses that were not achievable by immunizing with the non-NP-conjugated long-peptide. The partial responses were characterized by distinct phases of regression, stable disease, and relapse to progressive growth, establishing a platform to investigate adaptive resistance mechanisms. The efficacy of NP-E7LP could be further improved by therapeutic activation of the costimulatory receptor 4-1BB. This NP-E7LP formulation illustrates a "solid-phase" antigen delivery strategy that is more effective than a conventional free-peptide ("liquid") vaccine, further highlighting the potential of using such formulations for therapeutic vaccination against solid tumors. Cancer Immunol Res; 6(11); 1301-13. ©2018 AACR.
©2018 American Association for Cancer Research.

Polymorphisms impacting thymic function may decrease peripheral tolerance and hasten autoimmune disease. The NF-κB transcription factor subunit, RelB, is essential for the development and differentiation of medullary thymic epithelial cells (mTECs): RelB-deficient mice have reduced thymic cellularity and markedly fewer mTECs, lacking AIRE. The precise mechanism of this mTEC reduction in the absence of RelB is unclear. To address this, we studied mTECs and dendritic cells (DCs), which critically regulate negative selection, and thymic regulatory T-cells (tTreg) in RelB-/- mice, which have spontaneous multiorgan autoimmune disease. RelB-/- thymi were organized, with medullary structures containing AIRE- mTECs, DCs, and CD4+ thymocytes, but fewer tTreg. Granulocytes infiltrated the RelB-/- thymic cortex, capsule, and medulla, producing inflammatory thymic medullary atrophy, which could be treated by granulocyte depletion or RelB+ DC immunotherapy, with concomitant recovery of mTEC and tTreg numbers. These data indicate that central tolerance defects may be accelerated by autoimmune thymic inflammation where impaired RelB signaling impairs the medullary niche, and may be reversible by therapies enhancing peripheral Treg or suppressing inflammation.

Tumor associated macrophages and tumor infiltrating regulatory T cells greatly hamper host-protective antitumor responses. Therefore, we utilized a novel immunomodulator, heat-killed Mycobacterium indicus pranii (Mw), to repolarize TAM and an agonistic GITR antibody (DTA-1) to reduce intratumoral regulatory T cell frequency for generation of a host-protective antitumor response. Although, the combination of Mw and DTA-1was found to be effective against advanced stage tumors, however, Mw or DTA-1 failed to do so when administered individually. The presence of high level of regulatory T cells abrogated the only Mw induced antitumor functions, whereas only DTA-1 treatment was found to be ineffective due to its inability to induce TAM repolarization in vivo. The combination therapy was found to be effective since DTA-1 treatment reduced the frequency of regulatory T cells to such an extent where they could not attenuate Mw induced TAM repolarization in vivo. Therefore, the combination therapy involving Mw and DTA-1 may be utilized to the success of advanced stage solid tumor immunotherapies.

The ability of CD4(+)Foxp3(+) regulatory T-cells (Treg) to produce interleukin (IL)-10 is important for the limitation of inflammation at environmental interfaces like colon or lung. Under steady state conditions, however, few Tregs produce IL-10 ex vivo. To investigate the origin and fate of IL-10 producing Tregs we used a superagonistic mouse anti-mouse CD28 mAb (CD28SA) for polyclonal in vivo stimulation of Tregs, which not only led to their numeric expansion but also to a dramatic increase in IL-10 production. IL-10 secreting Tregs strongly upregulated surface receptors associated with suppressive function as compared to non-producing Tregs. Furthermore, polyclonally expanding Tregs shifted their migration receptor pattern after activation from a CCR7(+)CCR5(-) lymph node-seeking to a CCR7(-)CCR5(+) inflammation-seeking phenotype, explaining the preferential recruitment of IL-10 producers to sites of ongoing immune responses. Finally, we observed that IL-10 producing Tregs from CD28SA stimulated mice were more apoptosis-prone in vitro than their IL-10 negative counterparts. These findings support a model where prolonged activation of Tregs results in terminal differentiation towards an IL-10 producing effector phenotype associated with a limited lifespan, implicating built-in termination of immunosuppression.