Clonal expansion of t memory stem cells determines early anti-leukemic responses and long-term car t cell persistence in patients

ABSTRACT Low-affinity CD19 chimeric antigen receptor (CAR) T cells display enhanced expansion and persistence, enabling fate tracking through integration site analysis. Here we show that

integration sites from early (1 month) and late (>3 yr) timepoints cluster separately, suggesting different clonal contribution to early responses and prolonged anti-leukemic

surveillance. CAR T central and effector memory cells in patients with long-term persistence remained highly polyclonal, whereas diversity dropped rapidly in patients with limited CAR T

persistence. Analysis of shared integrants between the CAR T cell product and post-infusion demonstrated that, despite their low frequency, T memory stem cell clones in the product

contributed substantially to the circulating CAR T cell pools, during both early expansion and long-term persistence. Our data may help identify patients at risk of early loss of CAR T cells

and highlight the critical role of T memory stem cells both in mediating early anti-leukemic responses and in long-term surveillance by CAR T cells. Access through your institution Buy or

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LEUKAEMIA REMISSIONS WITH PERSISTENCE OF CD4+ CAR T CELLS Article 02 February 2022 DURABLE RESPONSE TO CAR T IS ASSOCIATED WITH ELEVATED ACTIVATION AND CLONOTYPIC EXPANSION OF THE CYTOTOXIC

NATIVE T CELL REPERTOIRE Article Open access 23 May 2025 TWO-STAGE CD8+ CAR T-CELL DIFFERENTIATION IN PATIENTS WITH LARGE B-CELL LYMPHOMA Article Open access 06 May 2025 DATA AVAILABILITY

The fastq files relative to sequencing of IS amplicons generated for this study are available through the NCBI repository (https://submit.ncbi.nlm.nih.gov/), BioProject accession number

PRJNA718947. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request. CODE

AVAILABILITY No new code was generated or used in this manuscript. REFERENCES * Sallusto, F., Lenig, D., Förster, R., Lipp, M. & Lanzavecchia, A. Two subsets of memory T lymphocytes with

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Gene Ther._ 28, 1122–1129 (2017). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS This work was funded the JP Moulton Foundation and supported by the National Institute

for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London. P.J.A. is a recipient of an NIHR Research

Professorship which also supported S.G. M.P. is supported by the National Institute of Health Research University College London Hospital Biomedical Research Centre. A.J.T. is a recipient of

a Wellcome Trust Senior Fellowship. The work of L.B. was supported by the Wellcome Trust (grant no. 104807/Z/14/Z, Principal Research Fellowship awarded to A.J.T.) and the National

Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK. L.B. is also currently Director of R&D for the

gene therapy company AVROBIO located in Cambridge, MA, USA (none of what is presented in this work is supported by or relates to AVROBIO). The work of L.B. was also in part performed using

the resources of the Gene Therapy Program of the Dana Farber/Boston Children’s Cancer and Blood Disorders Center. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Molecular and Cellular

Immunology Section, UCL Great Ormond Street Institute of Child Health, London, UK Luca Biasco, Natalia Izotova, Christine Rivat, Rachel Richardson, Aleks Guvenel, Adrian J. Thrasher & 

Persis J. Amrolia * Gene Therapy Program, Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, Harvard Medical School, Boston, MA, USA Luca Biasco * Molecular Haematology

Section, UCL Great Ormond Street Institute of Child Health, London, UK Sara Ghorashian * Department of Haematology, University College London Hospital, London, UK Rachael Hough * Department

of Bone Marrow Transplant, Royal Manchester Children’s Hospital, Manchester, UK Robert Wynn * CRUK UCL Cancer Trials Centre, University College London, London, UK Bilyana Popova & Andre

Lopes * University College London Cancer Institute, London, UK Martin Pule * Department of Bone Marrow Transplantation, Great Ormond Street Hospital, London, UK Persis J. Amrolia Authors *

Luca Biasco View author publications You can also search for this author inPubMed Google Scholar * Natalia Izotova View author publications You can also search for this author inPubMed 

Google Scholar * Christine Rivat View author publications You can also search for this author inPubMed Google Scholar * Sara Ghorashian View author publications You can also search for this

author inPubMed Google Scholar * Rachel Richardson View author publications You can also search for this author inPubMed Google Scholar * Aleks Guvenel View author publications You can also

search for this author inPubMed Google Scholar * Rachael Hough View author publications You can also search for this author inPubMed Google Scholar * Robert Wynn View author publications You

can also search for this author inPubMed Google Scholar * Bilyana Popova View author publications You can also search for this author inPubMed Google Scholar * Andre Lopes View author

publications You can also search for this author inPubMed Google Scholar * Martin Pule View author publications You can also search for this author inPubMed Google Scholar * Adrian J.

Thrasher View author publications You can also search for this author inPubMed Google Scholar * Persis J. Amrolia View author publications You can also search for this author inPubMed Google

Scholar CONTRIBUTIONS L.B. designed and co-supervised the study, performed computational analyses and wrote the manuscript. N.I. and C.R. performed cell isolation and molecular insertion

sites retrieval. S.G. and R.R. collected and provided clinical sample material for analysis. A.G. cryopreserved study samples and performed flow cytometric staining. R.H. and R.W. were

Principal Investigators for the clinical study. B.P. wrote study documentation and provided trial management. A.L. provided statistical analysis for the study. M.P. generated the CAR

construct and participated in its preclinical characterization. A.J.T. conceived the idea and participated in the experimental design and data analysis. P.J.A. supervised the study as PI and

wrote the manuscript. CORRESPONDING AUTHOR Correspondence to Persis J. Amrolia. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION

PEER REVIEW INFORMATION _Nature Cancer_ thanks Justin Eyquem, Alena Gros and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.' after the

Supplementary information section. PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. EXTENDED DATA

EXTENDED DATA FIG. 1 IMMUNOPHENOTYPING OF CD3+ CELLS IN PT4 AND PT6. Additional FACS plot on CD62L+CD45RA+CD95 expression in CAR+ T cells, subtype frequencies within total CD3+ cells and

relative summary of data on the CD3+ T cell composition overtime for Pt4 (top panels) and Pt6 (bottom panels). Percentages of CD62L/CD45RA compartments are showed inside each gate.

Longitudinal contribution of each subpopulations to the CD3+ cell compartment is shown below for each patient according to the color code legend on the right. Source data EXTENDED DATA FIG.

2 INTEGRATION SITES COLLECTED FROM PT4 AND PT6. Summary of number of IS (grey bars) and relative sequencing reads (white bars) in Pt4 (top panel) and Pt6 (bottom panel) (d = days after

treatment). Source data EXTENDED DATA FIG. 3 DISTRIBUTION OF INTEGRATION SITES COLLECTED FROM PT4 AND PT6. Distribution of IS with respect to TSS (left plots) or gene content of the loci

(right plots) in the cell product (red) or after infusion at early or late timepoints after treatment (blue) for Pt4 (top panels) and Pt6 (bottom panels). EXTENDED DATA FIG. 4 GENE

CATEGORIES RELATIVE TO INTEGRATION SITES COLLECTED FROM PT4 AND PT6. Plots on the left show word clouds of hit genes in the cell product (in red) and after infusion at early or late

timepoints after treatment (in blue) for Pt4 (top panels) and Pt6 (bottom panels). Relative gene enrichment analysis for top biological processes of hit genes relative to each word cloud in

the product (red bars) or after infusion (blue bars) is shown on the right plots (significance reported as -log10 binomial p-value from one-tailed tests decreasing from top to bottom).

EXTENDED DATA FIG. 5 RELATIVE ABUNDANCE AND DIVERSITY OF INTEGRATION SITES COLLECTED FROM PT4 AND PT6. Scattered dot plots showing relative abundance of IS in the product (A) and at early

(B) or late (C) timepoints after treatment in Pt4 (plots on the left) and Pt6 (plots on the right) (d = days after treatment, m = months after treatment). Mean percent abundance is shown as

a dotted line for each sample. Number of events in each dataset is equal to what reported in Extended Data Fig. 2. D) Longitudinal plots showing Gini/Simpson Diversity Index (left y-axis) of

IS overtime in TSCM (orange lines) and in TCM/TEM (dark blue) and in all T cells (green lines). The grey lines show the percentage of CAR cells overtime (right y-axis). Source data EXTENDED

DATA FIG. 6 CORRELATION OF INTEGRATION SITES AND NUMBER OF CELLS COLLECTED FROM PT4 AND PT6. Longitudinal plots showing number of IS collected (A) and number of cells collected (B) in TSCM

(orange lines) and in TCM/TEM (dark blue) at early timepoints for Pt4 (left panels) and Pt6 (right panels). The plot in (C) shows the correlation observed between number of cells collected

(y-axis) and clonal diversity for all samples and both patients (x-axis, Shannon Diversity Index) shown as blue dots. Interpolation with best fit curve and R squared value are shown in

black. Source data EXTENDED DATA FIG. 7 INTEGRATION SITES COLLECTED FROM PT10 AND PT17. Summary of number of IS (grey bars) and relative sequencing reads (white bars) in Pt10 (top panel) and

Pt17 (bottom panel) (d = days after treatment). Source data EXTENDED DATA FIG. 8 SHARING OF INTEGRATION SITES IN THE PRODUCT WITH THE CAR T POPULATIONS IN PT10 AND PT17 AFTER INFUSION. Ring

plots showing relative contribution from each subtype (coloured section of the ring) to the pool of IS detected in the product and at early timepoints in Pt10 (left) and Pt6 (right). The

total number of IS captured both in the product and after infusion is shown inside each plot. The relative percentage of IS belonging to each T cell subtype of the product that were shared

with samples after infusion is shown in white inside each section of each ring plot. SUPPLEMENTARY INFORMATION SUPPLEMENTARY DATA 1 Clinical study protocol. REPORTING SUMMARY SOURCE DATA

SOURCE DATA FIG. 1 Raw datasets relative to figure panels. SOURCE DATA FIG. 2 Raw datasets relative to figure panels. SOURCE DATA FIG. 4 Raw datasets relative to figure panels. SOURCE DATA

FIG. 5 Raw datasets relative to figure panels. SOURCE DATA FIG. 6 Raw datasets relative to figure panels. SOURCE DATA FIG. 7 Raw datasets relative to figure panels. SOURCE DATA FIG. 8 Raw

datasets relative to figure panels. SOURCE DATA EXTENDED DATA FIG. 1 Raw datasets relative to figure panels. SOURCE DATA EXTENDED DATA FIG. 2 Raw datasets relative to figure panels. SOURCE

DATA EXTENDED DATA FIG. 5 Raw datasets relative to figure panels. SOURCE DATA EXTENDED DATA FIG. 6 Raw datasets relative to figure panels. SOURCE DATA EXTENDED DATA FIG. 7 Raw datasets

relative to figure panels. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Biasco, L., Izotova, N., Rivat, C. _et al._ Clonal expansion of T memory stem

cells determines early anti-leukemic responses and long-term CAR T cell persistence in patients. _Nat Cancer_ 2, 629–642 (2021). https://doi.org/10.1038/s43018-021-00207-7 Download citation

* Received: 07 July 2020 * Accepted: 07 April 2021 * Published: 24 May 2021 * Issue Date: June 2021 * DOI: https://doi.org/10.1038/s43018-021-00207-7 SHARE THIS ARTICLE Anyone you share the

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