*4.3. Retargeted oHSVs Subvert TME Immunosuppression*

The purpose of this series of experiments was to provide evidence that the long-term distant protection was mediated by an immune response, documented as dramatic changes to the immunosuppressive TME. In these experiments, mice were treated i.t. with the R-337, and sacrificed a few days after the end of treatment, at a time when tumors were decreasing in size (Figure 5A–C). Analyses were carried out on tumor infiltrating lymphocytes and cytokines, on the reactivity of splenocytes and of serum antibodies to tumors cells, with the aim to detect local and systemic modifications. In R-115-treatred mice, the major modifications consisted in the tumor infiltration by CD4+, CD8+ and activated CD8+, NK (natural killer) and activated NK, Tregs (T-regulatory), along with the reduction in intratumoral CD11b+ leucocytes [55]. The immune landscape of LLC-1-HER2 TME is that of an immunologically desert tumor, characterized by low infiltration from anti-tumor immune subpopulations and low levels of immune activation markers, co-stimulatory molecules and pro-inflammatory cytokines [61]. In essence, the host immune system is unable to recognize and react against LLC-1 tumors. Figure 5D–K documents the modifications detected in R-337-treated mice. Worth noting are the increase in tumor infiltrating leucocytes, specifically CD4+, CD8+ and activated CD8+, DCs, and NK and activated NK cells. The CD11b-positive population, which includes the immunosuppressive myeloid derived suppressor cells, was decreased (Figure 5L). FoxP3+ cells, which include the Tregulatory cells, were also increased (Figure 5H), in agreement with previous reports [55]. Transcriptional analysis of the tumor specimens revealed an increase in IFNγ, IL-12 (most of which likely expressed from the viral genome), CXCL11 chemokine and t-bet transcrip-

tion factor (Figure 5M–Q), hallmarks of inflamed TME and polarization to activated Th1 cells. Analysis of the systemic effect was carried out on spleen samples. The modifications were essentially similar to those detected in the tumor samples (Figure 5R–W), except that the increase in NK cells was non statistically significant. The splenocyte reactivity and the antibody response to LLC-1-HER2 cells were essentially similar to those detected in mice sacrificed at about 100 days after primary tumor implantation (Figure 5X,Y). Altogether, i.t.- administered R-337 elicited a strong systemic and intratumoral immune response, and the inflammation of the LLC-1-HER2 TME.

**Figure 5.** Immune heating of TME and spleen modifications induced by intratumoral R-337 monotherapy. (**A**,**B**) Kinetics of tumor growth in HER2-TG C57BL/6 mice treated with vehicle (**A**) or R-337 (**B**), according to the schedule reported in Figure 4A. (**C**) Tumor volumes at d 24. Black (vehicle) and red (R-337) circles. (**D**–**L**) Immune cell populations in tumors. Single cell suspensions were prepared from freshly isolated LLC1-HER2 tumors at sacrifice. Tumors were minced in small pieces, digested with collagenase, passed through 70 µm cell strainer and rinsed with FACS buffer. For each sample, <sup>2</sup> <sup>×</sup> <sup>10</sup><sup>6</sup> cells were blocked with α-CD16/32 Ab (clone 93), and then reacted with the antibodies CD4-FITC (clone GK1.5), CD8a-PE (clone 53-6.7), CD45-Percp-Cy7 (clone 30-F11), CD335-APC (clone 29A1.4), FoxP3-PE (clone 150d/e4), CD11b-FITC (clone M1/70), CD11c-PE (clone N418) and CD69-PercP (clone H1-2F3). Data were acquired on BD C6 Accuri. CD4 (CD4+ cells), CD8 (CD8+ cells), NK (CD335+ cells) and myeloid cells (CD11b+ cells) were gated on CD45+ subpopulation. 142

Activated (CD69+) CD8 and NK cells were gated on CD8+ and CD335+ subpopulations, respectively. DC cells (CD11c+CD11b+) were gated on CD11b+ population. Tregs (FoxP3+CD4+) were gated on CD4+ population. (**M**–**Q**). Expression profile of cytokines, immune related transcription factor and immune markers. Tumor homogenates (a few mgs) were employed for total RNA purification and 1.2 µg of RNA was employed for the cDNA synthesis. Diluted cDNAs (1:4) were assayed by real-time PCR with Taqman probes. The levels of expression were determined using the ∆∆Ct method, normalized on the Rpl13a housekeeping gene and on the mean of the vehicle-treated group. (**R**–**W**) Immune cell populations in spleens. Sample preparation and staining as described for tumors. (**X**) Immune response in splenocytes to LLC-1-HER2 and LLC-1 cells was quantified as IFNγ secretion in the culture medium. For the details, see Figure 4. (**Y**) Serum antibody reactivity to LLC-1-HER2 and LLC-1 cells. For the details, see Figure 4C–Y Statistical significance was calculated by the t-test and expressed as \* = *p*-value < 0.05; \*\* = *p*-value < 0.01; \*\*\* = *p*-value < 0.001; \*\*\*\* = *p*-value < 0.0001, ns = not significant. Color code, mice treated with vehicle or R-337 are indicated in black or red, respectively.
