scholarly journals 178 Expansion of Tumor-Infiltrating Lymphocytes and Marrow-Infiltrating Lymphocytes from Pediatric Malignant Solid Tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A190-A190
Author(s):  
Jonathan Metts ◽  
Jonathan Hensel ◽  
Alejandro Alfaro ◽  
Brook Olmo ◽  
Shari Pilon-Thomas ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Solid Tumors ◽  
Memory T Cells ◽  
Tumor Infiltrating Lymphocytes ◽  
Standard Of Care ◽  
Rapid Expansion ◽  
Expansion Protocol ◽  
Specific Reactivity ◽  
Infiltrating Lymphocytes

BackgroundHigh-risk non-CNS pediatric malignant solid tumors (pMST) have unsatisfactory outcomes, and novel therapies are warranted. Adoptive cellular therapy (ACT) using tumor-infiltrating lymphocytes (TIL) has produced durable responses in melanoma, and improvements in TIL expansion have made ACT-TIL feasible for other solid tumors.1–3 Preclinical mouse models suggest that T-cells from bone marrow (marrow-infiltrating lymphocytes, MIL) have antitumor reactivity offering another source for ACT.4 5 To demonstrate feasibility of ACT in pMST we hypothesized that TIL/MIL can be expanded from these patients.MethodsPatients ≤21 years old undergoing standard-of-care pMST resection were enrolled on an IRB approved protocol. Fresh tumor (≥1 cm3) was collected and bone marrow (10 mL) was obtained when accessible from standard of care procedures. TIL/MIL were cultured in media containing IL-2 (6000 IU/mL). TIL were expanded from tumor fragment cultures (TFC, >1 mm3) or tumor digest. Select TIL samples were further expanded using a rapid expansion protocol (REP). Phenotype of expanded TIL (CD3, CD4, CD8 and CD56) was evaluated using flow cytometry. IFN- γ secretion, measured by ELISA assay, measured tumor-specific reactivity after co-culture with autologous tumor and TIL.ResultsTwenty samples were obtained between March 2019-May 2021. Two samples were ineligible (final pathology not pMST), leaving 18 samples for analysis. Five marrow samples were collected. TIL were expanded from 14/18 samples (78%) through TFC with median 5.17 x 10^6 cells (range 1.86 x 10^6–3.21 x 10^8). Average phenotype (%) of TFC-TIL were CD3 (63.17), CD4 (21.46), CD8 (46.19) and CD56 (32.68). 9/10 (90%) of samples successfully underwent REP with median 9.35 x 10^7 cells(range 2.49 x 10^7–5.86 x 10^8) final viable TIL and average fold-change 718.6 (median 458.6). Average phenotype (%) of post-REP TIL were CD3 (96.04), CD4 (75.04), CD8 (19.17) and CD56 (0.43). TIL were expanded from TFC of therapy-naïve (8/10, 80%) and pretreated (chemotherapy and checkpoint immunotherapy) samples (5/8, 63%). Seven samples had sufficient tissue to test tumor-specific reactivity; all were non-reactive. MIL pre-REP was expanded from four samples with median 9.55 x 10^6 cells (range 8.00 x 10^5–1.00 x 10^7). Average phenotype of expanded MIL (%) were CD3 (45.17), CD4 (24.46), CD8 (36.15) and CD56 (28.21) (table 1).Results of TIL and MIL expansion from 18 pMST samples. Abbreviations: Dx: diagnosis, pre-REP: pre-rapid expansion protocol, post-REP: post-rapid expansion protocol, PBMC: peripheral blood mononuclear cells, GNB: ganglioneuroblastoma, WT: Wilms tumor, OS: osteosarcoma, NB: neuroblastoma, IMT: inflammatory myofibroblastic tumor; ASPS: alveolar soft part sarcoma, SS: synovial sarcoma, ERMS: embryonal rhabdomyosarcoma, N: no systemic therapy, C: chemotherapy, I: immunotherapy, DNG: did not grow, N/A: not applicable, NR: non-reactiveAbstract 178 Table 1Expansion of TIL from pMSTConclusionsThis study demonstrates feasibility of pMST TIL expansion ex vivo. Due to tissue volume constraints inherent in pMST sampling, anti-tumor reactivity testing was not feasible for most patients. Determining optimal strategy for TIL-ACT in pMST will require further investigation regarding techniques for expanding tumor-specific TIL.AcknowledgementsThe authors would like to thank Swim Across America (www.swimacrossamerica.org) and the Ocala Royal Dames (www.ocalaroyaldames.org) for their generous support of this work.ReferencesRosenberg SA, Restifo NP. Adoptive cell transfer as personalized immunotherapy for human cancer. Science 2015;348(6230):62–68.Hall M, Mullinax JE, Royster E, et al. Expansion and characterization of tumor-infiltrating lymphocytes from human sarcoma. Journal of Immunotherapy of Cancer 2015;3(Suppl. 2):19.Mullinax JE, Hall M, Beatty M, et al. Expanded tumor-infiltrating lymphocytes from soft tissue sarcoma have tumor-specific function. J Immunother 2021;44(2):63–70.Feuerer M, Beckhove P, Bai L, et al. Therapy of human tumors in NOD/SCID mice with patient-derived reactivated memory T cells from bone marrow. Nat Med 2001;7(4):452–458.Feuerer M, Rocha M, Bai L, et al. Enrichment of memory T cells and other profound immunological changes in the bone marrow from untreated breast cancer patients. Int J Cancer 2001;92(1):96–105.Ethics ApprovalThis study was approved by the Johns Hopkins All Children’s Hospital IRB (#IRB00193453). Consent was obtained from the patient or parent, as appropriate for age, prior to participating in this study.

154 Marrow-infiltrating lymphocytes (MILs): A novel adoptive immunotherapy for hematological and solid tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A168-A168
Author(s):  
Eric Lutz ◽  
Lakshmi Rudraraju ◽  
Elizabeth DeOliveira ◽  
Amanda Seiz ◽  
Monil Shah ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Lung Cancer ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cells ◽  
Memory T Cells ◽  
Small Cell ◽  
Small Cell Lung ◽  
Blood Samples ◽  
Infiltrating Lymphocytes

BackgroundMarrow infiltrating lymphocytes (MILsTM) are the product of activating and expanding bone marrow T cells.1 The bone marrow is a specialized niche in the immune system enriched for antigen-experienced, memory T cells. In patients with multiple myeloma and other hematological malignancies that relapse post-transplant, MILs have been shown to contain tumor antigen-specific T cells and adoptive cell therapy (ACT) using MILs has demonstrated antitumor activity.2 3 The bone marrow has been shown to harbor tumor-antigen specific T cells in patients with melanoma,4 5 glioblastoma,6 breast,7 non-small-cell lung8 and pancreatic cancers.9 Here, we sought to determine if tumor-specific MILs could be expanded from the bone marrow of patients with a range of different solid tumors.MethodsBone marrow and blood samples were collected from patients with advanced and metastatic cancers. To date, samples have been collected from a minimum of four patients with non-small cell lung cancer (NSCLC), prostate cancer, head and neck cancer, glioblastoma, and breast cancer. Samples from patients with multiple myeloma were used as a reference control. Utilizing a 10-day proprietary process, MILs and peripheral blood lymphocytes (PBLs) were activated and expanded from patient bone marrow and blood samples, respectively. T cell lineage-specific markers (CD3, CD4 and CD8) were characterized by flow cytometry pre- and post-expansion.Tumor-specific T cells were quantitated in expanded MILs and PBLs using a previously described cytokine-secretion assay [2]. Briefly, autologous antigen-presenting cells (APCs) were pulsed with lysates from allogeneic cancer cell lines and co-cultured with activated MILs or PBLs. APCs pulsed with irrelevant mis-matched cancer cell line lysates or media alone were used as negative controls. Tumor-specific T cells were defined as the IFNgamma-producing population by flow cytometry.ResultsMILs were successfully expanded from all patient bone marrow samples tested, regardless of tumor type. Cytokine-producing tumor-specific CD4+ and CD8+ T cells were detected in each of the expanded MILs. In contrast, tumor-specific T cells were not detected in any of the matched activated and expanded PBLs.ConclusionsMILs have been successfully grown for all solid tumor types evaluated, including NSCLC, prostate, head and neck, glioblastoma and breast cancer. Clinical studies have been completed in patients with multiple myeloma and other hematological cancers. 2 3 A phase IIa trial to evaluate MILs in combination with a checkpoint inhibitor is underway in patients with anti-PD1/PDL1-refractory NSCLC (ClinicalTrials.gov Identifier: NCT04069936). The preclinical data presented herein demonstrate that expanding MILs is feasible. MILs-based therapies hold therapeutic promise across a wide range of tumor indications.Ethics ApprovalThis study was approved by each participating instituion’s IRB.ReferencesBorrello I and Noonan KA. Marrow-Infiltrating Lymphocytes - Role in Biology and Cancer Therapy. Front Immunol 2016 March 30; 7(112)Noonan KA, Huff CA, Davis J, et al. Adoptive transfer of activated marrow-infiltrating lymphocytes induces measurable antitumor immunity in the bone marrow in multiple myeloma. Sci. Transl. Med 2015;7:288ra78.Biavati L, Noonan K, Luznik L, Borrello I. Activated allogeneic donor-derived marrow-infiltrating lymphocytes display measurable in vitro antitumor activity. J Immunother 2019 Apr;42(3):73–80.Müller-Berghaus J, Ehlert K, Ugurel S, et al. Melanoma-reactive T cells in the bone marrow of melanoma patients: association with disease stage and disease duration. Cancer Res 2006;66(12):5997–6001.Letsch A, Keilholz U, Assfalg G, et al., Bone marrow contains melanoma-reactive CD8+ effector T Cells and, compared with peripheral blood, enriched numbers of melanoma-reactive CD8+ memory T cells. Cancer Res 2003 Sep 1;63(17):5582–5586.Chongsathidkiet P, Jackson C, Koyama S, et al., Sequestration of T cells in bone marrow in the setting of glioblastoma and other intracranial tumors. Nature Medicine 2018 Aug 13; 24:1459–1468.Feuerer M, Rocha M, Bai L, et al. Enrichment of memory T cells and other profound immunological changes in the bone marrow from untreated breast cancer patients. Int J Cancer 2001; 92(1):96–105.Safi S, Yamauchi Y, Stamova S, et al. Bone marrow expands the repertoire of functional T cells targeting tumor-associated antigens in patients with resectable non-small-cell lung cancer. Oncoimmunology 2019;8(12):e1671762.Schmitz-Winnenthal FH, Volk C, Z’Graggen K, et al. High frequencies of functional tumor-reactive T cells in bone marrow and blood of pancreatic cancer patients. Cancer Res 2005;65(21):10079–87.

773 Adoptive Cell Therapy Response in Melanoma is Mediated by Stem-like CD8 T cells

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A822-A822
Author(s):  
Sri Krishna ◽  
Frank Lowery ◽  
Amy Copeland ◽  
Stephanie Goff ◽  
Grégoire Altan-Bonnet ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Single Cell ◽  
Cd8 T Cells ◽  
Tumor Infiltrating Lymphocytes ◽  
High Dimensional ◽  
Tumor Control ◽  
Intrinsic Factors ◽  
Infiltrating Lymphocytes

BackgroundAdoptive T cell therapy (ACT) utilizing ex vivo-expanded autologous tumor infiltrating lymphocytes (TILs) can result in complete regression of human cancers.1 Successful immunotherapy is influenced by several tumor-intrinsic factors.2 3 Recently, T cell-intrinsic factors have been associated with immunotherapy response in murine and human studies.4 5 Analyses of tumor-reactive TILs have concluded that anti-tumor neoantigen-specific TILs are enriched in subsets defined by the expression of PD-1 or CD39.6 7 Thus, there is a lack of consensus regarding the tumor-reactive TIL subset that is directly responsible for successful immunotherapies such as ICB and ACT. In this study, we attempted to define the fitness landscape of TIL-enriched infusion products to specifically understand its phenotypic impact on human immunotherapy responses.MethodsWe compared the phenotypic differences that could distinguish bulk ACT infusion products (I.P.) administered to patients who had complete response to therapy (complete responders, CRs, N = 24) from those whose disease progressed following ACT (non-responders, NRs, N = 30) by high dimensional single cell protein and RNA analysis of the I.P. We further analyzed the phenotypic states of anti-tumor neoantigen specific TILs from patient I.P (N = 26) by flow cytometry and single cell transcriptomics.ResultsWe identified two CD8+ TIL populations associated with clinical outcomes: a memory-progenitor CD39-negative stem-like TIL (CD39-CD69-) in the I.P. associated with complete cancer regression (overall survival, P < 0.0001, HR = 0.217, 95% CI 0.101 to 0.463) and TIL persistence, and a terminally differentiated CD39-positive TIL (CD39+CD69+) population associated with poor TIL persistence post-treatment. Although the majority (>65%) of neoantigen-reactive TILs in both responders and non-responders to ACT were found in the differentiated CD39+ state, CR infusion products also contained a pool of CD39- stem-like neoantigen-specific TILs (median = 8.8%) that was lacking in NR infusion products (median = 23.6%, P = 1.86 x 10-5). Tumor-reactive stem-like T cells were capable of self-renewal, expansion, and persistence, and mediated superior anti-tumor response in vivo.ConclusionsOur results support the hypothesis that responders to ACT received infusion products containing a pool of stem-like neoantigen-specific TILs that are able to undergo prolific expansion, give rise to differentiated subsets, and mediate long-term tumor control and T cell persistence, in line with recent murine ICB studies mediated by TCF+ progenitor T cells.4 5 Our data also suggest that TIL subsets mediating ACT-response (stem-like CD39-) might be distinct from TIL subsets enriched for anti-tumor-reactivity (terminally differentiated CD39+) in human TIL.6 7AcknowledgementsWe thank Don White for curating the melanoma patient cohort, and J. Panopoulos (Flowjo) for helpful discussions on high-dimensional analysis, and NCI Surgery Branch members for helpful insights and suggestions. S. Krishna acknowledges funding support from NCI Director’s Innovation Award from the National Cancer Institute.Trial RegistrationNAEthics ApprovalThe study was approved by NCI’s IRB ethics board.ReferencesGoff SL, et al. Randomized, prospective evaluation comparing intensity of lymphodepletion before adoptive transfer of tumor-infiltrating lymphocytes for patients with metastatic melanoma. J Clin Oncol 2016;34:2389–2397.Snyder A, et al. Genetic basis for clinical response to CTLA-4 blockade in melanoma. N Engl J Med 2014;371:2189–2199.McGranahan N, et al. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science 2016;351:1463–1469.Sade-Feldman M, et al. Defining T cell states associated with response to checkpoint immunotherapy in melanoma. Cell 2019;176:404.Miller BC, et al. Subsets of exhausted CD8 T cells differentially mediate tumor control and respond to checkpoint blockade. Nat. Immunol 2019;20:326–336.Simoni Y, et al. Bystander CD8 T cells are abundant and phenotypically distinct in human tumour infiltrates. Nature 2018;557:575–579.Gros A, et al. PD-1 identifies the patient-specific CD8+ tumor-reactive repertoire infiltrating human tumors. J Clin Invest 2014;124:2246–2259.

scholarly journals Tumor microenvironment in triple-negative breast cancer: the correlation of tumor-associated macrophages and tumor-infiltrating lymphocytes

2021 ◽  
Author(s):  
H. Kuroda ◽  
T. Jamiyan ◽  
R. Yamaguchi ◽  
A. Kakumoto ◽  
A. Abe ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Tumor Microenvironment ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Cytotoxic T Cells ◽  
Tumor Infiltrating Lymphocytes ◽  
Tumor Associated Macrophages ◽  
Free Survival ◽  
Infiltrating Lymphocytes

Abstract Purpose Immune cells such as cytotoxic T cells, helper T cells, B cells or tumor-associated macrophages (TAMs) contribute to the anti-tumor response or pro-tumorigenic effect in triple negative breast cancer (TNBC). The interrelation of TAMs, T and B tumor-infiltrating lymphocytes (TILs) in TNBC has not been fully elucidated. Methods We evaluated the association of tumor-associated macrophages, T and B TILs in TNBC. Results TNBCs with a high CD68+, CD163+ TAMs and low CD4+, CD8+, CD20+ TILs had a significantly shorter relapse-free survival (RFS) and overall survival (OS) than those with low CD68+, CD163+ TAMs and high CD4+, CD8+, CD20+ TILs. TNBCs with high CD68+ TAMs/low CD8+ TILs showed a significantly shorter RFS and OS and a significantly poorer prognosis than those with high CD68+ TAMs/high CD8+ TILs, low CD68+ TAMs/high CD8+ TILs, and low CD68+/low CD8+. TNBCs with high CD163+ TAMs/low CD8+, low CD20 + TILs showed a significantly shorter RFS and OS and a significantly poorer prognosis than those with high CD163+ TAMs/high CD8+ TILs and high CD163+ TAMs /high CD20+ TILs. Conclusions Our study suggests that TAMs further create an optimal tumor microenvironment (TME) for growth and invasion of cancer cells when evasion of immunoreactions due to T and B TILs occurs. In TNBCs, all these events combine to affect prognosis. The process of TME is highly complex in TNBCs and for an improved understanding, larger validation studies are necessary to confirm these findings.

scholarly journals Tumor infiltrating lymphocytes in seminoma lesions comprise clonally expanded cytotoxic T cells

2006 ◽  
Vol 119 (4) ◽  
pp. 831-838 ◽  
Author(s):  
Sine Reker Hadrup ◽  
Otto Brændstrup ◽  
Grete Krag Jacobsen ◽  
Svend Mortensen ◽  
Lars Østergaard Pedersen ◽  
...  
Keyword(s):  
T Cells ◽  
Cytotoxic T Cells ◽  
Tumor Infiltrating Lymphocytes ◽  
Infiltrating Lymphocytes

scholarly journals Increased IL-15 Production and Accumulation of Highly Differentiated CD8+ Effector/Memory T Cells in the Bone Marrow of Persons with Cytomegalovirus

2017 ◽  
Vol 8 ◽  
Author(s):  
Luca Pangrazzi ◽  
Erin Naismith ◽  
Andreas Meryk ◽  
Michael Keller ◽  
Brigitte Jenewein ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Memory T Cells ◽  
Effector Memory ◽  
Effector Memory T Cells
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