7MO - Effect of anti-CTLA-4 immunotherapy on lymphocyte subset and activation profiles and clonal composition on the B16F0 mouse melanoma model

Background

One of most promising strategies for cancer immunotherapy is immune checkpoint blockade. However, the remarkable responses to the therapy are currently limited to a minority of patients and indications, highlighting the importance of understanding of immune mechanisms. The purpose of this study was to investigate the effect of anti-CTLA-4 immunotherapy on lymphocyte subset and activation profiles and clonal composition on the B16F0 mouse melanoma model.

Methods

The experiments were carried out on C57BL/6 mice bearing B16F0 mouse melanoma. Mice were treated with 250 μg anti-CTLA4 (Bio X Cell, USA). T-lymphocytes were obtained from tumor or lymphatic nodules (LN), analyzed by flow cytometry using a FACSAria III cell sorter, sorted and evaluated by RNA- and TCR (T cell receptor)-seq.

Results

We identified that Th СD4 subset appeared to be primary target of CTLA-4 therapy. The effect consisted of increased ratio of Th/CD8 and skew of Th cells from IFNγ-secreting towards proliferating cells within the tumor. Analysis of major lymphocyte subsets from LN revealed an increased percentage of activated CD69+/CD25+ cells from Th, CD8+ and B cells and IFNγ+ secretion by Th. We found that the number of TCR clusters significantly increased in Th after therapy. This indicates proliferation of certain Th clones, yet with unknown specificities. To identify the antigen specificity of these clones we immunized mice with melanoma peptide neoantigens and harvested T-cells from LN. Then T-cells were cocultured with APCs loaded with different melanoma peptides. We identified up to 75% CD69+/CD25+ activated T-cells to the specific peptides. We detected clones of TCR, which were found with a high frequency in cells reactivated to specific peptides and were not found in the control. Now the data set is in progress. Future experiments will be directed to compare B16F0-specific TCRs with proliferative clones after anti-CTLA4 blockade.

Conclusions

We found that anti-CTLA-4 immunotherapy leads to activation and clonal expansion of lymphocytes with similar TCRs. For detailed analysis, we developed the approach to identify the TCR specificity to specific B16F0 peptides.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

The authors.

Funding

Ministry of Science and Higher Education of the Russian Federation (grant # 14.W03.31.0005).

Disclosure

All authors have declared no conflicts of interest.