scholarly journals OP0328 A UNIQUE PD1+CD38+ CD8+ T CELL POPULATION CHARACTERIZES CHECKPOINT INHIBITOR-ASSOCIATED INFLAMMATORY ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 201.1-202
Author(s):  
R. Wang ◽  
K. K. Chan ◽  
A. Cunningham-Bussel ◽  
L. Donlin ◽  
G. Vitone ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Synovial Fluid ◽  
Cell Population ◽  
Cd8 T Cells ◽  
Inflammatory Arthritis ◽  
Mononuclear Cells ◽  
Fold Increase ◽  
Mass Cytometry ◽  
Independent Cohort

Background:Immune checkpoint inhibitors (CI) are monoclonal antibodies that block CTLA-4, PD-1 or PD-L1, resulting in cytotoxic T cell activation in the tumor microenvironment. They have revolutionized the management of metastatic cancer but unleash “immune related adverse events” in > 80% of treated patients, including inflammatory arthritis in ~4%1. CI-associated arthritis (CI-A) often presents as a symmetrical polyarthritis, phenotypically indistinguishable from rheumatoid arthritis (RA), but whether it shares cellular and molecular features of RA has not been determined.Objectives:To compare synovial fluid (SF) T cell populations from CI-A patients to those in patients with RA, phenotypically and functionally.Methods:We immunophenotyped SF mononuclear cells from patients with CI-A caused by anti-PD-(L)1 therapy (n=9), seropositive RA (n=5), and psoriatic arthritis (PsA) (n=5) using a 39-marker mass cytometry (CyTOF) panel. FlowSOM was used to cluster CD4 and CD8 T cells into 15 ‘metaclusters’ based on multidimensional phenotypes. We used Kruskal-Wallis or Mann-Whitney tests to identify significantly altered populations (p<0.05), which we confirmed by biaxial gating. Flow cytometry was used to confirm SF findings in an independent cohort, and to identify cells of interest in peripheral blood. Cytokine staining was performed on sorted T cells populations after CDCD3/CD28 stimulation for 72 hours, followed by 4 hour PMA/ION+BRA/MON restimulation.Results:In CI-A patients, T cells represented 50% of SF mononuclear cells (53% CD4, 40% CD8), followed by monocytes (24%) and NK cells (8%), comparable to RA and PsA. However, FlowSOM analysis revealed expansion of a distinct population of PD-1+CD38hiCD127-CD8 T cells (CD8 metacluster2) (Fig. 1). These cells comprised 30% of CD8+ SF T cells in CI-A, a 3.4-fold increase over RA/PsA, p=0.0002 (Fig. 2). Over 40% of these cells expressed Ki67 in CI-A, suggesting active proliferation. Flow cytometry on SF cells from an independent cohort of CI-A patients (n= 5) and RA/PsA comparators (n= 9) confirmed our findings. PD-1+CD38hiCD127-CD8 T cells were also expanded in the blood of CI-A patients, where they represented 4.6% of CD8 Tcells, a 2.8-fold increase over RA, p = 0.0057. In addition to expressing high levels of PD-1, CD38hiCD127-, these CD8 T cells express other immune checkpoint receptors including ICOS and TIGIT. After in vitro stimulation, CD38hiCD127-CD8 T cells produced granzyme B along with TNF and IFN-γ at comparable levels to other CD8 populations, suggesting that they are not functionally exhausted.Figure 1.Mass cytometry CD8+T cells (tSNE plots) with FlowSOM metaclusters.Figure 2.Synovial fluid PD-1+CD38hiCD127-CD8+T cellsFlowSOM analysis of SF CD4 T cells in CI-A patients revealed the expansion of a subpopulation of CD4 cells with a similar surface phenotype of PD-1+CD38hiCD127-(metacluster2, 10% of CD4s in CI-A, a 2.4-fold increase over RA/PsA, p=0.0047). In contrast, RA patients had a significantly expanded population of PD-1hiICOS+ CD4 T peripheral helper (Tph) cells (metacluster5, 30% of CD4s in RA, p=0.006), but these cells were not expanded in CI-A (Fig 3).Figure 3.Synovial fluid CD4+T peripheral helper cellsConclusion:CyTOF analysis of SF revealed a uniquely expanded PD-1+CD38hiCD127-CD8 T cell population in CI-A not present in RA or PsA, and a similar PD-1+CD38hiCD127-CD4 T cell population. These cells may contribute to the amplified immune response seen in CI-A patients. Further functional and transcriptional analysis of these cells will help to elucidate their function may reveal key mechanisms driving CI-associated immune related adverse events.References:[1]Kostine M. Ann Rheum Dis 2018;77(3):393-398Disclosure of Interests:Runci Wang: None declared, Karmela Kim Chan: None declared, Amy Cunningham-Bussel: None declared, Laura Donlin Consultant of: Consultant – Genentech/Roche, Gregory Vitone: None declared, Aidan Tirpack: None declared, Caroline Benson: None declared, Gregory Keras: None declared, A. Helena Jonsson: None declared, Michael Brenner: None declared, Anne Bass: None declared, Deepak Rao Grant/research support from: Has received research grant support from Celgene and Merck., Consultant of: Has received consulting fees or honoraria from Merck, Pfizer, GlaxoSmithKine, Bristol-Myers Squibb, Janssen, and Scipher Medicine

scholarly journals IMMU-30. UTILIZING A NOVEL MASS CYTOMETRY-BASED IMMUNOMONITORING PLATFORM FOR THE CHARACTERIZATION OF VACCINE-REACTIVE, EPITOPE-SPECIFIC CD8+ T-CELLS IN HLA-A*0201+ PATIENTS WITH K27M+ DIFFUSE MIDLINE GLIOMAS

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi125-vi125
Author(s):  
Jared Taitt ◽  
Payal Watchmaker ◽  
Takahide Nejo ◽  
Neil Almeida ◽  
Kaori Okada ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Time Course ◽  
Mononuclear Cells ◽  
Expression Profiles ◽  
Peptide Vaccine ◽  
Effector Memory ◽  
Mass Cytometry ◽  
Peripheral Blood Mononuclear

Abstract Diffuse midline glioma (DMG), including diffuse intrinsic pontine glioma (DIPG) constitutes up to 20% of pediatric brain cancer and has a median survival of less than one year. We have identified a novel HLA-A*02:01-restricted neoantigen epitope encompassing the H3.3K27M mutation and implemented a pilot clinical trial through the Pacific Pediatric Neuro-Oncology Consortium (PNOC007). Newly diagnosed DIPG patients who are HLA-A2+ and H3.3K27M+ underwent radiation therapy, and then received the H3.3K27M peptide vaccine and tetanus toxoid (TT) peptide emulsified in Montanide in combination with poly-ICLC every 3 weeks for a total of 24 weeks. Our objective is to characterize vaccine-induced H3.3K27M-specific T-cell subpopulations in peripheral blood mononuclear cells through the evaluation of surface markers correlated with activation, memory, and exhaustion phenotypes utilizing a novel H3.3K27M-specific dextramer-based mass cytometry method. Through this approach, the temporal expansion of vaccine-reactive CD8+ T-cells was observed in all of patients (n = 4) who completed a minimum of 18 weeks on the study. These T-cells were subsequently stratified into discrete clusters on a tSNE plot using canonical CD8+ T-cell markers. Resultant clusters were further classified by their expression profiles, revealing distinct effector memory and exhausted subpopulations. Chronological monitoring of these groups indicates the time course-dependent development and persistence of vaccine-reactive exhausted and effector memory CD8+ T-cells in 75% of patients analyzed. Furthermore, a comparative analysis of myeloid subpopulations revealed an inverse correlation between the expansion of monocytic myeloid-derived suppressor cells (M-MDSCs) and length of enrollment in the trial. Future plans include the analysis of regulatory T-cells (Tregs) and MDSCs of all enrolled patients to solidify the relationship between the length of stay on the study and prevalence of immunosuppressive populations. This methodology offers insight into the progression of vaccine-induced patient immune responses and exhibits promise as a platform that may be extrapolated to other immunotherapies.

Integrin and transcriptomic profiles identify a distinctive synovial CD8+ T cell subpopulation in spondyloarthritis

2019 ◽  
Vol 78 (11) ◽  
pp. 1566-1575 ◽  
Author(s):  
Zoya Qaiyum ◽  
Eric Gracey ◽  
YuChen Yao ◽  
Robert D Inman
Keyword(s):  
T Cells ◽  
T Cell ◽  
Synovial Fluid ◽  
Cd8 T Cells ◽  
Mononuclear Cells ◽  
Cd8 T Cell ◽  
Cell Subpopulation ◽  
Rna Seq ◽  
Cell Subpopulations ◽  
Trafficking Molecules

ObjectivesCurrent evidence suggests that immune events in the gut may impact joint inflammation in ankylosing spondylitis (AS) but the expression of gut-related trafficking molecules in the inflammed joint is poorly characterised. We aimed to (1) assess differential expression patterns of trafficking molecules between patients and controls, (2) generate joint-specific cellular signatures and (3) obtain transcriptomic profiles of noteworthy cell subpopulations.MethodsMale subjects under 40 years of age fulfilling the mNY criteria were recruited. The following cells were surface stained using a 36-marker mass cytometry antibody panel: (1) peripheral blood mononuclear cells from AS patients, and healthy controls; (2) synovial fluid mononuclear cells from AS and rheumatoid arthritis (RA) patients. Additionally, RNA-seq was performed on CD8+ T cell subpopulations from the synovial fluid (SF).ResultsMature CD8+ T cells were enriched in AS SF, with a distinct pattern of integrin expression (β7, CD103, CD29 and CD49a). RNA-seq analysis of SF-derived CD103+CD49a+CD8+ T cells revealed elevated TNFAIP3, GZMB, PRF1 and IL-10.ConclusionsWe have identified a novel integrin-expressing mature CD8+ T cell population (CD49a+CD103+β7+CD29+) that appears to be more prevalent in AS SF than RA SF. These cells seem to possess dual cytotoxic and regulatory profiles which may play a role in AS pathogenesis.

scholarly journals Targeting CD38 is lethal to Breg-like chronic lymphocytic leukemia cells and Tregs, but restores CD8+ T-cell responses

2020 ◽  
Vol 4 (10) ◽  
pp. 2143-2157 ◽  
Author(s):  
Alak Manna ◽  
Timothy Kellett ◽  
Sonikpreet Aulakh ◽  
Laura J. Lewis-Tuffin ◽  
Navnita Dutta ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Cd8 T Cells ◽  
Mononuclear Cells ◽  
Transforming Growth Factor ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Lymphocytic Leukemia ◽  
Dependent Manner

Abstract Patients with chronic lymphocytic leukemia (CLL) are characterized by monoclonal expansion of CD5+CD23+CD27+CD19+κ/λ+ B lymphocytes and are clinically noted to have profound immune suppression. In these patients, it has been recently shown that a subset of B cells possesses regulatory functions and secretes high levels of interleukin 10 (IL-10). Our investigation identified that CLL cells with a CD19+CD24+CD38hi immunophenotype (B regulatory cell [Breg]–like CLL cells) produce high amounts of IL-10 and transforming growth factor β (TGF-β) and are capable of transforming naive T helper cells into CD4+CD25+FoxP3+ T regulatory cells (Tregs) in an IL-10/TGF-β-dependent manner. A strong correlation between the percentage of CD38+ CLL cells and Tregs was observed. CD38hi Tregs comprised more than 50% of Tregs in peripheral blood mononuclear cells (PBMCs) in patients with CLL. Anti-CD38 targeting agents resulted in lethality of both Breg-like CLL and Treg cells via apoptosis. Ex vivo, use of anti-CD38 monoclonal antibody (mAb) therapy was associated with a reduction in IL-10 and CLL patient-derived Tregs, but an increase in interferon-γ and proliferation of cytotoxic CD8+ T cells with an activated phenotype, which showed an improved ability to lyse patient-autologous CLL cells. Finally, effects of anti-CD38 mAb therapy were validated in a CLL–patient-derived xenograft model in vivo, which showed decreased percentage of Bregs, Tregs, and PD1+CD38hiCD8+ T cells, but increased Th17 and CD8+ T cells (vs vehicle). Altogether, our results demonstrate that targeting CD38 in CLL can modulate the tumor microenvironment; skewing T-cell populations from an immunosuppressive to immune-reactive milieu, thus promoting immune reconstitution for enhanced anti-CLL response.

Class I–unrestricted noncytotoxic anti–HTLV-I activity of CD8+ T cells

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1994-1995 ◽  
Author(s):  
Masako Moriuchi ◽  
Hiroyuki Moriuchi
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Mononuclear Cells ◽  
Membrane Filter ◽  
Cd8 T Cell ◽  
Class I ◽  
Type I ◽  
Peripheral Blood Mononuclear ◽  
Permeable Membrane

Abstract Although it is widely believed that viral clearance is mediated principally by the destruction of infected cells by cytotoxic T cells, noncytolytic antiviral activity of CD8+ T cells may play a role in preventing the progression to disease in infections with immunodeficiency viruses and hepatitis B virus. We demonstrate here that (1) replication of human T-lymphotropic virus type I (HTLV-I) is more readily detected from CD8+ T-cell–depleted (CD8−) peripheral blood mononuclear cells (PBMCs) of healthy HTLV-I carriers than from unfractionated PBMCs, (2) cocultures of CD8− PBMCs with autologous or allogeneic CD8+ T cells suppressed HTLV-I replication, and (3) CD8+ T-cell anti-HTLV-I activity is not abrogated intrans-well cultures in which CD8+ cells are separated from CD8− PBMCs by a permeable membrane filter. These results suggest that class I-unrestricted noncytolytic anti–HTLV-I activity is mediated, at least in part by a soluble factor(s), and may play a role in the pathogenesis of HTLV-I infection.

scholarly journals Alterations in regulatory T cells and immune checkpoint molecules in pancreatic cancer patients receiving FOLFIRINOX or gemcitabine plus nab-paclitaxel

2021 ◽  
Author(s):  
L. Sams ◽  
S. Kruger ◽  
V. Heinemann ◽  
D. Bararia ◽  
S. Haebe ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cd8 T Cells ◽  
Mononuclear Cells ◽  
Progression Free Survival ◽  
Ductal Adenocarcinoma ◽  
Immune Checkpoints ◽  
Prognostic Information ◽  
Peripheral Blood Mononuclear

Abstract Purpose This pilot study aimed on generating insight on alterations in circulating immune cells during the use of FOLFIRINOX and gemcitabine/nab-paclitaxel in pancreatic ductal adenocarcinoma (PDAC). Patients and methods Peripheral blood mononuclear cells were isolated before and 30 days after initiation of chemotherapy from 20 patients with advanced PDAC. Regulatory T cells (FoxP3+) and immune checkpoints (PD-1 and TIM-3) were analyzed by flow cytometry and immunological changes were correlated with clinical outcome. Results Heterogeneous changes during chemotherapy were observed in circulating T-cell subpopulations with a pronounced effect on PD-1+ CD4+/CD8+ T cells. An increase in FoxP3+ or PD-1+ T cells had no significant effect on survival. An increase in TIM3+/CD8+ (but not TIM3+/CD4+) T cells was associated with a significant inferior outcome: median progression-free survival in the subgroup with an increase of TIM-3+/CD8+ T cells was 6.0 compared to 14.0 months in patients with a decrease/no change (p = 0.026); corresponding median overall survival was 13.0 and 20.0 months (p = 0.011), respectively. Conclusions Chemotherapy with FOLFIRNOX or gemcitabine/nab-paclitaxel induces variable changes in circulating T-cell populations that may provide prognostic information in PDAC.

scholarly journals IMMU-18. IMMUNOGENOMIC RESPONDER PHENOTYPE FROM A PHASE I TRIAL OF ANTI-LAG3 OR ANTI-CD137 ALONE AND IN COMBINATION WITH ANTI-PD-1 IN PATIENTS WITH RECURRENT GBM

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi122-vi123
Author(s):  
Christina Jackson ◽  
John Choi ◽  
JiaJia Zhang ◽  
Anna Piotrowski ◽  
Tobias Walbert ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Checkpoint Inhibitors ◽  
Lower Percentage ◽  
Radiographic Evaluation ◽  
Peripheral Blood Mononuclear ◽  
Initial Resection

Abstract BACKGROUND Immune checkpoint inhibitors (ICIs) are not uniformly effective in glioblastoma treatment. Immunogenomic determinants may identify patients who are most likely to benefit from these therapies. Therefore, we compared the immunogenomic phenotype of a responder to combination anti-LAG-3 and anti-PD-1 therapy to non-responders. METHODS We performed T cell receptor (TCR) sequencing and gene expression analysis on pre-treatment, post-chemoradiation, and post-immunotherapy tumor specimens of glioblastoma patients treated with anti-LAG3 in combination with anti-PD-1 after first recurrence (NCT02658981, ongoing). We evaluated T cell clonotypes and immunophenotype of serially collected peripheral blood mononuclear cells (PBMCs) during treatment using multi-parametric flow cytometry. RESULTS To date, six patients have been enrolled in the initial anti-LAG-3 and anti-PD-1 cohort. One patient demonstrated complete response, one had stable disease, and four had progressive disease by radiographic evaluation. The responder demonstrated substantially higher TCR clonality in the resected tumor at initial diagnosis compared to non-responders (mean 0.028 vs. 0.005). Shared tumor infiltrating clonotypes with pre-immunotherapy PBMCs exhibited an increase in frequency from initial resection (6.8%) to resection at recurrence (20%). The responder’s tumor at initial resection exhibited increased gene signatures of PD1low CD8+ T cells, chemokine signaling, and interferon gamma pathways. On PBMC phenotypic analysis, the responder demonstrated significantly higher percentages of CD137+ CD8+T cells (median 8.38% vs 3.24%, p=0.02) and lower percentages of Foxp3+CD137+ CD4+T cells compared to non-responders (median 18.5% vs. 38.5%, p=0.006). Interestingly, dynamic analysis of PBMCs showed that the responder demonstrated a lower percentage of PD1+ CD8+ T cells pre-immunotherapy (median 2.5% vs.12.4%, p=0.002), with persistent decrease over the course of treatment while non-responders showed no consistent pattern. CONCLUSION Our preliminary results demonstrate significant differences in tumor and peripheral blood immunogenomic characteristics between responder and non-responders to anti-LAG3 and anti-PD-1 therapy. These immunogenomic characteristics may help stratify patients’ response to combination ICIs.

scholarly journals Differential effects of IL-21 and IL-15 on perforin expression, lysosomal degranulation, and proliferation in CD8 T cells of patients with human immunodeficiency virus-1 (HIV)

Blood ◽  
2006 ◽  
Vol 109 (9) ◽  
pp. 3873-3880 ◽  
Author(s):  
Lesley White ◽  
Subramaniam Krishnan ◽  
Natasa Strbo ◽  
Huanliang Liu ◽  
Michael A. Kolber ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Highly Active Antiretroviral Therapy ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Cell Receptor ◽  
Peripheral Blood Mononuclear ◽  
Perforin Expression

Abstract An urgent need exists to devise strategies to augment antiviral immune responses in patients with HIV who are virologically well controlled and immunologically stable on highly active antiretroviral therapy (HAART). The objective of this study was to compare the immunomodulatory effects of the cytokines interleukin (IL)–21 with IL-15 on CD8 T cells in patients with HIV RNA of less than 50 copies/mL and CD4 counts greater than 200 cells/mm.3 Patient CD8 T cells displayed skewed maturation and decreased perforin expression compared with healthy controls. Culture of freshly isolated patient peripheral-blood mononuclear cells (PBMCs) for 5 hours to 5 days with IL-21 resulted in up-regulation of perforin in CD8 T cells, including memory and effector subsets and virus-specific T cells. IL-21 did not induce T-cell activation or proliferation, nor did it augment T-cell receptor (TCR)–induced degranulation. Treatment of patient PBMCs with IL-15 resulted in induction of perforin in association with lymphocyte proliferation and augmentation of TCR-induced degranulation. Patient CD8 T cells were more responsive to cytokine effects than the cells of healthy volunteers. We conclude that CD8 T cells of patients with HIV can be modulated by IL-21 to increase perforin expression without undergoing overt cellular activation. IL-21 could potentially be useful for its perforin-enhancing properties in anti-HIV immunotherapy.

Molecular Transfer of Human CD40 and OX40 Ligands to Leukemic Human B-CLL Cells Extensively Expands Tumor-Reactive Cytotoxic T-Lymphocytes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 951-951
Author(s):  
Ettore Biagi ◽  
Giampietro Dotti ◽  
Eric Yvon ◽  
Raphael Rousseau ◽  
Edward Lee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Fold Increase ◽  
Mean Value ◽  
Lymphocytic Leukemia ◽  
Molecular Transfer

Abstract CD40 ligand is an accessory signal for T-cell activation and can overcome T-cell anergy. The OX40-OX40 ligand pathway is involved in the subsequent expansion of memory T cells. We expressed both human CD40L and OX40L on B-Chronic Lymphocytic Leukemia (B-CLL) cells, by exploiting the phenomenon of molecular transfer from fibroblasts engineered to over-express both of these TNF-receptor superfamily members. We analyzed the effects of the modified B-CLL cells on the number, phenotype and cytotoxic function of autologous T cells in seven B-CLL patients. Transfer of CD40L and OX40L to B-CLL cells was observed in all patients (mean value from 1% pre to 76% post for CD40L; from 0.7% pre to 88% post for OX40L). Subsequent up-regulation of the costimulatory molecules CD80 (B7-1) and CD86 (B7-2) was obtained after engagement of the endogenous CD40 receptor on B-CLL by the transferred CD40L molecules (mean value from 8% pre to 64% post for CD80; from 36% pre to 95% post for CD86). Co-culture of modified and unmodified B-CLL cells with autologous T cells revealed profound differences in the immune responses they induced. With unmodified B-CLL cells, or cells expressing either CD40L or OX40L individually, less than a 10-fold expansion of autologous T cells was observed, with a <100-fold expansion in tumor reactive T cells (measured by IFN-gamma Elispot with autologous B-CLL cells as stimulators, and allogeneic B-CLL cells as controls). By contrast, co-culture with B-CLL cells expressing both CD40L and OX40L induced a >40 fold expansion of autologous T cells - including both CD8+ T cells and CD4+ T cells with a Th1 pattern of cytokine release - and a >2500-fold increase in leukemia-reactive T cells. These expanded T cells were also directly cytotoxic to B-CLL targets, producing a mean 48% B-CLL killing at an E:T ratio of 10:1. A proportion of these tumor-reactive CD8+ T cells were specific for survivin, a B-CLL associated tumor antigen. Hence the combination of CD40L and OX40L expression by B-CLL cells allows generation of potent immune responses to B-CLL, which may be exploitable either by using active immunization with CD40L/OX40L-modified tumor cells or by adoptive immunotherapy with CD40L/OX40L generated tumor-reactive T cells.

AAV-2 Capsid-Specific CD8+ T Cells Limit the Duration of Gene Therapy in Humans and Cross-React with AAV-8 Capsid.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 455-455 ◽  
Author(s):  
Federico Mingozzi ◽  
Marcela V. Maus ◽  
Denise E. Sabatino ◽  
Daniel J. Hui ◽  
John E.J. Rasko ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Cell Population ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Transgene Expression ◽  
Cd8 T Cell ◽  
Target Cells

Abstract Efforts to establish an adeno-associated viral (AAV) vector-mediated gene therapy for the treatment of hemophilia B have been hindered by an immune response to the viral capsid antigen. Preclinical studies in small and large animal models of the disease showed long-term factor IX (F.IX) transgene expression and correction of the phenotype. However, in a recent phase I/II clinical trial in humans (Manno et al., Nat. Med. 2006), after hepatic gene transfer with an AAV-2 vector expressing human F.IX transgene, expression lasted for only a few weeks, declining to baseline concurrently with a peak in liver enzymes. We hypothesized that T cells directed towards AAV capsid antigens displayed by transduced hepatocytes were activated and these mediated destruction of the transduced hepatocytes, thereby causing loss of transgene expression and a transient transaminitis. Peripheral blood mononuclear cells isolated from AAV-infused subjects were stained with an AAV capsid-specific MHC class I pentamer either directly or after in vitro expansion. Two weeks after vector infusion 0.14% of circulating CD8+ T cells were capsid-specific on direct staining, and five weeks after infusion the capsid-specific population had expanded to 0.5% of the circulating CD8+ T cells, indicating proliferation of this T cell subset. By 20 weeks after vector infusion, the capsid-specific CD8+ T cell population had contracted to the level seen at 2 weeks. The expansion and contraction of this capsid-specific CD8+ T cell population paralleled the rise and fall of serum transaminases in the subject observed. Subsequent ex vivo studies of PBMC showed the presence of a readily expandable pool of capsid-specific CD8+ T cells up to 2.5 years post vector-infusion. Similarly, we were able to expand AAV-specific CD8+ T cells from peripheral blood of normal donors, suggesting the existence of a T cell memory pool. Expanded CD8+ T cells were functional as evidenced by specific lysis of HLA-matched target cells and by IFN-γsecretion in response to AAV epitopes. It has been argued that potentially harmful immune responses could be avoided by switching AAV serotypes, however, capsid protein sequences are highly conserved among different serotypes, as are some immunodominant epitopes that we identified. Indeed, we demonstrated that capsid-specific CD8+ T cells from AAV-infused hemophilic subjects functionally cross-react with AAV-8. Moreover, cells expanded from normal donors with AAV-2 vector capsids proliferated upon culture with AAV-8 capsids, demonstrating that both vectors could be processed appropriately in vitro to present the epitopic peptide to capsid-specific T cells. This suggests that AAV-2-specific memory CD8+ T cells normally present in humans likely would expand upon exposure to AAV-8 capsid epitopes. We conclude that the use of immunomodulatory therapy may be a better approach to achieving durable transgene expression in the setting of AAV-mediated gene therapy.

Addition of Clofarabine to TLI/ATG Conditioning: Impact on Immune Reconstitution and Clinical Outcomes,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4066-4066
Author(s):  
Brett Glotzbecker ◽  
Heidi Mills ◽  
Jacalyn Rosenblatt ◽  
Robin Joyce ◽  
James Levine ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Median Time ◽  
Cd8 T Cells ◽  
Fold Increase ◽  
Disease Relapse ◽  
Platelet Recovery ◽  
Post Transplant ◽  
T Cell Reconstitution

Abstract Abstract 4066 The fundamental challenge in designing an effective conditioning regimen for allogeneic transplantation involves the prevention of disease relapse while minimizing the risk for Graft versus Host Disease (GVHD). Treatment with total lymphocyte irradiation (TLI) and anti-thymocyte globulin (ATG) has been shown to minimize the risk of GVHD through the biasing of the T cell reconstitution towards an inhibitory phenotype. However, disease relapse remains a significant concern. Clofarabine is a second generation nucleoside analog with potent cytoreductive capacity and demonstrates efficacy in hematological malignancies. In this study, we examined the combination of clofarabine, TLI and ATG with respect to T cell reconstitution, risk for GVHD and transplant outcome. Sequential cohorts of 5 patients were treated with TLI and ATG alone or in conjunction with 20 mg/m2, 30 mg/m2 or 40 mg/m2 of clofarabine for 5 days. Cyclosporine and mycophenolate mofetil were administered as GVHD prophylaxis. Twenty patients have been enrolled (5 AML/MDS, 2 ALL, 6 lymphoma, 2 CLL, 5 myeloma) and received HLA matched peripheral blood stem cells collected from related (N=11) and unrelated donors (N=9). Of 19 evaluable patients, 15 are alive with a median follow up of 665 days. Day 30 and 100 mortality was 0% for TLI and ATG and 0% and 10% for those receiving clofarabine. The maximum tolerated dose (MTD) of clofarabine was 30 mg/m2 as 2 patients experienced treatment related mortality at the 40 mg/m2 dose level. Grade 5 infections and multiorgan failure occurred in both patients. All patients demonstrated engraftment with mean bone marrow donor chimerism of 92.5% at Day 30. The first cohort's ANC did not drop below 500 cells/uL, while median time to neutrophil engraftment in the patients who received clofarabine was 9 days. The median time to platelet recovery was 11 and 12 days for patients receiving TLI and ATG alone or with clofarabine, respectively (p=0.39). T cell reconstitution studies demonstrated a significant decrease in CD4+ cells to (<200 cells/uL) persisting for more than 6 months and a more than a two fold increase in circulating CD56+ NK cells. No significant decrease in CD8 T cells in the early post-transplant period was seen in either group. The mean percentage of regulatory T cells (CD4+/25+/FoxP3+) rose in the early post-transplant period following TLI and ATG (5.5 to 14.2% from baseline to day 30; p=0.015), but not in those receiving clofarabine (8.1 to 6%; p=0.15). Assessment of T cell polarization at these time points demonstrated a two fold increase in CD8+ T cells expressing IL-4 at Day 30 in patients receiving TLI and ATG alone (p=0.04); but not following clofarabine containing conditioning. Consistent with these findings, the incidence of grade II-IV GVHD was 0% and 42% in those receiving TLI and ATG alone or in conjunction with clofarabine, respectively. cGVHD was seen in 20% and 42% of patients, respectively. In contrast, disease progression was seen in 60% of patients receiving TLI and ATG alone as compared to 27% receiving clofarabine, TLI, and ATG. In summary, the addition of clofarabine to TLI and ATG conditioning resulted in a decrease in circulating regulatory T cells, decreased CD8+ T cell expression of IL-4, and was associated with an increased risk of GVHD and a potential for a decrease in the risk of relapse. Disclosures: Chen: Genzyme: Membership on an entity's Board of Directors or advisory committees. Avigan:Genzyme: Research Funding.

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