Application of a Standardized Flow Cytometry Panel for Defining and Monitoring the Immunophenotype of CAR-T Cells

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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CXCR3 Is Required for the Efficient Recruitment of Bone Marrow-Resident Memory T Cells to Inflamed Tissues,

Blood ◽

10.1182/blood.v118.21.3242.3242 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3242-3242

Author(s):

Robbert van der Voort ◽

Claudia Brandao ◽

Thomas J. Volman ◽

Viviènne Verweij ◽

Klaas van Gisbergen ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Immune Responses ◽

Cd8 T Cells ◽

Memory T Cells ◽

Central Memory ◽

T Cell Subsets ◽

Effector Memory ◽

Memory T Cell

Abstract Abstract 3242 Although the importance of the bone marrow (BM) in hematopoiesis is well known, its function in adaptive immune responses has only recently been acknowledged. Currently it is known that the BM contains fully functional CD4+ and CD8+ T cells that can engage in both primary and secondary immune responses. Interestingly, most of these T cells belong to the memory T cell lineage, identifying the BM as one of the largest memory T cell reservoirs in the body. Since not much is known about the trafficking of BM T cells, we compared the homing phenotype and function of T cell subsets in the BM, blood, spleen and peripheral lymph nodes (pLN). In addition, we determined the expression of chemokine mRNA and protein levels in the BM and other lymphoid organs. We confirmed that at least 80% of the CD4+ and 60% of the CD8+ BM T cells have a memory phenotype, and that most CD4+ T cells belong to the effector memory lineage, while the CD8+ population predominantly consists of central memory T cells. Most BM T cells expressed the chemokine receptor CXCR3, the adhesion molecules P-selectin glycoprotein ligand 1 and VLA-4, and increased levels of CD44 and LFA-1, as compared to T cells from the spleen. In addition, L-selectin was absent from most CD4+ BM T cells, but present on virtually all CD8+ T cells. Notably, the percentage of CXCR3+ T cells within the effector memory and central memory subsets from BM was higher than within the same subsets from pLN. Furthermore, BM contained significant mRNA levels of the CXCR3 ligands CXCL9, CXCL10 and CXCL11. An in vivo migration assay using a mixture of fluorescent-labeled T cells from CXCR3-deficient mice and control mice indicated however that during homeostasis CXCR3 does not play a major role in BM entry or retention. These data suggest that CXCR3 expressed by memory T cells is rather involved in BM exit, than in BM entry. Indeed, we observed that, as compared to control mice, CXCR3−/− mice contained significantly more CD4+ and CD8+ T cells in their BM. Additional in vitro assays demonstrated that CD4+ and CD8+ BM T cells migrated vigorously in response to CXCL9 and CXCL10, generally released in high concentrations during inflammation. Finally, we demonstrate that CXCR3−/− effector/effector memory T cells, but not wild type T cells, accumulate in the BM of mice infected with lymphocytic choriomeningitis virus. Altogether, these data demonstrate that the BM is a major reservoir of memory T cells that employ CXCR3 to quickly respond to chemotactic signals from inflamed tissues. Disclosures: No relevant conflicts of interest to declare.

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BCG vaccination induces enhanced frequencies of memory T cells and altered plasma levels of common γc cytokines in elderly individuals

PLoS ONE ◽

10.1371/journal.pone.0258743 ◽

2021 ◽

Vol 16 (11) ◽

pp. e0258743

Author(s):

Nathella Pavan Kumar ◽

Chandrasekaran Padmapriyadarsini ◽

Anuradha Rajamanickam ◽

Perumal Kannabiran Bhavani ◽

Arul Nancy ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Cd8 T Cells ◽

Plasma Levels ◽

T Cell Subsets ◽

Effector Memory ◽

Elderly Individuals ◽

Bcg Vaccination ◽

Adaptive Immune

BCG vaccination is known to induce innate immune memory, which confers protection against heterologous infections. However, the effect of BCG vaccination on the conventional adaptive immune cells subsets is not well characterized. We investigated the impact of BCG vaccination on the frequencies of T cell subsets and common gamma c (γc) cytokines in a group of healthy elderly individuals (age 60–80 years) at one month post vaccination as part of our clinical study to examine the effect of BCG on COVID-19. Our results demonstrate that BCG vaccination induced enhanced frequencies of central (p<0.0001) and effector memory (p<0.0001) CD4+ T cells and diminished frequencies of naïve (p<0.0001), transitional memory (p<0.0001), stem cell memory (p = 0.0001) CD4+ T cells and regulatory T cells. In addition, BCG vaccination induced enhanced frequencies of central (p = 0.0008), effector (p<0.0001) and terminal effector memory (p<0.0001) CD8+ T cells and diminished frequencies of naïve (p<0.0001), transitional memory (p<0.0001) and stem cell memory (p = 0.0034) CD8+T cells. BCG vaccination also induced enhanced plasma levels of IL-7 (p<0.0001) and IL-15 (p = 0.0020) but diminished levels of IL-2 (p = 0.0033) and IL-21 (p = 0.0020). Thus, BCG vaccination was associated with enhanced memory T cell subsets as well as memory enhancing γc cytokines in elderly individuals, suggesting its ability to induce non-specific adaptive immune responses.

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Modulation of the central memory and Tr1-like regulatory T cells in multiple sclerosis patients responsive to interferon-beta therapy

Multiple Sclerosis Journal ◽

10.1177/1352458511427720 ◽

2011 ◽

Vol 18 (6) ◽

pp. 788-798 ◽

Cited By ~ 13

Author(s):

M Chiarini ◽

F Serana ◽

C Zanotti ◽

R Capra ◽

S Rasia ◽

...

Keyword(s):

Multiple Sclerosis ◽

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Interferon Beta ◽

Central Memory ◽

T Cell Subsets ◽

Individual Treatment ◽

Cell Phenotype ◽

Multiple Sclerosis Patients

Background: Interferon-beta is used to reduce disease activity in multiple sclerosis, but its action is incompletely understood, individual treatment response varies among patients, and biological markers predicting clinical benefits have yet to be identified. Since it is known that multiple sclerosis patients have a deficit of the regulatory T-cell subsets, we investigated whether interferon-beta therapy induced modifications of the two main categories of regulatory T cells (Tregs), natural and IL-10-secreting inducible Tr1 subset, in patients who are biologically responsive to the therapy. Methods: T-cell phenotype was determined by flow cytometry, while real-time PCR was used to evaluate interferon-beta bioactivity through MxA determination, and to measure the RNA for IL-10 and CD46 molecule in peripheral blood mononuclear cells stimulated with anti-CD46 and anti-CD3 monoclonal antibodies, which are known to expand a Tr1-like population. Results: Interferon-beta induced a redistribution of natural Treg subsets with a shift of naive Tregs towards the ‘central memory-like’ Treg population that expresses the CCR7 molecule required for the in vivo suppressive activity. Furthermore, in a subgroup of treated patients, the CD46/CD3 co-stimulation, probably through the Tr1-like subset modulation, increased the production of RNA for IL-10 and CD46. The same group showed a lower median EDSS score after two years of therapy. Conclusions: The selective increase of ‘central memory-like’ subset and the involvement of the Tr1-like population may be two of the mechanisms by which interferon-beta achieves its beneficial effects. The quantification of RNA for IL-10 and CD46 could be used to identify patients with a different response to interferon-beta therapy.

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Pomalidomide Induces Expansion of Activated and Central Memory CD4+ and CD8+ T Cells in Vivo in Patients with and without HIV Infection

Blood ◽

10.1182/blood.v124.21.4128.4128 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4128-4128 ◽

Cited By ~ 1

Author(s):

Mark N. Polizzotto ◽

Irini Sereti ◽

Thomas S. Uldrick ◽

Kathleen M. Wyvill ◽

Stig M. R. Jensen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Research Funding ◽

Kaposi Sarcoma ◽

Central Memory ◽

T Cell Subsets ◽

Effector Memory ◽

Significant Difference

Abstract Background: Despite antiretroviral therapy (ART), people with HIV continue to exhibit immune deficits including failure to fully reconstitute CD4 T cell numbers and function, resulting in increased risks of tumors and infections and reduced response to vaccination. Pomalidomide, a derivative of thalidomide (IMID), has immunomodulatory properties that may be beneficial in this setting. We explored its impact on lymphocyte number and activation in patients with and without HIV treated within a prospective clinical trial for Kaposi sarcoma. Methods: Patients received pomalidomide 5mg orally for 21 days of 28 day cycles. Assessments were performed every 4 weeks for lymphocyte numbers, Kaposi sarcoma associated herpesvirus (KSHV/HHV8) viral load (VL) and HIV VL and at 8 weeks for T cell subsets and activation by immunophenotyping of peripheral blood mononuclear cells (PBMC). KSHV VL in PBMC and HIV VL in plasma were assayed by quantitative PCR; for HIV VL we used an ultrasensitive single copy assay. Changes from baseline were evaluated using the Wilcoxon signed rank test with P<0.005 considered significant given multiple comparisons. Differences in changes between the HIV infected and uninfected groups were evaluated using the Wilcoxon rank sum test. Study registered as NCT1495598. Results: 19 patients (12 HIV infected, 7 uninfected) median age 50 years (range 32-74) were studied. All with HIV were receiving ART for median 48 months (7-227), HIV VL 1.5 copies/mL (<0.5–37), and CD4 378 cells/µl (135–752). At week 4 and 8 of therapy we observed significant increases in CD4 and CD8 counts, with a decline in CD19 B cells and no change in NK cells or HIV VL. A transient increase in KSHV VL was seen at week 4, not sustained at week 8: Abstract 4128. Table 1ParameterBaseline (cells/µl unless noted)Change to Week 4 (Med, range)PChange to Week 8 (Med, range)PCD31143 (525–2305)+264 (-419–1524)0.0028+210 (-496–1455)0.0020CD4429 (135–1171)+107 (-87–650)0.0009+86 (-37–491)0.0015CD8495 (259–1529)+108 (-271–915)0.0085+155 (-495–834)0.0046NK184 (28–557)+30 (-130–117)0.52+2 (-174–127)0.98CD19139 (9–322)-47 (-117–76)0.0039-79 (-169–62)<0.0001KSHV VL 0 copies/PBMC (0–8750)+23 (-92–5250)0.00980 (-92–20850)0.31Plasma HIV VL (infected pts)1.5 copies/mL (<0.5–37)+0.3 (-1.5–3.0)0.75+0.75 (0–28)0.13 In addition, at week 8 both CD4 and CD8 T cells showed significant increases in activation (CD38+, HLADR+ and DR+/38+) and decreases in senescence (CD57+). Both also showed a significant shift towards increased central memory (CM) and away from naive (N) and effector (E) phenotypes, with no change in effector memory (EM) cells: Abstract 4128. Table 2CD4 SubsetsBaseline (%) (med, range)Absolute Change in % at Week 8 (med, range)PRO- 27+ (N)32.6 (13.3–76.5)-6.6 (-35.8–21.6)0.002RO+ 27+ (CM)41.9 (13.6–63.6)+6.4 (-15.5–32.5)0.027RO+ 27- (EM)16.7 (4.6–31.7)+1.7 (-7.2–21.0)0.28RO- 27- (E)3.3 (0.4–14.3)-1.5 (-5.7–0.3)0.000438+34.5 (11.2–67.3)+4.3 (-13.0–19.4)0.024HLA DR+8.9 (3.3–25.0)+8.3 (0.7–19.5)<0.000138+ DR+2.5 (0.6–11.7)+2 (-1.0–8.1)<0.000157+6.3 (0.6–26.6)-1.34 (-16.2–7.6)0.034CD8 SubsetsRO- 27+ (N)21.0 (9.7–70.4)-5.1 (-13.7–14.3)0.019RO+ 27+ (CM)17.1 (2.5–37.9)+8.1 (-8.4–18.6)0.0047RO+ 27- (EM)18.4 (4.6–40.8)+1.0 (-9.4–44.9)0.35RO- 27- (E)31.8 (4.1-63.7)-6.1 (-47.3–22.5)0.0138+33.4 (8.3–66.0)+19.9 (-0.8–40.6)<0.0001HLA DR+19.6 (5.0–46.4)+11.6 (-4.7–32.1)0.000138+ DR+8.0 (0.4–33.3)+8.5 (0.1–22.6)<0.000157+30.8 (2.9–72.9)-11.0 (-28.5–6.1)<0.0001 There were no significant changes in Ki67 or PD-1 expression in either CD4 or CD8 cells. There was no significant difference between HIV infected and uninfected patient groups in the observed effects on any parameter including cell number and phenotype. Conclusions: Pomalidomide induced significant increases in the number of CD4 and CD8 T cells and the proportion of activated and central memory cells and decreased senescence in both HIV infected and uninfected subjects. Effects were not explained by alterations in HIV viremia. The transient early rise in KSHV VL may reflect reactivation of latent infection and enhance immune killing of KSHV infected cells. This analysis sheds light on possible mechanisms of IMID activity in viral-associated tumors. As the first study of immune modulation by IMIDs in vivo in people with HIV it also suggests exploration of IMIDs to augment immune responsiveness in HIV and other immunodeficiencies is warranted. Disclosures Polizzotto: Celgene Corporation: Research Funding. Off Label Use: Pomalidomide for Kaposi sarcoma. Uldrick:Celgene Corporation: Research Funding. Zeldis:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Yarchoan:Celgene Corporation: Research Funding.

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PD-1-Mediated T Cell Exhaustion Is Prevalent Among Patients with MPN-Associated Myelofibrosis Independent of JAK1/2 Inhibition

Blood ◽

10.1182/blood-2018-99-119428 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5487-5487

Author(s):

Ivo Veletic ◽

Taghi Manshouri ◽

Graciela M. Nogueras González ◽

Sanja Prijic ◽

Joseph E. Bove ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Central Memory ◽

T Cell Subsets ◽

Effector Memory ◽

Spleen Size ◽

Advisory Committees ◽

Cd8 Cells ◽

Circulating T Cells

Abstract Background: Primary myelofibrosis (PMF), post-polycythemia vera MF (post-PV MF) and post-essential thrombocythemia MF (post-ET MF) are characterized by expansion of the neoplastic clone and by progressive bone marrow (BM) fibrosis. Like in other hematologic malignancies, in most patients with MF the immune system is significantly deregulated: MF patients' plasma cytokine and chemokine levels are markedly increased and their normal T cell subset distribution is significantly altered. Although treatment with the Janus kinase (JAK)-1/2 inhibitor ruxolitinib significantly decreases cytokine/chemokine levels, reduces spleen size, and improves symptoms and quality of life, it does not reverse BM fibrosis nor does it halt the propagation of the neoplastic clone. The T cell immune checkpoint programmed cell death protein-1 (PD-1) promotes immune tolerance by binding to the tumor's cell surface PD-1 ligand (PD-L1). Whereas the importance of T cell-mediated immune tolerance in MF has been documented and trials evaluating clinical benefits of PD1/PD-L1 checkpoint inhibition are ongoing, little is known about the effect of ruxolitinib on PD-1 expression in T cell subsets. Therefore we systematically analyzed MF patients circulating T cells' surface marker expression prior to and during ruxolitinib treatment. Methods: Peripheral blood cells were obtained from well-characterized PMF, post-PV MF and post-ET MF patients prior to and during the course of treatment with ruxolitinib (n=47) and, as control, from age-matched healthy donors (n=28). The proportion of PD-1-expressing CD4+ and CD8+ cells was assessed using multiparameter flow cytometry. Naïve, central memory, effector memory, and effector T cell subsets were defined based on CD45RO and CD27 cell surface antigen expression. Results: A significantly high number of circulating T cells co-expressing CD4+/PD-1+ and CD8+/PD-1+ was found in MF patients compared to age-matched healthy individuals (5.3±4.1% vs. 3.4±1.7%, P=0.028; 7.1±4.4% vs. 3.8±2.3%, P=0.001). Whereas MF patients' naïve T cells harbored an increased number of cells co-expressing CD8+/PD-1+ (P=0.007), but not CD4+/PD-1+, their T central memory cells had a high proportion of cells co-expressing CD4+/PD-1+ and CD8+/PD-1+ (P<0.001; P<0.001). Similarly, a high proportion of circulating PD-1+ T effector memory cells (P<0.001; P<0.001), and T effector cells (P=0.013; P<0.001) was found in MF patients compared to the same cell subsets in healthy age-matched individuals. The proportions of PD-1+ T cells significantly correlated with LDH level and DIPSS score (CD4+ T cells), monocyte count (CD8+ T cells), and total leukocyte count and spleen size (both subsets). Remarkably, the percentage of PD-1+ cells within naïve and central memory CD8+ T cell populations was significantly higher in MF patients with circulating blasts (P=0.036). To determine the effects of ruxolitinib administration, we performed repeated flow cytometry analyses on MF patients' T cells prior to and during treatment (median duration: 4.3 years). Overall, no significant change in PD-1 expression levels in any of the different T cell subsets was detected over the entire treatment period. However, a significant reduction in percentage of cells co-expressing CD4+/PD-1+ and CD8+/ PD-1+ compared to treatment baseline (4.4±0.4% vs. 7.6±2.0%, P=0.011; 6.3±0.6% vs. 10.4±2.7%, P=0.021) was found in patients whose spleen size was reduced after 6 months of treatment. Conclusions: In patients with MF, circulating T cells express high levels of PD-1. While not restricted to a particular stage of T cell differentiation, the correlation between PD-1-expressing T cells and distinct clinical parameters suggests that increased PD-1 levels might induce immune exhaustion in T cell subsets in different ways. Although ruxolitinib significantly inhibits the JAK1/2 signaling pathway in a variety of hematopoietic cells, thereby lowering cytokine/chemokine levels in almost all MF patients, treatment with ruxolitinib did not affect PD-1 expression nor did it alter its distribution among the T cell subsets. Yet, the proportion of PD-1-expressing CD4+ and CD8+ cells was markedly reduced in patients who experienced a superior response to ruxolitinib as assessed by significant spleen size reduction. How disease burden and MF microenvironment affect PD-1 expression in T cells of patients with MF warrants further investigation. Disclosures Verstovsek: Incyte: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Italfarmaco: Membership on an entity's Board of Directors or advisory committees.

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145 Comparison of CAR-T cell manufacturing platforms reveals distinct phenotypic and transcriptional profiles

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.145 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A153-A153

Author(s):

Hannah Song ◽

Lipei Shao ◽

Michaela Prochazkova ◽

Adam Cheuk ◽

Ping Jin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Effector Memory ◽

Cell Phenotype ◽

Gene Clustering ◽

Car T Cells ◽

Car T Cell ◽

Cell Manufacturing ◽

Car T ◽

And Function

BackgroundWith the clinical success of chimeric antigen receptor (CAR)-T cells against hematological malignancies, investigators are looking to expand CAR-T therapies to new tumor targets and patient populations. To support translation to the clinic, a variety of cell manufacturing platforms have been developed to scale manufacturing capacity while using closed and/or automated systems. Such platforms are particularly useful for solid tumor targets, which typically require higher CAR-T cell doses that can number in the billions. Although T cell phenotype and function are key attributes that often correlate with therapeutic efficacy, it is currently unknown whether the manufacturing platform itself significantly influences the output T cell phenotype and function.MethodsStatic bag culture was compared with 3 widely-used commercial CAR-T manufacturing platforms (Miltenyi CliniMACS Prodigy, Cytiva Xuri W25 rocking platform, and Wilson-Wolf G-Rex gas-permeable bioreactor) to generate CAR-T cells against FGFR4, a promising target for pediatric sarcoma. Selected CD4+CD8+ cells were stimulated with Miltenyi TransAct, transduced with lentiviral vector, and cultured out to 14 days in TexMACS media with serum and IL2.ResultsAs expected, there were significant differences in overall expansion, with bag cultures yielding the greatest fold-expansion while the Prodigy had the lowest (481-fold vs. 84-fold, respectively; G-Rex=175-fold; Xuri=127-fold; average of N=4 donors). Interestingly, we also observed considerable differences in CAR-T phenotype. The Prodigy had the highest percentage of CD45RA+CCR7+ stem/central memory (Tscm)-like cells at 46%, while the bag and G-Rex cultures had the lowest at 16% and 13%, respectively (average N=4 donors). In contrast, the bag, G-Rex, and Xuri cultures were enriched for CD45RO+CCR7- effector memory cells and also had higher expression of exhaustion markers PD1 and LAG3. Gene clustering analysis using a CAR-T panel of 780 genes revealed clusters of genes enriched in Prodigy/de-enriched in bag, and vice versa. We are currently in the process of evaluating T cell function.ConclusionsThis is the first study to our knowledge to benchmark these widely-used bioreactor systems in terms of cellular output, demonstrating that variables inherent to each platform (such as such as nutrient availability, gas exchange, and shear force) significantly influence the final CAR-T cell product. Whether enrichment of Tscm-like cells in the final infusion product correlates with response rate, as has been demonstrated in the setting of CD19 CAR-Ts, remains to be seen and may differ for FGFR4 CAR-Ts and other solid tumors. Overall, our study outlines methods to identify the optimal manufacturing process for future CAR-T cell therapies.

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A BCL2L1 Armoured BCMA Targeting CAR T Cell to Overcome Exhaustion and Enhance Persistence in Multiple Myeloma

Blood ◽

10.1182/blood-2021-153871 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 327-327

Author(s):

Ranjan Maity ◽

Sacha Benaoudia ◽

Franz Zemp ◽

Holly Lee ◽

Elie Barakat ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Memory T Cells ◽

Antigenic Stimulation ◽

T Cell Subsets ◽

Effector Memory ◽

Car T Cells ◽

Car T Cell ◽

Chronic Antigenic Stimulation ◽

Car T

Abstract Chimeric antigen receptor (CAR) T cells targeting the B-cell maturation antigen (BCMA) have resulted in deep responses in patients with relapsed MM however most remissions are not sustained. While cellular and molecular mediators of relapse post CAR T therapy in MM are not fully delineated, current data suggest three possible mechanisms including the lack of persistence of the CAR T cell product, acquired exhaustion and less commonly loss of BCMA expression. Using CITE-seq we measured the expansion of variable T cell subsets, T cell specific activation and inhibitor markers and their functional states in serial blood and marrow samples (n=10) collected from patients treated with BCMA targeting CAR T cells. CAR T cells were identified by the expression of the chimeric CAR T cell transcript. With the exception of one patient where biallelic loss of BCMA was identified at relapse, CAR T cells of resistant patients were enriched with terminally exhausted CD45RA+ cells with loss of CD28, low BCL2L1 (gene encoding BCL-XL) expression, high CD57 with co-expression of checkpoint inhibitors (LAG3, TIGIT and PD1). The lack of persistence of the CAR T cells product was notable in all relapsing patients consistent with an activation induced cells death (AICD) specially in the setting of chronic antigenic stimulation. Cognizant of the role BCL-XL plays in T cells survival in response to CD28 co-stimulatory signaling, we postulated that increasing BCL-XL expression is a feasible strategy to enhance CAR T cell resistant to AICD, improve their persistence and anti-BCMA reactivity. To this goal, we designed a 2nd generation lentiviral CAR construct where the anti-BCAM scFV-41BBz CAR and the BCL2L1 cDNA were linked with self-cleaving 2A sequence. The efficiency in eradicating MM cells of this BCL-XL armored CAR (BCMA_BCL2L1_CAR) was compared to that of non-unarmored CAR (BCMA_CAR) in vitro and i n vivo studies. While BCMA_BCL2L1_CAR and BCMA_CAR were equally cytotoxic to OPM2 MM cells, in MM cell lines expressing the FAS death receptor ligand FASLG (MM1S, OCMY5 and H929) BCMA_BCL2L1_CAR viability and cytolytic activity was significantly superior to that of unarmored BCMA_CAR. Of note, the expression of FASLG, a known interferon response gene, was upregulated in H929 cells when co-cultured with CAR T cells. Importantly, under chronic antigenic stimulation conditions (FIG 1A), where CAR T cells were stimulated every 6 days over a 28 days period with irradiated OPM2 cells, we found no phenotypic difference between BCMA_BCL2L1_CAR and BCMA_CAR with respect to the composition of effector memory T cells (Tem: CCR7− CD45RO+ CD45RA−) or central memory T cells (Tcm: CCR7+CD45RO+CD45RA−) or terminal effector / exhausted T cells. However, under these chronic antigenic stimulation conditions, the CAR T cells viability, proliferation (FIG 1B) and anti-MM cytotoxic activities (FIG 1C) of the BCMA_CAR were dramatically reduced compared to that of the BCL2L1 armored CAR. Furthermore, in initial animal studies where NOD-SCID mice were tail vein injected with 2e6 OPM2 MM cells transduced with a luciferin reporter gene, followed 10 days later by control T cells, BCMA_CAR or BCMA_BCL2L1_CAR T cells IV injection, and despite a skewing to a larger initial disease burden in the BCMA-BCL2L1-CAR group, BCL2L1 armored CAR T cells resulted in more prolonged disease control and animal survival compared to the BCMA_CAR treated mice (FIG 1D). Our studies indicate that BCL2L1 blockade of AICD not only enhanced the viability and proliferation of BCMA targeting CAR T cells but surprisingly also reduced their functional exhaustion. Our findings provide an novel approach for CAR T optimization and overcoming disease relapse resulting from lack of persistence and/or T cells exhaustion. Figure 1 Figure 1. Disclosures Neri: Amgen: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria. Bahlis: Sanofi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Genentech: Consultancy; Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

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Altered T-Lymphocyte Biology Following High-Dose Melphalan and Autologous Stem Cell Transplantation With Implications for Adoptive T-Cell Therapy

Frontiers in Oncology ◽

10.3389/fonc.2020.568056 ◽

2020 ◽

Vol 10 ◽

Author(s):

Thomas Mika ◽

Swetlana Ladigan-Badura ◽

Abdelouahid Maghnouj ◽

Bakr Mustafa ◽

Susanne Klein-Scory ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Autologous Stem Cell Transplantation ◽

Cell Phenotype ◽

High Dose ◽

Car T ◽

The Impact

In relapsed and refractory multiple myeloma (MM), adoptive cell therapies (ACT) including CAR-T-cells are under clinical investigation. However, relapse due to T-cell exhaustion or limited persistence is an obstacle. Before ACT are considered in MM, high-dose (HD) melphalan followed by autologous stem-cell transplantation (autoSCT) has been administered in most clinical situations. Yet, the impact of HD chemotherapy on T-cells in MM with respect to ACT is unclear. In this study, T-lymphocytes’ phenotypes, expansion properties, lentiviral transduction efficacy, and gene expression were examined with special respect to patients following HD melphalan. Significant impairment of T-cells’ expansion and transduction rates could be demonstrated. Expansion was diminished due to inherent disadvantages of the predominant T-cell phenotype but restored over time. The quantitative fraction of CD27−/CD28− T-cells before expansion was predictive of T-cell yield. Following autoSCT, the transduction efficacy was reduced by disturbed lentiviral genome integration. Moreover, an unfavorable T-cell phenotype after expansion was demonstrated. In initial analyses of CD107a degranulation impaired T-cell cytotoxicity was detected in one patient following melphalan and autoSCT. The findings of our study have potential implications regarding the time point of leukapheresis for CAR-T-cell manufacturing. Our results point to a preferred interval of more than 3 months until patients should undergo cell separation for CAR-T therapy in the specific situation post-HD melphalan/autoSCT. Monitoring of CD27−/CD28− T-cells, has the potential to influence clinical decision making before apheresis in MM.

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Expression of Immune Memory Markers CD8 α α and Interleukin−7 Receptor on CD8+ T Cell Subsets among HIV−1 Infected Patients.

Blood ◽

10.1182/blood.v108.11.1256.1256 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1256-1256

Author(s):

Jean Pierre Routy ◽

Francois Mercier ◽

Ahmed Galal ◽

Med-Rachid Boulassel

Keyword(s):

T Cells ◽

T Cell ◽

Immune Activation ◽

Cd8 T Cell ◽

Central Memory ◽

T Cell Subsets ◽

Immune Memory ◽

Effector Memory ◽

Activation Markers ◽

Hiv 1

Abstract Evidence from animal models suggests that the expression of CD8α α homodimer on CD8+ T-cells plays a key role in the generation of long-lived memory cells. However, very little information is available in the human clinical setting. Here, we examined immunophenotypic patterns of CD8+ T-cell subsets expressing CD8α α with other markers involved in generating and maintaining memory cells such as interleukin-7 receptor (IL-7Rα ) and circulating levels of IL-7 and IL-15, in three well-defined groups of human immunodeficiency virus-1 (HIV-1)-infected individuals including aviremic (n=15), viremic (n=31) and slow-progressor (n=15). In addition, immunophenotypic patterns were correlated with immune activation markers (CD38/HLA-DR), which are known to be an important factor in HIV-1 disease pathogenesis. Cell-surface expression of CD8α α , IL-7Rα and CD38/HLA-DR on CD8+ naïve, central memory, pre-terminal and terminal effector memory T-cells was measured by eight-color flow cytometry on freshly peripheral blood samples. IL-7 and IL-15 levels were measured by ELISA and viral loads were assessed by PCR. Group differences in the CD8+ T-cell subsets expressing each antigen tested were evaluated using the unpaired nonparametric Mann Whitney U test. Correlations were determined by Spearman’s correlation tests. Compared to slow-progressor subjects, expression of CD8α α was significantly reduced in aviremic and viremic patients and this reduction occurred mainly within naïve and central memory T-cell subsets and not in effector memory compartments. In contrast, persistent antigenemia in viremic patients appeared to lead to IL-7Rα loss mainly on central and effector memory subsets and not on naive T-cells. Compared to aviremic and viremic patients, slow-progressor subjects had lower levels of circulating IL-7, normal levels of IL-15, CD8α α and IL-7Rα , and reduced activated T-cells. Overall, expression of CD8α α was not significantly related to IL-7Rα although negative associations were evidenced within all CD8+ T-cell subsets. However, in viremic patients, naïve and central memory cell subsets expressing CD8α α were positively correlated with viral load but not with CD8+ T-cell subsets expressing immune activation markers. Together, these results provide new insights into the role of CD8α α /IL-7Rα along with immune activation markers in maintaining memory populations during HIV-1 infection. The inter-relationships between these immune memory markers require further investigations, which may help understanding the mechanisms of antiviral control.

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Can Memory T Cell Monitoring Using Flow Cytometry Predict Survival in Haploidentical Hematopoietic Stem Cell Transplantation?

Blood ◽

10.1182/blood.v128.22.4591.4591 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4591-4591

Author(s):

Bohyun Kim ◽

Seongsoo Jang ◽

Yu-Jin Lee ◽

Young-Uk Cho ◽

Chan-Jeoung Park ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Hla Antigens ◽

Early Period ◽

Cell Subpopulation ◽

Lower Percentage ◽

Effector Memory ◽

Hematopoietic Stem ◽

Memory T Cell

Abstract Introduction:Haploidentical hematopoietic stem-cell transplantation (HHSCT) is an alternative transplant strategy for patients who lack a suitable HLA-matched donor. One of the advantages of HHSCT is the possibility to use donor cells for post-transplantation cell therapy. However, HHSCT has risks associated with HLA barrier, such as graft failure, severe graft-versus-host disease (GVHD), and delayed immune reconstitution. T cell receptor gamma delta (TCRγδ) T cells may have potent antileukemic effects. These cells can be preserved in a graft by negatively selecting only T cells that express an alpha beta (αβ) T cell receptor (TCR αβ). Moreover, γδ T cells are important effector cells, especially in situations where the function of adaptive immunity is impaired, such as those characterizing early immune recovery after HHSCT. In this study, we performed flow cytometry (FCM)-based T cell and TCRγδ memory T cell subpopulation analysis with anti-HLA antibodies to monitor the changes of memory T cell subpopulations after HHSCT according to clinical course. Methods:Peripheral blood samples of total eighty-one pediatric patients who underwent HHSCT in Asan Medical Center were collected between October 2011 and June 2016. Diagnoses were aplastic anemia (n=31), acute myeloid leukemia (n=20), acute lymphoblastic leukemia (n=15), non-Hodgkin's lymphoma (n=10), non-malignant hematologic disorders (n=5). Four patients received CD34-selected graft, 26 patients received CD3-depleted graft, and 51 patients received TCRαß-depleted graft. Patients who experienced graft loss or disease relapse were fifteen. Seventeen patients received zoledronate (Zol) and interleukin-2 (IL-2) to augment TCRγδ T cells after HHSCT. Twelve patients were expired. Nineteen patients were experienced GVHD. FCM analysis was performed using antibodies for HLA antigens and anti-CD45, CD3, CD4, CD8, CD45RA, CD45RO, CD62L and TCRγδ antibody to identify naïve, central memory (CM) and effector memory (EM) T cells. The anti-HLA antibody can be used to evaluate chimeric status in HHSCT based on disparity of HLA antigens between donor and recipient. FCM analysis was done according to regularly scheduled protocol from the start of stem cell infusion. We evaluated the differences of T cell and memory T cell subpopulations and Treg according to engraftment status, treatment strategy, survival status, GVHD status, and the types of underlying disease were analyzed. Results: At the early stage (0-30 day) after HHSCT, the absolute counts of TCRγδ CM, EM and naïve T cells were higher in engraftment group than graft failure (CM 2.71/µL vs. 0.19/µL, EM 0.63 vs. 0.00, and Naïve 2.50 vs. 0.37, P<0.05). The administration of Zol+IL-2 significantly increases TCRγδ memory cell absolute counts (CM 4.51/µL vs. 0.68/µL, EM 1.78 vs. 0.00, and Naïve 2.73 vs. 0.72, P<0.05) during early period after HHSCT. These drugs also resulted in higher donor T cell count (147.56 vs. 60.76, P=0.025) and donor TCRγδ T cell count (92.96 vs. 43.9, P=0.046). After 6 months of HHSCT, survived patients showed lower percentage of EM cells than non-survivors (CD4+EM 5.69% vs. 22.22%, and CD8+EM 11.13 vs. 66.79, P<0.05), and higher percentage of CM cells (CD4+CM 59.11% vs. 36.84%, P=0.02, and CD8+CM 29.73% vs. 1.83%, P=0.00) than expired patients. Patients with GVHD showed significantly lower percentage of Naïve T cells than patients without GVHD (CD4+Naïve 0.00% vs. 68.64%, and CD8+Naïve 1.77 vs. 41.31, P<0.05). The percentage of TCRγδ T cell was higher in malignant disease patients than non-malignant disease patients (15.61 vs. 4.75, P=0.008). Conclusions: The increase of TCRγδ memory cells in the early period after HHSCT could expect engraftment. The administration of Zol+IL-2 augmented TCRγδ memory cells during early period after HHSCT. Therefore, these drugs might be related with engraftment maintenance. The monitoring of reconstitution pattern of central and effector memory T cell after 6 months of HHSCT could predict survival. Our study suggests that close monitoring of FCM-based memory T cell subpopulation is useful to predict clinical outcome after HHSCT. Disclosures No relevant conflicts of interest to declare.

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