TGFβ Programs Central Memory Differentiation in Ex Vivo–Stimulated Human T Cells

T cells are essential for eradicating microorganisms and cancer and for tissue repair, have a pro-cognitive role in the brain, and limit Central Nervous System (CNS) inflammation and damage upon injury and infection. However, in aging, chronic infections, acute SARS-CoV-2 infection, cancer, chronic stress, depression and major injury/trauma, T cells are often scarce, exhausted, senescent, impaired/biased and dysfunctional. People with impaired/dysfunctional T cells are at high risk of infections, cancer, other diseases, and eventually mortality, and become multi-level burden on other people, organizations and societies. It is suggested that “Nerve-Driven Immunity” and “Personalized Adoptive Neuro-Immunotherapy” may overcome this problem. Natural Neurotransmitters and Neuropeptides: Glutamate, Dopamine, GnRH-II, CGRP, Neuropeptide Y, Somatostatin and others, bind their well-characterized receptors expressed on the cell surface of naïve/resting T cells and induce multiple direct, beneficial, and therapeutically relevant effects. These Neurotransmitters and Neuropeptides can induce/increase: gene expression, cytokine secretion, integrin-mediated adhesion, chemotactic migration, extravasation, proliferation, and killing of cancer. Moreover, we recently found that some of these Neurotransmitters and Neuropeptides also induce rapid and profound decrease of PD-1 in human T cells. By inducing these beneficial effects in naïve/resting T cells at different times after binding their receptors (i.e. NOT by single effect/mechanism/pathway), these Neurotransmitters and Neuropeptides by themselves can activate, rejuvenate, and improve T cells. “Personalized Adaptive Neuro-Immunotherapy” is a novel method for rejuvenating and improving T cells safely and potently by Neurotransmitters and Neuropeptides, consisting of personalized diagnostic and therapeutic protocols. The patient’s scarce and/or dysfunctional T cells are activated ex vivo once by pre-selected Neurotransmitters and/or Neuropeptides, tested, and re-inoculated to the patient’s body. Neuro-Immunotherapy can be actionable and repeated whenever needed, and allows other treatments. This adoptive Neuro-Immunotherapy calls for testing its safety and efficacy in clinical trials.

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Interleukin-15 Favors the Expansion of Central Memory CD8+ T Cells in Ex Vivo Generated, Antileukemia Human Cytotoxic T Lymphocyte Lines

Journal of Immunotherapy ◽

10.1097/cji.0b013e31816b1092 ◽

2008 ◽

Vol 31 (4) ◽

pp. 385-393 ◽

Cited By ~ 16

Author(s):

Liane Daudt ◽

Rita Maccario ◽

Franco Locatelli ◽

Ilaria Turin ◽

Lucia Silla ◽

...

Keyword(s):

T Cells ◽

T Lymphocyte ◽

Cd8 T Cells ◽

Cytotoxic T Lymphocyte ◽

Ex Vivo ◽

Central Memory ◽

Memory Cd8 T Cells ◽

Interleukin 15

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P01.13 MERTK signaling is critical for T cell proliferation and memory

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-itoc7.26 ◽

2020 ◽

Vol 8 (Suppl 2) ◽

pp. A14.2-A15

Author(s):

RM Powell ◽

MJW Peeters ◽

A Rachbech ◽

PT Straten

Keyword(s):

T Cells ◽

Cell Division ◽

T Cell ◽

Cd8 T Cells ◽

Central Memory ◽

T Cell Proliferation ◽

Flow Cytometric ◽

Donor T Cells ◽

Memory Differentiation

BackgroundOverexpression of TAM receptors, including MERTK, in some cancers are integral for chemoresistance, proliferation and metastasis.1 Our group has previously demonstrated that T cells also express MERTK and engagement of MERTK signaling is responsible for increased proliferation, functional capacity and metabolic fitness.2 It is therefore important to further study the effect of MERTK inhibition on T cell function in the context of cancer treatments where MERTK inhibitors may play a role. Here we provide evidence that MERTK inhibition impacts greatly on T cell proliferation, specifically reducing phosphorylated mTOR. We have also demonstrated that MERTK expression is increased on CD8 central memory subsets during longterm expansion providing evidence that this signaling pathway may be important for sustaining T memory responses.Materials and MethodsFlow cytometric analysis was used to investigate the effect of titration of MERTK small molecule inhibitor UNC2025 on healthy donor T cells activated with CD3/CD28 dynabeads. Cell trace dye was used to track proliferation of CD4 and CD8 T cells along with markers of memory differentiation (CCR7 and CD45RO), activation (CD137) and function (IFNy, Tnfa and IL-2). MERTK signaling was assessed using phospho flow cytometric methodology of phosphorylated mTOR, AKT, ERK1/2, p38-MAPK and STAT5. Long term cultures of donor T cells of up to 28 days were investigated for MERTK expression alongside memory differentiation.ResultsWe demonstrated that moderate concentrations of MERTK inhibitor reduced proliferation of activated T cells. Despite inhibition of cell division, cell size still increased 2 fold compared to resting cells and cell viability remained unchanged. Additionally, the proportion of central memory to effector memory populations and intracellular cytokine production was not impacted. Analysis of molecules involved in MERTK signaling revealed that phosphorylated mTOR was significantly modulated following the addition of MERTK inhibitor. Long term culture of CD8 T cells demonstrated MERTK was significantly increased following early and late re-stimulation, and expression of MERTK was strongly associated with central memory subsets.ConclusionsOur results demonstrate that inhibition of MERTK signaling on T cells reduces cell division where mTOR is significantly impacted. Despite this, other functional aspects, such as intracellular cytokine production remain unchanged. Therefore, interruption of MERTK signaling on T cells has a specific effect on cell division rather than cytotoxic function on a cell by cell basis. This has potential ramifications on the use of MERTK inhibitors to treat tumors where the ability to form substantial cytotoxic T cell populations might be reduced. In addition, increased MERTK expression on central memory subsets during long term culture suggests this signaling pathway could be critical for generating memory pools of T cells and provide new avenues for the improvement of adoptive T cell therapy protocols.ReferencesCummings CT, Deryckere D, Earp HS, Graham DK. Molecular pathways: MERTK signaling in cancer. Clin Cancer Res 2013;19(19):5275–5280.Peeters MJW, Dulkeviciute D, Draghi A, et al. MERTK Acts as a Costimulatory Receptor on Human CD8+T Cells. Cancer Immunol Res 2019;7(9):1472–1484.Disclosure InformationR.M. Powell: None. M.J.W. Peeters: None. A. Rachbech: None. P.T. Straten: None.

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Manufacture of Gene-Modified Human T-Cells with a Memory Stem/Central Memory Phenotype

Human Gene Therapy Methods ◽

10.1089/hgtb.2014.004 ◽

2014 ◽

Vol 25 (5) ◽

pp. 277-287 ◽

Cited By ~ 27

Author(s):

Raquel Gomez-Eerland ◽

Bastiaan Nuijen ◽

Bianca Heemskerk ◽

Nienke van Rooij ◽

Joost H. van den Berg ◽

...

Keyword(s):

T Cells ◽

Central Memory ◽

Memory Phenotype ◽

Human T Cells ◽

Central Memory Phenotype

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Naive and Ex Vivo Activated Human T Cells Generate Consistent Engraftment and Lethal Graft-Versus-Host Disease (GvHD) in NOD SCID β 2M Null Mice: A New Xenogeneic Model for GvHD.

Blood ◽

10.1182/blood.v106.11.3106.3106 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3106-3106

Author(s):

Bruno Nervi ◽

Michael P. Rettig ◽

Julie K. Ritchey ◽

Gerhard Bauer ◽

Jon Walker ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Ex Vivo ◽

Conditioning Regimen ◽

Activation Markers ◽

In Vivo Models ◽

Hematopoietic Stem ◽

Human T Cells ◽

The Impact

Abstract GvHD remains a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusion. The human GvHD pathophysiology includes recipient tissue destruction and proinflammatory cytokine production associated with the conditioning regimen; donor T cells become allo-activated, proliferate, and mediate tissue injury in various organs, including the liver, skin, and gut. Modern therapeutic strategies to control GvHD while maintaining the beneficial graft-versus-leukemia effects require ex vivo T cell stimulation and expansion. Multiple studies have demonstrated that these ex vivo expanded T cells exhibit decreased survival and function in vivo, including reduced alloreactivity and GvHD potential. Unfortunately no in vivo models exist to consistently examine the impact of ex vivo manipulation of human T cells (HuT) on T cell function. Naive HuT were compared to HuT activated using CD3/28 beads (XcyteTMDynabeads) with 50 U/ml IL-2 for 4 days (Act). We initially evaluated the HuT engraftment and GvHD potential of naive and Act in RAG2γ null mice (n=22) conditioned with clodronate liposomes on day −1 and 350cGy on day 0, as previously described by others. We injected 107 and 1.5x107 naive or Act HuT intravenously (iv). All mice exhibited low HuT engraftment and no lethal GvHD. NOD SCIDβ 2M null mice (β 2M) were next conditioned with 250cGy on day −1 (n=34), or 300cGy on day 0 (n=21). 107 naive vs Act HuT were injected retroorbitaly (ro). Lower HuT doses or iv injection resulted in no expansion or GvHD. Engraftment of HuT in peripheral blood of recipient mice was evaluated weekly by FACS and euthanasia was performed if mice lost > 20% body weight. 60% of the mice conditioned with 250cGy that received naive HuT developed lethal GvHD, in comparison to 75% of mice that received 300cGy and nave HuT, and 100% of mice that received 300cGy and Act HuT. Table 1 250cGy 300cGy Naive (n=34) Naive (n=8) Activated (n=13) *p<0.02 PB engraftment (%HuT) 20%±15 33%±21 59%±19 Lethal GvHD 60% 75% 100% All mice receiving 300cGy had well preserved CD4/CD8 ratios (1–1.5). Tissue infiltration was greatest in mice that had received 300cGy and Act HuT (spleen, liver, lung, kidney: 50–70%). Of interest, serum levels of hu IFNγ dramatically increased over time in all mice who went on to develop lethal GvHD (day 3=270 ug/ml and day 15=36,000 ug/ml) compared to mice that did not develop lethal GvHD (day 10=40 ug/ml and day 17=1,020 ug/ml)(p<0.05). Interestingly, the up-regulation of the activation markers CD25 and CD30 in HuT, and IFNγ production predicted lethal GvHD in β 2M null mice. In summary, we developed a xenogeneic model of lethal GvHD where naive or ex vivo Act HuT injected ro in sublethaly irradiated β 2M not only engraft, expand in vivo, but also infiltrate and damage different mouse target organs. HuT are allo-activated against mouse antigens and damage the target tissues, sharing the major characteristics of human GvHD and causing the death of mice. This model will allow us to study the effects of specific ex vivo T cell manipulation including transduction, selection, expansion, and the depletion or addition of various T cells and other cellular subsets on the outcome of GvHD, to determine improved therapeutic interventions.

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Induction of tolerance to bone marrow allografts by donor-derived host nonreactive ex vivo–induced central memory CD8 T cells

Blood ◽

10.1182/blood-2009-10-248716 ◽

2010 ◽

Vol 115 (10) ◽

pp. 2095-2104 ◽

Cited By ~ 20

Author(s):

Eran Ophir ◽

Yaki Eidelstein ◽

Ran Afik ◽

Esther Bachar-Lustig ◽

Yair Reisner

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Ex Vivo ◽

Memory Cell ◽

Central Memory ◽

Third Party ◽

High Survival

Abstract Enabling engraftment of allogeneic T cell–depleted bone marrow (TDBM) under reduced-intensity conditioning represents a major challenge in bone marrow transplantation (BMT). Anti–third-party cytotoxic T lymphocytes (CTLs) were previously shown to be endowed with marked ability to delete host antidonor T cells in vitro, but were found to be less effective in vivo. This could result from diminished lymph node (LN) homing caused by the prolonged activation, which induces a CD44+CD62L− effector phenotype, and thereby prevents effective colocalization with, and neutralization of, alloreactive host T cells (HTCs). In the present study, LN homing, determined by imaging, was enhanced upon culture conditions that favor the acquisition of CD44+CD62L+ central memory cell (Tcm) phenotype by anti–third-party CD8+ cells. These Tcm-like cells displayed strong proliferation and prolonged persistence in BM transplant recipients. Importantly, adoptively transferred HTCs bearing a transgenic T-cell receptor (TCR) with antidonor specificity were efficiently deleted only by donor-type Tcms. All these attributes were found to be associated with improved efficacy in overcoming T cell–mediated rejection of TDBM, thereby enabling high survival rate and long-term donor chimerism, without causing graft-versus-host disease. In conclusion, anti–third-party Tcms, which home to recipient LNs and effectively delete antidonor T cells, could provide an effective and novel tool for overcoming rejection of BM allografts.

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Dysregulated T cell expression of TIM3 in multiple sclerosis

Journal of Experimental Medicine ◽

10.1084/jem.20060210 ◽

2006 ◽

Vol 203 (6) ◽

pp. 1413-1418 ◽

Cited By ~ 160

Author(s):

Ken Koguchi ◽

David E. Anderson ◽

Li Yang ◽

Kevin C. O'Connor ◽

Vijay K. Kuchroo ◽

...

Keyword(s):

Multiple Sclerosis ◽

T Cells ◽

T Cell ◽

Ex Vivo ◽

Interleukin 12 ◽

Intrinsic Defect ◽

Control Subjects ◽

Human T Cells ◽

Ifn Γ ◽

Cell Expression

T cell immunoglobulin- and mucin domain–containing molecule (TIM)3 is a T helper cell (Th)1–associated cell surface molecule that regulates Th1 responses and promotes tolerance in mice, but its expression and function in human T cells is unknown. We generated 104 T cell clones from the cerebrospinal fluid (CSF) of six patients with multiple sclerosis (MS) (n = 72) and four control subjects (n = 32) and assessed their cytokine profiles and expression levels of TIM3 and related molecules. MS CSF clones secreted higher amounts of interferon (IFN)-γ than did those from control subjects, but paradoxically expressed lower levels of TIM3 and T-bet. Interleukin 12–mediated polarization of CSF clones induced substantially higher amounts of IFN-γ secretion but lower levels of TIM3 in MS clones relative to control clones, demonstrating that TIM3 expression is dysregulated in MS CSF clones. Reduced levels of TIM3 on MS CSF clones correlated with resistance to tolerance induced by costimulatory blockade. Finally, reduction of TIM3 on ex vivo CD4+ T cells using small interfering (si)RNA enhanced proliferation and IFN-γ secretion, directly demonstrating that TIM3 expression on human T cells regulates proliferation and IFN-γ secretion. Failure to up-regulate T cell expression of TIM3 in inflammatory sites may represent a novel, intrinsic defect that contributes to the pathogenesis of MS and other human autoimmune diseases.

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