31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Visualization of Intrapulmonary Lymph Vessels in Healthy and Inflamed Murine Lung Using CD90/Thy-1 as a Marker

<div><h3>Background</h3><p>Lymphatic vessels play a pivotal role in fluid drainage and egress of immune cells from the lung. However, examining murine lung lymphatics is hampered by the expression of classical lymph endothelial markers on other cell types, which hinders the unambiguous identification of lymphatics. The expression of CD90/Thy-1 on lymph endothelium was recently described and we therefore examined its suitability to identify murine pulmonary lymph vessels under healthy and inflammatory conditions.</p> <h3>Methodology/Principal Findings</h3><p>Immunohistochemistry with a monoclonal antibody against CD90.2/Thy-1.2 on 200 µm thick precision cut lung slices labeled a vascular network that was distinct from blood vessels. Preembedding immunostaining and electron microscopy verified that the anti-CD90.2/Thy-1.2 antibody labeled lymphatic endothelium. Absence of staining in CD90.1/Thy-1.1 expressing FVB mice indicated that CD90/Thy-1 was expressed on lymph endothelium and labeling was not due to antibody cross reactivity. Double-labeling immunohistochemistry for CD90/Thy-1 and α-smooth muscle actin identified two routes for lymph vessel exit from the murine lung. One started in the parenchyma or around veins and left via venous blood vessels. The other began in the space around airways or in the space between airways and pulmonary arteries and left via the main bronchi. As expected from the pulmonary distribution of lymph vessels, intranasal application of house dust mite led to accumulation of T cells around veins and in the connective tissue between airways and pulmonary arteries. Surprisingly, increased numbers of T cells were also detected around intraacinar arteries that lack lymph vessels. This arterial T cell sheath extended to the pulmonary arteries where lymph vessels were located.</p> <h3>Conclusions/Significance</h3><p>These results indicate that CD90/Thy-1 is expressed on lymphatic endothelial cells and represents a suitable marker for murine lung lymph vessels. Combining CD90/Thy-1 labeling with precision cut lung slices allows visualizing the anatomy of the lymphatic system in normal and inflamed conditions.</p> </div

Immunohistochemistry of CD90/Thy-1 in murine precision cut lung slices.

<p>A) The anti-CD90/Thy-1 antibody (green) stains a vascular system (arrowheads) in murine precision cut lung slices that is distributed throughout the lung. Red staining shows immunoreactivity for α-smooth muscle actin. AW: airways. B) Initial CD90/Thy-1-immunoreactive capillaries (arrowheads) in the alveolar region. C) The CD90/Thy-1-immunoreactive vascular network is interconnected. D) A CD90/Thy-1-immunoreactive valve (arrowheads). E) Only close to the hilum, α-smooth muscle actin-immunoreactive cells (red, arrow) were found on CD90/Thy-1-immuoreactive vessels.</p

T cell distribution in the murine lung after house dust mite sensitization and challenge.

<p>A, B) CD90/Thy-1-immunoreactive T cells are found (A) around veins (V) and (B) around intraacinar arteries (IA). C) Accumulations of T cells around arteries are continuous from intraacinar arteries to pulmonary arteries (PA). D) Around airways (AW), T cells preferentially accumulate around pulmonary arteries.</p

Identification of lymph vessels in the living trachea by <i>ex vivo</i> staining with an anti-CD90/Thy-1 antibody.

<p>Projection of a z-stack in a living murine trachea <i>ex vivo</i> recorded by multiphoton microscopy. Preincubation with an anti-CD90/Thy-1 antibody coupled to FITC stains a lymph vessel (white arrow = lymph vessel valve) and a cell with fibroblast morphology (red arrow) in a living trachea <i>ex vivo</i>. Other structures of the tissue are visualized by detection of tissue autofluorescence.</p

Distribution of lymph vessels in the murine lung with respect to blood vessels and airways.

<p>A–D and F) Double-labeling of precision cut lung slices with anti-CD90/Thy-1 antibody (green) and anti-α-smooth muscle actin antibody (red). A) CD90/Thy-1-immunoreactive lymph vessels are found around veins (V) and B) around muscularized airways (AW). C) Intraacinar arteries (IA) are not accompanied by lymph vessels. D, E) Lymph vessels are found frequently in the connective tissue between pulmonary arteries and airways. E) Paraffin section of murine lung stained with Masson Goldner stain. F) Frequently, accumulations of CD90/Thy-1-immunoreactive cells with lymphocyte morphology (arrows) are found around lymph vessels. Arrowheads = lymph vessels.</p