β-catenin does not regulate memory T cell phenotype

Chimeric antigen receptor T-cells (CAR-T) have emerged as an extremely promising therapy for hematological malignancies. The immunophenotype of apheresis material and the CAR-T cell product is known to be predictive of the likelihood of response to treatment of certain malignancies. Central memory and stem cell-like memory T cell phenotypes are associated with a more sustained proliferative response and long-term CAR-T persistence (Fraietta et al, Nature Medicine, 2018). There is an unmet need for standardized methods and reagents to reliably profile the memory phenotype of CAR-Ts to better evaluate product quality, and support improvements in CAR-T manufacturing. The BD Biosciences dried memory T-cell panel contains a pre-validated mixture of 7 antibodies for the identification of naïve, stem cell memory, central memory and effector memory CD4+ and CD8+ T cell subsets. The pre-mixed dried antibody tube offers consistency in staining profiles over time and reduces the risk of operator errors. Additional drop-in antibodies can complement the panel and enable more in-depth evaluation of the T cell phenotype. Here we demonstrate the use of this panel with drop-in markers to monitor changes in expression of PD-1, TIM-3, LAG-3, HLA-DR, CD45RO, and CXCR3 on T cells transduced to express our novel anti-CD37 CAR. Cells were stained at day 0 prior to transduction, day 7, and following resting and re-stimulation, and acquired on a 12 color BD FACS Lyric. The use of a standardized memory T-cell panel will allow us to more accurately evaluate how T-cell phenotype impacts on the efficacy and longevity of response in patients receiving CAR-T therapies. Disclosures Maus: INFO PENDING: Other: INFO PENDING. Bornheimer:BD Biosciences: Employment. Hanley:BD Biosciences: Employment. Frigault:Novartis: Patents & Royalties: Royalty; Arcellx, Celgene, Foundation Medicine, Kite/Gilead, Nkarta, Novartis, and Xenetic: Consultancy.

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The in vitro senescence of human T lymphocytes: Failure to divide is not associated with a loss of cytolytic activity or memory T cell phenotype

Mechanisms of Ageing and Development ◽

10.1016/0047-6374(93)90121-7 ◽

1993 ◽

Vol 67 (1-2) ◽

pp. 173-185 ◽

Cited By ~ 52

Author(s):

N.L. Perillo ◽

F. Naeim ◽

R.L. Walford ◽

R.B. Effros

Keyword(s):

T Cell ◽

T Lymphocytes ◽

Cytolytic Activity ◽

Cell Phenotype ◽

Memory T Cell ◽

Human T Lymphocytes

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Reply to: “β-catenin does not regulate memory T cell phenotype”

Nature Medicine ◽

10.1038/nm0510-514 ◽

2010 ◽

Vol 16 (5) ◽

pp. 514-515 ◽

Cited By ~ 5

Author(s):

Luca Gattinoni ◽

Yun Ji ◽

Nicholas P Restifo

Keyword(s):

T Cell ◽

Cell Phenotype ◽

Memory T Cell

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The impact of aging on memory T cell phenotype and function in the human bone marrow

Journal of Leukocyte Biology ◽

10.1189/jlb.0611299 ◽

2011 ◽

Vol 91 (2) ◽

pp. 197-205 ◽

Cited By ~ 54

Author(s):

Dietmar Herndler-Brandstetter ◽

Katja Landgraf ◽

Alexandar Tzankov ◽

Brigitte Jenewein ◽

Regina Brunauer ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Human Bone ◽

Human Bone Marrow ◽

Cell Phenotype ◽

Memory T Cell ◽

And Function ◽

The Impact

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A Case of Intravascular Malignant Lymphomatosis (Angiotropic Large-cell Lymphoma) Presenting Memory T Cell Phenotype and Its Expression of Adhesion Molecules

The Journal of Dermatology ◽

10.1111/j.1346-8138.1992.tb03223.x ◽

1992 ◽

Vol 19 (5) ◽

pp. 263-269 ◽

Cited By ~ 23

Author(s):

Mitsuru Setoyama ◽

Shimako Mizoguchi ◽

Tamiko Orikawa ◽

Masaaki Tashiro

Keyword(s):

T Cell ◽

Adhesion Molecules ◽

Cell Lymphoma ◽

Large Cell ◽

Cell Phenotype ◽

Memory T Cell ◽

Large Cell Lymphoma

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Short-Term Ionomycin Exposure Activates Naive Murine T-Cells and Induces a Rapid Phenotypic Shift to Memory T-Cell Status: Potential for Use as a Method To Reduce GvHD Activity of Allogeneic T-Cells.

Blood ◽

10.1182/blood.v110.11.2182.2182 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2182-2182

Author(s):

Mohammad Hossain ◽

Cynthia R. Giver ◽

Ned Waller

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

Memory T Cells ◽

Cell Phenotype ◽

Naive T Cells ◽

Memory T Cell ◽

Naïve T Cells

Abstract We are investigating methods to reduce the graft-versus-host disease (GVHD) potential of donor T-cells while retaining graft-versus-leukemia (GVL) activity in allogeneic HSCT. Previous investigations by our group and others in have shown that naive CD4 T-cells induce severe acute GVHD, while memory CD4 T-cells do not induce GVHD but retain GVL activity in murine transplant models. These findings have led to studies for the development of methods to increase the number of memory T-cells available for transplant. The calcium ionophore, ionomycin, is a T-cell activating agent and mitogen. By increasing intracellular Ca2+ levels, ionomycin is induces T-cell activation through signaling mechanisms including phospholipase C activation, hydrolysis of phosphoinositides, and activation of protein kinase C. Differences in memory and naive T-cell responses to ionomycin have been attributed to resistance of memory T-cells to increases in Ca2+. Memory T-cells lack intracellular Ca2+ stores, and are also resistant to influx of Ca2+. Brief low dose ionomycin exposure (20min, 2μM) of T-cells, leading to increased density of naive T-cells, has previously been exploited as a method for separating memory and naive T-cells by Percoll gradient separation. Since ionomycin exposure induces T-cell activation through native Ca2+ dependent signaling mechanisms, we hypothesized that ionomycin-treated T-cells would shift to an activated/memory T-cell phenotype. Murine splenic T-cells were treated with 1.3μM ionomycin for 4hr. Memory and naive T-cell subsets and activation markers were analyzed by flow cytometry. 75% and 85% of untreated CD4 and CD8 T-cells, respectively, had the CD62L+ naive phenotype. These numbers were dramatically reduced to 7% and 17% after ionomycin exposure, representing a shift to the memory T-cell phenotype. Viability of T-cells was not significantly affected. The majority of remaining CD62L+ naive T-cells expressed activation markers CD25 and CD69. The fraction of CD4+CD25+Foxp3+ regulatory T-cells was also determined by intracellular staining of the transcription factor and co-expression of surface markers. CD4+CD25+Foxp3+ regulatory T-cells represented 4% of untreated CD4 T-cells and 3% of ionomycin-treated CD4 T-cells. While ionomycin has been used for many years in studies of T-cell activation, to our knowledge this is the first demonstration of a rapidly-induced shift of naive T-cells to a memory phenotype. A pilot experiment was conducted testing the GVHD activity of ionomycin-treated splenocytes (SP) in B6→ (B6 × Balb/C)CB6F1 recipients. 5 × 106 T-cell depleted bone marrow cells (TCD-BM) were transplanted along with 10 × 106 treated or untreated SP. Mice that received untreated SP all died from acute GvHD by 34 days after transplant, while all recipients of ionomycin-treated SP survived until the experiement was terminated at day 49 (average weight loss was 25%, data not shown). Continuing experients will refine the dose to further reduce GVHD symptoms and also test GVL activity of the treated cells. Treatment of donor T-cells with ionomycin may represent a clinically applicaple method to engineer donor lymphocyte infusions that are safer for HSCT patients. Figure 1. Survival of CB6F1 recipients after transplant with 5 million B6 TCD-BM and 10 million B6 splenocytes that were either untreated or stimulated ex-vivo with a combination of PMA, ionomycin and brefeldin-A for 4 hours. 5 recipient animals per group. The experiment was terminated at day 49. Figure 1. Survival of CB6F1 recipients after transplant with 5 million B6 TCD-BM and 10 million B6 splenocytes that were either untreated or stimulated ex-vivo with a combination of PMA, ionomycin and brefeldin-A for 4 hours. 5 recipient animals per group. The experiment was terminated at day 49.

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HIV-1 infection alters CD4+memory T-cell phenotype at the site of disease in extrapulmonary tuberculosis

European Journal of Immunology ◽

10.1002/eji.201141927 ◽

2011 ◽

Vol 42 (1) ◽

pp. 147-157 ◽

Cited By ~ 28

Author(s):

Kerryn Matthews ◽

Mpiko Ntsekhe ◽

Faisal Syed ◽

Thomas Scriba ◽

James Russell ◽

...

Keyword(s):

T Cell ◽

Extrapulmonary Tuberculosis ◽

Cell Phenotype ◽

Memory T Cell ◽

Hiv 1

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Modified Vaccinia Ankara–Vectored Vaccine Expressing Nucleoprotein and Matrix Protein 1 (M1) Activates Mucosal M1-Specific T-Cell Immunity and Tissue-Resident Memory T Cells in Human Nasopharynx-Associated Lymphoid Tissue

The Journal of Infectious Diseases ◽

10.1093/infdis/jiz593 ◽

2019 ◽

Vol 222 (5) ◽

pp. 807-819 ◽

Cited By ~ 1

Author(s):

Suttida Puksuriwong ◽

Muhammad S Ahmed ◽

Ravi Sharma ◽

Madhan Krishnan ◽

Sam Leong ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Response ◽

Lymphoid Tissue ◽

Matrix Protein ◽

T Cell Response ◽

Effector Memory ◽

Cell Phenotype ◽

Memory T Cell ◽

Cell Immunity

Abstract Background Increasing evidence supports a critical role of CD8+ T-cell immunity against influenza. Activation of mucosal CD8+ T cells, particularly tissue-resident memory T (TRM) cells recognizing conserved epitopes would mediate rapid and broad protection. Matrix protein 1 (M1) is a well-conserved internal protein. Methods We studied the capacity of modified vaccinia Ankara (MVA)–vectored vaccine expressing nucleoprotein (NP) and M1 (MVA-NP+M1) to activate M1-specific CD8+ T-cell response, including TRM cells, in nasopharynx-associated lymphoid tissue from children and adults. Results After MVA-NP+M1 stimulation, M1 was abundantly expressed in adenotonsillar epithelial cells and B cells. MVA-NP+M1 activated a marked interferon γ–secreting T-cell response to M1 peptides. Using tetramer staining, we showed the vaccine activated a marked increase in M158–66 peptide-specific CD8+ T cells in tonsillar mononuclear cells of HLA-matched individuals. We also demonstrated MVA-NP+M1 activated a substantial increase in TRM cells exhibiting effector memory T-cell phenotype. On recall antigen recognition, M1-specific T cells rapidly undergo cytotoxic degranulation, release granzyme B and proinflammatory cytokines, leading to target cell killing. Conclusions MVA-NP+M1 elicits a substantial M1-specific T-cell response, including TRM cells, in nasopharynx-associated lymphoid tissue, demonstrating its strong capacity to expand memory T-cell pool exhibiting effector memory T-cell phenotype, therefore offering great potential for rapid and broad protection against influenza reinfection.

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