CXCR4 induces memory formation over exhaustion in CAR-T cells to achieve durable leukemia targeting

CXCR4 marks CD4+ memory T cells associated with AML remission

To identify features linked to durable anti-leukemic immunity, we profiled T cells from AML patients in remission or relapse following CB transplantation using Cytometry by Time-of-Flight (CyTOF) (Fig. 1a, Table S1-S2). Unsupervised clustering of T cell markers revealed subsets including CCR7⁺IL7RA⁺CD45RA⁺ T naïve/stem cell memory (Tn/scm), CCR7⁺IL7RA⁺CD45RA^– T central memory (Tcm), and CCR7⁺CD45RA^–GZMK⁺ T effector memory (Tem), amongst other T cell populations (Fig. 1b, c; Figure. S1a). Compared to remission, relapse samples showed marked depletion in the proportion of CD4⁺ Tn/scm cells (23.1-fold) and naïve T cells (10.6-fold), alongside a modest (3.7-fold) increase CD4⁺ effector cells (Fig. 1d; Figure. S1b). Consistent with observations in CD19 CAR-T clinical trials²⁸, the majority (86.6%) of memory cells were CD4⁺.

a Schematic of experimental design. T cells were isolated from peripheral blood (PB) or bone marrow (BM) of primary AML patients in remission following ablative radiation therapy and cord blood transplant (CBT). CD33⁻CD3⁺ T cells were analyzed by CyTOF (see Table S1). b UMAP projection of patient-derived T cells showing expression of CD4/CD8, memory/stemness, and exhaustion markers. c Unsupervised clustering of T cell populations based on select CyTOF markers. Cell subtype designations are based on marker expression patterns. Number of cells in each T cell subset across all n = 14 samples from 11 patients is shown. d Comparison of T cell subsets in remission and relapse samples. Forest plot shows mean log₂ fold-enrichment of each subtype in n = 11 remission samples versus n = 3 relapse samples, with 95% confidence intervals. Statistical analysis was performed using a two-tailed T-test. The non-adjusted p-value is reported. e Violin and box plots show CXCR4 protein expression across T cell subsets. Dashed lines indicate median CXCR4 expression in remission (blue) and relapse (red). f UMAP projection of CD4⁺ tumor-infiltrating lymphocytes (TILs) from a previously published scRNA-seq dataset³⁰, annotated by subtype. CXCR4 expression was imputed using ALRA. g Dot plot showing expression of canonical T cell marker genes across CD4⁺ TIL subtypes. h Violin and box plots showing CXCR4 expression across CD4⁺ TIL subtypes. i) Number of cells from each T-cell subtype is shown across different quartiles of CXCR4 expression (calculated across the entire dataset). For box and whisker plots in panels (d, h)central lines show median, lower and upper hinges show first and third quartiles (the 25th and 75th percentiles) and whiskers extend up to 1.5 * IQR. Source data are provided as a Source data file for (d). “Fig. 1a” created in BioRender. Liang, M. (https://BioRender.com/uexx577) licensed under CC BY 4.0.

Unsupervised clustering of CyTOF-derived protein expression identified CXCR4 as closely associated with memory markers CCR7, IL7RA, and CD45RA, despite not being used as a marker to define T cell subtypes (Fig. 1c). CXCR4 expression in remission samples correlated positively with memory markers CCR7 and IL7RA (r = 0.550 and 0.350, respectively), and negatively with exhaustion/effector markers GZMB and Tbet (r = −0.349 and −0.292) (p < 1.e-100 for all) (Fig. 1c). Consistent with the association between CXCR4 and central memory markers, CXCR4 expression was highest in CD4⁺ Tscm and CD8⁺ Tscm (Fig. 1e)²⁹.

In a published CD4+ tumor infiltrating lymphocyte (TIL) scRNA dataset spanning 9 different solid tumor types³⁰ (Fig. 1f), the cluster annotated as Tn/scm/cm was most similar in marker gene expression to CD4⁺ memory populations we defined by CyTOF (Fig. 1g). CXCR4 expression was elevated in Tn/scm/cm and EOMES^Hi populations and depleted in exhausted precursors and Th17 cells (Fig. 1g, h). High CXCR4-expressing cells were enriched for memory phenotypes and underrepresented in exhausted subsets (Fig. 1i). Together, our findings indicate a potential link between CXCR4 and CD4⁺ memory T cell populations in the acquisition of anti-tumoral immunity.

These data support a shared association between CXCR4 expression and CD4⁺ memory T cell identity, suggesting that CXCR4 may promote memory acquisition and persistence across tumor contexts. Based on these findings, we hypothesized that engineering of CXCR4 expression could be used to enhance CAR-T cell memory and antitumor function.

CD25 identified as a candidate CAR-T target with minimal off-tumor toxicity

To identify optimal CAR targets for CXCR4-engineered CAR-T cells, we screened for AML-specific surface antigens using bulk RNA-seq from AML blasts (n = 235 samples) and normal CD34⁺ HSPCs (n = 44 samples), selecting genes with high AML-specific expression (log₂FC > 2.5, padj < 0.001) and low expression in non-hematopoietic tissues (avg TPM < 5, max TPM < 10 across 31 tissues from GTEx) (Fig. 2b; Supplementary Data 1, 2). Four of six candidate targets identified in our cohort met the same criteria for expression level and DE significance in the BEAT AML cohort (Figure. S2a; Supplementary Data 3)^31,32.

a Differential gene expression analysis of AML blasts (n = 235 samples from 155 patients) versus normal hematopoietic stem/progenitor cells (HSPCs; n = 44 samples from 14 donors). Genes encoding surface proteins are highlighted in pink. Validated AML CAR targets (black) and candidate targets identified in our screen (blue) are annotated. log₂ fold-changes and FDR-corrected p-values from DESeq2 (Wald-test) are reported. b Heatmap of surface molecule expression across 31 non-hematopoietic tissues (GTEx dataset; median TPM for n = 4 to 803 donors per tissue). Candidate targets with low off-tumor expression and canonical AML CAR-T targets are annotated. Novel targets discovered in our cohort and replicated in beatAML are labeled in blue. c Representative flow cytometry histogram of CD25 expression in CD25⁻ and CD25⁺ AML patient samples (pink = anti-CD25 antibody, gray = isotype control). CD25⁺ AML was defined as > 70% CD25⁺ blasts by flow cytometry (n = 12/66 patients). d AML cohort ranked by CD25 mRNA expression (black dots, n = 155 patient samples). For a subset, CD25 surface levels were validated by flow cytometry (pink bars; n = 55 patient samples). Middle: Expression log₂ fold-change relative to HSPCs for candidate and select validated AML CAR-T targets. Bottom: Mutational landscape for individual samples. For some samples, multiple individual measurements were made for different rounds of xenotransplantation. e In vitro cytotoxicity of CAR-T cells assessed by co-culture with primary AML blasts. Effector CAR constructs include: ACTT and CD19 (negative controls); CD33 (positive control); CD25; and CD96. Sorted PDX BM-derived AML blasts for a representative CD25/CD96 dual-positive patient sample (P12) were used as target cells. Co-culture experiments were setup for multiple effector:target ratios and %AML reduction was evaluated at 24 h and 72 h. Experiments were replicated using CAR-T cells derived from n = 3 CB donors (rep1-3). Each data point corresponds to average of three technical repicates. Y-axis reports the percentage of hCD33+ cells compared to AML blasts alone. Error bars correspond to mean ± SD. The non-adjusted p-value reported for each CAR construct versus ACTT (two-tailed T-test). Source data are provided as a Source data file for panel(s) (a, d, e).

Amongst these, mRNA expression of IL-2 receptor alpha chain CD25/IL2RA was highly AML-specific and was associated with poor overall survival in the BEAT AML cohort (Figure. S2b). The minimal expression of CD25 other tissues and the absence of CD25 protein in hematopoietic populations other than CD4+ Tregs (Figure. S2c, d) supports a favorable safety profile. Furthermore, CD25 is a known marker of chemotherapy-resistant leukemia-initiating cells (LICs)³³ and is frequently reported as a marker of poor prognosis in AML^34,35. We therefore selected CD25 as a target based on its 1) AML-specific overexpression vs HSPCs; 2) low off-tumor toxicity risk; 3) enrichment in LICs and 4) potential to address unmet needs for high-risk, treatment-refractory disease.

CD25 expression varied across patients in our cohort, with high surface positivity ( > 70% of cells) detected in 18.2% (12/66) of AML cases (Fig. 2c). Among 155 samples with matched RNA-seq and mutational data, IL2RA mRNA expression was elevated in FLT3-ITD (6.7-fold, p = 1.6e-7) and NPM1 (3.4-fold, p = 0.0021) mutant AML (Fig. 2d, Figure. S2e). We selected four representative IL2RA-high samples for subsequent experiments (Figure. S3). Surface CD25 protein expression in these samples was highest in putative LIC-like CD34⁺CD38^– fractions (Figure. S3).

As an additional target, we selected CD96, a previously reported LSC marker³⁶ which also met the same criteria for high expression in AML blasts versus HSPCs and low expression in non-hematopoietic tissues. CD96 surface positivity was observed in 55.1% of AML samples and mRNA overexpression overlapped with 75% of IL2RA-high cases, offering broader coverage (Fig. 2d, Fig. 4a, b). We note that CLL1/CLEC12A, a previously validated AML CAR target, may also be a viable alternative based on overexpression in both our overall cohort (48.8%) and in IL2RA-high cases (43.8%) (Fig. 2d).

Generation of CAR-T from CB T cells

We generated CAR-T cells by lentiviral transduction of donor CD3⁺ T cells with second-generation CAR vectors, incorporating an extracellular CD8 leader, antigen-specific scFv, CD8 hinge, 4-1BB co-stimulatory, and CD3ζ signaling domains³⁷ (Figure. S4a). CXCR4-expressing CAR-T cells were created by dual transduction with an additional vector encoding full-length CXCR4 derived from either mouse (mCXCR4) or human (hCXCR4) (Figure. S4b). Murine and human CXCR4 and CXCL12 share a high degree of homology, with 91% and 99% similarity, respectively. Furthermore, human CXCR4 knock-in mice exhibit normal hematopoiesis in vivo, suggesting functional conservation of the CXCR4-CXCL12 axis between humans and mice in hematopoietic cells³⁸. Therefore, in our initial CD25-CAR experiments, we used mCXCR4 to facilitate monitoring of transduced CXCR4 expression in human CAR-T cells. In subsequent experiments, we transitioned to hCXCR4 to align with clinical translation goals, as humanized receptors are associated with a lower risk of immune rejection^39,40. See “Methods” and Data Availability for full vector sequences.

We considered several donor T cell sources for CAR-T cell generation, including autologous patient-derived peripheral blood (PB), healthy adult PB, and CB. We initially attempted to generate autologous CAR-T cells using patient PB samples isolated during active disease. However, patient T cells exhibited poor viability and minimal expansion in vitro. Viable T cell numbers never reached the threshold ( ~ 1 × 10⁵) required for lentiviral transduction, even after extended activation (Figure. S4b). Phenotypic analysis of the starting CD3⁺ populations revealed higher proportions of exhausted and lower proportions of naïve and memory subsets in patient samples (Figure. S4b), suggesting that poor quality of T cells severely limits the feasibility of generating functional autologous CAR-T cells from AML patients with relapse or refractory disease.

Given the poor expansion and exhaustion-prone phenotype of autologous T cells from AML patients, we explored alternative sources for CAR-T generation. While healthy adult PB is traditionally the most commonly used source of allogeneic immune cells, CB is increasingly recognized as a promising alternative, offering several advantages for adoptive immune cell transfer, including less differentiated T cell phenotype, an abundance of naïve T cells, reduced risk of graft-versus-host disease (GVHD), less stringent HLA matching requirements, and the potential for development of off-the-shelf CAR-T products^41,42. CB- and healthy donor PB-derived T cells both showed robust expansion in vitro and high transduction efficiency for CAR constructs and dual CAR/CXCR4 (Figure. S4c-f). In consideration of the superior T cell phenotype, expansion potential, and translational advantages, we selected CB as the primary donor source for CAR-T cell generation.

In vitro assessment of CD25-targeted CAR-T cytotoxicity and on-target off-tumor toxicity

We generated CD25-targeted CAR-T cells as above, substituting the scFv domain with an anti-CD25 scFv, which we developed in-house as previously described (sequence in Data Availability)⁴³ (Figure. S4c). We evaluated cytotoxicity and specificity of CD25-targeted CAR-T cells against patient-derived CD25⁺ AML blasts in vitro. Controls included non-specific activated T cells (ACTT), CD19-targeted CAR-T (negative), and CD33-targeted CAR-T (positive). CD25-targeted CAR-T cells showed potent killing of CD25-expressing primary AML cells, slightly weaker than CD33-targeted CAR-T (90.0 ± 3.6 % versus 99.9 ± 0.06 % AML reduction, respectively; 72 h; E:T = 3:1) but significantly greater than ACTT or CD19-CAR-T controls (Fig. 2e). Cytotoxicity against AML blasts was more pronounced at later time points and at higher effector:target ratio for all AML-specific CARs, confirming cytotoxicity is mediated by CAR-T cells. CD25-targeted CAR-T were ineffective against primary AML cells for which CD25 was knocked out, confirming antigen specificity (Figure. S2f). In vitro cytotoxicity data for additional patient samples are provided in (Figure. S2f).

Co-culture of CAR-T cells with autologous CB-derived CD34+ HSPCs resulted in comparable decrease in HSPC number between ACTT, CD19- and CD25-targeting CAR-T cells (Figure. S2g), suggesting that that off-tumor toxicity against HSPCs may be mediated by CAR-T-derived inflammatory cytokines rather than CD25-directed killing⁴⁴. Activated T-cells and regulatory T cells (Tregs) normally express CD25, posing a risk for on-target off-tumor toxicity (Figure. S2d). During CAR-T generation, no significant difference in T cell viability or number was observed between CD25- and CD19-targeted CAR-T or ACTT within the first 2–3 days post-transduction (Figure. S5a). In contrast, co-culture with autologous Tregs showed reduction in Treg counts after 3 days with CD25-targeted CAR-T (72.2 ± 11.6%) compared to CD19 CAR-T (0.1 ± 30.9%) (Figure. S5b). These results support the specificity of CD25-targeted CAR-T cells, with minimal on-target off-tumor toxicity against normal HSPCs and modest cytotoxicity against normal Tregs. The long-term in vivo impact of CD25-targeted CAR-T treatment on Tregs and other hematopoietic cells remains to be determined.

Structural assessment of CD25 CAR targeting specificity

Given difference in the extent of CD25-targeted CAR-T toxicity against AML and T cells, we investigated potential structural differences in CD25 presentation on AML blasts versus T cells. CD25/IL2RA forms part of the IL-2 receptor complex with CD122/IL2RB and CD132/IL2RG. IL2RB mRNA was ~11-fold lower in CD25⁺ AML blasts than in T cells (Figure. S5c), suggesting that AML cells predominantly express single-chain IL2RA (scIL2RA). In vitro reconstitution confirmed stable complex formation between our anti-CD25 scFv (S417 Fab) and both scIL2RA and the full IL-2R heterotrimer (Figure. S5d), but not with IL2RA and IL2RG alone in the absence of IL2RB (Figure. S5e). Single-particle cryo-EM analysis of the S417 Fab and IL-2R heterotrimer suggests that antibody binding does not interfere with IL-2 binding or complex assembly (Figure S5f).

CXCR4 improves therapeutic efficacy of CD25-targeted CAR-T cells in vivo

Having identified CD25 as a viable AML target, we next investigated whether CXCR4 overexpression enhances CD25-targeted CAR-T cell persistence and antitumor efficacy in vivo. We generated PDX models of CD25⁺ AML using four primary AML samples with high surface CD25 expression (75.7-85.5% of blasts, Figure. S3; and Table S1; see Supplementary Data 4 for full cohort details) via xenotransplantation of sorted AML-initiating cells into newborn NSG mice. After confirming successful AML engraftment (hCD45⁺CD33⁺) in PB, mice were given one intravenous dose (5 × 10⁵ − 4.6 × 10⁶ cells per mouse, see Supplementary Data 4) of CAR-T cells, followed by weekly monitoring by phlebotomy (Fig. 3a). mCXCR4(+) CD25-targeted CAR-T cells exhibited significantly improved clearance of AML from PB (mean %AML chimerism change = −6.2 ± 21.7% in mCXCR4(+) CD25-targeted CAR-T, versus +18.3 ± 34.3%, p = 0.002 and versus +63.1% ± 25.6%, p = 2.9e-5 in CXCR4(-) CD25-targeted CAR-T and untreated PDX, respectively) (Fig. 3b). AML clearance was also improved in BM, spleen, and liver (Fig. 3c).

a Schematic of study design. b Peripheral blood %AML chimerism pre-CAR-T treatment and at endpoint in untreated (n = 22 animals), CD25-targeted CAR-T-treated (n = 32 animals), and mCXCR4(⁺) CD25 CAR-T-treated (n = 27 animals). c AML chimerism in PB, spleen, bone marrow, and liver of PDX mice, stratified by treatment. Group sizes by treatment: untreated (n = 4, 9, 8, 15 animals), CXCR4(^⁻)CD25-targeted CAR-T (n = 10, 10, 8, 7 animals), and mCXCR4(^⁺) CD25-targeted CAR-T (n = 12, 8, 7, 8 animals), PDX mice of Patients 10-13, respectively. Statistical comparisons made using unpaired two-sided Mann-Whitney U tests. The non-adjusted p-value is reported. For b, c Box and whisker plots: central lines show median, lower and upper hinges show first and third quartiles (the 25th and 75th percentiles) and whiskers extend up to 1.5 * IQR. d Longitudinal kinetics of AML cells (hCD45⁺CD33⁺), CAR-T cells (hCD45⁺CD3⁺), and murine leukocytes (mCD45⁺) in nucleated cell fraction from PB (red cells were excluded by lysis pre-FACS). Lines and shaded regions indicate mean ± SD. The number of mice included at each time point is shown. e Kaplan-Meier survival curves for PDX animals stratified by treatment arm including CXCR4(-) CD25-targeted CAR-T (n = 27 animals), mCXCR4(+) CD25-targeted CAR-T (n = 32 animals), and mock/ACTT-treated (n = 10 animals) groups. Individuals removed for paired analysis or time-controlled sampling were censored (see Table S4). Shaded regions show 95% confidence intervals. f H&E and CD33 IHC staining of femur sections from PDX (P11). Representative images selected from sections prepared from n = 5 mice per treatment group (scale: 20 µm) g) Laser scanning microscopy (LSM) imaging and 3D reconstruction of liver sections acquired from PDX (P10), harvested 10 days after injection with CXCR4(-) or mCXCR4(+) CD25-targeted CAR-T. IF staining was performed for CXCL12 (green), CD3 (red) and DAPI (blue). Images were acquired on a Zeiss Axio Observer 7. Z-stack 3D reconstruction was performed using ImarisViewer. Representative images selected from sections prepared from n = 3 mice per treatment group (scale: 30 µm). Source data are provided as a Source data file for (b, c, d, e). “Fig. 3a” created in BioRender. Liang, M. (https://BioRender.com/5hfwv50) licensed under CC BY 4.0.

We tracked temporal changes in PB cell chimerism and observed transient reduction of murine CD45⁺ cells during peak AML and CAR-T cell chimerism, followed by recovery as AML burden declined (Fig. 3d). AML clearance correlated with a rise in CAR-T chimerism in both groups between weeks 2-4 post-injection. However, CXCR4(-) CD25-targeted CAR-T cells failed to persist, coinciding with AML relapse, while mCXCR4(+) CAR-T cells expanded until weeks 6-8 and were associated with durable AML suppression (Fig. 3d). We assessed percent survival in PDX post CAR-T injection, counting mice that either died or had to be sacrificed prior to a designated experiment endpoint (Fig. 3e). Although survival time could not be precisely determined due to our time-controlled experiment design, CD25-targeted CAR-T treated mice exhibited significantly better survival at 8 weeks post-treatment for both CXCR4(-) (n = 27; OS = 65%; 95% CI: 46-92%) and mCXCR4(+) (n = 32; OS = 92%; 95% CI: 82-100%) when compared to mock (ACTT-treated) mice (n = 10; OS = 20%; 95% CI: 4.1-97.1%). Furthermore, treatment with mCXCR4(+) versus CXCR4(-) CD25-targeted CAR-T resulted in an 83% reduction in mortality (Cox hazard ratio 0.17; 95% CI: 0.03-0.89).

Immunohistochemistry for human CD33 revealed near-complete clearance of AML and robust recovery of leukocytes, erythroid cells, and megakaryocytes in the BM of mCXCR4(+) CD25-targeted CAR-T-treated mice (Figure. S6a, b). In contrast, CXCR4(-) CAR-T-treated mice showed persistent AML and limited recovery of hematopoietic elements (Fig. 3f; and S6b). Notably, mCXCR4(+) CAR-T cells localized near CXCL12⁺ stromal cells in BM at day 10 post-infusion (Fig. 3g; and S6c). Mirroring our findings in post-CBT patients, T cells isolated from BM and spleen of animals treated with mCXCR4(+) CAR-T were predominantly CD4⁺ with a significantly higher proportion of CD4⁺ cells versus in CXCR4(-) CAR-T -treated animals (Figure. S6d).

mCXCR4(+) CD19-targeted CAR-T failed to eliminate CD25⁺CD19⁻ AML cells (Figure. S6e), showing that the improved anti-AML activity of mCXCR4(+) CAR-T was target-specific. In cases with ≥4/8 HLA matches between CB donors and AML, CD25-targeted CAR-T cells cleared AML while ACTT could not, indicating the effect is not due to alloreactivity (Figure. S6f). Prior studies suggest CXCR4 enhances effector cell trafficking to tumor sites via CXCL12^19,22,45, which is also expressed by BM stromal cells. We tested whether CXCR4 improved CAR-T trafficking by comparing animals receiving mCXCR4(+) CD25-targeted CAR-T with matched PDX littermates receiving CXCR4(-) CD25-targeted CAR-T. CAR-T chimerism was significantly higher in BM of mCXCR4(+) versus CXCR4(-) CAR-T-treated animals. However, these differences disappeared upon normalization for circulating (PB) CAR-T chimerism, indicating increased CAR-T presence in BM was due to overall survival and persistence, not selective BM trafficking/retention (Figure. S6g). Moreover, mCXCR4(+) CAR-T were not sequestered in BM but circulated systemically. Signs of graft versus host disease such as liver pathology, dermal thickening, elevated AST/ALT, and abnormal CD3⁺ infiltration were absent in both HSC- and AML-engrafted mice up to 8+ weeks post-CAR-T infusion (Figure. S7a, b).

Together, our findings demonstrate that CXCR4 expression enhances the anti-AML activity of CD25-targeted CAR-T cells in vivo, promoting leukemia clearance in PB, BM, and peripheral tissues, facilitating hematopoietic recovery, and increased survival.

CXCR4 improvement of CAR-T cell therapeutic efficacy in vivo is replicated against an independent CAR target, CD96

We constructed CD96-targeted CAR-T cells from CB-derived donor T cells as was done for CD25-targeted CAR-T cells, switching the anti-CD25 scFv sequence with a previously published anti-CD96 scFv sequence⁴⁶. For CD96 CAR-T cells, we substituted full-length murine CXCR4 with full-length human CXCR4 (hCXCR4).

In vitro co-culture revealed potent killing of CD96⁺ AML blasts by CD96-targeted CAR-T cells (97.2 ± 2.2 % AML reduction; 72 h; E:T = 3:1) as compared to ACTT and CD19-targeted CAR-T controls, with efficacy comparable to that of CD33- and CD25-targeted CAR-T cells (Fig. 2g). In PDX mice engrafted with CD96⁺ AML, treatment with hCXCR4(+) CD96-targeted CAR-T cells led to improved AML clearance in PB (%AML chimerism change = +7.7 ± 30.5% in hCXCR4(+) CD96-targeted CAR-T, versus +47.5 ± 40.2%, p = 0.01 in CXCR4(-) CD96-targeted CAR-T and versus +59.8 ± 21.3%, p = 2.7e-5 in untreated PDX, respectively), plus improved AML clearance in BM, spleen, and liver (Fig. 4c, d). IHC staining of BM revealed recovery of murine hematopoietic elements in mice treated with hCXCR4(+) CD96 CAR-T, while CXCR4(-) treated animals showed persistence of AML and impaired hematopoiesis (Fig. 4e).

a AML samples ranked by normalized CD96 mRNA expression (black dots, n = 155 patients). CD96 surface expression (flow cytometry) shown for a subset (blue bars, n = 95 patients). Bottom heatmap shows mutational profiles per sample, as in Fig. 2d.b Representative flow cytometry histogram showing CD96 surface expression in CD96⁻ and CD96⁺ patient-derived AML samples, as in Fig. 2c. Anti-CD96 antibody staining is shown in blue, isotype control in gray. 98 AML samples were analyzed; cases with >70% CD96⁺ leukemic blasts were defined as CD96⁺ (blue, n = 54), others as CD96⁻ (gray, n = 44). c Peripheral blood %AML chimerism pre-CAR-T injection and at endpoint in untreated (n = 21), CXCR4(-) CD96 CAR-T-treated (n = 23), and hCXCR4(+) CD96 CAR-T-treated (n = 18) animals. Red dots indicate animals that achieved complete remission (0% AML chimerism in PB). d AML chimerism in peripheral blood, spleen, bone marrow, and liver of PDX mice as in (c), stratified by treatment. Group sizes by treatment: untreated (n = 3, 9, 4, 5), CXCR4(-) CD96 CAR-T (n = 6, 9, 5, 3), and hCXCR4(+) CD96 CAR-T (n = 4, 6, 4, 4), PDX mice of Patients 12–15, respectively (See Table S5). Statistical comparisons were made using unpaired two-sided Mann-Whitney U tests. The non-adjusted p-value is reported. For (c, d) Box and whisker plots: central lines show median, lower and upper hinges show first and third quartiles (the 25th and 75th percentiles) and whiskers extend up to 1.5 * IQR. e) H&E and CD33 immunohistochemistry of bone marrow from Patient 13-derived PDX (untreated, CXCR4(-), or hCXCR4(+) CD96 CAR-T treated) (scale: 20 µm). Representative images selected from sections prepared from n = 3 animals per treatment group. Source data are provided as a Source data file for (a, c, d).

These findings demonstrate that CXCR4-driven enhancement of in vivo CAR-T cell efficacy extends beyond CD25 targeting, supporting a broader role for CXCR4 in improving CAR-T cell function against AML.

CXCR4 expression enhances memory CAR-T cell development in vitro and in vivo

To investigate how CXCR4 influences the differentiation of CAR-T cell subsets, we profiled memory and exhaustion markers on CD25-targeted CAR-T cells after extended in vitro co-culture (1 week) under normoxic (21% pO₂, mimicking peripheral circulation) and hypoxic (2.5% pO₂, mimicking the bone marrow microenvironment) conditions, with or without hCXCR4 overexpression (Fig. 5a). To assess whether the beneficial effects of CXCR4 are specific to CB-derived T cells, we also included CAR-T cells generated from healthy donor PB.

a Timeline of extended co-culture experiments. b Proportion of CD4+ Tcm cells (as % of CD3 + ) and (c) PD-1 surface expression (MFI) were measured by flow cytometry on day 6 of co-culture. Data shown for n = 3 donors for both cord blood (CB) and peripheral blood (PB). The average of 3 technical replicates per sample is plotted. Statistical analysis was performed using a paired two-tailed T-test. The non-adjusted p-value is reported. d (Left) Unsupervised clustering of CD25-targeted CAR-T cells based on CyTOF protein expression. Cells from bone marrow and spleen of PDX treated with CXCR4(-) or mCXCR4(+) CD25-targeted CAR-T (n = 5 mice per group). (Right) Expression of marker proteins defining each UMAP cluster. e Relative abundance of each cluster in CD3⁺ CAR-T population. Forest plot shows log₂ fold-enrichment of each subtype in CXCR4(-) or mCXCR4(+) treatment groups, with 95% confidence intervals. Diamonds indicate mean values; whiskers represent 95% confidence intervals. Statistical analysis was performed using a two-tailed T test. The non-adjusted p-value is reported. f Proportion of CD4⁺ naïve/T stem cell memory (Tscm) and central memory (Tcm) CAR-T cells in bone marrow and spleen of PDX treated with CXCR4(-) (n = 14 animals) or mCXCR4(+) (n = 30 animals) CD25-targeted CAR-T cells. g Frequencies of immune-surveillant (CD226⁺TIGIT⁻), memory (IL7RA⁺, TCF1⁺), and checkpoint-expressing (TIM3⁺TIGIT⁺) CD4⁺ CAR-T cells in bone marrow and spleen. mCXCR4(+) CD25-targeted CAR-T cells exhibited enhanced memory and immune-surveillant phenotypes, while checkpoint markers were enriched in CXCR4(-) CD25-targeted CAR-T cells. Data shown for n = 14 and n = 30 (n = 4 and n = 14 remission) animals for CXCR4(-) and mCXCR4(+) CD25-targeted groups, respectively. h Proportion of CD4⁺ naïve/Tscm and Tcm CAR-T cells in PDX treated with CXCR4(-) (n = 15) or hCXCR4(+) (n = 16) CD96-targeted CAR-T cells. i Frequency of immune-surveillant and memory phenotype CD4⁺ CAR-T cells in CD96 CAR-T-treated PDX, as in (g). Data shown for n = 15 (n = 2 remission) and n = 16 (n = 9 remission) animals for CXCR4(-) and hCXCR4(+) CD96-targeted groups, respectively. Note: TCF1 was not included in CD96-targeted CAR-T profiling. Graphs in panels (f–i) show mean ± SD. Statistical comparisons were performed using unpaired two-tailed t-test. Source data are provided as a Source data file for panel(s) (b, c, f–i).

Although in vitro T cell differentiation demonstrates large inter-donor variability, hCXCR4(+) CAR-T cells exhibited a trend of higher Tcm percentage compared to CXCR4(-) CAR-T cells under normoxia for both CB- and PB-derived cells (Fig. 5b). Additionally, we observed a reduction in PD-1 surface expression in hCXCR4(+) cells under normoxia (Fig. 5c). These effects were also seen in PB-derived hCXCR4(+) CD25-targeted CAR-T cells, suggesting that the effects of CXCR4 on memory acquisition and exhaustion resistance is may not limited to CB-derived T cells. The beneficial effects of CXCR4 co-expression were abrogated under hypoxic conditions, potentially suggesting that these cells require the specific bone marrow niche for memory acquisition in vivo.

We next assessed memory and exhaustion of CAR-T cells in vivo by performing high-dimensional proteomic profiling of functional T cell memory and exhaustion-associated markers on CAR-T cells isolated from AML PDX mice treated with CD25-targeted CAR-T cells (Figure. S8). Unsupervised clustering of CAR-T cells from bone marrow and spleen at 4-6 weeks post-infusion revealed distinct populations: naïve/stem-like T cells (CD4⁺ and CD8⁺ N/Tscm), central memory T cells (CD4⁺ Tcm), effector T cells (CD4⁺ and CD8⁺ Teff), and checkpoint-expressing/exhausted T cells (CD4⁺ and CD8⁺ Tex) (Fig. 5d).

mCXCR4(+) CAR-T cells exhibited higher proportions of CD4⁺ naïve/stem-like (3.3-fold increase) and central memory populations (3.9-fold increase), marked by elevated expression of canonical memory markers TCF1, IL7RA, and the immune-surveillance receptor CD226 (Fig. 5e). Additionally, BCL-2 expression was enriched in Tcm subsets, suggesting that CXCR4 signaling may enhance memory development through BCL-2-mediated survival pathways (Fig. 5d). Conversely, T cell populations expressing immune checkpoint and exhaustion markers, including CD4⁺ cells expressing TIM3, TIGIT, and PD-1, and CD8⁺ cells co-expressing LAG3 and TIM3, were diminished in mCXCR4(+) CAR-T cells.

Across both bone marrow and spleen, mCXCR4(+) CAR-T cells showed a consistently higher frequency of CD4⁺ cells with memory and immune surveillance phenotypes, and a lower frequency of cells expressing exhaustion and checkpoint molecules (Fig. 5f, g). In the small number of CXCR4(-) CD25-targeted CAR-T-treated PDX that successfully eliminated AML (n = 4), we found lower frequencies of central memory T cells and higher frequencies of exhausted T cells compared with mCXCR4(+) CD25-targeted CAR-T recipients, indicating that CXCR4-induced memory and evasion of exhaustion is not solely due to reduced AML burden (Fig. 5g, red labels).

These findings were corroborated in PDX models treated with CD96-targeted CAR-T cells, where hCXCR4 overexpression similarly promoted expansion of Tcm populations and CD4⁺ memory subsets, along with acquisition of a CD226⁺TIGIT⁻ immune-surveillant phenotype (Fig. 5h, i). Together, these results demonstrate that CAR-T cells can establish durable memory phenotypes through interaction with human AML cells in a xenogeneic context. Importantly, CXCR4 expression enables CAR-T cells to evade exhaustion and acquire memory, immune surveillance, and longevity features that are not restricted to a specific target antigen.

Single-cell RNA sequencing reveals T cell and AML transcriptional programs associated with CXCR4-driven therapeutic efficacy

To investigate the impact of CXCR4 on CAR-T cell fate at single-cell resolution, we performed single-cell RNA sequencing (scRNA-seq) on CD25-targeted CAR-T cells isolated from the bone marrow of PDX mice. We profiled CXCR4(-) CAR-T cells at 4 weeks (n = 3) and mCXCR4(+) CAR-T cells at both 4 weeks (n = 3) and 5 months (n = 2) post-injection (Fig. 6a; and Figure. S9). Unsupervised clustering revealed 13 T cell clusters, which we annotated using established lineage and state-specific marker genes (Fig. 6b, c; and Figure. S10a). Three CD4⁺ clusters expressed canonical memory-associated markers (e.g., TCF1/TCF7, CCR7, IL7R) and scored highly for memory gene modules (Figure. S10b), which we designated Tscm-cm1-3. These represent stem-like memory (Tscm) and central memory (Tcm) populations, which share transcriptional profiles²⁹. Notably, Tscm-cm1 and Tscm-cm2 clusters expressed high levels of CD226, a costimulatory receptor involved in tumor immune surveillance whereas Tscm-cm3 cells expressed EOMES, a transcriptional regulator of tissue-resident memory fate. Expression of CAR transgene (CD25) and mCXCR4 transgene persisted in CAR-T cells up to 5 months post-injection (Figure. S10c), indicating durable transgene expression in vivo.

a Schematic of experimental design detailing CAR-T cell collection for single-cell RNA sequencing. Cells were obtained from AML PDX BM, spleen, and liver at 2.5 weeks, 4 weeks, and 5 months post-injection. b UMAP projection of BM-derived CD25-targeted CAR-T cells collected at 4 weeks and 5 months post-injection. Cells are colored by CD4/CD8 identity, cell cycle score, and select memory/exhaustion markers. Cell type annotations were made based on canonical T-cell subtype markers (see Fig. S7c). c Relative abundance of each cluster across treatment conditions. Forest plot shows log₂ fold-enrichment of each subtype in CXCR4(-) or mCXCR4(+)treatment groups (n = 3 animals per group) at 4 weeks, with 95% confidence intervals. Diamonds indicate mean; whiskers represent 95% confidence intervals. Statistical comparisons were made using two-tailed t-tests. Non-adjusted p-values are reported. See also Figure. S7e. d Dot plot showing expression of representative marker genes across T-cell subtypes and treatment conditions. Additional markers and full subtype profiles are shown in Figure. S7f. e Differential expression (DE) analysis comparing CXCR4(-) versus mCXCR4(+) CD25-targeted CAR-T cells at 4 weeks and 5 months. Genes with base mean >15, FDR < 0.05, and log₂ fold-change ≥ 0.58 in at least one comparison are shown. Genes with early (4-week) or late (5-month) CXCR4-associated upregulation are highlighted in red and blue, respectively. f Gene set enrichment analysis (GSEA) of DE genes ranked by log₂ fold-change. GSEA p-values were calculated using the fgsea package in R. FDR-adjusted p-values are reported. Only pathways with FDR < 0.05 in at least one contrast and enrichment FDR < 0.01 are displayed. g UMAP visualization of liver- and spleen-derived CAR-T cells harvested at 2.5 weeks post-injection. Cells are colored by CXCR4 expression status and tissue origin. ALRA-imputed expression of select memory and exhaustion markers is shown. Source data are provided as a Source data file for (c, f). “Fig. 6a” created in BioRender. Liang, M. (https://BioRender.com/j4jhcy1) licensed under CC BY 4.0.

Quantitative analysis of T cell subtype populations revealed substantial enrichment of memory-associated CD4⁺ CAR-T cells in mCXCR4(+) samples at both 4 weeks and 5 months compared to CXCR4(-) counterparts (52.4% and 36.2% vs. 18.7% Tscm-cm cells, respectively; (Fig. 6c; and Figure. S10d, e) In contrast, proliferative and exhausted subsets were markedly overrepresented in CXCR4(-) samples (29.2% vs. 3.4% and 1.1% at 4 weeks and 5 months, respectively). Consistently, memory- and resting-state gene modules were more strongly expressed in mCXCR4(+) CAR-T cells, while expression of exhaustion-associated markers was reduced, particularly at the 5-month time point even within the same T cell subtypes (Fig. 6d; and Figure. S10f). CD8⁺ and proliferative/exhausted subsets declined over time in mCXCR4(+) CAR-T cells, whereas CD4⁺ Tscm-cm2 cells persisted, supporting a model in which long-lived CD4⁺ memory cells mediate durable immune surveillance following AML clearance.

Differential gene expression and gene set enrichment analysis comparing mCXCR4(+) and CXCR4(-) CAR-T cells at both 4 weeks and 5 months, as well as memory-enriched (Tscm-cm) versus non-memory subsets (Fig. 6e), revealed significant enrichment of T cell activation, proliferation, mTOR signaling, and cytotoxicity pathways in CXCR4(-) and non-memory populations. In contrast, mCXCR4(+) and Tscm-cm cells were enriched for TNFα signaling (Fig. 6f), a pathway implicated in T cell survival, memory maintenance, and anti-leukemic activity⁴⁷. Notably, TNFα target genes JUNB, FOS, MKP-1/DUSP1, and CD69, all key regulators of T cell self-renewal and long-term persistence, were prominently expressed and further upregulated at 5 months (Figure. S10g). Additionally differentially expressed genes included BCL2, ZFP36 (a negative regulator of T cell activation), and GADD34/PPP1R15A (an inhibitor of mTOR signaling), collectively suggesting a transcriptional program that favors memory formation by mitigating exhaustion and senescence.

To investigate the timing of memory and exhaustion switch, we performed scRNA-seq at 2.5 weeks post-injection (Fig. 6a,g; Figure. S9b), an early phase when CAR-T cells begin to engage AML and cytotoxic activity starts to decline. At this point, both CXCR4(-) and mCXCR4(+) CAR-T cells exhibited similar proportions of proliferating cells. However, mCXCR4(+) CAR-T cells were enriched for CD4⁺ T cells (65.5% vs. 5.8%) and expressed elevated levels of TCF7, IL7R, CCR7, and CD226, markers indicative of early memory commitment. While PD-1 expression was observed across all conditions, CXCR4(-) CAR-T cells showed greater expression of exhaustion markers, including LAG3, TIM3/HAVCR2, and 4-1BB/TNFRSF9, the latter associated with regulatory T cell activity in tumors^48,49 (Fig. 6g). These findings indicate that CXCR4 expression in CAR-T cells promotes CD4+ fate and memory gene expression as early as 2.5 weeks post-injection.

Collectively, these data demonstrate that mCXCR4 expression programs CAR-T cells toward a memory-enriched CD4⁺ phenotype characterized by reduced exhaustion and enhanced expression of survival and longevity-associated genes. This transcriptional reprogramming is evident as early as 2.5 weeks post-infusion and persists for at least 5 months, suggesting durable remodeling of T cell fate.

Stemness- and longevity-associated gene expression in mCXCR4(+) CAR-T cells

Quiescence, self-renewal, and extended longevity are defining features of both memory T cells and hematopoietic stem cells (HSCs)⁵⁰. To determine whether these lineages share common transcriptional programs, we analyzed a published scRNA-seq dataset of purified CD34⁺/CD45RA⁻ populations from cord blood. Long-term HSCs were identified by low expression of lineage/maturation markers (CD33, CD38) and high expression of canonical stem cell markers (CD90/THY1, CD45b/ITGA2, CD133/PROM1) (Figure. S11a).

Differential gene expression analysis comparing HSCs with non-HSC progenitors revealed substantial transcriptional overlap with the signatures observed in mCXCR4(+) versus CXCR4(-) CAR-T cells, as well as in Tscm-cm versus non-memory CAR-T subsets (Figure. S11b). Unsupervised clustering of shared DEGs showed that genes downregulated across both comparisons were enriched for cell cycle and mitotic processes, whereas upregulated genes were associated with oxidative stress response (Figure. S11c). Notably, these included regulators of stress tolerance and survival (FOS, FOSB, TXNIP), quiescence-promoting transcription factors (NURR2/NR4A2, HLF, KLF2), and stemness-associated factors (CITED2, SESN1). Together, these findings suggest that memory T cells and HSCs achieve persistence through shared transcriptional programs that suppress proliferation and enhance resistance to oxidative stress.

Memory-favoring metabolic programs in mCXCR4(+) CAR-T cells reflect adaptation to hypoxic bone marrow conditions

T cell fate is tightly regulated by metabolic state: while effector and exhausted T cells (Teff, Texh) predominantly rely on glycolysis⁵¹, while memory populations such as Tscm and preferentially utilize oxidative phosphorylation (OXPHOS) and fatty acid oxidation⁵². The BM, a physiologically hypoxic environment, imposes unique metabolic constraints on infiltrating T cells ^53,54,55,56. Consistent with their quiescent phenotype, Tscm-cm subsets displayed enriched expression of OXPHOS-related genes, alongside upregulation of oxidative stress response pathways shared with hematopoietic stem cells (Fig. 6f; and Figure. S11c).

To investigate this further, we assessed metabolite and metabolism-related gene expression changes in mCXCR4(+) versus CXCR4(-) CAR-T cells. Unsupervised clustering of CAR-T cell subpopulations based on glycolytic, TCA cycle, and OXPHOS gene expression clearly separated exhausted T cells (Texh) from other subsets, characterized by high expression of glycolytic enzymes (HK, PGM) and lactate transporters (SLC16A1, SLC16A4) and low expression of OXPHOS/TCA cycle genes (Figure. S12a). Similarly, CXCR4(-) CAR-T cells showed elevated expression of glycolytic genes (GAPDH, ALDOC, TPI1) and reduced expression of TCA cycle enzymes (OGDH, SDHD, ACO1, ACO2, CS), (Figure. S12a–c), together supporting a link between metabolic programming and memory differentiation.

We measured oxygen saturation and lactate levels in non-engrafted NSG mice and in AML PDX mice with or without CAR-T treatment. As expected, untreated PDX BM was more hypoxic than that of non-engrafted NSG mice; however, lactate levels were comparable, suggesting that AML alone does not significantly elevate BM lactate production (Figure. S12d, e). In contrast, BM from CXCR4(-) CAR-T-treated PDX mice showed markedly elevated lactate compared to all other groups, including non-engrafted, untreated PDX, and mCXCR4(+) CAR-T-treated mice whereas lactate levels in mCXCR4(+) CAR-T-treated mice remained similar to baseline (Figure. S12e). These results suggest that CXCR4(-) CAR-T cells rely more heavily on anaerobic glycolysis, possibly due to impaired TCA cycle activity, leading to increased lactate production, metabolic exhaustion, and reduced therapeutic efficacy. Notably, CXCR4 overexpression enhanced memory formation most prominently under hypoxic conditions (Fig. 5b), further supporting a model in which CXCR4 facilitates memory differentiation by promoting metabolic adaptation to the bone marrow microenvironment.

CXCR4 expression promotes long-term functional immune memory and durable protection against AML rechallenge in vivo

Lastly, we investigated the formation of functional immune memory in vivo. In PDX models treated with mCXCR4(+) CD25-targeted CAR-T cells, TCF1⁺ memory T cells were observed in close proximity to CXCL12⁺ stromal cells in the spleen, while CCR7⁺ memory T cells localized near CXCL12⁺ stromal cells in the liver beyond one month post-transfusion, suggesting persistent maintenance of central memory T cells within peripheral tissue niches (Fig. 7a, b).

a HE (left) and TCF1 IHC (middle) staining in Patient 13-derived PDX spleens harvested 30 days post-treatment with mCXCR4(+) CD25-targeted CAR-T cells. CXCL12 IHC staining (right) in spleens of untransplanted NSG mice reveals stromal expression. Inset panels show 20x magnification. Scale: 20 µm. Representative images selected from sections prepared from n = 3 animals (b) CXCL12, CCR7 and CD3 IHC staining in Patient 11-derived PDX liver harvested 40 days post-treatment with mCXCR4(+) CD25-targeted CAR-T cells. CCR7-expressing naïve/Tscm and Tcm cells are observed to co-localized CXCL12-expressing stromal cells in the liver. Representative images selected from sections prepared from n = 5 animals. Scale: 50 µm c) Frequencies of immune-surveillant (CD226⁺TIGIT⁻), memory (IL7RA⁺, TCF1⁺), and checkpoint-expressing (TIM3⁺TIGIT⁺) CD4⁺ CAR-T cells in bone marrow and spleen of PDX mice treated with mCXCR4(+) CD25-targeted CAR-T cells, stratified by time post-injection. Short-term (ST) samples (n = 8 animals) were collected <30 days post-treatment; long-term (LT) samples (n = 22 animals) were collected >90 days post-treatment. Graphs show mean ± SD. Statistical analysis performed using an unpaired two-tailed t-test. d In vivo re-challenge of AML PDX previously cleared of peripheral blood AML following mCXCR4(+) CD25-targeted CAR-T treatment. Patient-derived CD25⁺ AML cells failed to engraft or expand in previously treated animals (pink lines and dots) across four independent experiments, whereas robust AML proliferation occurred in control littermates lacking CAR-T therapy. PDX IDs correspond to those in Supplementary Data 4. Source data are provided as a Source data file for (c, d).

Immunophenotypic analysis of CAR-T cells isolated at short-term ( < 30 days) and long-term ( > 90 days) time points post-treatment revealed sustained and increasingly pronounced CXCR4-associated advantages. These included elevated frequencies of CD226⁺TIGIT⁻ immune-surveillant T cells and IL7RA⁺ Tscm-cm populations, alongside reduced proportions of TIGIT⁺TIM3⁺ exhausted CD4⁺ T cells and Ki67⁺ proliferating cells (Fig. 7c).

To directly assess functional memory, we performed an AML rechallenge experiment. PDX mice that achieved complete remission following mCXCR4(+) CD25-targeted CAR-T treatment were re-injected with CD25-expressing AML cells 4-5 weeks post-remission, a stage when CAR-T cell numbers typically contract (Fig. 3f). AML cells failed to engraft or initiate disease (Fig. 7d), demonstrating functional memory and long-lasting anti-leukemic activity of mCXCR4(+) CAR-T cells.

Together, these findings show that CXCR4 expression supports the formation and maintenance of long-lived, immune-surveillant memory CAR-T cells capable of preventing relapse through sustained functional persistence. CXCR4 signaling thus plays a critical role in overcoming T cell exhaustion and enabling long-term disease suppression in vivo.