The calreticulin (CALR) exon 9 mutations are found in ∼30% of patients with essential thrombocythemia and primary myelofibrosis. Recently, we reported spontaneous immune responses against the CALR mutations. Here, we describe that CALR-mutant (CALRmut)-specific T cells are able to specifically recognize CALRmut cells. First, we established a T-cell culture specific for a CALRmut epitope. These specific T cells were able to recognize several epitopes in the CALRmut C terminus. Next, we established a CALRmut-specific CD4 T-cell clone by limiting dilution. These CD4 T cells recognized autologous CALRmut monocytes and hematopoietic stem cells, and T-cell recognition of target cells was dependent on the presence of CALR. Furthermore, we showed that the CALRmut response was human leukocyte antigen (HLA)-DR restricted. Finally, we demonstrated that the CALRmut-specific CD4 T cells, despite their phenotype, were cytotoxic to autologous CALRmut cells, and that the cytotoxicity was mediated by degranulation of the T cells. In conclusion, the CALR exon 9 mutations are targets for specific T cells and thus are promising targets for cancer immune therapy such as peptide vaccination in patients harboring CALR exon 9 mutations.

Tumor cells and tumor-infiltrating macrophages produce the chemokine CCL22, which attracts regulatory T cells (Tregs) into the tumor microenvironment, decreasing anticancer immunity. Here, we investigated the possibility of targeting CCL22-expressing cells by activating specific T cells. We analyzed the CCL22 protein signal sequence, identifying a human leukocyte antigen A2-(HLA-A2-) restricted peptide epitope, which we then used to stimulate peripheral blood mononuclear cells (PMBCs) to expand populations of CCL22-specific T cells in vitro. T cells recognizing an epitope derived from the signal-peptide of CCL22 will recognize CCL22-expressing cells even though CCL22 is secreted out of the cell. CCL22-specific T cells recognized and killed CCL22-expressing cancer cells. Furthermore, CCL22-specific T cells lysed acute monocytic leukemia cells in a CCL22 expression-dependent manner. Using the Enzyme-Linked ImmunoSPOT assay, we examined peripheral blood mononuclear cells from HLA-A2+ cancer patients and healthy volunteers for reactivity against the CCL22-derived T-cell epitope. This revealed spontaneous T-cell responses against the CCL22-derived epitope in cancer patients and in healthy donors. Finally, we performed tetramer enrichment/depletion experiments to examine the impact of HLA-A2-restricted CCL22-specific T cells on CCL22 levels among PMBCs. The addition or activation of CCL22-specific T cells decreased the CCL22 level in the microenvironment. Activating CCL22-specific T cells (e.g., by vaccination) may directly target cancer cells and tumor-associated macrophages, thereby modulating Treg recruitment into the tumor environment and augmenting anticancer immunity.

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