Bone Marrow-derived CD8+ T Cells From Pediatric Leukemia Patients Express PD1 and Expand Ex Vivo Following Induction Chemotherapy

Abstract There is currently significant interest in the transplant field to develop adoptive-transfer strategies utilizing T cell populations to provide immediate immune function as well as long-term immune reconstitution following hematopoietic cell transplantation (HCT). Presumably, these pre-selected T cell populations could then be further expanded in the transplant recipient as a consequence of lymphopenia-induced proliferation. However, clinical application of adoptive transfer strategies has been limited by practical (time, expense) and technical (isolation and expansion of antigen-specific T cell populations) difficulties, hence more efficient approaches need to be identified. Recent reports have demonstrated the feasibility for the rapid ex vivo generation of transgenic memory CD8 populations. We investigated the potential of applying this ex vivo approach to generate and expand an immunodominant antigen-specific memory population from primary CD8 T cells. CD8 cells recognizing the mouse H60 epitope were selected as the antigen-specific CD8 population. The H60 glycoprotein is the ligand for NKG2D and the LTFNYRNL peptide is an immunodominant minor transplantation antigen. H60 is expressed by BALB.B (H2b) hematopoietic cells and recognized by C57BL/6 (B6) CD8 cells within the context of the H2Kb molecule. CD8 T cells from normal B6 spleens were positively selected using Miltenyi beads. The purified CD8 cells (97%) were then cultured with bone marrow-derived B6 DC, rmIL-2, and H60 peptide (1μM) for 3 days. Cells were harvested and re-cultured with rmIL-15 for 2–4 days. The resultant CD8 population was enriched 10 fold for tetramer-stained H60+ CD8 T cells (average: 3.0% of CD8 T cells). The H60+ CD8 cells displayed a memory phenotype as characterized by CD44+, Ly6C+, CD62Lintermed, and CD25lo expression. We hypothesized these H60+ CD8 T cells could be further expanded in transplant recipients by lymphopenia-induced proliferation. To determine the expansion and persistence of H60+ TM post-HCT, H60+-enriched CD8 cells were co-transplanted with T cell-depleted B6 bone marrow into 9.0Gy-conditioned syngeneic recipients. The phenotype and number of H60+ cells in recipient spleens and bone marrow were assessed beginning 3 days post-HCT. Notably, the H60+ CD8 cells maintained their memory phenotype and persisted at least 2 months post-transplant. The ex vivo-generated H60+ TM underwent a relative expansion of 1.5–2 fold as assessed in recipient spleens, similar to the post-transplant expansion of H60+ CD8 TM derived in vivo from B6 mice primed to BALB.B cells. Moreover, this post-HCT expansion was also similar to that by an ex vivo-generated, transgenic CD8 TM population. Both (ex vivo and in vivo generated) H60+ TM populations also exhibited expansion (1.5–2 fold) in the bone marrow. In total, an immunodominant antigen-specific CD8 TM population was selectively generated and enriched ex vivo and found to undergo expansion following transplant into ablatively conditioned HCT recipients. The similarities in expansion and persistence between ex vivo generated H60 and in vivo primed H60 populations suggest this approach may have useful applications towards the development of successful adoptive transfer strategies.

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Blocking VIP Signaling during Allogeneic Bone Marrow Transplantation Separates the Graft Versus Leukemia from Graft Versus Host Disease Activity of Donor CD8 T-Cells

Blood ◽

10.1182/blood.v126.23.1877.1877 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1877-1877

Author(s):

Jian-Ming Li ◽

Christopher T Petersen ◽

Jingxia Li ◽

Cynthia R. Giver ◽

Bruce R. Blazar ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Cd8 T Cells ◽

Ex Vivo ◽

Allogeneic Bone Marrow Transplantation ◽

Tumor Burden ◽

Cytolytic Activity ◽

Allogeneic Bone ◽

Donor Cells ◽

Donor T Cells

Abstract Background: Separating graft versus leukemia (GvL) from graft versus host disease (GvHD) is a critical issue in allogeneic bone marrow transplantation (allo BMT) for malignant hematopoietic cancers. Vasoactive intestinal peptide (VIP) is capable of inducing tolerogenic dendritic cells (DC) from bone marrow progenitors that limit GvHD (Blood 2006 107:3787). We have published that use of donor mice deficient in VIP or pharmacological blockade of VIP-signaling both increased cellular anti-viral activity in murine allo BMT models of CMV infection (JI 2011 187:1057, Blood 2013 121:2347). We hypothesized that blockade of VIP could enhance GvL activity without increasing GvHD in recipients of allo BMT. Methods: We used the B10BR to B6 and B6 to B10BR murine allo BMT models, with luciferase-expressing C1498 acute myeloid leukemia and LBRM acute T-lymphoblastic lymphoma tumor cells, respectively, to study the effect of VIP-signaling on GvL and GvHD. Murine recipients of WT TCD-BM plus splenic allografts (5 x106 TCD-BM plus 0, 0.5, 1.0 and 3.0 x106 splenocytes) were treated with 7 daily s.c. injections of VIPhyb, a VIP antagonist, on d-1 to d+5 (early treatment) or d+6 to d+12 (late treatment), or PBS. Tumor cells were administered by i.v. injection one day before transplant (d-1), and tumor burden was monitored by bioluminescence imaging (BLI). Expression of activation immune cell markers, intracellular cytokines, and ex vivo cytolytic activity against tumor were measured by flow cytometry. TCR-β clono-types were analyzed by TCR-β CDR3 deep sequencing. Results: Recipients of VIP-KO donor cells and VIPhyb-treated recipients of WT donor cells did not have more GvHD than untreated recipients of equivalent numbers of splenocytes from WT donors. VIPhyb-treatment significantly increased survival of transplant tumor recipients in the B10.BR to B6 model with C1498 (2 replicate experiments, n= 10 in VIPhyb early-treatment, p<0.001; n=15 in VIPhyb late-treatment, p=0.005 vs. control, n=14; Figure 1A) and in the B6 to B10.BR model with LBRM (3 replicate experiments in 0.5 x 106 splenocyte dose, n=10, p =0.001 in VIP-KO, n=20, p=0.01 in VIPhyb-treatment, n=40 in control; Figure 1B). All recipients of TCD-BM alone died of tumors within 60 days post-BMT. VIPhyb-treatment, and transplantation of VIP-KO donor cells, led to significant decreases in tumor-burden as measured by BLI. Tumor-free recipients following VIPhyb treatment re-challenged with a 10x dose of LBRM had 100% survival compared with 40% survival among saline-treated recipients (p=0.009). Secondary transplantation of splenocytes from tumor-free VIPhyb-treated recipients resulted in 100% tumor-free survival compared with 30% survival among recipients of naive donor T cells (p=0.001). Splenic T cells, and cDC and pDC from VIPhyb-treated mice expressed lower levels of PD-1, and PD-L1, respectively, compared with saline-treated control (Figure 2). Donor T cells from leukemia-bearing mice treated with VIPhyb expressed higher-levels of ICOS and IFN-γ compared with T cells from saline-treated mice. Both cDC and pDC from VIPhyb-treated mice had increased expression of ICOS-L and MHC-II. Donor T-cells recovered from mice treated with VIPhyb had increased levels of granzyme B and enhanced cytolytic activity against tumor targets but not against non-tumor recipient-type splenocytes or third party splenocytes. Genetic or ex vivo depletion of donor NK cells or CD8+ T-cells abrogated the enhanced GvL activity seen with VIP antagonist-treatment. VIPhyb had no direct anti-proliferative activity against LBRM in vitro using a range of concentrations that exceeded predicted in vivo concentration. VIPhyb-treated transplant recipients had expansion of oligo-clonal T-cell subsets that conferred leukemia protection to secondary recipients and expressed distinct TCR-β sequences compared with T cells from donor or GvHD mice. Conclusions: Blocking VIP signaling enhanced GvL activity without increasing GvHD. Donor CD8+ T-cells and NK cells were indispensable for the enhanced GvL activity. The ability of VIP antagonist treatment to down-regulate PD-1/PD-L1 signaling and enhance adaptive cellular immunity is a novel approach to treat and prevent relapse in allo BMT. Figure 1. VIPhyb-treatment enhanced the GvL activity (5 x 106 TCD BM and 0.5 x 106 splenocytes from WT or VIP-KO donors) Figure 1. VIPhyb-treatment enhanced the GvL activity (5 x 106 TCD BM and 0.5 x 106 splenocytes from WT or VIP-KO donors) Figure 2. VIPhyb-treatment reduced expression of PD-1 on T cells and PD-L1 on DC Figure 2. VIPhyb-treatment reduced expression of PD-1 on T cells and PD-L1 on DC Disclosures No relevant conflicts of interest to declare.

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High Frequencies of Anti-Host Reactive CD8+ T Cells Ignore Non-Hematopoietic Antigen after Bone Marrow Transplantation in a Murine Model

Cellular Physiology and Biochemistry ◽

10.1159/000443078 ◽

2016 ◽

Vol 38 (4) ◽

pp. 1343-1353 ◽

Cited By ~ 1

Author(s):

Asmae Gassa ◽

Halime Kalkavan ◽

Fu Jian ◽

Vikas Duhan ◽

Vishal Khairnar ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Bone Marrow Transplantation ◽

Cd8 T Cells ◽

Marrow Transplantation ◽

Ex Vivo ◽

Cell Damage ◽

Lymphocytic Choriomeningitis ◽

Host Cells ◽

Transgenic Mouse Line

Background: Graft versus host disease (GvHD) occurs in 20% of cases with patients having an MHC I matched bone marrow transplantation (BMT). Mechanisms causing this disease remain to be studied. Methods: Here we used a CD8+ T cell transgenic mouse line (P14/CD45.1+) and transgenic DEE mice bearing ubiquitously the glycoprotein 33-41 (GP33) antigen derived from the major lymphocytic choriomeningitis virus (LCMV) epitope to study mechanisms of tolerance in anti-host reactive CD8+ T cells after BMT. Results: We found that anti-host reactive CD8+ T cells (P14 T cells) were not negatively selected in the thymus and that they were present in wild type (WT) recipient mice as well as in DEE recipient mice. Anti-host reactive CD8+ T cells ignored the GP33 antigen expressed ubiquitously by host cells but they could be activated ex vivo via LCMV-infection. Lipopolysaccharides (LPS) induced transient cell damage in DEE mice bearing anti-host reactive CD8+ T cells after BMT, suggesting that induction of host inflammatory response could break antigen ignorance. Introducing the GP33 antigen into BM cells led to deletion of anti-host reactive CD8+ T cells. Conclusion: We found that after BMT anti-host reactive CD8+ T cells ignored host antigen in recipients and that they were only deleted when host antigen was present in hematopoietic cells. Moreover, LPS-induced immune activation contributed to induction of alloreactivity of anti-host reactive CD8+ T cells after BMT.

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Bone Marrow T Cells Are Driven into Exhaustion By Acute Leukemia in Pediatric Patients Based on Protein and Transcriptome Analysis

Blood ◽

10.1182/blood-2018-99-112811 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3722-3722

Author(s):

Semjon Willier ◽

Paula Rothaemel ◽

Jonas Wilhelm ◽

Dana Stenger ◽

Theresa Kaeuferle ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Acute Leukemia ◽

Cd8 T Cells ◽

Pediatric Patients ◽

Healthy Controls ◽

Rna Seq ◽

Pediatric Leukemia ◽

Healthy Donors ◽

Significant Difference

Abstract Introduction: Acute leukemia is the most common malignancy in children and develops within the bone marrow. Consequently, bone marrow derived T cells of leukemia patients can be defined as tumor infiltrating lymphocytes (TILs). Dysfunctional TILs have been described in several other malignancies. However, in pediatric patients the interaction between leukemic blasts and TILs remains largely unknown. In order to understand the impact of leukemic blasts on bone marrow T cells we profiled T cells in the bone marrow of pediatric leukemia patients by surface marker and transcriptome wide analysis. Methods: First, artificial changes in marker expression due to cryopreservation and thawing were ruled out (n=5). Then, cryopreserved bone marrow samples from both pediatric patients with acute leukemia (n= 77; BCP-ALL: 18, TCP-ALL: 23, AML: 36) and age-matched healthy bone marrow donors (HD, n=23) were identified in a local biobank. Multicolor flow cytometry was performed to quantify co-inhibitory markers on CD4 and CD8 T cells in primary (n=49) and relapse leukemia samples (n=28). As we could not detect surface CTLA4 expression on T cells, CTLA4 was stained intracellularly. Additionally, RNA-Seq on sorted bone marrow derived CD8 T cells (n=48; TCP-ALL: 12, AML: 20, HD: 16) was performed. Analysis of RNA-Seq data was based on Reads Per Kilobase Million (RPKM) normalization and False Discovery Rate (FDR, Benjamini-Hochberg) statistics. 172 differentially expressed genes were found when comparing bone marrow derived CD8 T cells from healthy donors (n=16) and leukemia patients (n=32) using the following criteria: RPKM>2 in both groups, fold change>2 and FDR<0.05). Results: The frequency of bone marrow T cells was reduced in patients with acute leukemia in comparison with healthy controls (5.9% vs. 24.4%, mean values, p<0.001). This reduction was more pronounced in BCP-ALL than in AML (0.9% vs. 8.4%, p<0.001). LAG3 and CTLA4 protein expression of T cells was increased in leukemia patients vs. healthy controls (LAG3: CD4: 2.6% vs. 0.7%, p<0.001; CD8: 8.6% vs. 2.2%, p<0.001; CTLA4: CD4: 7.3% vs. 3.8%, p=0.001; CD8: 1.2 vs. 0.3%, p<0.001). For CD8 T cells, those findings could be confirmed by RNA-Seq of sorted CD8 T cells (LAG3: 60.4 vs. 23.3 (RPKM), FDR=0.0044; CTLA4: 28.7 vs 4.7 (RPKM), FDR=0.046). Equally, TIM3 on T cells showed higher expression in leukemia patients vs. healthy controls (CD4: 3.7% vs. 1.3%, p=0.002; CD8: 8.5% vs. 3.3%, p<0.001). However, the same analysis of RNA-Seq data on sorted CD8 T cells did not yield a significant difference (18.1 vs. 5.6 (RPKM), FDR=0.29). PD1 was the only surface marker found to be more highly expressed in relapse samples than in primary diagnosis samples than in healthy controls (CD4: 42.3% vs. 28.9% vs. 19.8%, p<0.001; CD8: 45.2% vs. 33.3% vs. 26.5%, p=0.002). For CD8 T cells, RNA-Seq did not recapitulate this finding as no significant difference of PD1 transcript abundancy could be observed between leukemia patients and healthy donors by RNA-Seq (21.4 vs. 16.9 (RPKM), FDR=0.92). Finally, RNA-Seq on sorted CD8 T cells showed a pronounced overexpression of genes that are involved in the cytotoxic granule machinery in leukemia patients indicating an increase of effector phenotype in those cells. Contrarily, genes crucial for T cell function and memory formation were significantly downregulated in CD8 T cells from leukemia patients. Conclusion: By analyzing bone marrow samples from pediatric leukemia patients and healthy controls we confirm that bone marrow T cells of leukemia patients show signs of exhaustion compared to healthy individuals. Importantly, PD1 surface expression on T cells was identified as a marker that correlates with disease status (relapse > primary > healthy). A significant increase of exhaustion markers could be demonstrated both on protein and transcriptome level (LAG3, CTLA4) or on protein level only (TIM3, PD1). Moreover, we observed an increase of many elements of the cytotoxic granule machinery which is compatible with a loss of naïve/memory CD8 T cells. Additionally, genes essential for T cell memory formation were found to be downregulated in CD8 T cells from leukemia patients. These findings reflect an insufficient immune surveillance of pediatric leukemia by bone marrow T cells and may provide a rationale for future therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

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Clinical Trial Evaluating DC/AML Fusion Cell Vaccination In AML Patients

Blood ◽

10.1182/blood.v122.21.3928.3928 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3928-3928 ◽

Cited By ~ 6

Author(s):

Jacalyn Rosenblatt ◽

Richard M. Stone ◽

Lynne Uhl ◽

Donna S. Neuberg ◽

Baldev Vasir ◽

...

Keyword(s):

Clinical Trial ◽

Bone Marrow ◽

T Cells ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Ex Vivo ◽

Fold Increase ◽

Vaccine Site ◽

Fusion Cell ◽

The Mean

Abstract We have developed a promising leukemia vaccine in which patient derived AML cells are fused with autologous dendritic cells (DCs), presenting a broad array of antigens. We are conducting a clinical trial in which AML patients who are not candidates for allogeneic transplantation undergo vaccination with DC/AML fusion cells following chemotherapy induced remission. Twenty-six patients (14 males, 12 females) underwent collection of AML cells at disease presentation for vaccine generation and immune monitoring studies. Median age of the patients is 66 years. Tumor was collected from either a bone marrow aspirate (N=16), 20 cc of peripheral blood (N=7), or leukapheresis product (N=3) at the time of presentation with newly diagnosed AML (N=25) or first relapsed AML (N=1). The mean yield of AML cells was 109 x 106 cells with a mean viability of 91%. Eligible patients achieving CR following chemotherapy (N=16) underwent leukapheresis for DC generation and vaccine preparation. Adherent peripheral blood mononuclear cells were isolated, cultured in the presence of GM-CSF and IL-4 for 5-7 days, and exposed to TNFα for 48-72 hours to generate mature DCs. The mean yield of DCs was 177 x106 cells with a mean viability of 89%. Fusion cells were generated by co-culture of DCs with AML cells in the presence of 50% polyethylene glycol and identified as cells co-expressing antigens that were unique to the DC and tumor population. Mean fusion efficiency and viability was 38% and 85%, respectively. As a measure of their activity as antigen presenting cells, the capacity of fusion cells to stimulate allogeneic T cell proliferation ex vivo was quantified. In contrast to the leukemia preparation (mean stimulation index (SI) 3.81), the DC and fusion cell preparation were potent stimulators (mean SI 19.61 and 13.48, respectively). Vaccination with DC/leukemia fusion cells was initiated within 12 weeks from count recovery following the final cycle of chemotherapy. 13 patients received at least two monthly vaccinations at a dose of 5x106 fusion cells. 8 patients had intermediate risk cytogenetics, 3 patients had good risk cytogenetics, and 2 patients had a complex karyotype. Vaccination was well tolerated, and importantly, was not associated with clinically significant auto-immunity. Possibly related adverse events were transient and of grade 1-2 intensity, including vaccine site reactions, pruritis, arthalgias, myalgias, eosinophilia, leukopenia, thrombocytopenia. Biopsy of vaccine site reactions demonstrated a dense infiltrate of CD4 and CD8 T cells consistent with recruitment of reactive T cell populations to the vaccine bed. To date, 9 patients remain in remission (69%), with a mean follow up of 23 months. Peripheral blood samples were collected prior to each vaccination and at 1, 3, and 6 months following completion of vaccination. Vaccination resulted in the potent induction of leukemia specific immunity as measured by an increase in CD8 T cells expressing IFNγ in response to ex vivo exposure to autologous leukemia cell lysates (mean fold increase 8, n=6). Bone marrow derived T cells were isolated prior to and following vaccination in patients who are HLA2.1+. Vaccination resulted in the expansion of bone marrow infiltrating T cells recognizing MUC1 (9 fold increase), WT1 (5 fold increase), PRAME (12 fold increase) tumor antigens by tetramer analysis (n=2). In conclusion, DC/AML fusion cell vaccination results in the potent expansion of leukemia reactive T cells and durable remissions following chemotherapy. Enrollment to a second cohort is being initiated, in which patients with be treated with DC/AML fusion cell vaccination in conjunction with PD1 blockade. Disclosures: No relevant conflicts of interest to declare.

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Ex Vivo Evidence for the Combination of Immune Checkpoint Inhibition with TGF-β Blockade to Enhance Anti-Tumor T Cell Responses in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-117816 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3198-3198

Author(s):

Yoon Seok Choi ◽

Minsuk Kwon ◽

Ik-Chan Song ◽

Deog-Yeon Jo ◽

Eui-Cheol Shin

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Immune Checkpoint ◽

Plasma Cells ◽

Ex Vivo ◽

Immune Checkpoint Blockade ◽

High Level

Abstract Immunosuppressive milieu of multiple myeloma is associated with various cellular and non-cellular factors that foster immune escape leading to tumor progression. While immune checkpoint inhibitors have achieved significant clinical success in many types of solid tumors, recent clinical trials of immune checkpoint blockade performed in patients with multiple myeloma failed to demonstrate significant anti-tumor efficacy. To enhance the clinical efficacy of immune checkpoint blockade in multiple myeloma, elaborate characterization of tumor antigen-specific T cells is an essential prerequisite. In the present study, we investigated the immunophenotypic and functional characteristics of tumor antigen-specific T cells in patients with multiple myeloma. In addition, using direct ex vivo experimental techniques, we tried to examine how to manipulate the immunosuppressive microenvironment to maximize anti-myeloma responses of the tumor-specific T cells. We first tried to define and characterize CD8+ T cell population capable of specifically recognizing and reacting to myeloma cells in bone marrow of myeloma patients using MHC multimer technique. In selected patients, we could successfully define CD8+ T cell population specifically recognizing the HLA-A*0201-restricted epitopes (either "SLLMWITQC" or "LLLGIGILV"), included in myeloma tumor antigens NY-ESO-1 and HM1.24. The vast majority of myeloma-antigen specific CD8+ T cells expressed high level of PD-1 and also co-expressed other types T cell inhibitory receptors. More strikingly, PD-1+ myeloma-specific CD8+ T cells had a distinct pattern of transcriptional factor expression, high level of Eomes and low level of T-bet (EomeshiT-betlo), indicating that they were profoundly exhausted functionally and that a simple blockade of PD-1/PD-L1 binding might not be enough to reinvigorate their anti-myeloma activity. Consistent with the immunophenotypes of myeloma-specific CD8+ T cells, malignant plasma cells in bone marrow of myeloma patients, defined as CD14-CD19-CD138+CS1+CD56hi, also expressed PD-L1 abundantly, compared to normal plasma cells, suggesting that PD-1/PD-L1 axis plays a major role in making myeloma-recognizing T cells unresponsive to TCR stimulation. Interestingly, in addition to tumor cells, various types of immune cells comprising myeloma microenvironment also highly express PD-L1. Indeed, in response to ex vivo TCR stimulation with anti-CD3, CD8+ T cells from myeloma bone marrow showed lower proliferation and reduced production of anti-tumor effector cytokines (INF-γ and TNF-α), compared to marrow-infiltrating CD8+ T cells of diffuse large B cell lymphoma and Hodgkin lymphoma patients with extensive bone marrow involvement. However, even in the presence of anti-PD-1, myeloma-specific responses of marrow-infiltrating CD8+ T cells was only modestly improved in terms of proliferation and cytokine production. Next, we investigated whether blocking TGF-β signaling in combination with PD-1/PD-L1 axis blockade could restore the function of marrow-infiltrating CD8+ T cells of myeloma patients, since TGF-β produced by clonal plasma cells and bone marrow stromal cells is critical in immunosuppressive microenvironment of multiple myeloma. To this end, we found that combination of TGF-β signaling blockade (either anti-TGF-β1 or Galunisertib, a small molecule inhibitor of TGF-β receptor I) with anti-PD-1 significantly increased the frequencies of IFN-γ- and/or TNF-α-producing CD8+ T cells in response to ex vivo TCR stimulation, compared to a single PD-1 or a single TGF-β blockade. Likewise, myeloma antigen-specific proliferation of CD8+ T cells was significantly enhanced with addition of TGF-β signaling blockade. Taken together, although PD-1/PD-L1 axis acts as a major component of immunosuppressive milieu in multiple myeloma, the efficacy of PD-1 blockades in multiple myeloma might be hampered by complicated microenvironment consisting of T cell-intrinsic and -extrinsic factors. Our results provide an ex vivo evidence of incorporating TGF- β signaling blockade to immune checkpoint inhibition to enhance anti-tumor T cell responses in multiple myeloma Disclosures No relevant conflicts of interest to declare.

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Faculty Opinions recommendation of CD8 T-cells from most HIV-infected patients lack ex vivo HIV-suppressive capacity during acute and early infection.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717996871.793475361 ◽

2013 ◽

Author(s):

Susana Asin ◽

Christiane Rollenhagen

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Ex Vivo ◽

Early Infection ◽

Suppressive Capacity

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

Blood Advances ◽

10.1182/bloodadvances.2019001091 ◽

2020 ◽

Vol 4 (10) ◽

pp. 2143-2157 ◽

Cited By ~ 1

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.

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602 STING agonist-based treatment promotes vascular normalization and tertiary lymphoid structure formation in the therapeutic melanoma microenvironment

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0602 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A637-A637

Author(s):

Manoj Chelvanambi ◽

Ronald Fecek ◽

Jennifer Taylor ◽

Walter Storkus

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Immune Cell ◽

Ex Vivo ◽

In Vitro Assays ◽

Type I ◽

Vascular Normalization ◽

S 100 ◽

Transcriptional Changes

BackgroundThe degree of immune infiltration in tumors, especially CD8+ T cells, greatly impacts patient disease course and response to interventional immunotherapy. Hence, enhancement of TIL prevalence is a preferred clinical endpoint, one that may be achieved via administration of agents that normalize the tumor vasculature (VN) leading to improved immune cell recruitment and/or that induce the development of local tertiary lymphoid structures (TLS) within the tumor microenvironment (TME).MethodsLow-dose STING agonist ADU S-100 (5 μg/mouse) was delivered intratumorally to established s.c. B16.F10 melanomas on days 10, 14 and 17 post-tumor inoculation under an IACUC-approved protocol. Treated and control, untreated tumors were isolated at various time points to assess transcriptional changes associated with VN and TLS formation via qPCR, with corollary immune cell composition changes determined using flow cytometry and immunofluorescence microscopy. In vitro assays were performed on CD11c+ BMDCs treated with 2.5 μg/mL ADU S-100 (vs PBS control) and associated transcriptional changes analyzed via qPCR or profiled using DNA microarrays. For TCRβ-CDR3 analyses, CDR3 was sequenced from gDNA isolated from enzymatically digested tumors and splenocytes.ResultsWe report that activation of STING within the TME leads to slowed melanoma growth in association with increased production of angiostatic factors including Tnfsf15 (Vegi), Cxcl10 and Angpt1, and TLS inducing factors including Ccl19, Ccl21, Lta, Ltb and Tnfsf14 (Light). Therapeutic responses from intratumoral STING activation were characterized by increased vascular normalization (VN), enhanced tumor infiltration by CD8+ T cells and CD11c+ DCs and local TLS neo-genesis, all of which were dependent on host expression of STING. Consistent with a central role for DC in TLS formation, ex vivo ADU S-100-activated mCD11c+ DCs also exhibited upregulated expression of TLS promoting factors including lymphotoxin-α (LTA), IL-36, inflammatory chemokines and type I interferons. TLS formation was associated with the development of a therapeutic TIL TCR repertoire enriched in T cell clonotypes uniquely detected within the tumor but not the peripheral circulation in support or local T cell cross-priming within the TME.ConclusionsOur data support the premise that i.t. delivery of STING agonist promotes a pro-inflammatory TME in support of VN and TLS formation, leading to the local expansion of unique TIL repertoire in association with superior anti-melanoma efficacy.

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Outcome of alloanergized haploidentical bone marrow transplantation after ex vivo costimulatory blockade: results of 2 phase 1 studies

Blood ◽

10.1182/blood-2008-03-143636 ◽

2008 ◽

Vol 112 (6) ◽

pp. 2232-2241 ◽

Cited By ~ 48

Author(s):

Jeff K. Davies ◽

John G. Gribben ◽

Lisa L. Brennan ◽

Dongin Yuk ◽

Lee M. Nadler ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Bone Marrow Transplantation ◽

T Cell ◽

Marrow Transplantation ◽

Viral Infections ◽

Ex Vivo ◽

Chronic Gvhd ◽

Costimulatory Blockade ◽

Donor T Cells

AbstractWe report the outcomes of 24 patients with high-risk hematologic malignancies or bone marrow failure (BMF) who received haploidentical bone marrow transplantation (BMT) after ex vivo induction of alloantigen-specific anergy in donor T cells by allostimulation in the presence of costimulatory blockade. Ninety-five percent of evaluable patients engrafted and achieved full donor chimerism. Despite receiving a median T-cell dose of 29 ×106/kg, only 5 of 21 evaluable patients developed grade C (n = 4) or D (n = 1) acute graft-versus-host disease (GVHD), with only one attributable death. Twelve patients died from treatment-related mortality (TRM). Patients reconstituted T-cell subsets and immunoglobulin levels rapidly with evidence of in vivo expansion of pathogen-specific T cells in the early posttransplantation period. Five patients reactivated cytomegalovirus (CMV), only one of whom required extended antiviral treatment. No deaths were attributable to CMV or other viral infections. Only 1 of 12 evaluable patients developed chronic GVHD. Eight patients survive disease-free with normal performance scores (median follow-up, 7 years). Thus, despite significant early TRM, ex vivo alloanergization can support administration of large numbers of haploidentical donor T cells, resulting in rapid immune reconstitution with very few viral infections. Surviving patients have excellent performance status and a low rate of chronic GVHD.

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