WHAT IS ALREADY KNOWN ON THIS TOPIC

The manufacturing of autologous chimeric antigen receptor (CAR)-T cells is subject to several limitations, including failure rates, extended manufacturing times, complex logistical challenges, variability in both the starting materials and final products, high costs, and the potential for lower quality CAR-T cells due to the influence of the patient’s disease and prior chemotherapy treatments.

Clinical trials and animal studies indicate that optimizing the frequency of naïve and stem cell memory T cells in CAR-T cells leads to better clinical response and persistence.

Allogeneic, off-the-shelf CAR-T products may offer an alternative option. However, the source of T cells may introduce heterogeneity among CAR-T cell products. Here, we compare healthy adult peripheral blood T cells to placental circulating T cells for the generation of CAR-T drug products.

WHAT THIS STUDY ADDS

CAR-T derived from placental circulating T cells possess a transcriptomic signature consistent with a less differentiated T cell, which is more resistant to exhaustion than CAR-T derived from healthy, adult peripheral blood mononuclear cells (PBMCs).

CAR-T derived from placental circulating T cells have a more favorable cytokine profile with interleukin 2 (IL-2)-skewed naïve helper T cells, that favors IL-2 over interferon gamma secretion, which may lower the potential for toxicity.

CAR-T derived from placental circulating T cells demonstrate improved and prolonged in vivo efficacy and persistence compared with healthy, adult PBMC derived CAR-T.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

This study presents a novel, allogeneic T cell platform which enables large scale manufacturing of off-the-shelf CAR-T cells with homogeneous and preserved T-cell stemness to enhance efficacy and improve accessibility of cell therapy to patients.

Introduction

Immunotherapy using autologous gene-modified T cells expressing a chimeric antigen receptor (CAR) has changed the lymphoma treatment landscape, achieving complete remissions in heavily pretreated refractory patients.1 However, a number of patients fail to respond or relapse following CAR-T therapy. This may be due to inferior quality of CAR-T cells, variability of starting material and/or T-cell exhaustion from resistant disease.2 Since autologous T cell starting material across patients with cancer is variable and CAR-T expansion or potency can fail during manufacture, strategies to identify and expand the T cell subpopulations that correspond with CAR-T potency are an active area of investigation. Defining T cell specifications that improve successful CAR-T manufacture and maximize clinical efficacy is a shared goal in both the autologous and allogeneic CAR-T spaces.

T cell stemness, characterized by the ability for self-renewal, multipotency across various T cell lineages, and long-lasting persistence, plays a crucial role in the establishment of durable T cell memory throughout an individual’s lifespan.3 4 Early CAR-T studies revealed that in contrast to T effector (TE) and T effector memory (TEM) populations, CAR-T derived from CD8+ central memory (TCM) T cells were most effective in the presence of naïve (TN) or TCM CD4+ T cell help.5 Within the memory population, a less differentiated T stem cell memory (TSCM) population possesses even greater antitumor activity.4 6 Because TN and TSCM cells in adult peripheral blood mononuclear cells (PBMCs) are less abundant, additional efforts to enrich TSCM or TCM populations during CAR-T manufacture have been performed through selection and/or use of cytokines.7–9 Preclinical models have demonstrated the antitumor benefit of TSCM-enriched CAR-T cells compared with non-enriched cells.9 10 However, the scalability and reproducibility of autologous TSCM expansion across patients and indications can have some limitations.11 Additionally, patients with cancer have a reduced TSCM compartment compared with healthy donors and often have depleted T cell counts due to prior chemotherapy and radiation.12 The remaining T cells manifest higher rates of cell exhaustion, compelling the field to find robust alternatives to autologous therapy. Allogeneic CAR-T approaches can better address product consistency and provide immediate availability for patients as an “off-the-shelf” therapeutic.

While allogeneic T cells from healthy adult peripheral blood are currently being used, T cells from postpartum placenta/umbilical cord may offer potential phenotypical and functional advantages as a CAR-T cell drug product.13 Cord blood derived T cells have shown promise as the starting material for allogeneic CAR-T cell therapy.14 15 However, a direct comparison of adult peripheral blood T cells to placental circulating T (P-T) cells for CAR-T has not been adequately investigated and is the objective of the present study. In this study, we show that blood from the placenta and umbilical cord are a rich source of T cells whose TN enrichment could prove advantageous to CAR-T manufacture. While CAR-T manufactured from both cell sources demonstrate comparable in vitro CD19-directed cytotoxicity, P-T derived CD19 CAR-T cells (P-T CD19 CAR-T) resist activation-induced maturation and immune checkpoint expression during manufacture. We further show that P-T CD19 CAR-T maintains a CD4 T cell population which favors interleukin 2 (IL-2) over interferon gamma (IFN-γ) secretion. Transcriptome analysis of CAR-T drug product derived from P-T cells and adult PBMCs revealed a diverging CAR-T identity, with cells derived from the placenta displaying some clear phenotypical advantages.16 P-T CD19 CAR-T had attenuated cytokine and chemokine activation, naïve T cell identity, an early memory phenotype, and a unique CD4 T cell signature. Conversely, adult PBMC-CD19 CAR-T cells were enriched for exhaustion and stimulated memory T cell signatures. In line with these phenotypical advantages, P-T CD19 CAR-T cells demonstrated superior and more durable CD19+ lymphoma control in a lymphoma NSG mouse model. Altogether, our findings demonstrate that placental circulating T cells are a promising source for allogeneic CAR-T cell therapy and are enriched for stemness attributes which benefited their in vivo persistence and long-term durable activity.

MethodsT cell isolation

PBMCs were isolated from consenting healthy donor whole blood using Ficoll gradient centrifugation (donors age 49.3±9.9 years). Placental units (collected within <48 hours postdelivery of normal, healthy, full-term pregnancy) were obtained from consenting mothers. The circulating cells collected from the placental units (placenta and umbilical cord blood) were washed using spinning membrane filtration (Lovo). Mononuclear cells were depleted of monocytes and regulatory T cells (Tregs) using Miltenyi CD14 and CD25 microbeads using CliniMACS (Miltenyi) magnetic bead-based separation or LS columns. Cell fractions were then positively selected for T cells using CD4 and CD8 microbead selection. Eluted cells were frozen using controlled-rate cell freezing, then cryopreserved in liquid nitrogen vapor phase.

CD19 CAR-T cell generation

Isolated T cells were activated using 1% TransAct (Miltenyi) and expanded for 3 days in G-Rex plates (Wilson Wolf) containing AIM-V medium (Gibco) supplemented with 5% human serum (Millipore Sigma) and 100 IU/mL of IL-2 (Life Technologies). Transduction with CD19 CAR gene-expressing retrovirus was performed on day 3 in RetroNectin (TaKara Biotech)-coated plates. The CD19 CAR construct contained the anti-CD19 FMC63-based scFv, a hybrid CD8a/CD28 hinge region, a CD28 transmembrane domain, a CD28 costimulatory domain, and the CD3z signaling domain. Transduced cells were expanded from day 4 through day 13. On day 7, T cells underwent CRISPR-Cas9-mediated T-cell Receptor Alpha Chain constant (TRAC) knock-out using the MaxCyte ATx electroporation system, followed by depletion of T-cell receptor (TCR)-expressing cells using TCR α/β microbeads (Miltenyi) on day 12. Cells were cryopreserved on day 13 and stored in liquid nitrogen vapor phase. For in vitro functional experiments, CD19 CAR-T cells were thawed, then recovered for 24 hours in AIM-V complete media at 37°C, 5% CO2.

Cytotoxicity assay

Electrical impedance monitoring using xCELLigence (ACEA Biosciences) measured real-time target cell index for determining cytotoxicity. Daudi cells were immobilized to E-Plates (Agilent Technology) coated with anti-CD40 (Agilent Technology). Recovered CAR-T cells were added to Daudi cells at defined effector-to-target (E:T) ratios in RPMI 1640 media with 10% fetal bovine serum incubating at 37°C, 5% CO2. Each donor was analyzed in duplicate, measuring percent cytotoxicity at 4, 8, 12, and 24 hours of coculture time.

Cytokine release assay

Recovered CAR-T cells were cocultured with Daudi cells at 1:1 E:T ratio in duplicate in RPMI 1640 media with 10% fetal bovine serum for 24 hours at 37°C, 5% CO2. Collected supernatant was analyzed for IL-2, IFN-γ, and tumor necrosis factor alpha (TNF-α) concentration using the MSD (Meso Scale Diagnostics) platform according to manufacturer’s protocol. Data were analyzed using MSD Discovery Workbench V.4.0 software.

Telomere length measurement

Cell telomere length was measured using Telomere PNA Kit (Dako) according to manufacturer’s protocol. The 1301 cell line (Sigma-Aldrich) was used as a control for intra-assay and interassay variability. Quantum Molecules of Equivalent Soluble Fluorochrome (Bangs Laboratories) beads were used for the standardization of fluorescence intensity units to perform absolute fluorescence quantification.

In vivo model

To assess efficacy in vivo, P-T and PBMC-derived CD19 CAR-T cells (both without TRAC knockout) were evaluated in a disseminated Daudi (Burkitt’s) lymphoma xenograft model in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (Jackson Laboratory). In vivo studies were conducted at a contract research organization, Invivotek LLC, where fifteen 9-week-old female mice were housed together in cages and acclimated for 2 weeks. Based on previous studies, five experimental mice per group were used to determine statistical significance of treatment effect on group survival. There was no exclusion of experimental mice. Study personnel administering treatments to mice were aware of groups receiving vehicle control versus CAR-T cells but had no prior knowledge of key differences between the cell types administered.

All mice were preconditioned with a 30 mg/kg dose of busulfan 7 days prior to Daudi inoculation. On day 0, mice received a 3×106 dose of luciferase-labeled Daudi (Daudi-luc+) cells via intravenous administration. Seven days post-tumor cell inoculation, mice were randomized based on body weight and treated with vehicle (n=5; control group), 3×106 PBMC-CD19 CAR+-T (n=5), or 3×106 P-T CD19 CAR-T (n=5) cells by intravenous injection. CAR-T antitumor efficacy was monitored via bioluminescence imaging and survival. On day 122, surviving mice (n=5) were rechallenged with 3×106 Daudi-luc+ cells via intravenous administration. Age-matched NSG mice (n=5) served as controls and were injected with the same dose of Daudi-luc+ cells. Blood, spleen, and bone marrow samples were collected on days 185–215 from the remaining mice (n=4) and analyzed by flow cytometry for the presence and phenotype of persisting CAR-T cells. This study was repeated with additional CAR-T donors (n=5 mice per treatment group). An additional animal study was performed to compare P-T CD19 CAR-KO (with TRAC knockout; n=5) versus P-T CD19 CAR-NT (without TRAC knockout; n=5) with similar conditions as those described earlier with the exception that 6×106 CAR-T cells were infused into mice.

Statistical analysis

Statistical analysis was performed using GraphPad Prism V.9.0.2 (GraphPad). Unpaired two-tail t-test calculations were used for comparisons between placental circulating and PBMC-derived T cells. Comparisons of multiple groups were performed using one-way analysis of variance with Dunnett post-hoc test. A log-rank (Mantel-Cox) test was used to compare survival between different groups of mice. Data are represented as mean±SD or SEM as indicated in each figure’s legend. The significance level for all statistical tests was set at p=0.05.

All reagent details are listed in online supplemental table 2.

ResultsPlacental circulating T cells are enriched for CD45RA+CCR7+ TN/TSCM

CD4/CD8 T cell ratios have been reported to impact CAR-T performance5 17 18 leading us to assess any differences in the composition of P-T versus adult PBMC-T cells. P-T and PBMC-T showed an equivalent 2:1 ratio of CD4+ T cells to CD8+ T cells among total CD3+ T cells (figure 1A,B). We next compared the T cell subset composition of adult PBMCs to the post partum placenta. Low percentage (<1.2%) of γδ T cells+ was detected in both PBMC-T and P-T starting materials (online supplemental figure 2). CD45RA, CCR7, and CD27 staining by flow cytometry distinguished effector and memory T cells from naïve and stem cell memory subsets. Using CD45RA and CCR7 to identify TN/TSCM, P-T cells were >90% enriched compared with approximately 40% among PBMC-T cells (figure 1C,D). CD27, a lymphocyte costimulatory molecule, was uniformly expressed at high levels among all P-T cells (figure 1E,F) compared with PBMC-T (86±15.4 vs 99.8±0.17%) consistent with their identity as TN/TSCM as previously reported.19 The CD4:CD8 ratio did not vary between multiple evaluated donors (figure 1B) while the observed distribution of T cell differentiation status between P-T and PBMC-T was statistically significant (figure 1D). PBMC CD4+ T cells were comprised of a mix of TN/TSCM (CD45RA+CCR7+), TCM (CD45RA−CCR7+), TEM (CD45RA−CCR7−) cells, with limited TE (CD45RA+CCR7−) population, while CD8+ T cells contained all four populations. A clear difference was seen in the P-T cells where all cells, whether CD4+ or CD8+ T cells, were TN/TSCM. This distinction within the starting material highlights how differences in T cell subpopulation distribution may influence T cell expansion and phenotype during CAR-T expansion. CD57, a glycan whose expression is associated with differentiated effector memory T cell subsets20 and associated with aging21 was expressed in PBMC-T and no expression was observed in P-T cells (10.6±9.5 vs 0.09±0.05%) (figure 1G,H). Higher expression of exhaustion markers including PD-1 (26.6±4 vs 7.2±3.2) and TIGIT (17.7±11.63 vs 1.2±0.8) was observed in PBMC-T compared with P-T, while no difference was observed in LAG3 and TIM-3 (figure 1I,J).

Figure 1

Placental circulating T cell donor stocks are enriched for naïve/stem cell memory T cells compared with adult derived PBMC T cells. (A) Representative pseudocolor plots of CD4+ and CD8+ T cell distribution among CD56−CD3+ lymphocytes present in P-T and PBMC-T donor stocks. (B) Comparison of CD4+ and CD8+ T cell distribution among different PBMC donors (black, n=6) and P-T donors (green, n=9). (C) Representative pseudocolor plots of CD45RA and CCR7 T cell staining among CD56−CD3+CD4+ lymphocytes present in P-T and PBMC T donor stocks. (D) Comparison of TN/SCM, TCM, TEM, and TE subset distribution among multiple PBMC-T (black) and P-T (green) cell donors based on CD45RA and CCR7 staining. (E) Representative histograms of CD27 expression on total T cells from PBMC-T and P-T donor stocks. (F) Comparison of CD27 expression on total T cells within PBMC-T (black) and P-T (green) donor stocks. (G) Representative pseudocolor plots of CD57 expression on total T cells from PBMC-T and P-T donor stocks. (H) Comparison of CD57 expression on total T cells within PBMC-T (black) and P-T (green) donor stocks. (I) Representative pseudocolor plots of PD-1 and TIM-3 expression and representative histogram of TIGIT expression on total T cells. (J) Comparison of exhaustion markers PD-1, TIM-3, LAG3, and TIGIT expression on total T cells within PBMC-T (black) and P-T (green) donor stocks. Numbers indicate the percentage of cells in each gate. Individual data points shown with mean value±SD. Unpaired Student’s t-test was performed for all statistical analyses. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. PBMCs, peripheral blood mononuclear cells; P-T, placental circulating T cells.

CD19 CAR-T generated from placental circulating T cells retain stemness characteristics

We next evaluated CAR-T cells postexpansion as T cell phenotype may shift following activation, CAR transduction, and cell proliferation. Figure 2 depicts the P-T CD19 CAR-T manufacturing process. The 1:1 ratio of CD4/CD8 CAR-T cells has been shown to confer superior antitumor reactivity in vivo, indicating the synergistic antitumor effects of the two subsets.5 18 22 While P-T CD19 CAR-T maintained a more balanced ratio of CD4+ and CD8+ T cells, adult PBMC-CD19 CAR-T preferentially expanded CD8+ T cells (figure 3A). CD19 CAR transduction efficiency was similar between the two groups (figure 3B). P-T CD19 CAR-T cells retained longer telomeres compared with PBMC-CD19 CAR-T postexpansion (9.1±1.0% vs 15.2±1.3%) (figure 3C), suggesting better T cell proliferative potential and possibly better telomerase activity during T cell activation.23 24 T cell subset (TSCM, TCM, TEM, TE) evaluation of CAR-T products by flow cytometry yielded similar phenotype distribution for both CD4+ and CD8+ T cells in PBMC-CD19 CAR-T and P-T CD19 CAR-T due to IL-2 stimulation and activation-induced CCR7 downregulation (online supplemental figure 3).25 Loss of CD27 has been associated with effector cell differentiation among CD4+ and CD8+ T cells.26 27 Consistent with the profile of P-T starting material, high levels of CD27 costimulatory molecule expression were maintained on P-T CD19 CAR-T (76.8±13%), while CD27 expression was reduced among PBMC-CD19 CAR-T (40.3±16.1%) (figure 3D). CD57 expression was expressed at a significantly lower frequency on P-T CD19 CAR-T cells compared with PBMC-CD19 CAR-T (30.3±11.3% vs 13.1±7.1%) (figure 3E), suggesting that placental T cells better maintain a naïve-like T cell state post-CAR-T expansion. T cell differentiation involving T-bet and EOMES is responsible for effector and memory cell maturation.28 Expression of both transcription factors was significantly diminished in P-T CD19 CAR-T cells compared with PBMC-CD19 CAR-T (EOMES: 44.5±16.9 vs 10.6±12.6 and T-bet: 69.6±6.5 vs 60.1±7.6) (figure 3F). Expression of costimulatory molecule GITR, which supports proliferation and T cell activation by inducing IL2R and IL-2,29 was increased in P-T CD19 CAR-T compared with PBMC-CD19 CAR-T (53.4±12 vs 82.6±8.5) (figure 3G). Immune checkpoint molecules PD-1, TIM3, LAG3, and TIGIT are negative regulators that dampen T cell activation.30 PD-1 and TIM3 were also induced on T cells following TCR-mediated activation and in response to common γ-chain cytokines including IL-2, respectively.30 31 P-T CD19 CAR-T expressed reduced levels of LAG3 (9.7±5.6 vs 0.5±0.2%) and TIGIT (47.4±9.5 vs 13.2±2.7%) compared with PBMC-CD19 CAR-T cells, which were increased in PBMC-CD19 CAR-T (figure 3H,I). While no difference was observed in TIM-3, PD-1 was elevated among P-T CD19 CAR-T (27.1±4.2 vs 49.5±6.0%). These data collectively demonstrate improved maintenance of a less differentiated phenotype among placental circulating T cells following the genetic engineering and expansion of CAR-T cells.

Figure 2

P-T CD19 CAR-T manufacturing scheme. Placental circulating cells are collected at the hospital from normal, healthy, full-term pregnancy then shipped to the manufacturing facility. In upstream manufacturing, ≤48 hours postdelivery placental circulating cells are processed using the Lovo automated system followed by monocyte and regulatory T cell depletion and CD4+CD8+ T cells enrichment. The starting material T cells are then cryopreserved. In downstream manufacturing, isolated placental T cells are activated and expanded for 3 days then transduced with CD19 CAR retrovirus. On day 7, T-cell Receptor Alpha Chain Constant (TRAC) is knocked-out and the cells are allowed to expand for an additional 5 days. On day 12 any residual TCR-expressing cells are depleted, and the cells are left to expand for an additional day. On day 13 P-T CD19 CAR-T cells are cryopreserved and stored in liquid nitrogen vapor phase. CAR, chimeric antigen receptor; P-T, placental circulating T cells; TCR, T-cell receptor.

Figure 3

P-T CD19 CAR-T cells better retain stemness markers than PBMC-CD19 CAR-T cells following culture expansion. (A) CD4+ and CD8+ T cell frequencies in post-thaw PBMC-CD19 CAR-T (black, n=6 donors) and P-T CD19 CAR-T (green, n=9 donors) drug product. (B) Representative pseudocolor plots and bar graphs of CD19 CAR expression on post-thaw PBMC-CD19 CAR-T (black) and P-T CD19 CAR-T (green) drug product. (C) Relative telomere length of total cells post-thaw of PBMC-CD19 CAR-T (black) and P-T CD19 CAR-T (green) cell products. (D) Representative histograms and frequencies of CD27+ in total CD5+CAR+ T cells of PBMC-CD19 CAR-T (black) and P-T CD19 CAR-T (green) products. (E) Representative pseudocolor plots and frequencies of CD57+ in CAR+ T cells of PBMC-CD19 CAR-T (black) and P-T CD19 CAR-T (green) products. (F) Intracellular staining of transcription factors EOMES and T-bet on CAR+ T cells from PBMC-CD19 CAR-T (black) and P-T CD19 CAR-T (green). (G) Surface staining of immunoregulatory receptors GITR and CD25, and from PBMC-CD19 CAR-T (black) and P-T CD19 CAR-T (green) products. (H) Representative pseudocolor plots or histograms of exhaustion markers expression PD-1, TIM3, LAG3, and TIGIT on CAR+ T cells from PBMC-CD19 CAR-T (black) and P-T CD19 CAR-T (green) (I) Frequencies of exhaustion markers expression PD-1, TIM3, LAG3, and TIGIT on CAR+ T cells from PBMC-CD19 CAR-T (black) and P-T CD19 CAR-T. Individual data points shown with mean value±SD. Unpaired Student’s t-test was performed for all statistical analyses. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. CAR, chimeric antigen receptor; PBMCs, peripheral blood mononuclear cells; P-T, placental circulating T cells; TCR, T-cell receptor.

Recent publications have shown that TCR knockout may negatively impact persistence and phenotype of CAR-T cells,32 33 however, we did not observe phenotypic differences between CD19 CAR-T cells with intact TCR (CAR-NT) and TCR knockout cells (CAR-KO) in either PBMC-T or P-T CD19 CAR-T cells including the expression of exhaustion and activation markers (CD57, PD-1, TIM-3, LAG-3, TIGIT, EOMES, CD25) or T cell differentiation status (online supplemental figure 4). Moreover, we used an immunodeficient murine model to compare P-T CD19 CAR-T with intact (CAR-NT) or with deletion of TCR (CAR-KO). NSG mice were preconditioned with Busulfan, followed by Luciferase-expressing Daudi lymphoma inoculation 1 week later. P-T CD19 CAR-T cells with or without TCR deletion were administered intravenously after another week, and mice were monitored for tumor burden using bioluminescence imaging and survival over time. Similar survival was observed between groups indicating that the deletion of TCR does not impact P-T CD19 CAR-T efficacy or CAR-T cells fitness (online supplemental figure 4).

Transcriptional profiling of CAR-T products identifies distinct differentiation and inflammatory pathways associated with placental starting material

The significant phenotypic differences that were observed between PBMC-CD19 CAR-T and P-T CD19 CAR-T despite using identical expansion protocols prompted us to analyze the transcriptomic signatures to better capture distinct product characteristics. Full list of differentially expressed genes (DEGs) and normalized counts are shown in online supplemental tables 3 and 4. Next generation RNA sequencing (RNA-seq) data generated from PBMC-CD19 CAR-T (n=6) and P-T CD19 CAR-T (n=7) donors16 clustered in divergent populations corresponding to starting material source following principal component analysis (figure 4A). When comparing DEGs between PBMC-CD19 CAR-T and P-T CD19 CAR-T cohorts, significant differences between many gene targets were observed (figure 4B). Notably, the greatest differences in gene expression were in genes associated with immune function. PBMC-CD19 CAR-T enriched genes associated with the Th17 axis (IL17A, IL17F, IL21, IL22, IL26), inflammation (IFNG, IFNg, IL3, IL5, GZMB, GZMH), chemokines (CCL1, CCL4, CCL3, CXCL2, CXCL8, CXCL10), and immune checkpoints (TIGIT, LAG3) (figure 4B,C, online supplemental figure 5 and table 6). P-T CD19 CAR-T cells were enriched for TLR2, FCER1G, SYK, and IKZF2 (HELIOS) (figure 4B), all genes implicated in T cell activation and maturation state.34–36 P-T CD19 CAR-T also expressed KIT, which is typically expressed in hematopoietic stem cells and progenitor cells but not among adult T cells.37 Unbiased cluster analysis confirmed that DEGs were most strongly associated with starting material source among all donors evaluated (figure 4C, online supplemental figure 5 and table 6). Consistent with reduced IFN-γ levels, STAT1, JAK2, IFN-γ pathways were all significantly reduced in P-T CD19 CAR-T compared with PBMC-CD19 CAR-T donors (figure 4D,E and online supplemental figure 6). Gene signature sets for naïve/stemness were assessed for gene enrichment showing preservation of T-cell stemness in P-T CD19 CAR-T compared with PBMC-CD19 CAR-T cells (online supplemental table 5 and figures 7 and 8). Transcription factors EOMES andTBX21 (T-bet), as well as checkpoint molecules TIGIT and LAG3 were significantly reduced among P-T CD19 CAR-T (figure 4F,G) confirming protein data we previously observed.

Figure 4

Transcriptional profiles of CAR-T cellular products identify distinct differentiation and inflammatory pathways associated with neonatal starting material. (A) Principal component analysis of PBMC-CD19 CAR-T (black, n=6 donors) and P-T CD19 CAR-T (green, n=7 donors) and based on differentially expressed genes (DEGs). (B) Volcano plot showing DEGs of P-T CD19 CAR-T versus PBMC-CD19 CAR-T. Genes with p value <0.05 and gene fold change ≥1.5 are marked in black with genes not meeting the cut-off shown in gray. Annotated genes of interest are highlighted in red. (C) Clustering heat map of the top 100 DEGs between PBMC-CD19 CAR-T versus P-T CD19 CAR-T using a cut-off z score=3. Gene list is shown in online supplemental table 6 and online supplemental figure 5. (D) IFN-γ signaling pathway gene counts of IFNG, STAT1, and JAK2 in PBMC-CD19 CAR-T in comparison to P-T CD19 CAR-T. (E) Single sample (ss)GSEA score summarizing IFN-γ signaling signatures between PBMC-CD19 CAR-T versus P-T CD19 CAR-T. (F) EOMES and TBX21 (T-bet) gene counts in PBMC-CD19 CAR-T in comparison to P-T CD19 CAR-T. (G) Gene counts of exhaustion markers in PBMC-CD19 CAR-T in comparison to P-T CD19 CAR-T. (H) Heat map of selected GSEA pathways enriched significantly upregulated or downregulated in PBMC-CD19 CAR-T versus P-T CD19 CAR-T. For each pathway, a single sample enrichment score was calculated and the mean score was calculated per group. The color indicates pathways enriched in induced (red) or repressed (blue) genes. (I) ssGSEA scores from pathways in (H) for each donor replicate between PBMC-CD19 CAR-T and P-T CD19 CAR-T For normalized gene counts, DEG p-adjusted value is shown. Wilcoxon signed-rank test was performed in E and G. *p<0.05, **p<0.01, ***p<0.001. CAR, chimeric antigen receptor; GSEA, gene set enrichment analysis; IFN-γ, interferon gamma; PBMCs, peripheral blood mononuclear cells; P-T, placental circulating T cells; TCR, T-cell receptor.

A recent study revealed gene signature differences in CAR-T cells from chronic lymphocytic leukemia (CLL) patients classified as responders (complete remission and partial response with transformed disease) versus non-responders38 according to CLL guidelines.39 Single sample (ss) gene set enrichment analysis revealed that P-T CD19 CAR-T product was enriched for genes upregulated in autologous CAR-T drug product from responding patients (figure 4H,I). These P-T CD19 CAR-T enriched gene signatures are involved in early memory formation, naïve, resting CD4+ T cells, and central memory subsets (figure 4H,I, online supplemental figures 7 and 8 and table 5). In contrast, PBMC-CD19 CAR-T product was enriched for gene signatures involved in immune exhaustion, stimulated, and effector memory T cell subsets (figure 4H,I, online supplemental figures 7 and 8 and table 5). These data collectively show the naïve/stem cell memory gene signature of P-T CD19 CAR-T cells and the potentially exhausted and inflammatory nature of PBMC-CD19 CAR-T cells.

P-T CD19 CAR-T cells are cytotoxic but maintain an IFN-γ-deficient CD4 T cell population

Due to the diverging phenotype of PBMC-CD19 CAR-T versus P-T CD19 CAR-T products, CD19-specific cytotoxicity and cytokine effector potential required evaluation. Following product expansion and cryopreservation, thaw-recovered P-T CD19 CAR-T cells demonstrated comparable cytotoxicity kinetics and activity as PBMC-CD19 CAR-T against CD19+ Daudi lymphoma (figure 5A). Minimal background killing was observed using controls generated in the absence of CD19 CAR transduction indicating CAR-directed antigen specificity. Following coculture with Daudi lymphoma, IFN-γ and TNF-α levels were significantly elevated in cultures containing PBMC-CD19 CAR-T cells when compared with cocultures with P-T CD19 CAR-T cells (IFN-γ: 261.2±78.4 vs 114.2±66.0 ng/mL and TNF-α: 4.45±2.0 vs 2.6±0.86 ng/mL) (figure 5B). Both P-T CD19 CAR-T and PBMC-CD19 CAR-T secreted similar levels of IL-2 (figure 5B). To determine if differential cytokine production is isolated to CD4+ or CD8+ T cells, CAR-T cells were stimulated with PMA-ionomycin in the presence of Golgi transport inhibition (figure 5C–E). Intracellular cytokine staining showed that TNF-α was universally produced by CD4+ and CD8+ T cells, indicating similar activation between P-T CD19 CAR-T and PBMC-CD19 CAR-T. CD4+ and CD8+ PBMC-CD19 CAR-T cells strongly coexpressed IFN-γ, while IFN-γ was largely absent among CD4+ P-T CD19 CAR-T (29.7±12.3% vs 4.2±1.5%) but instead favored IL-2 production (73±8.1% vs 91.2±4.5%) (figure 5C–E). These dynamics were not observed among the CD8+ subset. Across multiple donors, the differences in the CD4+ helper T cell population were consistent and significant (figure 5D). The differences among the CD4+ T cell populations are in accordance with the transcriptome data describing altered CD4+ T cell subsets and IFN-γ pathways previously discussed. To understand whether our observed differences in IFN-γ directly affect PBMC-CD19 CAR-T or P-T CD19 CAR-T cytotoxicity, we included anti-IFN-γ blocking antibodies during Daudi lymphoma cell coculture (figure 5F). Compared with isotype controls, IFN-γ blockade had no impact on CD19-mediated cytotoxicity of CAR-T cells (figure 5G). Our data are consistent with others demonstrating IFN-γ does not directly impact CAR-directed cytotoxicity in hematology.40 Overall, these data demonstrate that P-T CD19 CAR-T cells have similar acute effector function to PBMC-CD19 CAR-T cells but produce limited IFN-γ, which does not negatively impact CAR-T cytotoxicity.

Figure 5

P-T CD19 CAR-T have similar cytotoxicity to PBMC-CD19 CAR-T but maintain an IFN-γ-attenuated helper T cell population. (A) Cytotoxicity of PBMC-CD19 CAR-T and P-CD19 CAR-T products against Daudi lymphoma cells at effector-to-target (E:T) ratio of 2.5:1. Non-transduced (NT) PBMC T cells (n=6) and P-T cells (n=9) were included as negative control. (B) Secreted IL-2 (left), IFN-γ (middle), and TNF-α (right) measured from supernatant of PBMC-CD19 CAR-T (black) and P-T CD19 CAR-T (green) cocultured for 24 hours with Daudi cancer lines at E:T ratio of 1:1. (C) Representative pseudocolor plots of CD4 and CD8 cell subsets for IL-2, TNF-α, IFN-γ intracellular cytokine staining (ICCS) from 4-hour stimulated CAR-T cells. Lymphocytes were gated on SSC-A versus FSC-A for debris exclusion followed by doublet discrimination on FSC-> doublet discrimination on SSC ->gating on live cells (Aqua-) ->CD5+ ->CD4+ or CD8+ cells. (D) Frequencies of IL-2, IFN-γ, and TNF-α positive populations by ICCS among CD4+ and CD8+ PBMC-CD19 CAR-T (black) or P-T CD19 CAR-T (green) for individual donors tested from (C). (E) Pie charts illustrating differences in IL-2 and IFN-γ distribution in P-T CD19 CAR-T and PBMC CAR-T CD4+ and CD8+ T cells.(F) IFN-γ secretion from PBMC-CD19 CAR-T (n=3) and P-T CD19 CAR-T (n=3) cells against Daudi cancer lines at E:T ratio of 1:1 post-24-hour coculture after treatment with IFN-γ antagonist antibody or isotype control. (G) Cytotoxicity of CAR-T products against Daudi cancer line at E:T ratio of 2.5:1 after IFN-γ blockade. Mean±SEM is presented. Unpaired Student’s t-test was performed for all statistical analyses. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. CAR, chimeric antigen receptor; GSEA, gene set enrichment analysis; IFN-γ, interferon gamma; IL-2, interleukin 2; PBMCs, peripheral blood mononuclear cells; P-T, placental circulating T cells; TCR, T-cell receptor; TNF-α, tumor necrosis factor alpha.

P-T CD19 CAR-T cells clear lymphoma and respond to re-challenge in vivo

We have reported differences in the stemness and functional phenotype of P-T CD19 CAR-T and PBMC-CD19 CAR-T cells in acute settings. To evaluate how in vitro activity translates to efficacy for targeting lymphoma, we used an immunodeficient murine model. NSG mice were preconditioned with Busulfan, followed by Luciferase-expressing Daudi lymphoma inoculation 1 week later. CD19 CAR-T cells were administered intravenously after another week, and mice were monitored for tumor burden using bioluminescence imaging and survival over time (figure 6A). In the absence of any CD19 CAR-T administration, Daudi lymphoma was detected systemically with tumor burden intensity increasing until all mice succumbed to disease burden by day 35 (31.2±4.5 days). Administration of PBMC-CD19 CAR-T delayed the onset of detectable lymphoma by about 14–21 days, but mice progressed with advanced disease by day 67 (58.3±10.3 days) (figure 6B,C,E). Only the mice dosed with P-T CD19 CAR-T cells exhibited 100% survival and cleared lymphoma with no detectable systemic bioluminescence signal observed during the 100-day observation period (figure 6B,C and E). All P-T CD19 CAR-T-treated mice, exhibiting long-term lymphoma control beyond 120 days, were given a second inoculation of Daudi lymphoma on day 122 to evaluate any recall response and antitumor activity of persisting P-T CD19 CAR-T from the original infusion. An additional group of age-matched NSG mice was also infused with Daudi lymphoma cells as vehicle control. Of note, one mouse from the P-T CD19 CAR-T-treated re-challenge group died on d