IL-7 coupled with IL-12 increases intratumoral T cell clonality, leading to complete regression of non-immunogenic tumors.

Immune checkpoint inhibitors against PD-1, PD-L1 and CTLA-4 have altered the treatment paradigm for various types of cancers in the past decade. However, they offer clinical benefits to only a subset of patients. Evaluation and identification of an appropriate therapeutic approach to improve intratumoral immune status are needed for better treatment outcomes. We previously demonstrated that intratumoral expression of IL-7 and IL-12 increased tumor-infiltrating lymphocytes in poorly immunogenic tumors, resulting in a higher tumor regression rate than IL-12 alone. However, the mechanism underlying the difference in efficacy with and without IL-7 remains unclear. Here, we identified a previously unknown effect of IL-7 on the T cell receptor (TCR) repertoire of intratumoral CD8+ T cells, which is induced in the presence of IL-12. While IL-7 alone increased the diversity of intratumoral CD8+ T cells, IL-7 with IL-12 increased a limited number of high-frequency clones, conversely augmenting IL-12 function to increase the clonality. The proportion of mice with multiple high-frequency clones in tumors correlated with that achieving complete tumor regression in efficacy studies. These findings provide a scientific rationale for combining IL-7 and IL-12 in anticancer immunotherapy and unveil a novel IL-7 function on intratumoral TCR repertoire.

Oncolytic vaccinia virus induces a novel phenotype of CD8+ effector T cells characterized by high ICOS expression.

Characterization of the intratumoral immune status is important for developing immunotherapies and evaluating their antitumor effectiveness. CD8+ T cells are one of the most important cell types that directly and indirectly contribute to antitumor efficacy by releasing cytolytic molecules and inflammatory cytokines in the tumor microenvironment. Previously, we engineered a tumor-selective oncolytic vaccinia virus that encodes interleukin-7 (IL-7) and IL-12 and demonstrated its usefulness as an agent for in situ vaccination against tumors, with data showing that antitumor efficacy was reliant upon CD8+ T cells recruited by viral treatment. Here, we investigated the phenotypic changes in intratumoral CD8+ T cells caused by this oncolytic virus and found increased expression of inducible co-stimulator (ICOS) in PD-1-CD8+ T cells. Unlike previously reported ICOS+CD8+ T cells, a subset of ICOS+PD-1-CD8+ T cells showed effector function characterized by granzyme B expression. ICOS expression was induced by the backbone virus, which did not encode any immune transgenes and was independent of upregulation of the type I interferon pathway. Not only did we identify a novel effector cell subset characterized by ICOS expression, but our findings also shed light on a potential unknown aspect of the mechanism of oncolytic vaccinia virotherapy.

Intratumoral expression of IL-7 and IL-12 using an oncolytic virus increases systemic sensitivity to immune checkpoint blockade.

The immune status of the tumor microenvironment is a key indicator in determining the antitumor effectiveness of immunotherapies. Data support the role of activation and expansion of tumor-infiltrating lymphocytes (TILs) in increasing the benefit of immunotherapies in patients with solid tumors. We found that intratumoral injection of a tumor-selective oncolytic vaccinia virus encoding interleukin-7 (IL-7) and IL-12 into tumor-bearing immunocompetent mice activated the inflammatory immune status of previously poorly immunogenic tumors and resulted in complete tumor regression, even in distant tumor deposits. Mice achieving complete tumor regression resisted rechallenge with the same tumor cells, suggesting establishment of long-term tumor-specific immune memory. Combining this virotherapy with anti-programmed cell death-1 (PD-1) or anti-cytotoxic T lymphocyte antigen 4 (CTLA4) antibody further increased the antitumor activity as compared to virotherapy alone, in tumor models unresponsive to either of the checkpoint inhibitor monotherapies. These findings suggest that administration of an oncolytic vaccinia virus carrying genes encoding for IL-7 and IL-12 has antitumor activity in both directly injected and distant noninjected tumors through immune status changes rendering tumors sensitive to immune checkpoint blockade. The benefit of intratumoral IL-7 and IL-12 expression was also observed in humanized mice bearing human cancer cells. These data support further investigation in patients with non-inflamed solid tumors.

Contact-dependent promotion of cell migration by the OL-protocadherin-Nap1 interaction.

OL-protocadherin (OL-pc) is a transmembrane protein belonging to the cadherin superfamily, which has been shown to accumulate at cell-cell contacts via its homophilic interaction, but its molecular roles remain elusive. In this study, we show that OL-pc bound Nck-associated protein 1 (Nap1), a protein that regulates WAVE-mediated actin assembly. In astrocytoma U251 cells not expressing OL-pc, Nap1 was localized only along the lamellipodia. However, exogenous expression of OL-pc in these cells recruited Nap1 as well as WAVE1 to cell-cell contact sites. Although OL-pc expression had no effect on the motility of solitary U251 cells, it accelerated their movement when they were in contact with one another, causing concomitant reorganization of F-actin and N-cadherin at cell junctions. OL-pc mutants lacking the Nap1-binding site exhibited no such effect. N-cadherin knockdown mimicked OL-pc expression in enhancing cell movement. These results suggest that OL-pc remodels the motility and adhesion machinery at cell junctions by recruiting the Nap1-WAVE1 complex to these sites and, in turn, promotes the migration of cells.

OL-Protocadherin is essential for growth of striatal axons and thalamocortical projections.

The ventral telencephalon in the embryonic brain is thought to provide guidance cues for navigation of thalamocortical axons, but the mechanisms involved remain largely elusive. OL-protocadherin (OL-pc), a member of the cadherin superfamily, is highly expressed by striatal neurons in the developing ventral telencephalon. Here we show that OL-pc-deficient (Pcdh10(-/-)) mice have defects in axon pathways through the ventral telencephalon; for example, thalamocortical and corticothalamic projections cannot cross the ventral telencephalon. In the ventral telencephalon, striatal axons fail to grow out, and, concomitantly, the caudal portion of the globus pallidus and the associated 'corridor' thought to be important for thalamocortical fiber navigation do not form. The inability of the striatum to extend axons is also observed in vitro. These results show that OL-pc is essential for both elongation of striatal axons and patterning of the putative guidance cues for thalamocortical projections.

Distribution of OL-protocadherin in axon fibers in the developing chick nervous system.

OL-protocadherin (OL-pc) is a homophilic cell adhesion molecule that belongs to the cadherin gene superfamily. We cloned and characterized the chicken homologue of OL-pc and examined its expression pattern in chick embryos mainly from embryonic day (E) 3.5 to E6.5. The structure of chick OL-pc was found to be essentially the same as that of mammalian OL-pc's except for some small deletions and insertions in the amino acid sequence. OL-pc protein was detected prominently along developing axonal fibers in the brain and also in the peripheral nervous system. In addition, it was detected in some mesenchymal cells and in the embryonic ectoderm of the mandible and limb bud. In the spinal cord, OL-pc was specifically expressed in motor neurons, and the protein was distributed along motor nerves. Motor nerves merged gradually with sensory nerves showing negative/faint OL-pc expression, but their fibers remained separated as small bundles in the nerves. Interestingly, OL-pc-positive motor nerves such as those to the sternocoracoideus became segregated from OL-pc-faint/weak motor nerves at the plexus region. Moreover, OL-pc was distributed along the path of the branchial nerves. These results suggest that OL-pc might play some roles in axon navigation such as in axon elongation, selective fasciculation, and pathfinding in the early stage of neural development.