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Harnessing immune system to treat cancer requires simultaneous generation of tumor-specific CTLs and curtailment of tumor immunosuppressive environment. Here, we developed an immunotherapeutic regimen capable of eliminating large established mouse tumors using HMGN1, a DC-activating TLR4 agonist capable of inducing anti-tumor immunity. Intratumoral delivery of HMGN1 with low dose of Cytoxan cured mice bearing small (∅ ≈ 0.5 cm), but not large (∅ ≈ 1.0 cm) CT26 tumors. Screening for activators capable of synergizing with HMGN1 in activating DC identified R848. Intratumoral delivery of HMGN1 and R848 plus Cytoxan eradicated large established CT26 tumors. The resultant tumor-free mice were resistant to subsequent challenge with CT26, indicating the generation of CT26-specific protective immunity. This immunotherapeutic regimen caused homing of tumor-infiltrating DC to draining lymph nodes and increased infiltration of T cells into tumor tissues. Cytoxan in this regimen could be replaced by anti-CTLA4) or anti-PD-L1. Importantly, this immunotherapeutic regimen was also curative for large established mouse Renca and EG7 tumors. Thus, we have developed a curative therapeutic vaccination regimen dubbed ‘TheraVac’ consisting of HMGN1 and R848 plus a checkpoint inhibitor, that can, without using exogenous tumor-associated antigen(s), eliminate various large tumors and induce tumor-specific immunity.

There is now compelling evidence that TNFR2 is constitutively expressed on CD4(+) Foxp3(+) regulatory T cells (Tregs) and TNF-TNFR2 interaction is critical for the activation, expansion and functional stability of Tregs. However, we showed that the expression of TNFR2 was also up-regulated on CD4(+) Foxp3(-) effector T cells (Teffs) upon TCR stimulation. In order to define the role of TNFR2 in the pathogenic CD4 T cells, we compared the effect of transferred naïve CD4 cells from WT mice and TNFR2(-/-) mice into Rag 1(-/-) recipients. Transfer of TNFR2-deficient Teff cells failed to induce full-fledged colitis, unlike WT Teffs. This was due to defective proliferative expansion of TNFR2-deficient Teff cells in the lymphopenic mice, as well as their reduced capacity to express proinflammatory Th1 cytokine on a per cell basis. In vitro, the proliferative response of TNFR2 deficient naïve CD4 cells to anti-CD3 stimulation was markedly decreased as compared with that of WT naïve CD4 cells. The hypoproliferative response of TNFR2-deficient Teff cells to TCR stimulation was associated with an increased ratio of p100/p52, providing a mechanistic basis for our findings. Therefore, this study clearly indicates that TNFR2 is important for the proliferative expansion of pathogenic Teff cells.

There is now compelling evidence that TNFR2 is constitutively expressed on CD4+ Foxp3+ regulatory T cells (Tregs) and TNF-TNFR2 interaction is critical for the activation, expansion and functional stability of Tregs. However, we showed that the expression of TNFR2 was also up-regulated on CD4+ Foxp3− effector T cells (Teffs) upon TCR stimulation. In order to define the role of TNFR2 in the pathogenic CD4 T cells, we compared the effect of transferred naïve CD4 cells from WT mice and TNFR2−/− mice into Rag 1−/− recipients. Transfer of TNFR2-deficient Teff cells failed to induce full-fledged colitis, unlike WT Teffs. This was due to defective proliferative expansion of TNFR2-deficient Teff cells in the lymphopenic mice, as well as their reduced capacity to express proinflammatory Th1 cytokine on a per cell basis. In vitro, the proliferative response of TNFR2 deficient naïve CD4 cells to anti-CD3 stimulation was markedly decreased as compared with that of WT naïve CD4 cells. The hypoproliferative response of TNFR2-deficient Teff cells to TCR stimulation was associated with an increased ratio of p100/p52, providing a mechanistic basis for our findings. Therefore, this study clearly indicates that TNFR2 is important for the proliferative expansion of pathogenic Teff cells.