Combination of a MIP3α-antigen fusion therapeutic DNA vaccine with treatments of IFNα and 5-Aza-2’Deoxycytidine enhances activated effector CD8+ T cells expressing CD11c in the B16F10 melanoma model

Previous studies in the B16F10 mouse melanoma model have demonstrated that combining a DNA vaccine comprised of regions of gp100 and tyrosinase-related protein 2 fused to Macrophage-inflammatory protein 3-alpha (MIP3α) with recombinant Interferon alpha (IFN) and 5-Aza-2’-Deoxycytidine (5Aza) treatments resulted in significantly greater anti-tumor activity and immunogenicity in the tumor microenvironment (TME). This brief report details that the combination of vaccine with treatments IFN and 5Aza results in both the upregulation of genes expressing CD11c-interacting proteins and an increase in the TME of a distinct CD11c+ CD8+ T cell population. This cell population correlates with tumor size, is primarily comprised of effector or effector memory T cells, and has a more robust response to ex vivo stimulation as compared to CD11c− CD8+ T cells as measured by surface activation markers 4-1BB (CD137) and KLRG1 (Killer cell lectin-like receptor G1) and intracellular IFNγ production. In conclusion, this combination therapy results in greater presence of highly active effector CD8+ T-cells expressing CD11c in the TME that correlate with and are likely primary contributors to treatment efficacy.

CD11c is a type I transmembrane protein forming part of the complement receptor 4 and has been shown to play a role in phagocytosis, cell migration, cytokine production, and T-cell proliferation.A subset of CD8+ T cells expressing CD11c+ have been described in various tissues, infections, and cancers [9][10][11] with the ability to become suppressor or effector cells [10,12,13].The current study shows that levels of CD8+ T cells expressing CD11c are increased in the TME with combination treatment and are correlated with tumor size in vaccinated groups.To our knowledge, this study is the rst to show the presence of CD8+CD11c+ T cells in a tumor-associated-antigen based therapeutic cancer vaccine system, and the data suggest that these cells are in higher numbers, are more robust, and are more potent with the combination treatments compared to vaccine alone or treatments alone.
These cells are therefore likely to be critical components in the tumor suppression observed with the therapeutic regimen we have employed against the B16F10 melanoma.

Materials and Methods
Animals 6-12-week-old female C57BL/6 (Charles River, Wilmington, MA) mice were challenged with a lethal dose of B16F10 melanoma (5×10 4 cells, >95% viability) administered intradermally on the mouse ank on day 0 of therapy [2,3,8].Tumor size was recorded by calipers every 1-3 days as square mm (L × W).Mice were monitored in accordance with IACUC protocols and were removed from the study once tumo diameterr>1cm or if mice showed signs of distress.

Immuno uorescence Staining
Tumor tissues were collected at the time of harvest and were immediately xed in 10% neutral-buffered formalin for 48 hours and were then processed, embedded into para n (FFPE), and cut onto slides.The slides were blocked, stained with αCD3 antibody, washed, stained with AF488 secondary antibody, washed, and repeated for αCD11c antibody and AF647 secondary.The nuclei were counterstained with DAPI and mounted.Three slides were blindly selected from each group for analysis.Images were acquired across ten random elds per slide under the 40x objective using a Leica THUNDER computational clearing wide eld microscope through LAS X software (Leica Microsystems, Wetzlar, Germany).

Database Collection and Statistics
The correlations between CD11c (ITGAX) expression and in ltrating immune cell subsets in the tumor microenvironment were assessed through TIMER

CD8+11c+ T cells in the TME
In our model combining vaccine with IFN and 5Aza (Fig. 1A), the CD8+CD11c+ T-cell population was signi cantly higher following combination therapy vs. vaccine alone (p=0.0271) or IFN+5Aza (p=0.0007) (Fig. 1B).Of note, when strati ed across groups, the presence of the CD11c+ CD8+ T-cell subpopulation was also correlated with tumor size in vaccinated groups, whereas CD11c-CD8+ T cell numbers were not correlated (Fig. 1C-D).Complementing the ow cytometry data, the presence and distribution of T cells in the intratumoral areas were determined by immuno uorescence microscopy (Fig. 1E).A signi cantly higher number of CD11c+ T cells were observed in the group that received combination therapy as compared with IFN+5Aza (P=0.0118) or vaccine alone (P=0.0234) (Fig. 1F).The relationship between CD11c (ITGAX) expression and CD8+ T cell in ltration was further explored by analyzing publicly available datasets of melanoma patients (Fig. 1G).The results suggest a signi cant positive correlation between CD11c expression and the overall in ltration level of CD8+ T cells (rho= 0.23, P= 4.03e-08).

Activation Markers of CD8+11c+ T cells in the Spleen
splenocytes were to further assess the subset of CD11c+ CD8+ T cells for markers of effector function and activation: 4-1BB, Killer cell lectin-like receptor G1 (KLRG1), and IFNγ.
Following ex-vivo stimulation with vaccine peptides, we show that co-expression of KLRG1 and 4-1BB was signi cantly higher in CD8+CD11c+ T cells (p<0.001) compared to CD11c-cells (Fig. 2C).Further, when strati ed by treatment group, expression of 41BB and KLRG1 on the surface of CD8+CD11c+ T cells was increased signi cantly following combination therapy relative to treatment with IFN+5Aza (p=0.0020) or vaccine alone (p=0.0250) (Fig. 2D).Using intracellular staining methods, IFNγ expression was greater in the combination group compared to the IFN+5Aza group (p= 0.0393) and the vaccine group (p= 0.0426) (Fig. 2E).Additionally, after stratifying the CD8+ T cells by CD11c expression, the CD11c+ population carries the phenotype of higher cytokine production in the combination group compared to IFNα+5Aza (p=0.0110) and vaccine (p= 0.0319) (Fig. 2F).Moreover, the CD11c+ cells had a signi cantly greater proportion of cells expressing IFNγ in the combination group compared to their CD11c-counterparts (p=0.0019) (Fig. 2F).

Discussion
The current study provides evidence that CD8+CD11c+ T cells are increased with combination therapy, are correlated with tumor size (Fig. 1), and express more markers associated with activation and effector function (Fig. 2).4-1BB is hypothesized to be a necessary costimulatory marker for CD8+CD11c+ T cells and is known to promote T-cell survival and enhance expression of cytokines such as IFNγ [14].KLRG1 is induced on highly cytolytic, short-lived effector CD8+ T cells and is hypothesized to play a role in CD8+CD11c+ T-cell activity [15].Interestingly, the majority of the IFNγ production induced by our vaccination protocol is attributable to the CD8+CD11c+ and not the CD8+CD11c-T-cell population (Fig. 2F).Some studies have pointed to stronger regulatory activity of this cell poopulation [10], but others have shown, similar to results here, that CD8+CD11c+ T cells aid in successful antitumoral responses via enhanced effector functions [9], including direct anti-tumor cytotoxicity [13].Our studies have identi ed an immunization protocol that is particularly effective in activating this T cell population.
Analysis of human melanoma trials from publicly available databases show that intratumoral CD11c expression correlates with increased CD8+ T-cell in ltration (Fig. 1F), especially effector/memory subtypes (Fig. 2B), and that patients with more expression have improved survival outcomes (Supp Fig. 3) and better responses to immune checkpoint inhibitor therapy (Supp Fig. 4).We hypothesize that the stronger Tcm correlation in patients compared to our results in the mouse model may be attributable to the more prolonged time frame of melanoma persistence in the clinical setting.Importantly, clinical data supports the interaction of CD11c with CD8+ T-cells and with improved outcomes in melanoma.
Our results show that vaccine-induced, activated, IFNγ-secreting, effector/memory CD8+CD11c+ T cells signi cantly correlate to the antitumoral effect elicited by our combination therapy and are dependent on the combination of vaccine and drug treatments, as removing either arm limits the induction of these cells.These results combined with the literature [9,[13][14][15] suggest that the vaccine-induced CD8+CD11c+ T cells are primary effectors of the anti-tumor immune response.Future studies will further delineate whether these T-cells 1) are relevant to other tumor models; 2) tra c into the tumor or develop at the site 3) vary in number and potency over time; 4) interact with other cells in the tumor; and 5) have direct anti-tumor cytotoxicity.

Figures Figure 1
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Figure 2 Analysis
Figure 2 Whitney tests for two groups, Scatter plots were analyzed by linear regression with Pearson correlation coe cient test.Grouped experiments were analyzed by 2-way ANOVA with Sidak's multiple comparison test.Sample sizes are de ned in the gure legends.GraphPad Prism™ 10 (GraphPad Software, Inc.San Diego, CA) was utilized for statistical analyses and gure creation.Error bars represent the estimation of the standard error of the mean and midlines the group mean.α≤0.05 2.0 web server (http://timer.cistrome.org/)and the xCell algorithm.Purity-adjusted Spearman's rho and statistical signi cance were used to evaluate correlations.Datasets were tested for normality by D'Agostino & Pearson test and followed by by oneway ANOVA with Tukey's or Dunn's multiple comparison test for multiple groups, or by unpaired Student's T test or Mann-