case_images: PMC9310037_01
case_images: Flow cytometry of cells isolated from fine needle aspiration of the patient's left prescapular lymph node and stained for CD5-PE, CD79a-APC and CD20-BV421 as previously described.
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case_images: F1
case_images: Flow cytometry at this time demonstrated preserved CD20 expression by the neoplastic cells (Figure 1), confirming eligibility for autologous CD20-specific CAR-T cell therapy.
case_images: Flow cytometry of CAR-T cell product 12 days after T cell activation. Gated on live, CD5+ cells that were stained for CAR using a biotinylated rabbit anti-mouse IgG H + L antibody and isotype control antibody with a secondary APC-streptavidin conjugate as previously described.
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case_images: F2
case_images: T cells, cells were washed and resuspended in Plasma-Lyte A (Baxter). Transduction was quantified flow cytometrically (Figure 2) using previously described protocols and a dose of ~5 x106 CAR-T/kg (a total of ~50 x106 total T cells/kg) of patient's body weight was attained, this represented a ten-fold increase in CAR-T dose compared to our previously published study.
case_images: Clinical summary of patient's response to CAR-T therapy. Therapeutic timeline (A); monitoring of body temperature and in vivo CAR-T cell levels (B); tracking of target node dimensions (C); circulating white blood cell and neutrophil counts (D); circulating lymphocyte and monocyte counts (E); tracking of circulating T and B cell counts determined by the product of the frequencies CD5+ or CD79a+ live cells in the lymphogate quantified flow cytometrically, and the number of circulating lymphocytes obtained by complete blood count (F) and flow cytometry performed on PBMCs stained for CD5-PE and CD79a-APC at day-14 and day 7 post-CAR-T (G). WPRE- woodchuck hepatitis virus post-transcriptional regulatory element, used for detection of genomic copies of CAR by qRT-PCR as previously described; CR- complete response.
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case_images: F3
case_images: Four days prior to CAR-T cell infusion the patient received 500 mg/m2 cyclophosphamide (Baxter) intravenously for lymphodepletion (Figure 3A). On the day of CAR-T cell infusion (Figure 3A) the patient received intramuscular diphenhydramine (Hikma) (2 mg/kg) and intravenous ondansetron (West Ward) (0.2 mg/kg) premedication followed by slow intravenous CAR-T cell infusion. The patient tolerated the infusion well and remained hemodynamically stable throughout the infusion and during the post-infusion observation period after which he was discharged for planned routine follow up (Figure 3A). Physical examination confirmed pyrexia at 105.6 F (Figure 3B), and a moderate tachycardia (160 BPM) with accompanying hyperdynamic pulses noted. Lymph nodes were small and soft on palpation (Figure 3C). Complete blood counts revealed grade 1 non/pre-regenerative anemia (HCT 39.7%), a grade 4 neutropenia (0.03 x103/muL), a grade 2 thrombocytopenia (53 x 3/muL), a moderate lymphopenia (0.48 x 3/muL), a monocytopenia (0.09 x 3/muL) and eosinopenia (0.01 x 3/muL) (Figures 3D,E). Both the red cell and platelet counts normalized over the following 2 weeks, body temperature (Figure 3B) and hemodynamic status remained within normal limits. The patient's lymph nodes were noted to be mildly enlarged 14 days post-CAR-T and this was progressive leading to the prescription of a CCNU based rescue regimen 27 days post-CAR-T (Figures 3A,C). A non-durable partial response to the rescue regimen was documented (Figure 3C). Flow cytometry performed on peripheral blood as described previously revealed a depletion of circulating B cells temporally coinciding with normal peripheral nodal measurements (Figures 3C,F,G). At disease relapse numbers of both circulating B and T cells increased (Figure 3F). Notably CAR-T cell levels peaked 3 days post-CAR-T administration and at the time of documentation of pyrexia (Figure 3B). Following peak peripheral CAR-T cell signal at day 3, circulating CAR-T cell numbers contracted and were below the limit of detection on day 14 at which point the lymph nodes were increasing in dimension consistent with relapse (Figures 3B,C).
case_images: Serial multiplexed analysis of serum cytokines. Dashed lines represent lower limits of detection, dashed lines are superimposed with x-axes for those plots where they are not visible.
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case_images: F4
case_images: Serum IL-6, MCP-1 and KC-like all increased and peaked in concentration at day 3 post-CAR-T cell administration prior to decreasing to varying degrees (Figure 4). Serum IFNgamma, IL-10, GM-CSF, IL-2, IL-15, IL-7, and IL-18 all increased and peaked in concentration at day 7 post-CAR-T prior to decreasing (Figure 4). Serum IL-8 initially decreased at day 3 following CAR-T prior to returning to pre-CAR-T concentration and subsequently contracting (Figure 4). CAR-T and was then detected just above the limit of detection from day 7 onwards (Figure 4) and TNFalpha was below the limit of detection at all time points (data not shown). Importantly, all changes in serum cytokine levels occurred at the time of, or immediately after peak CAR-T levels in the blood and coincided with B cell depletion and normal peripheral nodal diameters. Collectively, these findings, together with the clinical signs exhibited by the patient at day 3 post-CAR-T infusion, were consistent with grade 2 CRS as per the VCOG-CTCAE v2 scheme.