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385 Selection of CD8+CD103+CD31- tumor infiltrating lymphocytes (TIL) from bulk cultures enriches tumor-specific reactivity in a patient-agnostic manner
  1. Nivedita Ratnam,
  2. Frank Lowery,
  3. Hyunmi Halas,
  4. Stephanie Goff and
  5. Steven A Rosenberg
  1. National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Adoptive transfer of autologous T cells derived from tumor fragments cultured in IL-2 have been successful in treating melanomas and select epithelial cancers.1 2 However, tumor-reactive T cells are a small percentage of all TIL while most are bystander cells.3 Identification of neoantigen-reactive TIL for treatment involves time-intensive testing of TIL cultures against patient-specific candidate neoantigens, resulting in selection of TIL at the fragment rather than cell level and relying on tumor sequencing and testing of patient-specific reagents.4 5

Methods In this study, we cultured multiple fragments from resected tumors together in GREX100 flasks with IL-2 (n=14 patients). In 3 weeks, these cultures yielded a median of 3.09e8 (1.4e8-6e8) TIL. The large cell yield allowed the study of cell surface marker expression for sorting tumor-reactive TIL by FACS.

To identify candidate markers, we performed single-cell RNA-, CITE- and T cell receptor (TCR)-sequencing on TIL from a human rectal cancer lung metastasis. We cloned and expressed high frequency CD8 TCRs in peripheral blood lymphocytes (PBL) and screened them for tumor reactivity. Twelve TCRs showed functional reactivity to tumor neoantigens and 3 TCRs were reactive to CMV/EBV/Flu peptides (indicating true bystanders). Projecting these relevant TCRs onto the transcriptomic map indicated that cells with tumor-reactive TCRs clustered separately from bystanders (figure 1A). Antibody expression analysis showed CD103 as the top marker for reactive cells and CD31 for bystander cells (figure 1B).

Results Applying these findings to 4 prospective patients with metastatic epithelial cancer, we analyzed the expression of CD103 and CD31 within CD8+ TIL from pooled fragment cultures. The percentage of CD103+CD31- cells in these patients ranged from 2.12% to 29.6%. We sorted TIL according to CD103/CD31 expression and performed a rapid expansion protocol (REP). The CD103+CD31- population had a median expansion of 833.5 (285–1081) fold. Subsequent screens against candidate neoantigens demonstrated enriched reactivity among CD103+CD31- CD8+ TIL in all 4 patients compared to CD103- and CD31+ cells. In one patient, reactivity (% 4–1BB+ TIL following specific antigenic stimulation [5]) increased from 6% in the bulk, unsorted and un-REPed cultures to 25% among the REPed CD103+CD31- cells.

Conclusions Taken together, this study can help identify reactive TIL from bulk cultures of tumor fragments without requiring patient-specific reagents for testing. Studies are ongoing in scalability of this approach to generate high numbers of these TIL for patient treatment. If successful, this approach has the potential to decrease the time between resection to treatment.


  1. Kim SP, et al. Adoptive Cellular Therapy with Autologous Tumor-Infiltrating Lymphocytes and T-cell Receptor-Engineered T Cells Targeting Common p53 Neoantigens in Human Solid Tumors. Cancer Immunol Res, 2022;10(8):932–946.

  2. Lennerz V, et al. The response of autologous T cells to a human melanoma is dominated by mutated neoantigens. Proc Natl Acad Sci U S A, 2005;102(44):16013–8.

  3. Lowery FJ, et al. Molecular signatures of antitumor neoantigen-reactive T cells from metastatic human cancers. Science, 2022;375(6583):877–884.

  4. Tran E, PF Robbins, SA Rosenberg. ‘Final common pathway’ of human cancer immunotherapy: targeting random somatic mutations. Nat Immunol, 2017;18(3):255–262.

  5. Parkhurst MR, et al. Unique Neoantigens Arise from Somatic Mutations in Patients with Gastrointestinal Cancers. Cancer Discovery, 2019;9(8):1022–1035.

Ethics Approval This study was approved by National Institutes of Health (NIH) Internal Review Board (IRB); approval number 03-C-0277

Abstract 385 Figure 1

(A) UMAP of TIL clustered according to single-cell transcriptomic states. Map in gray is overlaid with corresponding scTCR data, with CMV/EBV/Flu-reactive bystander clones highlighted in red and tumor/neoantigen-reactive clones in blue. (B) TIL UMAPs from (A) overlaid with antibody expression data of top tumor-relevant (CD103, top) and bystander (CD31, bottom) markers

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