Gut Homing Potential of Human Naïve and Memory Alloreactive T Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2864-2864
Author(s):  
Michael W. Boyer ◽  
Kelly McQuown ◽  
Lindsay Hendey ◽  
Thomas G. Gross
Keyword(s):  
T Cells ◽  
T Cell ◽  
Memory T Cells ◽  
Human Memory ◽  
Cell Manipulation ◽  
Integrin Expression ◽  
Donor Pool ◽  
Tissue Specific ◽  
Relative Contribution

Abstract T cells are either naïve, having never encountered cognate antigen, or memory, with a history of activation, proliferation, and acquistion of effector specialization including tissue specific homing properties. We hypothesized that memory T cells would contain a subpopulation of alloantigen specific cells that might have acquired tissue specific homing characteristics such as upregulation of α4β7 integrin that directs T cells to gut lymphoid and non-lymphoid tissue. In mice, α4β7 integrin dependent migration to the peyer’s patch is essential to instigate lethal GVHD in MHC mismatched BMT models. To test this hypothesis, immunomagnetic sorting for CD45RO and CD45RA was used to obtain populations either depleted or enriched for naïve T cells respectively. Using CFDA dye dilution and CD69 upregulation, alloantigen specific T cells were tracked with flow cytometry at days 3, 5, and 7 of a mixed lymphocyte reaction, with an autologous control. ModFit LT software was used to estimate precursor frequencies, which showed that approximately 1 in 200 CD4 and CD8 positive T cells are alloreactive (n=9) regardless of whether they were CD45RO or CD45RA selected, demonstrating that alloreactivity could be potentially recruited from either the naïve or memory donor pool in a GVHD reaction. Analysis of α4β7 integrin coexpression before alloantigen stimulation demonstrated that for both CD4 and CD8 positive T cells, the memory pool had two-fold higher levels of coexpression that were statistically significant compared to the naive pool, with CD8 memory T cells demonstrating the highest coexpression (24 +/− 12%). When examined at day 5 of the MLR, both CD4 and CD8 positive alloantigen specific T cells from the CD45RO selected group maintained levels of α4β7 integrin coexpression similar to baseline (14% and 25% respectively). In contrast, CD4 and CD8 positive alloantigen specific T cells from the CD45RA selected group had levels of α4β7 integrin coexpression that were four-fold higher compared to baseline, with 20% and 48% coexpression respectively (n=6). The relative contribution of α4 and β7 integrins was examined by comparing the mean flourescent intensity (MFI) of the alloantigen specific T cells to the resting T cells within the same day 5 MLR. The greatest increase in expression was seen for β7 integrin on the CD45RA selected CD4 and CD8 positive T cells with 3.1 and 3.2 times higher expression respectively (both p<.001). In contrast, the CD45RO selected CD4 and CD8 positive T cells had 1.78 (p<.01) and 1.35 (p<.05) times higher β7 expression levels respectively. With regard to α4 integrin expression, CD45RA selected CD4 and CD8 positive T cells had 2.2 and 2.0 times higher expression respectively (both p<.01). The α4 integrin expression on CD45RO selected CD4 and CD8 positive T cells was 2.0 (p<.05) and 1.43 (p= NS) times higher respectively. These data suggest that both naïve and memory CD4 and CD8 positive T cells may contribute to alloreactivity, although there are differences in the regulation of α4β7 integrin expression which could significantly affect in vivo T cell homing and therefore instigation of GVHD. Based upon murine studies, it has been demonstrated that memory T cells do not contribute to GVHD; our data suggest in contrast that human memory T cells could contribute in a significant way to GVHD and further study is necessary before development of successful T cell manipulation strategies aimed at attenuating GVHD.

scholarly journals A novel humanized mouse model to study the function of human cutaneous memory T cells in vivo in human skin

2018 ◽  
Author(s):  
Maria M. Klicznik ◽  
Ariane Benedetti ◽  
Laura M. Gail ◽  
Suraj R. Varkhande ◽  
Raimund Holly ◽  
...  
Keyword(s):  
T Cells ◽  
Candida Albicans ◽  
T Cell ◽  
Human Skin ◽  
Skin Diseases ◽  
Memory T Cells ◽  
Human Memory ◽  
Human Pbmc ◽  
Human T Cell

AbstractHuman skin contains a population of memory T cells that support tissue homeostasis and provide protective immunity. The study of human memory T cells is often restricted to in vitro studies and to human PBMC serving as primary cell source. Because the tisse environment impacts the phenotype and function of memory T cells, it is crucial to study these cells within their tissue. Here we utilized immunodeficient NOD-scid IL2rγnull (NSG) mice that carried in vivo-generated engineered human skin (ES). ES were generated from human keratinocytes and fibroblasts and is initially devoid of skin-resident immune cells. Upon adoptive transfer of human PBMC this reductionist system allowed to study human T cell recruitment from a circulating pool of T cells into non-inflamed human skin in vivo. Circulating human memory T cells preferentially infiltrated ES and showed diverse functional profiles of T cells found in fresh human skin. The chemokine and cytokine microenvironment of ES closely resembled that of non-inflamed human skin. Upon entering the ES T cells assumed a resident memory T cell-like phenotype in the absence of infection, and a proportion of these cutaneous T cells can be locally activated upon injection of monocyte derived dendritic cells (moDCs) that presented Candida albicans. Interestingly, we found that CD69+ memory T cells produced higher levels of effector cytokines in response to Candida albicans, compared to CD69- T cells. Overall, this model has broad utility in many areas of human skin immunology research, including the study of immune-mediated skin diseases.

scholarly journals IL-7 and IL-21 are superior to IL-2 and IL-15 in promoting human T cell–mediated rejection of systemic lymphoma in immunodeficient mice

Blood ◽  
2010 ◽  
Vol 115 (17) ◽  
pp. 3508-3519 ◽  
Author(s):  
John C. Markley ◽  
Michel Sadelain
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Memory T Cells ◽  
Tumor Rejection ◽  
Effector Memory ◽  
Transfer Model ◽  
Human T Cell

Abstract The γc-cytokines are critical regulators of immunity and possess both overlapping and distinctive functions. However, comparative studies of their pleiotropic effects on human T cell–mediated tumor rejection are lacking. In a xenogeneic adoptive transfer model, we have compared the therapeutic potency of CD19-specific human primary T cells that constitutively express interleukin-2 (IL-2), IL-7, IL-15, or IL-21. We demonstrate that each cytokine enhanced the eradication of systemic CD19+ B-cell malignancies in nonobese diabetic/severe combined immunodeficient (NOD/SCID)/γcnull mice with markedly different efficacies and through singularly distinct mechanisms. IL-7– and IL-21–transduced T cells were most efficacious in vivo, although their effector functions were not as enhanced as IL-2– and IL-15–transduced T cells. IL-7 best sustained in vitro T-cell accumulation in response to repeated antigenic stimulation, but did not promote long-term T-cell persistence in vivo. Both IL-15 and IL-21 overexpression supported long-term T-cell persistence in treated mice, however, the memory T cells found 100 days after adoptive transfer were phenotypically dissimilar, resembling central memory and effector memory T cells, respectively. These results support the use of γc-cytokines in cancer immunotherapy, and establish that there exists more than 1 human T-cell memory phenotype associated with long-term tumor immunity.

scholarly journals Human memory CD8 T cell effector potential is epigenetically preserved during in vivo homeostasis

2017 ◽  
Vol 214 (6) ◽  
pp. 1593-1606 ◽  
Author(s):  
Hossam A. Abdelsamed ◽  
Ardiana Moustaki ◽  
Yiping Fan ◽  
Pranay Dogra ◽  
Hazem E. Ghoneim ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Human Memory ◽  
Memory Cd8 T Cells ◽  
Memory Cd8 T Cell ◽  
Cell Effector

Antigen-independent homeostasis of memory CD8 T cells is vital for sustaining long-lived T cell–mediated immunity. In this study, we report that maintenance of human memory CD8 T cell effector potential during in vitro and in vivo homeostatic proliferation is coupled to preservation of acquired DNA methylation programs. Whole-genome bisulfite sequencing of primary human naive, short-lived effector memory (TEM), and longer-lived central memory (TCM) and stem cell memory (TSCM) CD8 T cells identified effector molecules with demethylated promoters and poised for expression. Effector-loci demethylation was heritably preserved during IL-7– and IL-15–mediated in vitro cell proliferation. Conversely, cytokine-driven proliferation of TCM and TSCM memory cells resulted in phenotypic conversion into TEM cells and was coupled to increased methylation of the CCR7 and Tcf7 loci. Furthermore, haploidentical donor memory CD8 T cells undergoing in vivo proliferation in lymphodepleted recipients also maintained their effector-associated demethylated status but acquired TEM-associated programs. These data demonstrate that effector-associated epigenetic programs are preserved during cytokine-driven subset interconversion of human memory CD8 T cells.

Hemato Nanotechnology: Artificial APC System for T Cell Adoptive and Active Immunotherapy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3907-3907
Author(s):  
H. De La Pena ◽  
J. A. Madrigal ◽  
M. Bencsik ◽  
G. W.V. Cave ◽  
R. C. Rees ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Viral Infections ◽  
Ex Vivo ◽  
Nano Particles ◽  
Cell Manipulation ◽  
Active Immunotherapy ◽  
Effector Memory ◽  
Artificial System

Abstract One of the main problems of T cell mediated immunotherapy in delivering significant clinical impact and benefit to patients with malignant diseases and life threatening viral infections is the expansion of adequate numbers of functional antigen specific cytotoxic T cells. The current approaches for expanding T cells possess significant drawbacks in terms of timing, reproducibility and reliability. Many if not all these approaches rely on ex-vivo cell manipulation, which often leads to short T cell survival in-vivo after infusion. In-vivo artificial systems should be the ideal. There is no artificial APC system capable of both ex-vivo and, more importantly, in-vivo antigen specific T cell expansion. In order to address this we have developed a novel artificial nanotechnology system capable of priming and expanding antigen specific T cells in-vivo. As defined by the NIH, nanotechnology uses nanoscale injectable, targeted and traceable devices capable of important immunological/clinical functions. This nano-system was constructed using the latest generation of nanoscale immuno liposomes (100nm; 50 times smaller than average cells and same size as most human viruses), approved for in-vivo human use since they are non-toxic, biodegradable, avoid fast recognition by the reticulo-endothelial system, are safe in terms of size, have good stability and favourable pharmacokinetic behaviour for safe in-vivo trafficking. We have coated these liposomes with an optimised number of MHC Class I / peptide complexes and a specific and selected range of ligands for adhesion (ICAM-1), early activation (CD28, CD27), late activation (4-1BB) and survival receptors (CD40L). We have made these immuno liposomes traceable, either via a fluorescent lipid or iron oxide nano particles (13nm each), which make them traceable in vivo using Magnetic Resonance Imaging. Production of this system in a ready to use form is achievable in less than 48 hrs. We are currently working on an HLA-A2 transgenic mouse model to validate in-vivo behaviour of the system. After ex-vivo stimulation with this artificial system (using CMV pp65 as model antigen), we have measured successful expansions of high antigen specific T cell numbers (55 to 80 fold) in CMV positive individuals, which are superior when compared with other systems such as DCs (30 fold), beads (non antigen specific) and soluble tetramers and antibodies (30 fold). Expanded T cells are functional; they produce INF-γ and are predominantly of effector-memory and memory phenotype. We have demonstrated by double fluorescent staining that these liposomes are recognised directly on CD8+ T cells in an antigen specific fashion and also indirectly by being incorporated on the surface of the natural APCs as exosomes do. When tested in naive individuals, this system is also capable of priming naive T cells without additional adjuvants, as other APC systems use. In conclusion we have established the optimal conditions for an efficient artificial APC system, which embodies a powerful, controllable and superior approach with enormous potential for T cell immunotherapy in vivo. Figure Figure

Anti-Thymocyte Globulin (ATG) Differentially Depletes naïve and Memory T Cells and Permits Memory-Type Regulatory T Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4670-4670
Author(s):  
Chang-Qing Xia ◽  
Anna Chernatynskaya ◽  
Clive Wasserfall ◽  
Benjamin Looney ◽  
Suigui Wan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Memory T Cells ◽  
Conflicts Of Interest ◽  
T Cell Subsets ◽  
Hematopoietic Stem

Abstract Abstract 4670 Anti-thymocyte globulin (ATG) has been used in clinic for the treatment of allograft rejection and autoimmune diseases. However, its mechanism of action is not fully understood. To our knowledge, how ATG therapy affects naïve and memory T cells has not been well investigated. In this study, we have employed nonobese diabetic mouse model to investigate how administration of anti-thymocyte globulin (ATG) affects memory and naïve T cells as well as CD4+CD25+Foxp3+ regulatory T cells in peripheral blood and lymphoid organs; We also investigate how ATG therapy affects antigen-experienced T cells. Kinetic studies of peripheral blood CD4+ and CD8+ T cells post-ATG therapy shows that both populations decline to their lowest levels at day 3, while CD4+ T cells return to normal levels more rapidly than CD8+ T cells. We find that ATG therapy fails to eliminate antigen-primed T cells, which is consistent with the results that ATG therapy preferentially depletes naïve T cells relative to memory T cells. CD4+ T cell responses post-ATG therapy skew to T helper type 2 (Th2) and IL-10-producing T regulatory type 1 (Tr1) cells. Intriguingly, Foxp3+ regulatory T cells (Tregs) are less sensitive to ATG depletion and remain at higher levels following in vivo recovery compared to controls. Of note, the frequency of Foxp3+ Tregs with memory-like immunophenotype is significantly increased in ATG-treated animals, which might play an important role in controlling effector T cells post ATG therapy. In summary, ATG therapy may modulate antigen-specific immune responses through modulation of naïve and memory T cell pools and more importantly through driving T cell subsets with regulatory activities. This study provides important data for guiding ATG therapy in allogenieic hematopoietic stem cell transplantation and other immune-mediated disorders. Disclosures: No relevant conflicts of interest to declare.

scholarly journals 008 Multiple human memory T cell subsets can give rise to resident memory T cells in skin

2016 ◽  
Vol 136 (5) ◽  
pp. S2
Author(s):  
T.R. Matos ◽  
A. Gehad ◽  
J. Teague ◽  
J.T. O’Malley ◽  
E.L. Lowry ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Memory T Cells ◽  
Human Memory ◽  
T Cell Subsets ◽  
Memory T Cell ◽  
Resident Memory T Cells

scholarly journals Mobilization of CD8+ Central Memory T-Cells with Enhanced Reconstitution Potential in Mice By a Combination of G-CSF and GMI-1271-Mediated E-Selectin Blockade

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 512-512 ◽  
Author(s):  
Ingrid G Winkler ◽  
Valerie Barbier ◽  
Kristen J Radford ◽  
Julie M Davies ◽  
Jean-Pierre Levesque ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Memory T Cells ◽  
Central Memory ◽  
Donor Blood ◽  
Central Memory T Cells

Abstract T-cells are critical mediators of immune defense against pathogens and cancer. Adoptive T cell immunotherapy and T-cell engineering have promising clinical applications but T cell survival and exhaustion are current limitations. Central memory cells (TCM CD62L+ CCR7+) and their precursors, stem central memory T-cells (TSCM) possess the stem-like properties needed to reconstitute and prolong an effective immune response long-term. These cells have been shown to significantly improve therapeutic efficacy of adoptive T-cell therapy. The challenge remains to harvest good quality TCM-cells for these immunotherapy approaches. The bone marrow (BM) is the major reservoir of CD8+ TCM and their precursors. We have previously shown that E-selectin is expressed in the BM vasculature and drives activation and differentiation of hematopoietic stem cells during G-CSF induced mobilization to the blood. We find therapeutic blockade of E-selectin promotes HSC self-renewal and reconstitution in vivo. We now examine the impact of E-selectin blockade on CD8+ T cell mobilization from the bone marrow to the blood and hypothesize that E-selectin blockade may also dampen the activation/differentiation of this subset. First we administered a standard G-CSF regime (filgastim 250ug/kg/day for 3 days) to mice and then dosed some cohorts with GMI-1271 (40mg/kg BID) from 12 to 72 hours within this 3 day period. Administration of G-CSF alone results in a near complete disappearance of bone marrow resident CD8+ TCM cells, and their apparent migration (increase in numbers) to the blood, while CD8+ subsets in the lymph nodes and spleen were barely affected by G-CSF. Furthermore among T-cell subsets, CD8+ but not CD4+ TCM were specifically mobilized into the blood when GMI-1271 was co-administered for the last 12 to 24 hours of G-CSF. These findings are consistent with reports demonstrating the bone marrow to be a major reservoir for CD8+ but not CD4+ central memory T-cells. Administration of GMI-1271 caused a marked enhancement in mobilization into the blood of CD8+ TCM/SCM (CD62Lhi, CCR7+) cells over treatment with G-CSF alone (p<0.05). To determine the functional consequences of this skewed mobilization following GMI-1271 co-administration, 25 uL of mobilized blood was transplanted into irradiated congenic B6.SJL recipients together with 2x105 congenic BM cells to analyze long-term donor T-cell engraftment in the recipient mice. We found G-CSF mobilized donor blood did not contribute CD8+ TCM cells that can persist post-transplant (<0.5% at 20 weeks post-transplant). In contrast when donor mice were mobilized with G-CSF together with E-selectin blockade (GMI-1271), we found elevated levels of donor blood derived CD8+ T-cells demonstrating robust long-term CD8+ T-cell persistence / regeneration (5.3 ±3.2% of total recipient T-cells, p=0.04). This dramatic boost in donor CD8+ T-cell reconstitution in mobilized blood following GMI-1271 co-administration is likely to be due to the long-term persistence and in vivo amplification of CD8+ TCM cells from donor mobilized blood. Similar in vivo enhancing effects of GMI-1271 were also observed with other mobilizing agents such as combined CXCR4 and VLA-4 blockade and GM-CSF resulting in a significant 4.9-fold boost in donor CD8+ reconstitution with GMI-1271. Importantly, only 12 hours of E-selectin blockade was sufficient to achieve this boost in CD8+ TCM numbers in the blood following G-CSF. In a previous report we have shown that therapeutic blockade of E-selectin promotes HSC self-renewal in vivo. Thus, it is possible that E-selectin blockade boosts mobilization of CD8+ TCM/SCM with stem-like properties into the blood by loosening factors retaining CD8+ TCM/SCM in the bone marrow and/or blocking the E-selectin-mediated activation and differentiation of this T-cell subset. In summary, our studies identify E-selectin blockade as a novel target to improve harvesting of CD8+ TCM/SCM cells with stem-like properties. Blockade of this target with GMI-1271 significantly improves the in vivo reconstitution potential and regenerative properties of CD8+ T-cells from donor blood allowing a valuable source of desired T-cells for use in adoptive immunotherapy and T-cell engineering. Disclosures Winkler: GlycoMimetics Inc: Research Funding. Barbier:GlycoMimetics Inc: Research Funding. Davies:GlycoMimetics Inc: Research Funding. Smith:GlycoMimetics, Inc.: Employment. Fogler:GlycoMimetics, Inc.: Employment. Magnani:GlycoMimetics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Phase I Study of Adoptive Immunotherapy with HA-1-Specific CD8+ and CD4+ Memory T Cells for Children and Adults with Relapsed Acute Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation (HCT): Trial in Progress

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 45-46 ◽  
Author(s):  
Elizabeth F Krakow ◽  
Corinne Summers ◽  
Ann Dahlberg ◽  
Merav Bar ◽  
Melinda Ann Biernacki ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Phase I ◽  
Research Funding ◽  
Phase I Trial ◽  
Memory T Cells ◽  
Cell Immunotherapy ◽  
T Cell Immunotherapy

Background: Donor T cells specific for minor histocompatibility (H) antigens can deliver potent, selective anti-leukemic effects after allogeneic HCT when the antigen is negligibly or not expressed by non-hematopoietic tissues, not present in the donor, and expressed by the recipient. We reported a new minor H antigen-directed T-cell therapy that can be deployed after HCT to manage persistent or recurrent measurable residual hematologic malignancies or overt relapse (Blood 2018;131(1):108). We developed a transgene with 4 components: 1) a high-affinity T-cell receptor (TCR) specific for the hematopoietic-restricted minor H antigen, HA-1 that is presented on HLA-A*02:01; 2) a CD8 co-receptor to enhance function of the class I-restricted TCR in CD4+ T cells so they promote cytotoxic CD8+ T cell function and survival; 3) an inducible caspase-9 safety switch, which can be triggered by the drug rimiducid in case of in vivo toxicity; and 4) a CD34-CD20 epitope to facilitate selection of the engineered product during manufacturing and track HA-1 TCR T cells in the recipient. The 21-day manufacturing process entails CD45RA+ naïve T cell depletion (minimizes the risk of GvHD), and subsequent CD4+ and CD8+ separation (provides a consistent 1:1 CD4:CD8 ratio). The separate cultures are transduced with the lentivirus construct iCasp9-HA1-TCR2-RQR-CD8, expanded, and selected using the CD34 marker to ensure removal of untransduced T cells. Study Design and Methods: The single-center phase I trial (NCT03326921) evaluates the feasibility and safety of infusion of HA-1 TCR T-cell immunotherapy. Primary end points are 1) Feasibility of manufacturing and administering HA-1 TCR CD8+ and CD4+ memory T cells and 2) Dose-limiting toxicity of HA-1 TCR T cells. Major inclusion criteria are: HLA-A*02:01-positive, HA-1-positive patients who underwent HCT for acute leukemia, myelodysplastic syndrome, BPDCN, CML, CMML or JMML from a HLA-A*-02:01+/HA-1-negative donor or HLA-A*02:01-negative haploidentical or mismatched donor (excluding umbilical cord). HA-1 genotype screening is performed on patient and donor blood, hair follicle or cheek swab samples shipped to Fred Hutchinson Cancer Research Center. To be eligible for treatment, patients must develop measurable residual disease or overt relapse after HCT but may receive other standard or investigational therapies prior to treatment with HA-1 TCR T-cell immunotherapy if clinically indicated. Some systemic immunosuppression may be continued, but prior grade IV acute GVHD and prior severe chronic GVHD are key exclusions. Two groups, &lt;16 and ≥16 years, will be treated at dose levels ranging from 3 x 106 to30 x 106 cells/kg, in cohorts of 3-6 subjects, up to approximately 24 subjects in total. Fludarabine lymphodepletion will be used in most subjects, followed by a single T-cell infusion, with an option for a subsequent infusion(s) if the subject demonstrates an initial response without severe toxicity. Bone marrow aspirations are performed prior to T-cell infusion and at several time points following infusion. Recruitment and Patient Characteristics: To date, 3 subjects have been treated on the phase I clinical trial and received a total of 5 infusions (Table 1). HA-1 TCR T cell persistence in blood and bone marrow has been documented from &gt;3 months to &gt;13 months. Clear in vivo anti-leukemic activity was observed at the first dose level, including in a subject with aggressive, highly refractory T-ALL and early post-HCT relapse. Outlook: Minor H antigen-specific T-cell immunotherapy may offer effective management of post-HCT relapse while avoiding GvHD and other off-target effects. Due to population genetics of HA-1 and HLA-A*02:01, HA-1 TCR T-cell immunotherapy is applicable to 10-15% of HCT recipients with various hematological malignancies. The ongoing phase I trial is actively recruiting patients. Development of T-cell immunotherapy targeting other minor H antigen/HLA combinations is also underway to increase the broad applicability of minor H antigen-targeted T-cell immunotherapy. Disclosures Krakow: HighPass Bio: Research Funding. Cunningham:HighPass Bio: Research Funding. Vartanian:HighPass Bio: Research Funding. Bleakley:HighPass Bio: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Quality of Leukemia-Derived Dendritic Cells (DC) and T-Cells Are Predictive for the Specific Anti-Leukemic Potential of DC-Trained T-Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4883-4883
Author(s):  
Helga Schmetzer ◽  
Christine Grabrucker ◽  
Anja Liepert ◽  
Andreas Kremser ◽  
Julia Loibl ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Clinical Response ◽  
Memory T Cells ◽  
Ex Vivo ◽  
Cell Activity ◽  
Lytic Activity ◽  
Gm Csf

Abstract The presentation of leukemic antigens can be improved by in vitro conversion of leukemic cells in leukemia-derived DC (DCleu), thereby forming a platform for the generation of leukemia-specific cytotoxic lymphocytes (CTL). DC/ DCleu can be quantified by combination of suitable blast and DC-antigens (Schmetzer 2007). Now we want to enlight the role of the quality of DC/ DCleu and (DC-trained) T-cells to mediate leukemia-cytotoxic reactions ex vivo or to predict or correlate the clinical response to a DC/DLI-based immunotherapy in vivo. Methods: DC were generated with the best of 3 DC-generating methods(‘MCM-mimic’, Lee 2003;’Ca-Ionophore’, Houtenbos 2003; ‘Picibanil’, Sato 2003; Kufner S. 2005 I-III) and used to train T-cells in a ‘Mixed lymphocyte culture’ (MLC) for 10 days in the presence of IL-2 and restimulated with patient-derived DC every 3 days. Co-expression of T-cell-antigens on T-cells was measured before and after MLC. The antileukemic cytotoxic activity of DC-trained (or blast trained or untrained) T-cells against naïve blasts was quantified. We could show, that DC can be generated in every case of AML. In 65% of the cases T-cells gained a leukaemia-lytic activity after 24h training with DC, in 35% an increase of blasts was seen. The T-cell training efficacy with DC was superior to a blast training given rise to specific leukaemia-cytotoxic cells. A comparison of cases with a gain of lytic T-cell activity (n=11)with those without a lytic activity (n=6) showed 78 vs 51% DCleu, 55 vs 34% mature and 32 vs 18% migratory DC and 50vs40% proliferating T-cells, 53 vs 46% memory T-cells, 68vs56% CD4 and 38 vs 60% CD8 pos T-cells. Moreover we could evaluate cut-off values: 90% of DC-trained T-cells could gain a lytic activity if > 65% DCleu were in the MLR. In AML-patients who had presented with a relapse after SCT we could demonstrate a better ex vivo convertibility of blasts to DCleu if patients had successfully responded to a GM-CSF/DLI-based therapy of their relapse after SCT compared to cases with no response (72 vs 36% blasts convertible to DCleu; 44 vs 29% generable DC). Summary: The generation of DC/DCleu is possible in every AML/MDS-patient. Ex vivo convertibility of blasts to DCleu could predict a clinical response to a GM-CSF/DLI-based therapy or indirectly prove, that GM-CSF in vivo could contribute to produce DC/DCleu in vivo. A successful DC-training of T-cells is associated with high matureDC/ DCleu counts and high rates of proliferating, CD4 and Memory-T-cells. The lytic activity of DC-trained T-cells is predictable by quantities of DCleu generable in individual cases. So the generability of DC/DCleu and of DC/MNC-trained T-cells could contribute to predict the clinical course of the disease and could help to create specific anti-leukemic T-cells for immunotherapy of AML.

Engraftment Fitness of Human Central Memory-Derived Effector CD8+ T Cells In Immunodeficient Mice

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1019-1019
Author(s):  
Xiuli Wang ◽  
Berger Carolina ◽  
Stanley R. Riddell ◽  
ChingLam W Wong ◽  
Stephen Forman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Tumor Response ◽  
Memory T Cells ◽  
Effector T Cells ◽  
Caspase Activity ◽  
Effector Cells ◽  
Nog Mice

Abstract Abstract 1019 Development of T cell products that have engineered specificity for CD19 has broad application to adoptive transfer therapy for B-lineage lymphoma and leukemia. Clinical studies have demonstrated the safety and feasibility of T cell transfer as a therapy for patients. But the potency of this strategy has proven challenging, primarily due to issues relating to a lack of persistence of the adoptively transferred cells in patients. The repertoire of memory T cells is heterogeneous with respect to phenotypic, functional, and epigenetic attributes. Memory T cells are divided into sub-populations of 1) effector memory (TEM) cells that distribute to tissue beds and exhibit immediate cytolytic effector functioning, and 2) central memory (TCM) cells that home to lymph nodes based on CD62L/CCR7 expression and are capable of extensive proliferative activity upon re-encountering antigen. Thus the cell-intrinsic programming of distinct memory T cell subtypes, such as TEM and TCM, likely dictate divergent fates of their derived effector cells. To address this important issue, a clear functional dichotomy between TCM- and TEM-derived CD8+ CTLs was recently delineated in a nonhuman primate model, where it was found that virus-specific CD8+ CTL clones derived from TCM, but not TEM precursors, establish persistent and functional memory following adoptive transfer. Here, we extended these studies to human effector T cells using CMV as antigen model system to investigate the engraftment of human CMVpp65-specific CD8+ effector T cells derived in vitro from either sort purified CD45RO+CD62L+ TCM or CD45RO+CD62L- TEM precursors in NOD/Scid IL-2RγCnull (NOG) mice. TCM-derived effector cells (TE(CM)) and TEM-derived effector cells (TE(EM)) were adoptively transferred (i.v) into NOG mice reconstituted with human IL-15 and T cell levels in circulation were evaluated at different time points by FACS. 20% CD8+ TE(CM) and 3% CD8+ TE(EM) were detected on day 14. Then after, engraftment of the CD8+ TE(CM) remained at a steady state of approx 2% of circulating mononuclear cells for 100 days while TE(EM) remained at or below the level of detection, indicating that TE(CM) were superior in their ability to engraft in response to IL-15 as compared to TE(EM) after adoptive transfer (P<0.05). The long-term (100 days) persisting CD8+ TE(CM), harvested from primary recipient mice were found to be capable of engrafting secondary recipients. TcR Vβ analysis of persisting cells demonstrated that CD8+ TE(CM) engraftment was polyclonal, suggesting that homeostatic engraftment fitness is a general feature of these cells. To delineate the mechanism(s) by which TE(CM) exhibit superior in vivo engraftment, TE(CM) and TE(EM) were first labeled with CFSE before in vivo administration. CFSE profiles appear that the TE(EM) proliferated more extensively than TE(CM) early after adoptive transfer as indicated by the percent of cells which diluted CFSE on day 9 (i.e., 80% vs. only 25%, respectively). However, using D2R cleavage as a measure of caspase activity as a surrogate for apoptosis, 5.8% of engrafting TE(CM) were positive for activated caspase activity compared to 31.6% of TE(EM), suggesting that in NOG mice both CD8+ TE(CM) and TE(EM) proliferate in response to IL-15 whereas TE(CM) are intrinsically resistant to caspase activation and apoptosis. We also evaluated the antigen specific responsiveness of engrafted cells. Weekly infusions of irradiated pp65+/A2+ LCL as antigen significantly augmented the levels of circulating CD8+ TE(CM) as compared to no antigen stimulation (P<0.05), whereas CD8+ TE(EM) did not respond to antigen challenge. Moreover, when CMVpp65 specific CD8+ TE(CM) or TE(EM) were infused into CMVpp65+ tumor bearing mice, tumor cells progressed in mice receiving TE(EM) at a rate similar to untreated control mice over a ten day observation period, whereas TE(CM) significantly controlled tumor progression (P<0.05), indicating that CD8+ TE(CM) but not TE(EM) are able to mediate an anti-tumor response. Together these studies confirm that human CD8+ effector T cells derived from TCM precursors are capable of persistence after infusion, can proliferate in in vivo in response to antigen, can mediate an anti-viral or anti tumor response, and are likely the preferred T cells for antigen specific anti-tumor adoptive T cell therapy . Disclosures: No relevant conflicts of interest to declare.

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