对于CALR突变（CALRmut）骨髓增生性肿瘤（MPN）患者的突变型CALR的治疗性癌症疫苗在诱导强大的抗突变CALR T细胞反应的同时未能表现出临床活性。肿瘤特异性T细胞渗入肿瘤微环境是实现临床应答的治疗性癌症疫苗所需。确定接种后，来自接种患者的CALRmut特异性T细胞是否在骨髓中富集，并探索富集缺乏的可能解释。从十名接种患者中扩增、富集和分析CALRmut特异性T细胞，并通过T细胞受体测序（TCRSeq）进行分析。所确定的T细胞受体被用作CALRmut特异性T细胞的指纹。从四名患者处获得基线和试验结束时的骨髓抽样。从骨髓抽样中富集T细胞，并通过TCRSeq进行分析，以确定两个不同时间点CALRmut特异性T细胞的存在和比例。进行了计算模拟，以计算CALRmut MPN患者中转化细胞与效应细胞之间的比率。在接种试验结束时，骨髓中的CALRmut特异性T细胞比例没有增加。一般来说，骨髓中的T细胞库通过接种试验保持相对恒定。强化和扩增的CALRmut特异性T细胞能够识别外周血自体CALRmut细胞。计算模拟分析表明，外周血中转化细胞和CALRmut特异性效应T细胞之间的比例失衡较高。治疗性癌症肽疫苗对CALRmut特异性T细胞在骨髓中富集效果不佳。特异性T细胞能够识别自体外周血源CALRmut细胞。计算模拟分析显示，存在外周血中转化细胞数量与CALRmut特异性效应T细胞数量之间的高度不平衡。我们认为，在转化细胞数量高于效应细胞数量的情况下，特异性T细胞在骨髓中富集的能力可能受到影响。版权所有 © 2023 Holmström, Andersen, Traynor, Ahmad, Lisle, Handlos Grauslund, Skov, Kjær, Ottesen, Gjerstorff, Hasselbalch and Andersen.

Therapeutic cancer vaccination against mutant calreticulin (CALR) in patients with CALR-mutant (CALRmut) myeloproliferative neoplasms (MPN) induces strong T-cell responses against mutant CALR yet fails to demonstrate clinical activity. Infiltration of tumor specific T cells into the tumor microenvironment is needed to attain a clinical response to therapeutic cancer vaccination.Determine if CALRmut specific T cells isolated from vaccinated patients enrich in the bone marrow upon completion of vaccination and explore possible explanations for the lack of enrichment.CALRmut specific T cells from four of ten vaccinated patients were expanded, enriched, and analyzed by T-cell receptor sequencing (TCRSeq). The TCRs identified were used as fingerprints of CALRmut specific T cells. Bone marrow aspirations from the four patients were acquired at baseline and at the end of trial. T cells were enriched from the bone marrow aspirations and analyzed by TCRSeq to identify the presence and fraction of CALRmut specific T cells at the two different time points. In silico calculations were performed to calculate the ratio between transformed cells and effector cells in patients with CALRmut MPN.The fraction of CALRmut specific T cells in the bone marrow did not increase upon completion of the vaccination trial. In general, the T cell repertoire in the bone marrow remains relatively constant through the vaccination trial. The enriched and expanded CALRmut specific T cells recognize peripheral blood autologous CALRmut cells. In silico analyses demonstrate a high imbalance in the fraction of CALRmut cells and CALRmut specific effector T-cells in peripheral blood.CALRmut specific T cells do not enrich in the bone marrow after therapeutic cancer peptide vaccination against mutant CALR. The specific T cells recognize autologous peripheral blood derived CALRmut cells. In silico analyses demonstrate a high imbalance between the number of transformed cells and CALRmut specific effector T-cells in the periphery. We suggest that the high burden of transformed cells in the periphery compared to the number of effector cells could impact the ability of specific T cells to enrich in the bone marrow.Copyright © 2023 Holmström, Andersen, Traynor, Ahmad, Lisle, Handlos Grauslund, Skov, Kjær, Ottesen, Gjerstorff, Hasselbalch and Andersen.