Aris, Mariana Bravo, Alicia Inés Alvarez, Heli Magalí Garcia Carri, Ibel Podaza, Enrique Blanco, Paula Alejandra Rotondaro, Cecilia Bentivegna, Sofia Nielsen, Morten Barrio, María Marcela Mordoh, José Image_3_Immunization With the CSF-470 Vaccine Plus BCG and rhGM-CSF Induced in a Cutaneous Melanoma Patient a TCRβ Repertoire Found at Vaccination Site and Tumor Infiltrating Lymphocytes That Persisted in Blood.pdf <p>The CSF-470 cellular vaccine plus BCG and rhGM-CSF increased distant metastases-free survival in cutaneous melanoma patients stages IIB-IIC-III relative to medium dose IFN-α2b (CASVAC-0401 study). Patient-045 developed a mature vaccination site (VAC-SITE) and a regional cutaneous metastasis (C-MTS), which were excised during the protocol, remaining disease-free 36 months from vaccination start. CDR3-TCRβ repertoire sequencing in PBMC and tissue samples, along with skin-DTH score and IFN-γ ELISPOT assay, were performed to analyze the T-cell immune response dynamics throughout the immunization protocol. Histopathological analysis of the VAC-SITE revealed a highly-inflamed granulomatous structure encircled by CD11c<sup>+</sup> nested-clusters, brisk CD8<sup>+</sup> and scarce FOXP3<sup>+</sup>, lymphocytes with numerous Langhans multinucleated-giant-cells and macrophages. A large tumor-regression area fulfilled the C-MTS with brisk lymphocyte infiltration, mainly composed of CD8<sup>+</sup>PD1<sup>+</sup> T-cells, CD20<sup>+</sup> B-cells, and scarce FOXP3<sup>+</sup> cells. Increasing DTH score and IFN-γ ELISPOT assay signal against the CSF-470 vaccine-lysate was evidenced throughout immunization. TCRβ repertoire analysis revealed for the first time the presence of common clonotypes between a VAC-SITE and a C-MTS; most of them persisted in blood by the end of the immunization protocol. In vitro boost with vaccine-lysate revealed the expansion of persistent clones that infiltrated the VAC-SITE and/or the C-MTS; other persistent clones expanded in the patient's blood as well. We propose that expansion of such persistent clonotypes might derive from two different although complementary mechanisms: the proliferation of specific clones as well as the expansion of redundant clones, which increased the number of nucleotide rearrangements per clonotype, suggesting a functional antigenic selection. In this patient, immunization with the CSF-470 vaccine plus BCG and rhGM-CSF induced a T-cell repertoire at the VAC-SITE that was able to infiltrate an emerging C-MTS, which resulted in the expansion of a T-cell repertoire that persisted in blood by the end of the 2-year treatment.</p> cutaneous melanoma;CSF-470 vaccine;vaccination site;cutaneous metastasis;tumor infiltrating lymphocytes;TCRβ repertoire 2019-09-18
    https://frontiersin.figshare.com/articles/figure/Image_3_Immunization_With_the_CSF-470_Vaccine_Plus_BCG_and_rhGM-CSF_Induced_in_a_Cutaneous_Melanoma_Patient_a_TCR_Repertoire_Found_at_Vaccination_Site_and_Tumor_Infiltrating_Lymphocytes_That_Persisted_in_Blood_pdf/9874187
10.3389/fimmu.2019.02213.s004