Relevance of αβ-DNTs as Prognostic Factor on Clinical Outcome and Role on Tumor Surveillance in Haematological Diseases and Solid Tumor: Preliminary Evaluations

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3840-3840
Author(s):  
Giacoma De Tullio ◽  
Carla Minoia ◽  
Margherita Gigante ◽  
Sabino Strippoli ◽  
Simona Serratì ◽  
...  
Keyword(s):  
T Cells ◽  
Clinical Outcome ◽  
Ex Vivo ◽  
Granzyme B ◽  
Therapy Response ◽  
Tumor Burden ◽  
Tumor Surveillance ◽  
Functional Studies ◽  
Anti Tumor Activity

Abstract The mutual interactions between the host immune system and cancer cells may either promote or control oncogenesis. Cancer immunotherapies remain viable approaches to sustain patient survival. However, positive clinical response of phase I/II clinical trials remains still to low. The Double negative T cells (DNTs) have emerged as new subset of T cells that contributes specifically to anti- tumor immunity since involved in immune regulation and tolerance acting as regulatory T cells (Treg) and/or cytotoxic T cells. DNTs express either αβ or γδ T-cell receptors (TCR) or lack CD4, CD8 and CD56. Functional studies showed that DNTs might have a direct in vitro anti-tumor activity against lymphoma and melanoma cells. However no data are available on their prognostic significance, modulation during the therapy response and their ability to kill tumor cells in cooperation with other immune cells such as DCs to explain their role in human anti-lymphoma immunity. Knowledge of the DNTs role in tumor surveillance and as prognostic factor for clinical outcome has a strong clinical valence and might allow us to design new approaches of therapeutic strategy. The aim of this study was to evaluate the DNTs during therapy response in lymph nodes (LN), bone marrow (BM) and peripheral blood (PB) of both lymphoma (Ly), Renal Cell Carcinoma (RCC) and Metastatic Melanoma (MM) patients (pts) in order to assess their role on clinical outcome, progression and tumor surveillance. PB and BM samples of 90 Ly pts, with NHL and cHL were collected at diagnosis and during the follow-up (1-6-12 months after chemo- or immuno-chemotherapy therapy). PB samples of 16 healthy donors were collected as control. Twenty fresh LN tissues from pts clinically suggestive of Ly were quickly processed. To evaluate the interaction of DNTs with tumor burden either 22 samples from RCC pts and 30 samples from MM pts were collected. The ontogeny, functional attitude and TCR clonality of DNT subsets (TCRαβ+ and TCRγδ+) were evaluated by following antibodies: CD3, CD4, CD8, CD56, CD45, TCRαβ, CD45Ra, CD45Ro, CCR7, CD27, CD28, CD30, CD69, GITR, CD95, CD178, CD152, IFN-γ, TNF-α, perforin and granzyme B. For functional studies, DNTs were purified from PBMCs through a negative selection and then cultured for 2 weeks. Data were acquired by 8-colour flow cytometer and analysed using Kaluza software. Other immune cells such as DCs, MDSCs and Treg was also detected to evaluate the correlation with DNTs. 7 cases of LNs received a finally diagnosis of reactive follicular hyperplasia (RFH) so they were considered as controls. All pts provided their informed consent in accordance with the Declaration of Helsinki. We observed a significant decrease (p =0.006) of circulating αβ-DNTs in untreated Ly pts (20.5 cells/ul ± 4.8) as compared with healthy controls (31.3 cells/ul ± 3.4) (fig.1-2) and their number seemed to be modulated during the follow-up. Their frequency in 1-month post-cht or disease relapsed pts was significantly decreased (p=0.006) as compared with the diagnosis as well as when compared indolent with aggressive histotypes (p=0.0001). In HL pts the frequency of αβ-DNT was significantly increased as compared with other histotypes (p=0.005) (Fig.3) Furthermore, the αβ-DNTs in LNs of Ly pts were significantly reduced as compared to to RFH-LNs (p=0.006) (fig.5) and are related to the aggressiveness of the disease. When evaluated either in MM and RRC pts the αβ-DNTs were significantly decreased (p=0.001) as compared with healthy controls and more interestingly when compared with Ly pts (p=0.001) given the greater immunological impairment of RCC/MM tumor burden (fig.6). Finally, ex vivo expanded DNTs acquired an immunomodulatory cytokine profile, characterized by the secretion of IFN-gamma and granzyme B, which are known as central components of anti-tumor immune responses supporting the hypothesis of αβ-DNT potential use in immunotherapeutic strategies (fig.4) Our preliminary data, showed an inverse correlation between the frequency of circulating αβ-DNTs and the tumor condition as well as that they could play an important role in both the development and the progression of lymphomas. This is the first study that compares the frequency of αβ-DNTs in three different pathologies correlating to tumor burden. In addition, it is likely that ex-vivo expanded DNTs exert an anti-tumor activity thus suggesting their possible use as a new strategy for adoptive immune-therapy. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

The Functional Attitude and The Predictive Role Of An Unconventional Subset Of T Cells In Clinical Outcome Of Lymphoma Patients: αβ-Double Negative T Cells, Preliminary Data Of a Prospective Study

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4506-4506 ◽  
Author(s):  
Giacoma De Tullio ◽  
Carla Minoia ◽  
Simona Serratì ◽  
Francesca Merchionne ◽  
Giacomo Loseto ◽  
...  
Keyword(s):  
T Cells ◽  
Clinical Outcome ◽  
Ex Vivo ◽  
Double Negative ◽  
Murine Models ◽  
Disease Relapse ◽  
Healthy Donors ◽  
Ifn Γ ◽  
Anti Tumor Activity

Background Many aspects of lymphoma pathophysiology indicate mutual interactions between the host immune system and lymphoma cells. These interactions may either promote or control lymphomagenesis. An unconventional subset of CD4-CD8- double-negative T cells (DNTs) has been recently described to contribute specifically to anti-tumor immunity. Indeed, DNTs are involved in immune regulation and tolerance as well as in host defense and inflammation, acting as regulatory T cells and/or cytotoxic T cells. DNTs are T lymphocytes which express either αβ or γδ T-cell receptors (TCR) and lack CD4, CD8 and CD56. In healthy human donors and murine models, they constitute about 1-5% of the lymphocytes in peripheral blood and in lymphoid organs. No data are available on the role of DNT cells in human anti-lymphoma immunity. Information from murine models suggests that expanded DNT cells would not impair host immunity against lymphoma and would perhaps stimulate it. DNT cells have also demonstrated to have a direct in vitro anti-tumor activity against lymphoma. Few data are available on the prognostic significance of DNTs in lymphomas, on their interaction with other immune cells, and on their functional attitude. Aims The aim of this study was to assess the frequency and the functional attitude of circulating DNTs in Lymphoma patients and healthy donors as controls, in order to assess the role of DNTs on clinical outcome and progression. Methods To test this population as prognostic factor on clinical outcome and progression of lymphoma disease, peripheral blood (PB) and bone marrow (BM) samples of 46 Lymphoma patients (pts), with non-Hodgkin's Lymphomas and classical Hodgkin Lymphoma were selected and prospectively collected at diagnosis and after one month till the end of chemo- or immuno-chemotherapy therapy. Blood samples were collected also at the time of relapse or progression. As control PB samples of 16 healthy donors were collected. Circulating DNT subsets (TCRαβ+ and TCRγδ+) were characterized for their ontogeny, tolerogenic or cytotoxic attitude and TCR clonality by staining with the following conjugated monoclonal antibodies (MoAbs) for surface and intracellular markers: CD3, CD4, CD8, CD56, CD45, TCRαβ, CD45Ra, CD45Ro, CCR7, CD27, CD28, CD30, CD69, GITR, CD95, CD178, CD152, IFN-γ, TNF-α, granzyme B, and perforin. Isotype-matched MoAbs were used as staining controls. For functional studies, DNTs were purified from PBMCs of patients through a negative selection by using specific MACS microbeads and then cultured for 2 weeks in complete medium supplemented with anti-CD3 (OKT3), rhIL-2 and rhIL-4. Data were acquired using an 8-colour flow cytometer and analyzed using Kaluza software. Data were compared among the groups using the Mann-Whitney non-parametric test or Kruskal–Wallis one-way analysis of variance. The study was approved by the local Ethics Committee and all patients provided their informed consent in accordance with the Declaration of Helsinki. Results The percentage (mean + SE) of DNTs in BM (2.367 ± 0.5891) of Lymphoma pts was lower than in PB samples (3.421 ± 0.981). Moreover we observed a significant decrease (p = 0.006) of circulating αβ-DNTs in pts with untreated lymphoma (23.7 ± 3.7) as compared with healthy controls (31.3 ± 3.4), and their number seemed to be modulated by disease relapse/progression or disease treatment. (fig.1). In Hodgkin's Lymphoma circulating αβ-DNTs were significantly increased as compared with other histotypes (p = 0.0001) (fig.2). Circulating αβ-DNTs were significantly decreased (p=0.006) in serial samples collected after treatment or at the time of disease relapse. Interestingly, after ex vivo expansion, DNTs acquired an immunomodulatory cytokine profile, characterized by the secretion of IFN-γ and granzyme B which are known as central components of anti-tumor immune responses (fig.3). Conclusions To date, no data have been reported on DNT phenotypic and functional characterization in Lymphoma patients. Our study has demonstrated for the first time that αβ-DNTs could play an important role in both the development and the progression of lymphomas. In addition, based on our preliminary results, it is likely that ex-vivo expanded DNTs exert an anti-tumor activity thus suggesting their possible use as a new strategy for adoptive immune-therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals PD-L1 Checkpoint Blockade Augments Anti-Tumor Immune Response of GD2 or HER2-Bsab Ex Vivo Armed T-Cells (EVAT) Therapy in Osteosarcoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1959-1959
Author(s):  
Jeong A Park ◽  
Hong fen Guo ◽  
Hong Xu ◽  
Nai-Kong V. Cheung
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Immune Checkpoint ◽  
Bispecific Antibody ◽  
Ex Vivo ◽  
Activated T Cells ◽  
The Impact ◽  
Anti Tumor Activity

Background Ex Vivo Armed T-cells (EVAT) carrying zeptomoles (10-21M) of T-cell engaging GD2-bispecific antibody (GD2-EVAT) or HER2-bispecific antibodies (HER2-EVAT) have potent anti-tumor activity against GD2(+) and/or HER2(+) solid tumors. Strategies to further optimize this approach are highly relevant. PD-1 is a key immune checkpoint receptor expressed mainly by activated T-cells and mediates immune suppression by binding to its ligands PD-L1 or PD-L2. Upregulation of PD-L1 has been found in many cancers including osteosarcoma and associated with aggressive disease and poor outcome. While the use of immune checkpoint inhibitors (ICIs) seems logical, the ideal timing when combined with T-cell engaging bispecific antibody (T-BsAb) or EVAT has yet to be defined. Here, we described the effects of anti-PD-1 or anti-PD-L1 antibodies on GD2-EVAT or HER2-EVAT therapy and explored the impact of its timing in the treatment of osteosarcoma which is GD2(+), HER2(+) and PD-L1(+). Methods GD2-BsAb and HER-BsAb were built using the IgG(L)-scFv format (Can Immunol Res, 3:266, 2015, Oncoimmunology, PMID:28405494). T-cells from healthy volunteer donors were isolated, and cultured ex vivo in the presence of CD3/CD28 beads plus 30 IU/mL of interleukin 2 (IL-2). Between day 7 and day 14, activated T-cells (ATCs) were harvested and armed for 20 minutes at room temperature with GD2-BsAb or HER2-BsAb. In vivo anti-tumor activity against GD2(+), HER2(+), and PD-L1(+) osteosarcoma cell line xenografts was tested in BALB-Rag2-/-IL-2R-γc-KO mice. Anti-human PD-1 antibody (pembrolizumab, anti-PD-1) or anti-human PD-L1 antibody (atezolizumab, anti-PD-L1) were tested for synergy with GD2-EVAT or HER2-EVAT therapy. Results The PD-1 expression increased among T-cells that circulated in the blood, that infiltrated the spleen or the tumor after EVAT therapy. While anti-PD-L1 combination therapy with GD2-EVAT or HER2-EVAT improved anti-tumor response against osteosarcoma (P=0.0123 and P=0.0004), anti-PD-1 did not (all P>0.05). The addition of anti-PD-L1 significantly increased T-cell survival in blood and T-cell infiltration of tumor when compared to GD2-EVAT or HER2-EVAT alone (all P<0.0001). Treatment of GD2-EVAT or anti-PD-L1 plus GD2-EVAT downregulated GD2 expression on tumors, but anti-PD-1 plus GD2-EVAT did not. For the next step we tested the impact of different combination schedules of ICIs on GD2-EVAT therapy. Concurrent anti-PD-1 (6 doses along with GD2-EVAT therapy) interfered with GD2-EVAT, while sequential anti-PD-1 (6 doses after GD2-EVAT) did not make a significant effect (P>0.05). On the other hand, while the concurrent use of anti-PD-L1 did not show benefit on GD2-EVAT, sequentially administered anti-PD-L1 produced a significant improvement in tumor control when compared to anti-PD-L1 or GD2-EVAT alone (P=0.002 and P=0.018). When anti-PD-L1 treatment was extended (12 doses after GD2-EVAT), the anti-tumor effect was most pronounced compared to GD2-EVAT alone (P <0.0001), which translated into improved survival (P=0.0057). These in vivo anti-tumor responses were associated with increased CD8(+) tumor infiltrating lymphocytes (TILs) of tumor. Conclusion In the arming platform, large numbers of target-specific T-cells can be generated, and this EVAT therapy is a highly effective cellular treatment with high potency in preclinical models. In addition, the advantage of ex vivo cytokine release following T-cell arming and activation could reduce or avoid life threatening cytokine storm if such activation was to proceed in vivo. Adoptive T-cell therapy induced immune response upregulates the inhibitory immune checkpoint PD-1/PD-L1 pathway, and combination treatment with anti-PD-L1 antibody, especially when combined as sequential therapy and continuously treated, significantly improved anti-tumor effect of EVAT, partly through increase in CD8(+) TILs infiltration. Disclosures Xu: MSK: Other: co-inventors in patents on GD2 bispecific antibody and HER2 bispecific antibody. Cheung:Ymabs: Patents & Royalties, Research Funding.

scholarly journals Early Stage Follicular Lymphoma. Predictive Role of Minimal Residual Disease (MRD) and Impact of MRD-Driven Treatment with Radiotherapy and Rituximab on Clinical Outcome

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2959-2959 ◽  
Author(s):  
Alessandro Pulsoni ◽  
Irene Della Starza ◽  
Maria Elena Tosti ◽  
Luca Vincenzo Cappelli ◽  
Giorgia Annechini ◽  
...  
Keyword(s):  
Clinical Outcome ◽  
Molecular Marker ◽  
Follicular Lymphoma ◽  
Early Stage ◽  
Tumor Burden ◽  
Stage I ◽  
Predictive Role ◽  
Qualitative Pcr

Abstract Background. In localized follicular lymphoma (FL, stage I-II), BCL2/IGH+ cells can be detected in the peripheral blood (PB) and/or bone marrow (BM) in 66.7% of cases (Pulsoni et al, BJH 2007). We hereby analyzed the prognostic impact of MRD in localized FL and explored the possibility of a MRD-guided therapeutic approach on a series of patients with a long follow-up. Methods. Between April 2000 and February 2015, 67 consecutive patients with a confirmed histologic diagnosis of stage I/II FL followed at our Center were enrolled in the study. PB and BM samples were collected at enrollment in all patients and investigated by qualitative PCR to identify the presence of a BCL2/IGH rearrangement. Paraffin-embedded lymph nodes (LN) were studied when available. Patients who proved positive at baseline were studied for MRD every 6 months. Real-Time Quantitative PCR (RQ-PCR) was retrospectively performed according to material availability. All patients were treated with involved field radiotherapy (RT) (24-30 Gy); from 2005, patients who were MRD+ after RT received rituximab (R) (375 mg/m2, 4 weekly administration). The median follow-up is 67 months (17-183); 21 patients (31%) have relapsed after a median of 37 months (17-165) from diagnosis. Results. At baseline, a clonal marker was found by qualitative PCR in 48/67 cases (72%): 36 were MBR+ (54%), 6 mcr+ (9%), 6 showed a minor BCL2 rearrangement (9%), while 19 (28%) were negative. Fifteen of the latter 19 were analyzed by RQ-PCR and 4 proved MBR+. Of the 13 available LNs, 11 showed the same molecular marker identified in the PB/BM; 2 cases, negative in the PB/BM, showed a rearrangement in the LN only. After RT, 40/42 MBR+/mcr+ patients were analyzed: 20 resulted MRD-, while 20 persisted MRD+. Regardless of the post-RT MRD status, an equal number of relapses was recorded in both groups (7 each). R treatment was administered to the 20 MRD+ patients after RT. Sixteen (80%) achieved a MRD- status after R: over time, 7/16 patients converted to MRD+ and 4 relapsed, whilst 9/16 patients (56.2%) remain persistently MRD- and none has relapsed so far. To evaluate the impact of R, we considered a series of 27 patients MRD+ after RT or who were MRD- and became MRD+ during the follow-up. Of the 19 patients who received R (1 could not be studied), 15 (79%) did not relapse, while of the 8 untreated patients (pre-2005), 6 (75%) relapsed (p=0.025). Progression-free survival (PFS) was significantly longer for R-treated patients (p=0.0412) (Fig. 1). To define the predictive role of MRD in the entire cohort regardless of post-RT treatment, we considered the 39 patients with molecular follow-up. Thirteen have relapsed: 10/13 (77%) were MRD+ in the follow-up, including the pre-relapse time point, while 3 resulted persistently MRD-. Contrariwise, of the 26/39 patients in continuous remission, 18 (69%) were persistently MRD- while 8 were MRD+ (p=0.015). PFS was significantly better for MRD- patients (p=0.0163) (Fig. 2). RQ-PCR was performed in 30 MBR+ patients: 17 (57%) showed a tumor burden ≥10-5 and 13 <10-5. Tumor burden at diagnosis predicted the MRD clearance following RT: 9/13 (69%) cases with low tumor burden resulted MRD- after RT compared to 2/17 (12%) cases with high tumor burden (p=0.0027). Contrariwise, tumor burden did not predict the occurrence of relapse. Conclusions. Early stage FL at diagnosis can have a heterogenous disease extension: 2 of our cases were truly localized, showing a molecular marker only in the LN. However, in most cases the use of combined qualitative approaches, including canonical MBR/mcr and minor rearrangements, together with RQ-PCR has allowed to identify circulating BCL2/IGH+ cells (52/67 cases: 77.6%), despite a negative BM biopsy. RT induced a MRD negativity in 50% of BCL2/IGH+ patients, but this did not impact on clinical outcome. The administration of R in MRD+ patients decreased significantly the risk of a subsequent relapse and improved PFS. Regardless of treatment, MRD positivity during the follow-up is a predictor of relapse and PFS. Tumor burden at diagnosis is associated with MRD clearance after RT. We support the use of a MRD-driven treatment with anti-CD20 monoclonal antibodies in patients with localized FL after RT. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

206 An immune-competent tumor organoid platform to test novel immune checkpoint combinations targeting the receptor CD47 in triple negative breast cancer

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A222-A222
Author(s):  
Elizabeth Stirling ◽  
Ethan Willey-Shelkey ◽  
Adam Wilson ◽  
Aleksander Skardal ◽  
Pierre Triozzi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cells ◽  
Granzyme B ◽  
Tumor Burden ◽  
Cancer Center ◽  
Murine Models ◽  
Patient Response ◽  
Tumor Immunogenicity ◽  
Cellular Bioenergetics ◽  
Glycolytic Rate

BackgroundImmune checkpoint blockade therapy targeting PD-L1 has recently been approved for metastatic triple negative breast cancer (TNBC) patients. However, a 7% response rate calls for better models and strategies to stimulate TN-tumor immunogenicity to increase patient response. Overexpression of the receptor CD47 impairs innate and adaptive tumor immunosurveillance when engaged to its counter receptor SIRPα or ligand thrombospondin-1. Co-expression of CD47 and PD-L1 is implicated with disease progression in TNBC patients. We examined through murine models and tumor organoid platforms whether targeting CD47 sensitizes TNBC tumors to PD-L1 therapy, focusing on the modulation of cellular bioenergetics as a potential mechanism and potentially predict response.MethodsThe effects of targeting CD47 and PD-L1 were examined through orthotopic syngenic 4T1 and EMT-6 TNBC murine models. Due to predicting patient therapeutic response challenges, tumor organoid platforms investigated mechanisms of tumor sensitization to anti-PD-L1 by targeting CD47. Organoids were constructed by embedding murine TNBC tumor tissue and AH1 CD8+ T cells in a specialized ECM mimicking hydrogel. Immunohistochemistry was performed on organoid, human and murine TNBC tumor tissue. Cellular bioenergetics was analyzed through Seahorse® bioanalyzer.ResultsStaining of human TNBC biopsies found elevated CD47 expression, signifying a potential therapeutic target. Targeting CD47 or in combination with anti-PD-L1 resulted in decreased tumor volume and weight in a TNBC murine model. The decrease in tumor burden was correlated with increased intratumoral granzyme B secreting CD8+ T cells. Additionally, targeting CD47 within organoids increased IFNγ and granzyme B released, indicating enhanced CD8+ T cell cytolytic capacity. Differential cellular bioenergetics was observed between cancer and T cells suggesting a shift in metabolism in the tumor microenvironment. CD47 targeted T cells had an increased glycolytic rate compared to WT T cells. Conversely CD47 targeted TNBC cells had a decreased glycolytic rate, which may be correlated with decreased PD-L1 expression.ConclusionsTargeting CD47 enhanced granzyme B and IFNγ expression suggesting potential mechanisms to increase tumor immunogenicity. CD47 targeted monotherapy or combination with anti-PD-L1 preserves T cell bioenergetics and antitumor function, resulting in decreased TNBC tumor burden. Alternatively, CD47 targeted TNBC had a decreased glycolytic rate and decreased PD-L1 expression, which is reported to regulate glycolysis through Akt/mTOR signaling. Targeting CD47 on T cells enhances their bioenergetics and antitumor function while decreasing TNBC cell bioenergetics, making them more susceptible to immune cell killing. Our data indicates that CD47 targeted monotherapy or combination with anti-PD-L1 may enhance TNBC patient response and improve overall survival.AcknowledgementsDSP and SS are supported by the Wake Forest Comprehensive Cancer Center Breast Cancer Center of Excellence Pilot Award. DSP is also supported by the ASTRO-BCRF Career Development Award (637969) while ERS is supported by NIGMS T32 (GM127261).Ethics ApprovalAnimal studies were approved by the Institutional Care and Use Committee, Wake Forest Health Sciences.

scholarly journals Longitudinal Analysis of Dengue Virus–Specific Memory T Cell Responses and Their Association With Clinical Outcome in Subsequent DENV Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Luis Alberto Sanchez-Vargas ◽  
Kathryn B. Anderson ◽  
Anon Srikiatkhachorn ◽  
Jeffrey R. Currier ◽  
Heather Friberg ◽  
...  
Keyword(s):  
T Cells ◽  
Clinical Outcome ◽  
Dengue Virus ◽  
Memory T Cells ◽  
Ex Vivo ◽  
Secondary Infection ◽  
Denv Infection ◽  
Memory T Cell ◽  
Cell Responses ◽  
Specific Memory

Memory T cells resulting from primary dengue virus (DENV) infection are hypothesized to influence the clinical outcome of subsequent DENV infection. However, the few studies involving prospectively collected blood samples have found weak and inconsistent associations with outcome and variable temporal trends in DENV-specific memory T cell responses between subjects. This study used both ex-vivo and cultured ELISPOT assays to further evaluate the associations between DENV serotype-cross-reactive memory T cells and severity of secondary infection. Using ex-vivo ELISPOT assays, frequencies of memory T cells secreting IFN-γ in response to DENV structural and non-structural peptide pools were low in PBMC from multiple time points prior to symptomatic secondary DENV infection and showed a variable response to infection. There were no differences in responses between subjects who were not hospitalized (NH, n=6) and those who were hospitalized with dengue hemorrhagic fever (hDHF, n=4). In contrast, responses in cultured ELISPOT assays were more reliably detectable prior to secondary infection and showed more consistent increases after infection. Responses in cultured ELISPOT assays were higher in individuals with hDHF (n=8) compared to NH (n=9) individuals before the secondary infection, with no difference between these groups after infection. These data demonstrate an association of pre-existing DENV-specific memory responses with the severity of illness in subsequent DENV infection, and suggest that frequencies of DENV-reactive T cells measured after short-term culture may be of particular importance for assessing the risk for more severe dengue disease.

scholarly journals Eltrombopag: More Than Just a Thrombopoietin Receptor Agonist (TPO-RA) in Immune Thrombocytopenia (ITP)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2364-2364
Author(s):  
Anwar A. Sayed ◽  
Amna Malik ◽  
Grace Ayoola ◽  
Elisa Lucchini ◽  
Sasfia Candrianita ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Ex Vivo ◽  
Granzyme B ◽  
Immunomodulatory Effect ◽  
Dependent Manner ◽  
Advisory Committees ◽  
Dose Dependent

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by a skewed proinflammatory T cell profile. Thrombopoietin-receptor agonists (TPO-RA) have largely replaced immunosuppressants in the management of this disorder, with some patients achieving remission after a period of treatment with TPO-RA. The potential immune modulatory role of TPO-RA has not been fully investigated. The two current TPO-RA licensed for use in ITP; Eltrombopag (Elt) and Romiplostim (Romi) act on different parts of the TPO-R and have similar response rates. However, patients can respond to one agent but not the other. Elt has been described to have a strong iron chelating effect, and hence we propose that it may have an additive immunomodulatory effect on the T cells, absent in Romi. We determined the immunomodulatory effect of Elt by assessing the proliferation and functionality of T-cell lines and primary T-cells. T cell proliferation was assessed using both CFSE proliferation assay and MTT cell viability assay. T cell phenotype and functionality were assessed by multicolor surface and intracellular flow cytometric staining. Cells were co-cultured with Elt and Romi in vitro and ex vivo with both Jurkat and DG75 cells lines as well as primary cells, respectively. Deferoxamine (DFX) was used as a positive control for iron-chelation, and human TPO was used as a positive control for TPO-RA. All treatment doses were based on their calculated therapeutic serum levels. Mann Whitney U and Kruskal-Wallis H statistical tests were applied where applicable, and a P value of less than 0.05 were considered significant. Elt significantly decreased Jurkat T cells proliferation in a dose-dependent manner compared to no treatment and Romi. DFX, an iron chelator, also decreased Jurkat T cell proliferation to comparable levels of Elt. Interestingly, this anti-proliferative effect of Elt was only observed on Jurkat T cells, but not DG75 B cell line. Ex vivo CFSE proliferation assay was performed on primary CD4 and CD8 T cells assessing the antiproliferative effect of Elt. Elt significantly reduced proliferation compared to no treatment. DFX exhibited a similar antiproliferative effect on primary T cells, however, less potent compared to Elt. Neither Romi nor TPO affected the proliferation of Jurkat cells, DG75 cells or primary T cells. The functionality of CD4 and CD8 T cells was assessed based on the capacity of T cells to produce intracellular TNFα, IFNγ and Granzyme B. Elt significantly reduced the percentages of TNFα+/IFNγ+ CD4+ and CD8+ T cells in a dose-dependent manner. This reduction was also observed, albeit to a lesser extent, when T cells were treated with DFX. Furthermore, Granzyme B expression in CD8+ T cells was significantly reduced when cells were treated Elt, compared to no treatment. Romi did not affect the frequency of CD8+ TNFα+/IFNγ+ populations nor the expression of Granzyme B in CD8+ T cells. CD4+ and CD8+ T cells did not express TPO-R on their surface. To confirm the immunomodulatory role of Elt in vivo, the terminally-differentiated effector (CD45RA+CD62L-) CD8+ T cells were assessed in 13 Elt-treated patients and 11 Romi-treated patients. Patients on Elt had significantly reduced frequency of effector CD8 T cells compared to Romi-treated patients (44% vs 76.8%; p<0.01). Taken together, these novel findings suggest an off target immunomodulatory nature of Elt besides its thrombopoietic effect. This dose-dependent immunomodulatory effect is not TPO-R dependent and targets T cells primarily. This study is the first to display such property of Elt and could explain why there is a differential response to Elt and Romi. We hypothesise that Elt may be more effective in patients with T cell mediated disease, whilst patients with predominantly antibody mediated disease are more likely respond to Romi. These findings can also offer an explanation for Elt effectiveness in other T cell-mediated autoimmune conditions such as Aplastic Anemia. Disclosures Cooper: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Principia: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Adoptive Immunotherapy with Autologous CD25-Depleted and CD3/CD28-Costimulated T-Cells (ACTC) Enhances Numeric and Functional Lymphocyte Recovery after Cyclophosphamide - Fludarabine Chemotherapy in Patients with Low-Grade Follicular Lymphoma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1505-1505
Author(s):  
Charalambos Andreadis ◽  
Bruce L. Levine ◽  
Julio Cotte ◽  
Zhaohui Zheng ◽  
Rebecca Mair ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Follicular Lymphoma ◽  
Adoptive Immunotherapy ◽  
Ex Vivo ◽  
Delayed Type Hypersensitivity ◽  
Hematologic Toxicity ◽  
Low Grade ◽  
Median Increase

Abstract Fludarabine-based chemotherapy combinations are highly effective in patients (pts) with low-grade follicular lymphoma (FL), but cause severe and long-lasting immunosuppression due to depletion of normal CD4 T-cells. Aside from increasing the risk of serious infections, this toxicity may limit the ability of the immune system to eliminate minimal residual disease. Adoptive immunotherapy using autologous CD25-depleted, CD3/CD28-costimulated T-cells expanded ex vivo (ACTC) may enhance immune reconstitution and improve disease control. We initiated a phase I study in pts with purine analog-naive relapsed/refractory FL (grades 1 and 2). After leukapheresis, pts receive 4 cycles of fludarabine (25 mg/m2) days 1–3 and cyclophosphamide (250 mg/m2) days 1–3. Four weeks after last chemotherapy, responding patients (CR, CRu, PR) receive escalating doses of ACTC prepared ex vivo from autologous T-cells collected prior to chemotherapy and depleted of regulatory CD4+/CD25+ cells, then expanded and activated using anti-CD3 and anti-CD28. Eight pts have been enrolled to date. Median age is 40.5 y (range: 32–64). Median number of prior therapies is 2 (range: 1–3). Two pts were withdrawn from the study due to hematologic toxicity related to the chemotherapy; one patient has not completed chemotherapy. Of the 5 pts completing chemotherapy, 3 pts achieved a CR and 2 pts achieved a PR; 4 pts received 5 x 109 and 1 patient received 1 x 1010 CD3+ ACTC. There have been no adverse events related to T-cell infusions. Median follow-up after ACTC infusion is 9 months (range: 2–15 months). Median time to CD4 count &gt;200 /uL was 29 days following T-cell infusion (range 28–127 days). CD4 counts increased in all patients by 1 month after T-cell infusion, with a median increase of 126% from baseline (range 50 to 484%). CD8 counts also increased, with a median increase of 82% (range −4 to 976%). All 5 pts receiving ACTC were anergic to candida antigen by Delayed Type Hypersensitivity (DTH) skin testing before chemotherapy. Three pts developed a positive DTH response to candida antigen 60 days after ACTC infusion. From the start of therapy for patients receiving T-cells, median follow-up is 14 months (range 7–20 months) with median progression-free survival not reached; 4 pts remain in remission and 1 patient had progression of disease. ACTC results in significant CD4+ lymphocyte recovery in previously treated pts receiving cyclophosphamide-fludarabine chemotherapy and compares very favorably with historical controls. T-cell dose escalation is ongoing.

Adaptive Immunotherapy with Autologous CD25-Depleted and CD3/CD28-Costimulated T-Cells Enhances Lymphocyte Recovery after Cyclophosphamide - Fludarabine Chemotherapy in Patients with Low-Grade Follicular Lymphoma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2717-2717 ◽  
Author(s):  
Charalambos Andreadis ◽  
Bruce L. Levine ◽  
Sunita D. Nasta ◽  
Julio Cotte ◽  
Zhaohui Zheng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Follicular Lymphoma ◽  
Dose Level ◽  
Ex Vivo ◽  
Delayed Type Hypersensitivity ◽  
Hematologic Toxicity ◽  
Low Grade ◽  
Median Increase

Abstract BACKGROUND: Fludarabine-based chemotherapy combinations are highly effective in patients (pts) with low-grade follicular lymphoma (FL), but cause severe and long-lasting immunosuppression due to depletion of normal CD4+ T-cells. Aside from increasing the risk of serious infections, this toxicity may limit the ability of the immune system to eliminate minimal residual disease. Adoptive immunotherapy using autologous CD25-depleted, CD3/CD28-costimulated T-cells (ACTC) expanded ex vivo may enhance immune reconstitution and improve disease control. METHODS: We initiated a phase I study in pts with purine analog-naive relapsed/refractory FL (grades 1 and 2). After leukapheresis, pts receive 4 cycles of fludarabine (25 mg/m2) days 1–3 and cyclophosphamide (250 mg/m2) days 1–3. Four weeks after the last cycle, responding patients (CR/CRu, PR) receive escalating doses of ACTC prepared ex vivo from autologous T-cellss collected prior to chemotherapy and depleted of regulatory CD4+/CD25+ cells, then expanded and activated using anti-CD3 and anti-CD28. RESULTS: Eleven pts have been enrolled to date. Median age is 49 y (range: 33–64y). Median number of prior therapies is 2 (range: 1 – 3). Two pts were withdrawn from the study due to hematologic toxicity related to chemotherapy, one patient was withdrawn for progressive disease during chemotherapy, and one patient has not yet completed chemotherapy. Of the 7 pts completing chemotherapy and proceeding to T-cell infusion, 5 pts achieved a CR/CRu and 2 pts achieved a PR; 5 pts received 1 – 5 x 109 CD3+ cells and 2 patients received 5 – 10 x 109 CD3+ cells. There have been no adverse events related to T-cell infusions. Median follow-up after ACTC infusion is 19 mos (range: 3 – 26 mos). CD4+ counts increased in all patients by 1 month after T-cell infusion, with a median increase of 3.2 fold (range: 1.5 – 70, p= .028)(see figure). For patients at dose level 1, the median increase was 2.2 fold (n=4; range: 1.5 – 3.3); at dose level 2 it was 37 fold (n=2; range: 3.8 – 70). CD8+ counts also increased, with a median increase of 8.1 (range: 1.0 – 30, p= .046)(see figure). All 7 pts receiving ACTC were anergic to candida antigen by delayed type hypersensitivity (DTH) skin testing before chemotherapy. Four pts developed a positive DTH response to candida antigen 60 days after ACTC infusion. For patients receiving ACTC, median follow-up is 24 mos (range: 7 – 31 mos) with a median progression-free survival of 18 months which is significantly longer than the time to progression from last therapy (p= .024). CONCLUSIONS: ACTC infusion results in significant CD4+ and CD8+ numerical and functional lymphocyte recovery after cyclophosphamide-fludarabine chemotherapy in pts with low-grade FL. This compares very favorably to historical controls treated with fludarabine-based regimens. T-cell dose escalation is ongoing. Figure Figure

Targeted ‘Off-the-Shelf’ Tumor Immunotherapy Using Allogeneic T-Cell Precursors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 579-579
Author(s):  
Johannes L. Zakrzewski ◽  
David Suh ◽  
Odette M. Smith ◽  
Christopher King ◽  
Gabrielle L. Goldberg ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Genetic Engineering ◽  
Adoptive Transfer ◽  
Ex Vivo ◽  
Precursor Cells ◽  
Host Cells ◽  
Anti Tumor Activity ◽  
Selection Of

Abstract T cell deficiencies can occur in many settings, including aging, autoimmune and genetic disorders, hematological malignancies, infectious diseases, and exposure to cytotoxic/cytostatics agents. Notch-based culture systems can be utilized for ex vivo generation of large numbers of T lineage committed lymphoid precursor cells, and we recently reported that the adoptive transfer of T cell precursors significantly enhances T cell reconstitution and function after allogeneic T cell depleted HSCT. The aim of this study was to evaluate if allogeneic T cell precursors are safe and effective when used for adoptive transfer across MHC barriers in the absence of allogeneic HSCs to overcome radiation injury, enhance T cell function and improve anti-tumor activity in immunosuppressed recipients. We found that positive selection of adoptively transferred allogeneic (C57BL/6) precursor cells ± syngeneic (BALB/c) HSCs in irradiated hosts (BALB/c) is dependent on MHC molecules on non-hematopoietic host cells, resulting in the in vivo development of host-MHC restricted allogeneic T cells. We demonstrate that negative selection of these cells is mediated by donor-derived antigen presenting cells (APCs), resulting in host-tolerance. Adoptively transferred allogeneic T cell precursors significantly improved survival of BALB/c mice after irradiation (675 cGy) and enhanced anti-tumor activity against liquid (A20 lymphoma) and solid (renal cell carcinoma) tumors in syngeneic HSCT recipients. Furthermore, we demonstrate the feasibility of genetic engineering of antigen-specific T cell precursors, by transducing them to express a chimeric antigen receptor (CAR) targeting human CD19. Transduction efficiencies were routinely in the range of 50%–70%. Adoptive transfer of CAR-expressing T cell precursors resulted in the in vivo generation of high numbers of appropriately selected CAR-expressing T cells with significantly enhanced anti-tumor activity (compared with CAR-negative T cell precursors) against a CAR-sensitive tumor, but without any undesirable auto/alloreactivity. We conclude that adoptively transferred allogeneic T cell precursors develop into host-MHC restricted and host-tolerant T cells characterized by selection of a functional TCR repertoire even in a fully mismatched thymic epithelial MHC environment. This strategy overcomes important limitations of conventional adoptive T cell therapies: rejection, alloreactivity and impaired antigen recognition due to restriction to MHC disparate from the one expressed on APCs. The use of allogeneic instead of autologous cells eliminates the risk of contamination with residual malignant patient cells and allows the generation and storage of virtually unlimited quantities of precursor cells for ‘off-the-shelf’ immunotherapy. This procedure has not only substantial logistic advantages, but it facilitates ex vivo manipulation, in particular genetic engineering, to generate antigen-specific or otherwise enhanced designer cells. Adoptive transfer of MHC mismatched and genetically enhanced T cell precursors therefore represents a promising novel strategy for targeted ‘off-the-shelf’ immunotherapy in immunosuppressed patients.

CD4+ Regulatory T Cells Specific for the WT1 Antigen Are Present in Acute Myeloid Leukemia Patients: Implication for Immunotherapy.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1933-1933
Author(s):  
Said Dermime ◽  
Cynthia Lehe ◽  
Hazem Ghebeh ◽  
Abdullah Al-Sulaiman ◽  
Ghofran Al Qudaihi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cytotoxic Activity ◽  
Cd8 T Cells ◽  
Ex Vivo ◽  
Granzyme B ◽  
Critical Role ◽  
Leukemic Cells ◽  
Cell Contact

Abstract Compelling evidences indicate a key role for regulatory T cells (Tregs) on the host response to cancer and recent studies indicated that the generation of effective WT1-specific cytotoxic T cells can be largely affected by the presence of Tregs. This is the first study to describe human Tregs generated specifically against the WT1 antigen which is overexpressed in several human leukemias and provide the mechanism by which these anti-WT1 Tregs inhibit the immune response in leukemia patients. We have generated T cell lines and clones that specifically recognized a WT1-84 peptide in an HLA DRB1*0402/TCR-Vb8-restricted manner. Importantly, they recognized HLADRB1* 04-matched fresh leukemic cells expressing the WT1 antigen. These clones exerted a Th2 cytokine profile, had a CD4+CD25+Foxp3+GITR+CD127− Tregs phenotype, and significantly inhibited the proliferative activity of allogeneic T cells independently of cell-contact. Priming of allo-reactive T cells in the presence of Tregs strongly inhibited the expansion of NK; NK-T and CD8+ T cells, had an inhibitory effect on NK/NK-T cytotoxic activity but not on CD8+ T cells. Furthermore, priming of T cells with the WT1- 126 HLA-A0201-restricted peptide in the presence of Tregs strongly inhibited the induction of anti-WT1-126 CD8+ CTL responses as evidenced by both very low cytotoxic activity and IFN-g production. Moreover, these Tregs clones specifically produced Granzyme-B and selectively induced apoptosis in WT1-84 pulsed-autologous APCs but not in apoptoticresistant DR4-matched leukemic cells. Importantly, we have also detected anti-WT1-84 IL-5+/Granzyme-B+/Foxp3+ CD4+ Tregs in 5 out of 8 HLA-DR4+ AML patients. These findings suggest a critical role for anti-WT1 Tregs in the inhibition of T cell responses against leukemia. This study may have important implications for the clinical manipulation of Tregs such as immuno-targeting of TCR-Vb-8 with mAbs to eliminate anti-WT1 Tregs in leukemia patients in order to enhance GVL before vaccination with the WT1 antigen. On the other hand, leukemia patients with GVHD should be clinically-tried for vaccination with the current WT1-84 peptide or adoptively-treated with ex-vivo anti-WT1 Treg cells to specifically enhance their frequency, which is known to be very low in these patients.

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