case_images: PMC10749197_01
case_images: Characteristics of CD5 expression on normal lymphocytes in bone marrow at diagnosis and kinetics of CAR-T cells and inflammatory cytokines post CAR-T cells infusion. (A) Evaluation of CD5 antigen expression in normal lymphocytes of bone marrow before CAR-T cell treatment. (B) Structure of anti-CD5 CAR molecular. (C) Kinetic of CAR gene in vivo by using ddPCR. (D) Kinetic of CAR-T cells and CD5 positive cells in vivo by using flowcytometry. (E) Tumor necrosis factor alpha dynamics post CAR-T cells infusion. (F) Levels of IL-6 and ferritin in serum after CAR-T cell treatment.
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case_images: Expression of the CD5 antigen on the lymphocytes of bone marrow was measured by flow cytometry, which showed that 98% of CD3 positive cells expressed CD5 antigen ( Figure 1A ). The CAR structure ( Figure 1B ), as well as the manufacturing and preparation of CAR-T cells, were described previously. Figure 1C , the copy number of the CAR-T product was 23 copies/mug DNA on day 0 and reached its peak level (88623 copies/mug DNA) on day 14. Flow cytometry was also conducted in the analysis of CAR-T cells and CD5 positive targeted cells during treatment. T cells, while the proportion of CD5+ target cells in lymphocytes began to decrease on day 1 and reached to 0.00% on day 11 ( Figure 1D ). The absolute number of white blood cells, platelets, and hemoglobin level fluctuated within the normal range ( Supplemental Figure 1 ). These results demonstrate that CAR-T cells but not for CD3+CD5+ T cells can be effectively expanded in patients, and the routine blood indicators including white cell, platelet and hemoglobin were basically normal. TNF-alpha began to increase on day 1, reached its peak level (489 pg/mL) on day 13, and gradually decreased thereafter ( Figure 1E ). Ferritin levels remained elevated throughout the treatment, with the highest level recorded on day 105 ( Figure 1F ).
case_images: Hyperplastic T cells in inguinal lymph node turned out to be expanded CAR-T cells. (A) Histopathological microphotograph (hematoxylin and eosin, 40x and 400x). Normal structure of lymph node disappeared, and no lymph sinus and lymph follicles were found. The hyperplastic T cells are large in size, rich in cytoplasm, vacuolated nuclei with obvious nucleoli, and have a certain degree of atypia. (B) Immunohistochemical analysis of cervical lymph node biopsy for CD3, CD4, CD5, CD8, CD30 and Ki67 expression. (C) Immunophenotypic analysis of the inguinal lymph node biopsy. (D) Lymohotrack IGH assays in premanufactured leukapheresis sample, peripheral blood cells (59 days post CAR-T cells infusion) and lymph node biopsy sample detected by next generation sequencing. (E) TCRB V-gene usage and Shannon Diversity for peripheral blood cells (68 days post CAR-T cells infusion) and premanufactured leukapheresis sample were detected by immune repertoire assay.
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case_images: The hyperplastic T cells displayed enlarged size, abundant cytoplasm, vacuolated nuclei with prominent nucleoli, and exhibited a certain degree of atypia, consistent with features typically associated with refractory ALCL ( Figure 2A ). TIA1, CD30 (with scattered positivity), Ki67 (80% labeling index), and negativity for CD4, CD5, CD20, EBER, and ALK ( Figure 2B  and  Supplemental Figure 2A ). This immunophenotype is inconsistent with the previously observed ALK-positive ALCL profile. Flow cytometry results from the inguinal lymph node biopsy showed that abnormal lymphocytes were negative for CD5 expression ( Figure 2C ), indicating the proficiency of anti-CD5 CAR-T cells in eliminating CD5 positive cells. Clonal rearrangement IGHA and IGHB genes were evident in both the lymph node biopsy and peripheral blood cells, but not in the premanufactured leukapheresis sample ( Figure 2D ). In contrast, in the premanufactured leukapheresis, the Shannon diversity and clone numbers were calculated to be 14.47 and 53808, respectively ( Figure 2E  and  Supplemental Figure 2C ).