Granzyme B and perforin produced by SEC2 mutant-activated human CD4+ T cells and CD8+ T cells induce apoptosis of K562 leukemic cells by the mitochondrial apoptotic pathway

2021 ◽  
Author(s):  
Guojun Zhang ◽  
Guoliang Zheng ◽  
Fengli Jiang ◽  
Tianyi Wu ◽  
Lizhao Wu
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Granzyme B ◽  
Leukemic Cells ◽  
Apoptotic Pathway ◽  
Mitochondrial Apoptotic Pathway

WT1-Specific T Cells Exhibiting a Non-Exhausted, Functional Phenotype Can Be Generated From The Natural Repertoire Of Healthy Donors For Clinical Use

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4504-4504 ◽  
Author(s):  
Sabine Schmied ◽  
Anne Richter ◽  
Mario Assenmacher ◽  
Juergen Schmitz
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Leukemic Cells ◽  
Target Cells ◽  
Clinical Use ◽  
Healthy Donors ◽  
Effector Cytokines

Background The Wilms tumor antigen 1 (WT1) is a self-antigen expressed at high levels in leukemic cells, but not in healthy tissue. As WT1 expression in leukemic cells drives leukemogenesis, it is a favorable target antigen for immunotherapy, e.g. adoptive transfer of allogeneic T cells, to prevent or treat leukemic relapse after stem cell transplantation (Cheever et al., Clin Cancer Res 2009;15(17)). WT1-specific CD8+ T cells have been detected in healthy individuals at low frequencies (Rezvani et al., Blood 2003;102). However, a comprehensive characterization of CD4+ and CD8+WT1-specific T cells is missing and the efficient expansion of a polyclonal WT1-reactive T cell population for clinical use has remained a major challenge. In this study we aim to directly ex vivo characterize WT1-specific T cells present in the blood of healthy donors at high-resolution and to develop a rapid method for the generation of functionally potent, polyclonal CD4+ and CD8+WT1-specific T cells for clinical use. Methods For direct ex vivo analysis of CD4+ WT1-specific T cells peripheral blood mononuclear cells (PBMC) of healthy blood donors were in vitro stimulated with a pool of overlapping peptides spanning the WT1 protein for 7 hours. Subsequently CD154 (CD40L)-expressing cells were magnetically enriched and flow cytometrically examined for expression of effector cytokines and their differentiation status. Presence and phenotype of CD8+ WT1-specific T cells have been studied after stimulation of presorted naïve and memory T cell populations with WT-1 peptide pool for 30 hours, magnetic enrichment of CD137+ (4-1BB) cells and subsequent staining using pMHCI-Tetramers. For the generation of polyclonal WT1-specific CD4+ and CD8+ T cells PBMC were in vitro activated with WT-1 peptide pool for 30 hours. CD137+cells were magnetically selected and expanded for 9 days in the presence of the cytokines IL-7, IL-15 and IL-21 at low doses. Expanded T cells were analyzed for their phenotype, the expression of co-stimulatory and exhaustion markers and were tested for their functionality and cytotoxicity by restimulation experiments with antigen-loaded target cells. Results Ex vivo frequencies of WT1-specific T cells are low, 1 to 10 WT1-specific CD154+ CD4+ T cells can be detected within 1x106 CD4+ T cells. In about 80% of healthy donors (n=15) a CD4+ memory response, accompanied by production of effector cytokines like IFNγ, TNFα and IL-2, against WT1 peptides is present. Additionally, in all donors naïve WT1-specific CD4+ T cells can be detected. In contrast, detected CD137+CD8+ WT1-reactive T cells exhibit a naïve phenotype (CD45RA+CCR7+) in all donors (n=5), no WT1-reactive CD8+T cells could be enriched from presorted memory T cells. To evaluate the usefulness of our improved short-term expansion protocol to generate potent WT1-specific T cell cultures for clinical use, we characterized CD137 enriched and expanded T cells. Notably, a high frequency of CD4+ and CD8+ T cells show specific reactivity against WT1-presenting autologous cells as detected by production of effector cytokines like IFNγ, TNFα and IL-2 after antigen-specific restimulation. Cytotoxic activity against antigen-loaded target cells could be shown by direct flow-cytometry-based cytotoxicity assays and antigen-specific upregulation of the degranulation marker CD107a. Stainings using multiple WT1-MHCI-tetramers furthermore confirmed antigen-specificity and suggested polyclonality within the CD8+T cell population. In contrast to previous expansion protocols our polyclonally expanded T cells exhibit a favourable, unexhausted memory phenotype, express co-stimulatory markers CD27 and CD28 and the IL7R-a chain (CD127) which has been shown to mark cells with stem T cell like properties. Furthermore exhaustion markers like CD279 (PD-1), CD178 (FasL) and CD57 are scarcely expressed. Conclusions Functional, polyclonal, CD4+ and CD8+ WT1-specific, reactive T cells can be efficiently enriched directly ex vivo from the natural repertoire by magnetic separation of T cells after antigen-specific stimulation. Phenotypic and functional characterization revealed a non-exhausted phenotype of expanded WT1-specific T cells, thereby suggesting good persistence and functionality of the obtained T cell product in vivo. Thus, our approach holds great potential for the GMP-compliant generation of WT1-specific T cells for future clinical use. Disclosures: Schmied: Miltenyi Biotec GmbH: Employment. Richter:Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec: Employment.

scholarly journals Tumor-targeted delivery of childhood vaccine recall antigens by attenuated Listeria reduces pancreatic cancer

2019 ◽  
Author(s):  
Benson Chellakkan Selvanesan ◽  
Dinesh Chandra ◽  
Wilber Quispe-Tintaya ◽  
Arthee Jahangir ◽  
Ankur Patel ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Targeted Delivery ◽  
Cd4 T Cells ◽  
Granzyme B ◽  
Pancreatic Tumors ◽  
Specific Memory ◽  
Kpc Mice

ABSTRACTPancreatic ductal adenocarcinoma is highly metastatic, poorly immunogenic, and immune suppression prevents T cell activation. We developed a microbial-based immunotherapeutic concept for selective delivery of a highly immunogenic tetanus toxoid protein (TT856-1313) as an alternative for neoantigens, into tumor cells by attenuated Listeria monocytogenes, and reactivation of pre-existing TT-specific memory T cells (generated during childhood) to kill infected tumor cells. Thus, TT here functions as the tumor antigen. Treatment of KPC mice with Listeria-TT resulted in TT accumulation inside tumor cells, and attraction of the reactivated TT-specific memory CD4 T cells. Gemcitabine (GEM) combined with Listeria-TT significantly improved the migration of CD4 T cells into tumors and the production of perforin and granzyme B, turning cold into immunological hot tumors. The number of CD8 T cells in the KPC tumors was 3-fold lower than that of CD4 T cells. Moreover, lymph node like structures (LNS) were observed in close contact with the pancreatic tumors exhibiting CD4 T cells (and less abundantly CD8 T cells) of all treatment groups, but most frequently in KPC mice treated with Listeria-TT or Listeria-TT+GEM. Notably, the production of granzyme B (and less of perforin) was observed in the LNS of Listeria-TT+GEM only. The Listeria-TT+GEM treatment significantly reduced pancreatic tumors and metastases by 80% in Panc-02 and KPC mouse models, with minimal side effects. Our results unveil new mechanisms of Listeria and GEM improving immunotherapy for PDAC.

scholarly journals CD4+ T cells sustain aggressive chronic lymphocytic leukemia in Eμ-TCL1 mice through a CD40L-independent mechanism

2021 ◽  
Vol 5 (14) ◽  
pp. 2817-2828
Author(s):  
Matteo Grioni ◽  
Arianna Brevi ◽  
Elena Cattaneo ◽  
Alessandra Rovida ◽  
Jessica Bordini ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Antigen Specificity

Abstract Chronic lymphocytic leukemia (CLL) is caused by the progressive accumulation of mature CD5+ B cells in secondary lymphoid organs. In vitro data suggest that CD4+ T lymphocytes also sustain survival and proliferation of CLL clones through CD40L/CD40 interactions. In vivo data in animal models are conflicting. To clarify this clinically relevant biological issue, we generated genetically modified Eμ-TCL1 mice lacking CD4+ T cells (TCL1+/+AB0), CD40 (TCL1+/+CD40−/−), or CD8+ T cells (TCL1+/+TAP−/−), and we monitored the appearance and progression of a disease that mimics aggressive human CLL by flow cytometry and immunohistochemical analyses. Findings were confirmed by adoptive transfer of leukemic cells into mice lacking CD4+ T cells or CD40L or mice treated with antibodies depleting CD4 T cells or blocking CD40L/CD40 interactions. CLL clones did not proliferate in mice lacking or depleted of CD4+ T cells, thus confirming that CD4+ T cells are essential for CLL development. By contrast, CD8+ T cells exerted an antitumor activity, as indicated by the accelerated disease progression in TCL1+/+TAP−/− mice. Antigen specificity of CD4+ T cells was marginal for CLL development, because CLL clones efficiently proliferated in transgenic mice whose CD4 T cells had a T-cell receptor with CLL-unrelated specificities. Leukemic clones also proliferated when transferred into wild-type mice treated with monoclonal antibodies blocking CD40 or into CD40L−/− mice, and TCL1+/+CD40−/− mice developed frank CLL. Our data demonstrate that CD8+ T cells restrain CLL progression, whereas CD4+ T cells support the growth of leukemic clones in TCL1 mice through CD40-independent and apparently noncognate mechanisms.

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.

scholarly journals Granzyme B secretion by human memory CD4 T cells is less strictly regulated compared to memory CD8 T cells

2014 ◽  
Vol 15 (1) ◽  
Author(s):  
Lin Lin ◽  
Jacob Couturier ◽  
Xiaoying Yu ◽  
Miguel A Medina ◽  
Claudia A Kozinetz ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Granzyme B ◽  
Human Memory ◽  
Memory Cd8 T Cells ◽  
Memory Cd4 T Cells

Lack of IL-21 Signal Attenuates Graft-Versus-Leukemia Effect In the Absence of CD8 T-Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3739-3739
Author(s):  
Katsutoshi Ozaki ◽  
Akiko Meguro ◽  
Keiko Hatanaka ◽  
Iekuni Oh ◽  
Haruko Matsu ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Line ◽  
Dose Reduction ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Luciferase Activity ◽  
Critical Role ◽  
Leukemic Cells ◽  
Dose Titration ◽  
Wild Type

Abstract Abstract 3739 Introduction: IL-21 is a pleiotropic cytokine belongs to a common cytokine receptor g chain family of cytokines. IL-21 is mainly produced by activated CD4+ T cells and acts on T-, B-, NK-cells, and other lineages. IL-21 can drive Th17 differentiation and contributes to the development of autoimmune disease. Previously, we have shown that IL-21R−/− (KO) splenocytes ameliorate GVHD as compared to wild type (WT) splenocytes (BMT 2010), indicating a critical role of IL-21 in GVHD. Bucher et al. reported that IL-21 neutralization resulted in the same results as ours and it did not attenuate GVL effect (Blood 2009). However, they did not titrate the required T-cells for GVL effect, making it impossible to determine if the GVL strength was similar in the normal and the IL-21 neutralized conditions. Here, we sought to quantify and compare the strength of GVL effect between WT and KO splenocytes (SP), and moreover, analyze the contributions of CD4 and CD8 cells to GVL effect. Methods: GVL experiments were performed by co-transplantation of P815 leukemic cell line, T-cell depleted wild type bone marrow cells (TCD-BM), and either KO-SP vs. WT-SP. C57BL/6-DBA F1 strain mice were used as recipients. To make the leukemia visible in alive mice, we used luciferase transduced P815 cell line and IVIS® imaging system. Results: Previously (ASH 2008), we have shown that IL-21R−/− splenocytes (KO-SP) retained GVL effect and that IL-21 decoy receptor treatment retained GVL effect. To confirm previous results, here we decided to perform dose-reduction experiment to determined the number of splenocytes required to eliminate leukemic cells after transplantation, as otherwise it is impossible to compare the strength of GVL effect between the WT and KO cells. We first started with 1 × 107 splenocytes. Without co-infusion of splenocytes, control mice died around 30–40 days after transplantation with a marked increase of luciferase activity, whereas recipients of both WT-SP and KO-SP demonstrated an eradication of P815 leukemic cells. In addition, more mice died in recipients of WT because of more severe GVHD. Secondly, we used 5 ×106 splenocytes; with this number, almost no mice died due to GVHD anymore, but still graft eradicated leukemic cells completely. Thirdly, we used 5 × 105 and 5 × 104; with these numbers, graft could not eliminate leukemic cells anymore and mice died due to leukemia. Taken together, the threshold to eradicate P815 cells in our experimental conditions was between 5 × 105 and 5 × 106 of splenocytes, regardless of genotype, WT or KO. In the experiments above with bulk splenocyte, GVL effect with KO-SP was almost comparable to that with WT-SP even throughout in the titration. However, our previous experiments demonstrated that IL-21R−/− CD4+ T-cells are defective in GVH reaction after transplantation (J Immunol. 2010). It was therefore of great interest to determine whether IL-21R−/− CD4+ T-cells are also defective in GVL effect. To evaluate the contribution of CD4+ T-cells to GVL effect, we performed transplantation with CD8-depleted splenocytes with dose-reduction as above. Because CD8 T-cells compose of only ≂f10% of splenocyte, we chose CD8-depletion rather than CD4 purification, so we could use the same dose of CD8-depleted splenocytes as in the case of bulk splenocytes. CD8-depleted KO-SP at the dose of 5 × 107 demonstrated attenuated GVHD and prolonged survival, consistent with our previous results. However, interestingly, CD8-depleted KO-SP at the dose of 5 × 106 demonstrated diminished GVL effect. Higher luciferase activity and more deaths in the KO-SP group indicated leukemic deaths. According to luciferase activity, with CD8-depleted KO-SP at the dose of 5 × 106 cells, 10 out of 21 recipients showed leukemic growth at day 21, whereas for CD8-depleted WT-SP, only 3 out of 21 mice showed leukemic growth at day 21; only 7 out of 21 mice survived in recipients of KO-SP but 13 out of 21 mice survived in recipients of WT-SP at day 100 after transplantation. Conclusion: Here we demonstrated that IL-21R−/− splenocytes (KO-SP), which ameliorate GVHD, do not attenuate GVL effect even in the splenocyte-dose-titration. In the further detailed analysis with CD8-depleted splenocytes, GVL effect with IL-21R−/− CD8-depleted splenocytes was significantly diminished compared to that with wild type, suggesting that IL-21R−/− CD4 cells have lower GVL activity than wild type cells. Disclosures: Ozawa: Nippon Shinyaku Co., Ltd.: Research Funding.

The Mutated Region of Cytoplasmatic Nucleophosmine 1 (NPM1) Elicits Both CD4+ and CD8+ T Cell Responses

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2569-2569
Author(s):  
Jochen Greiner ◽  
Yoko Ono ◽  
Susanne Hofmann ◽  
Vanessa Schneider ◽  
Anita Schmitt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Healthy Volunteers ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Granzyme B ◽  
T Cell Responses ◽  
Interferon Γ ◽  
Cell Responses ◽  
Hla Dr

Abstract Abstract 2569 Introduction In AML, mutations in the nucleophosmin (NPM1) gene are one of the most frequent molecular alterations and predominantly occur in AML with normal cytogenetics. Patients with NPM1 mutation without FLT3-ITD mutation show a favourable prognosis of their disease. The functional role of mutated NPM1 for the improved clinical outcome is under evaluation. Immune responses might be involved in the clinical outcome of the disease. In this work, we demonstrate both CD4+ and CD8+ T cell responses against the mutated region of NPM1. Methods The entire amino acid sequences of the NPM1 wild type protein as well as of the mutated cytoplasmic NPM1 types A, B, C and D were screened for HLA-A*0201 binding T cell epitopes using the algorithms of the SYFPEITHI, the Rankpep and the HLA-Bind software programs. Ten peptides with most favourable characteristics were subjected to ELISpot analysis for interferon-γ and granzyme B in 22 healthy volunteers and 27 AML patients to test specific T cell responses of CD8+ T cells. Tetramer assays against the two most interesting epitopes have been performed and chromium release assays have been used to show the cytotoxicity of peptide-specific T cells to lyse T2 cells and leukemic blasts. Moreover, HLA-DR binding epitopes were screened in algorithmic analysis and HLA-DR*0701 binding peptides were exploited to stimulate CD4+ T cells. In the presence of overlapping peptide stimulated CD4+ T cells, NPM1-A specific CD8+ T cells revealed augmented interferon-γ and granzyme B secretion and up-regulation of intracellular interferon-γ. CD4+, CD4-CD8+, CD4-CD8- cell fractions were separated from PBMCs of HLA-A2+DR*0701+ healthy volunteers using a combination of CD4 and CD8 MicroBeads. Results Two epitopes (P3 and P9) derived from the NPM1-mutated protein showed specific T cell responses in healthy volunteers and AML patients. In NPM1-mutated AML patients 33% showed immune responses of CD8+ T cells against peptide P3 and 42% against peptide P9. Specific lysis was detected in chromium release assays NPM1 peptide-primed effector T cells generated from NPM1-mutated AML patients. Tetramer assays showed peptide-specific T cells. To obtain a robust and effective immune response against tumor cells, the activation of CD4 + helper T cells is crucial. Thus NPM1-peptide-A overlapping MHC class II epitopes were searched by primary structure analysis program. Based on plenary search, eight favourable overlapping peptides OL 1–8 were synthesized and exploited for CD4+ T cell stimulation. In granzyme B ELISPOT assay, OL8 co-pulsed NPM1-A CD8+ T cells indicated notable S.I., in contrast other OL1-7 disabled to increase granzyme B secretion. To ensure that Th1 cytokine secretion, under the condition of CD8+ and CD4+ T cells mixed culture, was resulted from NPM1-A CD8+ T cells but not HLA-DR epitope stimulated CD4+ T cells activation, HLA-A2 blocking effect was confirmed in ELISPOT assay. NPM1-A CD8+ T cells co-pulsed with OL6, 7 and 8 showed lesser interferon-γ secretion after HLA-A2 blocking antibody exposure as 73, 35 and 57%. Of note, 83–94% of granzyme B secretion levels were reduced by HLA-A2 blockade administration, and by which NPM1-A CD8+ T cells seemed to be the most probable IFN-gamma and granzyme B producers and CD4+ T cells to interfere with CD8+ T cells. Conclusion Taken together, mutated NPM1 is a promising target structure for specific immunotherapies in AML patients. Disclosures: No relevant conflicts of interest to declare.

Immune Cell Dysfunction in Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3875-3875 ◽  
Author(s):  
Marion E Cole ◽  
Alexander MacFarlane ◽  
Mowafaq Jillab ◽  
Mitchell R Smith ◽  
Adam D Cohen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Nk Cell ◽  
Granzyme B ◽  
Immune Dysfunction ◽  
T Cell Subsets ◽  
Cell Compartment

Abstract Abstract 3875 Introduction: Immunologic environment influences progression of lymphoid malignancies. Specifically, shifts in subsets of natural killer (NK) and T cells as well as tumor expression of inhibitory ligands may contribute to ability to evade host detection. Immune dysfunction may be particularly important in CLL/SLL, as prevalent circulating tumor cells engage in persistent, widespread interactions with immune cells; commonly-used mAb therapies (e.g. rituximab, alemtuzumab) rely upon ADCC mediated by NK cells and other innate effectors; and disease course is highly variable and not fully accounted for by tumor-intrinsic prognostic factors. Therefore, to better characterize the immune system in CLL/SLL, we prospectively assessed NK and T cell frequency, phenotype, and function in a series of CLL/SLL patients. Methods: Serial blood samples (up to 3 samples each, 3–6 months apart) were collected from 31 untreated CLL/SLL patients (median age 66) and 15 healthy age-matched controls (HC), and peripheral blood lymphocytes (PBL) analyzed directly ex vivo by multiparameter flow cytometry (160 distinct parameters evaluated, primarily on T and NK cells). NK cell-mediated natural and antibody-dependent cytotoxicity were also assessed by CD107a degranulation assay following PBL co-culture with rituximab, 721.221 EBV-transformed lymphoma cells, or both. Differences in parameters between patients and controls, or between progressors and non-progressors [categorized based on updated NCI-WG criteria (Blood 2008;111:5446)] were analyzed by Wilcoxon rank-sum test. All subjects signed IRB approved informed consent forms. Results: CLL/SLL VS. HC: CLL/SLL samples displayed a marked decrease in the ability of the cytolytic CD56dim NK cells to degranulate in response to tumor, both with or without rituximab (Table 1). CD56dim NK cells from CLL/SLL patients also displayed a more immature phenotype (↓CD57, ↓NKG2D, ↑CD27, ↓KIR) than those from HC, suggesting either a block in differentiation or elimination of the most-differentiated cells. NK cell expression of NKp44, CD69, CD62L, CD137, granzyme B, perforin, or PD-1, as well as tumor-induced NK cell production of IFNγ, did not differ. CLL/SLL patients had increased total T cells with a decreased CD4:CD8 ratio, associated with increased total number of CD8 T cells, greater activation of naive CD4 T cells and transition to a memory phenotype. Treg (CD4+CD25+FoxP3+) frequency was significantly higher in CLL/SLL patients (4.5% vs. 1.8% of CD4 T cells, p=0.005), as was PD-1 expression on both CD4 and CD8 T cells, while CD137 and ICOS expression was similar in both groups. PROGRESSORS VS. NON-PROGRESSORS: With median follow-up of 16.5 months (range 1–37), 7 of 31 patients have met criteria for progression. Compared to non-progressors, progressors showed changes in the CD56bright NK cell compartment suggestive of increased activation and accelerated differentiation, with increased expression of CD69, granzyme B, perforin, CD16, and KIR. However, no significant functional differences in NK cells, or consistent differences in T cell subsets, have been observed to date. Conclusions: CLL/SLL patients have a shift toward less mature NK cells, associated with deficits in NK cell degranulation against tumor targets, compared with healthy donors. Those CLL/SLL patients who progressed had greater CD56 bright NK cell phenotypic aberrancies than non-progressors, though these findings require confirmation with a larger cohort. Taken together, our findings support the hypothesis that immune dysfunction in CLL/SLL may be due in part to a block in NK cell differentiation or loss of more mature cells, and current studies are exploring these possibilities and potential mechanisms. Given these findings, along with the immunosuppressive changes observed in the T cell compartment (↑Tregs, ↑PD-1), these data support therapeutic strategies in CLL/SLL aimed at augmenting NK and/or T cell function. Disclosures: No relevant conflicts of interest to declare.

Melanoma-released exosomes directly activate the mitochondrial apoptotic pathway of CD4+ T cells through their microRNA cargo

2018 ◽  
Vol 371 (2) ◽  
pp. 364-371 ◽  
Author(s):  
Ji Zhou ◽  
Yi Yang ◽  
WenWen Wang ◽  
Yuan Zhang ◽  
ZhengRong Chen ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Apoptotic Pathway ◽  
Mitochondrial Apoptotic Pathway

Proliferation and Activation Patterns of Naïve, Memory and Regulatory T Cells in Patients with Multiple Myeloma During Thalidomide, Interferon-α and Bortezomib Maintenance Therapy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3880-3880
Author(s):  
Michael Hundemer ◽  
Isabelle Herth ◽  
Tobias Meissner ◽  
Dirk Hose ◽  
Anthony D Ho ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Regulatory T Cells ◽  
Maintenance Therapy ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Granzyme B ◽  
Effector Memory ◽  
Control Group ◽  
Ifn Γ

Abstract Abstract 3880 Poster Board III-816 Hundemer and Herth (Contributed equally) Introduction In patients with Multiple myeloma, maintenance therapy after high-dose chemotherapy and autologous stem cell transplantation is performed with the aim to prolong remission duration and survival. Beside IFN-α, thalidomide and bortezomib are increasingly applied in maintenance protocols. In this prospective study we have analysed the implication of the various types of maintenance therapy on the patients T cell pattern and activation status. Patients and Methods T cells from 63 patients in clinical remission were analyzed. The median duration of remission was 38,6 months. Eighteen patients were treated with IFN-α, 22 with thalidomide, 7 with bortezomib and 16 patients received no maintenance therapy (control group). Peripheral blood mononuclear cells were isolated and stimulated with CD3/CD28 beads. Activated and nonactivated T cells were analyzed by flow cytometry (CD45RA, CD45RO, CCR7, CD28, CD200R, CD95, CD279, CD69, CD134 and TCRγ/δ) and ELISA (IFN-γ, perforine and granzym B). Furthermore the rate of IFN-γ-producing and regulatory T cells were analyzed by intracytoplasmatic staining and flow cytometry. Results All groups including the control group showed an up-regulation of CD69 and CD134 on CD4+ and CD8+ T cells after activation (p<0,001), on CD8+ T cells in the bortezomib-group only CD69 was upregulated (p=0,008). Patients treated with IFN-α showed a high rate of naïve T cells (CD45RA- and CCR7-positive), while in the thalidomide-group a high rate of effector memory T-cells (CD45RA- and CCR7-negative) were observed (CD45RA on CD8+ and CD4+ T cells: p<0,001, CCR7 on CD8+ T cells: p=0,03, CCR7 on CD4+ T cells: p=0,003). Regarding the surface marker CD28 on CD8+ T cells the IFN-α-group demonstrated a significant higher expression than the control-group (p=0,04) and the bortezomib-group a significant lower expression than the IFN-α- and the thalidomide-group (p=0,006 and p=0,02). Furthermore the rate of IFN-γ-producing CD4+ T cells was significant higher in the thalidomide-group than in the IFN-α-group after activation (p=0,02). On the basis of the cytoplasmatic staining of Foxp3 there was a trend to a higher amount of regulatory T cells in the thalidomide-group compared to the IFN-α-group (p=0,07). Analysis of IFN-y secretion by ELISA, an increases IFN-γ secretion could be demonstrated in all groups after activation (control group: p=0,002, IFN-α-group: p<0,001, thalidomide-group: p<0,001, bortezomib group: p=0,01), furthermore in all groups despite the bortezomib-group an increase of the granzyme B-production can be observed (control group: p=0,003, IFN-α-group: p=0,03, thalidomide-group: p<0,001). Regarding the activated state of the T cells the production of IFN-γ, perforine and granzyme B was significant higher in the thalidomide-group than in the IFN-α-group (IFN-γ: p=0,05, perforine: p=0,02, granzyme B: p=0,04). Furthermore the nonactivated and the activated T cells of the patients treated with thalidomide showed a significant higher production of granzyme B than the T cells of the control group (p=0,0003 and p=0,006). Conclusion During maintenance therapy, thalidomide promotes maturation and proliferation of effector memory T cells and regulatory T cells, while IFN-α treatment increases the number of naïve T cells and subsequently, the T cell activation in the thalidomide group was significantly higher than in the IFN-α group. These results have profound impact on the development of novel immunomodulating therapy strategies in the treatment of multiple myeloma. Disclosures: No relevant conflicts of interest to declare.

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