P16.06 Immunophenotyping of tumor-infiltrating T cells in primary CNS lymphoma

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii57-ii57
Author(s):  
S Schliffke ◽  
C Maire ◽  
M Holz ◽  
C Bokemeyer ◽  
M Westphal ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Primary Cns Lymphoma ◽  
Great Promise ◽  
Cns Lymphoma ◽  
High Dose ◽  
Dismal Prognosis

Abstract BACKGROUND Primary CNS lymphoma represents a malignant disease with dismal prognosis. Standard of care is high dose chemotherapy and radiation. However, this combination cannot be applied to the elderly and fragile population. Immunotherapy holds great promise to be effective in these patients. This study therefore aims to explore the phenotype of tumor-infiltrating lymphocytes (TIL) in order to analyze the potential for immune checkpoint inhibition. MATERIAL AND METHODS We performed ex vivo multicolor flow-cytometry on surgical specimens of nine patients with intracerebral lymphoma, including seven with primary CNS lymphoma after isolation of TILs following standard protocols. Data was analyzed using a Fortessa LSR flow cytometer and Diva software. The study was approved by the local ethics committee (PV4904). RESULTS Our ex vivo phenotyping demonstrated a predominant infiltration of CD8+ T cells, which outnumber CD4+ T cells by a ratio of 2:1 (p<0.01). Regulatory T cells (Tregs) were not increased in the tumor microenvironment and the NK cell frequency was reduced compared to the peripheral blood. While CD4+ T helper cells displayed significantly increased surface expression of multiple activation and checkpoint markers, including TIGIT, PD-1, Tim3 and CD57, cytotoxic CD8+ T cells predominantly expressed only TIGIT and PD-1. On average 70% and 80% of CD8+ T cells expressed PD-1 and TIGIT, respectively, compared to 35% and 60% of PD-1 and TIGIT on CD4+ T cells (p<0.05). CD8+ T cells furthermore showed an increased expression of CD39 and a simultaneous downregulation of CD73, both ectoenzyms involved in the modulation of intratumoral ATP, thereby indicating a metabolic immune modulation by the tumor. CONCLUSION Taken together, our study demonstrates a strong infiltration of cytotoxic CD8+ T cells into cerebral lymphoma, which potentially can be disinhibited using checkpoint immunotherapy. Our profiling suggests that PD-1 and TIGIT present appealing targets for such kind of immune disinhibition.

Ex Vivo Fludarabine Selectively Depletes Human Naive T-Cell Subsets: A Step Toward Modifying Donor Lymphocyte Infusion.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1071-1071
Author(s):  
Melody M. Smith ◽  
Cynthia R. Giver ◽  
Edmund K. Waller ◽  
Christopher R. Flowers
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Memory T Cells ◽  
Ex Vivo ◽  
T Cell Subsets ◽  
Effector Memory ◽  
Naïve T Cell ◽  
Naive T Cell

Abstract Ex vivo modification of donor lymphocytes with purine analogs (mDL) may help to minimize graft versus host disease (GvHD) while providing beneficial graft versus leukemia (GvL) effects. In a murine model system, we have shown that allogeneic donor splenocytes, treated with fludarabine ex vivo have significantly reduced GvHD activity when transferred to irradiated recipient mice, and retain anti-viral and GvL activities (Giver, 2003). This effect appears to be mediated by relative depletion of donor CD4 CD44low, “naive” T-cells. As a first step toward developing mDL for use in patients, we sought to evaluate the effects of ex vivo fludarabine exposure on human T-cell subsets, and to determine the minimum dose of fludarabine required to achieve this effect. Methods: Peripheral blood mononuclear cell samples from 6 healthy volunteers were evaluated at 0, 24, 48, and 72 hour time points after ex vivo incubation in varying dosages of fludarabine: 2, 5, and 10(n=3) mcg/ml. Fludarabine incubated samples were compared to samples that received no fludarabine (untreated). The total viable cell number was determined and the fractions and absolute numbers of viable CD4 and CD8 naïve and memory T-cells were determined using flow cytometry after incubation with 7-AAD (dead cell stain), CD4, CD8, CD45RA, CD62L, and CCR7 antibodies, and measuring the total viable cells/ml. Results: The numbers of viable CD4 and CD8 T-cells remained relatively stable in control cultures. Without fludarabine, the average viability at 72 hr of naive and memory T-cells were 92% and 77% for CD4 and 86% and 63% for CD 8 (Fig. 1A). Naive CD4 T-cells were more sensitive to fludarabine-induced death than memory CD4 cells. At 72 hr, the average viability of fludarabine-treated naive CD4 T-cells was 33% at 2 mcg/ml (8.2X the reduction observed in untreated cells) and 30% at 5 mcg/ml, while memory CD4 T-cells averaged 47% viability at 2 mcg/ml (2.3X the reduction observed in untreated cells) (Fig. 1B) and 38% at 5 mcg/ml. The average viability of naive CD8 T-cells at 72 hr was 27% at 2 mcg/ml and 20% at 5 mcg/ml, while memory CD8 T-cell viability was 22% at 2 mcg/ml and 17% at 5 mcg/ml. Analyses on central memory, effector memory, and Temra T-cells, and B-cell and dendritic cell subsets are ongoing. The 5 and 10 mcg/ml doses also yielded similar results in 3 initial subjects, suggesting that 2 mcg/ml or a lower dose of fludarabine is sufficient to achieve relative depletion of the naive T-cell subset. Conclusions: Future work will determine the minimal dose of fludarabine to achieve this effect, test the feasibility of using ex vivo nucleoside analog incubation to reduce alloreactivity in samples from patient/donor pairs, and determine the maximum tolerated dose of mDL in a phase 1 clinical trial with patients at high risk for relapse and infectious complications following allogeneic transplantation. Figure Figure

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.

NKG2D, An NK Cell Activating Receptor on CD8+ T Cells, Plays An Essential Role In Killing Myeloma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2087-2087 ◽  
Author(s):  
Laleh Talebian ◽  
Kenneth Meehan ◽  
Megan E Patey ◽  
Dawn A Fisher ◽  
Zbigniew Szczepiorkowski ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Cell Transplant ◽  
Mhc I ◽  
Myeloma Cells ◽  
Post Transplant ◽  
Autologous Transplant ◽  
Activating Receptor

Abstract Abstract 2087 Autologous stem cell transplant for myeloma improves response rates, but the immunologic mechanisms contributing to this improvement are unknown. Our results indicate that this beneficial effect may be due to a subpopulation of CD8+T cells that express an NK cell activating receptor, called NKG2D. NKG2D is present on some CD8+ T cells that mediate TCR-independent and non-MHC restricted tumor cell killing. Three NKG2D ligands (MICA, ULBP1, and ULBP3) are expressed on patients’ myeloma cells. Our data indicate that NKG2D expression can be up-regulated on some CD8+T cells making these cells highly effective at killing myeloma cells. We previously developed an ex vivo expansion method that enriches for NKG2D+CD8+T cells using mobilized blood progenitor cells (Cytotherapy 2008). These ex vivo expanded NKG2D+CD8+ T cells aggressively lysed myeloma cells and blocking the NKG2D receptor significantly inhibited this killing (p<0.0009). Due to these intriguing laboratory results, we conducted a phase II trial using adoptive cellular immunotherapy following autologous transplant, using ex vivo expanded cells enriched for NKG2D+CD8+ T cells. Myeloma patients received high-dose melphalan (200mg/m2) followed by an autologous transplant. Low dose IL-2 (6×105 IU/m2/d × 20 days) and GM-CSF (250 μg/m2/d) were administered following transplant. The ex vivo expanded cells (1 × 109 CD3+ T cells per infusion) were administered at weeks 1, 2, 3, and 8 following transplant. Nineteen of twenty-three patients are evaluable. Median engraftment of neutrophils was 13 days (range: 12–16 days) and 16 days for platelets (range: 12–26 days). All nineteen patients completed the full course of post-transplant IL-2. There were no treatment-related deaths. Due to the required CD3+T cell number in each of the ex vivo expanded cell infusions, fifteen patients received the ex vivo expanded cells (4 infusions n = 9; 3 infusions n = 3; 2 infusions n = 2; 1 infusion n = 1). Transplant-related adverse effects (> Grade 3) included nausea/vomiting (n=2), fever (n=7), elevated AST/ALT (n=2), anorexia (n=2), pneumonia (n=2), enteritis (n=1), diarrhea (n=2), pulmonary embolism (n=1), or typhlitis (n=2).There was an increased number and function of NKG2D+CD8+T cells circulating in vivo post-transplant. At 1 month post-transplant, there was an increase in the number of NKG2D+CD8+T cells (p < 0.0004), CD3+CD8+ T cells (p < 0.027), CD8+CD56+T cells (p < 0.0086), and NKG2D+CD56+ T cells (p < 0.0036). The circulating NKG2D+CD8+T cells recognized and lysed autologous myeloma cells (p<0.005) and lysis was significantly inhibited by blocking NKG2D (p<0.0014). NKG2D ligand expression on patients’ myeloma cells strongly correlated with % cell lysis. These results suggest that NKG2D+CD8+ T cells recognize and kill autologous myeloma cells in an NKG2D-dependent manner. Since myeloma cells down regulate MHC-I, NKG2D+CD8+T cells’ MHC-I unrestricted killing of myeloma cells may improve outcomes in transplanted myeloma patients. Ongoing experiments will identify the kinetics of NKG2D+CD8+ T cells following transplant and the molecular mechanisms of cytotoxicity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Immunological Monitoring of CML Patients during First-Line Bosutinib and Imatinib Treatment

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3069-3069
Author(s):  
Anna Kreutzman ◽  
Perttu Koskenvesa ◽  
Kasanen Tiina ◽  
Ulla Olsson-Strömberg ◽  
Jesper Stentoft ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Nk Cell ◽  
T Cell Subsets ◽  
Imatinib Treatment ◽  
First Line

Abstract Background: Tyrosine kinase inhibitors (TKIs) used in the treatment of chronic myeloid leukemia (CML) are not entirely selective for the BCR-ABL1 kinase but also inhibit a variety of other kinases, sometimes triggering unpredicted biological effects. As an example, the TKIs dasatinib and bosutinib both inhibit Src-kinases, which are important mediators of T-cell function. Earlier in vitro data has shown that dasatinib can suppress activation and proliferation of T and NK cells, but it can also elicit signs of immunostimulation in patients, including rapid mobilization of lymphocytes and LGL lymphocytosis. No extensive analyses of the immunological in vivoeffects of bosutinib have been performed thus far. Therefore, we aimed at characterizing T and NK cell phenotypes and functional features in CML patients in a clinical setting in the context of first-line bosutinib and imatinib treatment. Methods:Peripheral blood samples were obtained from newly diagnosed CML CP patients enrolled in the BFORE clinical trial (NCT02130557), receiving bosutinib (n=13) or imatinib (n=20) as frontline TKI treatment. Samples were drawn at diagnosis and following 3 and 12 months of therapy. Detailed immunophenotyping of NK and T cells was performed with multicolor flow cytometry. In addition, mononuclear cells were used to study the function of NK and T cells (CD107ab degranulation upon stimulation with K562 cells and detection of IFN-γ/TNF-α secretion after stimulation with anti-CD3/anti-CD28 antibodies, respectively). Moreover, blood differential counts were taken before and 2 hours after drug intake at 3 and 12 months to examine the direct effects on lymphocyte counts (mobilization). Results: No significant changes were observed in absolute white blood cell or lymphocyte counts directly (2 hours) after bosutinib or imatinib intake, in contrast to what has been observed in dasatinib treated patients. Analysis of T cell subsets during bosutinib treatment revealed that the proportion of CD4+ cells increased after the start of treatment (median dg. 60.0% vs. 3 months 62.0% p=0.06; vs. 12 months 72.8% p=0.03), but no significant changes were observed in the phenotype. Correspondingly, the proportion of CD8+ T-cells decreased moderately (dg. 31.6% vs. 3 months 25.5% p=0.01) after the therapy start. Interestingly, the proportion of PD1+ (dg. 19.6% vs. 3 months 11.9%, p=0.06; vs. 12 months 14.3%, p=0.11) and DNAM+ CD8+ T-cells decreased (dg. 73.1% vs. 3 months 66.2% p=0.004; vs. 12 months 64.6% p=0.02). No changes in the cytokine production of any of the studied subgroups of T-cells was observed. Moreover, the proportion, phenotype and function of NK-cells were not affected by bosutinib treatment. In contrast, during imatinib treatment the proportion of CD56+CD16+ NK-cells significantly increased (dg 4.3% vs. 3 months 9.9% p=0.0005; vs 12 months 14.4% p=0.002; 8.1% in bosutinib treated patients). Moreover, in imatinib patients NK-cells downregulated CD27 (dg 9.0% vs. 3 months 5.2% p=0.004; vs. 12 months 4.9%; p=0.002). Further, NK-cells from imatinib-treated patients expressed more CD107ab upon stimulation with K562 at 3 and 12 months, when compared to samples from diagnosis (dg 13.0% vs. 3 months 16.1%, p=0.01; vs. 12 months 23.2%, p=0.008). The proportion of CD4+ T-cells increased 3 months after the start of imatinib treatment (dg 60.1% vs. 3 months 63.5% p=0.01), whereas the percentage of CD8+ T-cells decreased (dg. 38.6% vs. 3 months 31.5% p=0.02). Decreased expression of DNAM (dg 73.5% vs. 3 months 67.9% p=0.0008; vs. 12 months 62.4% p=0.002) was observed in the CD4+ T-cells. Similarly as in bosutinib treated patients, the proportion of PD1+ CD8+ cells decreased during imatinib treatment (dg 18.2% vs. 3 months 14.7%, p=0.02; vs. 12 months 14.8%, p=0.03). Both CD4+ and CD8+ T-cell subsets from imatinib-treated patients secreted less cytokines after the start of treatment when compared to the pre-treatment samples. Conclusions: Despite of the Src-kinase inhibitory profile of bosutinib, no major changes were observed in T- or NK-cell phenotype or function during first-line bosutinib treatment. In contrast, in imatinib treated patients the proportion of NK-cells increased and their degranulation responses were significantly higher than in untreated CML patients. Comparison of these data with the clinical variables and treatment outcome is warranted. Disclosures Stentoft: Novartis: Research Funding; Bristol-Myers-Squibb: Research Funding; Pfizer: Research Funding; Ariad: Research Funding. Gjertsen:BerGenBio AS: Consultancy, Research Funding. Janssen:Pfizer: Honoraria; Novartis: Research Funding; Ariad: Honoraria; BMS: Honoraria. Brümmendorf:Pfizer: Research Funding; Novartis: Research Funding. Richter:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Mustjoki:Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

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.

scholarly journals 195 Stimulation of tumor infiltrating B-cells improves ex-vivo TIL expansion for melanoma immunotherapy

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A206-A206
Author(s):  
Renata Rossetti ◽  
Leticia Tordesillas ◽  
Matthew Beatty ◽  
Yian Ann Chen ◽  
Dongliang Du ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Tumor Infiltrating Lymphocytes ◽  
Effector Memory ◽  
Cancer Center ◽  
High Dose ◽  
Standard Protocol ◽  
Melanoma Tumor

BackgroundThe immunogenic nature of melanoma has been exploited for the development of adoptive transfer of ex-vivo expanded tumor infiltrating lymphocytes (TIL). This adoptive cell transfer therapy has overall response rates of around 50%. Multiple factors may determine the quality of the TIL product including components of the tumor microenvironment. B-cells are frequently found in melanoma metastasis, and display signs of antigen experience. Recently, B-cell tumor infiltration has been associated with improved clinical responses to immune checkpoint inhibitors,1 2 but their role in TIL therapy remains unexplored. Considering the potential role of B cells, we aim to develop strategies to enhance the quality of TIL products through B-cell stimulation during ex-vivo TIL expansion.MethodsWe stimulated melanoma infiltrating B-cells using human recombinant CD40L on the first day of ex-vivo TIL expansion. Thirteen samples were expanded from melanoma tumor single cell suspensions, in high dose IL-2 alone (standard protocol), or in high dose IL-2 plus CD40L. After up to four weeks of expansion, the TIL phenotype was analyzed by flow cytometry.ResultsThe expansion success rate from the frozen tumor digests was 69% (95% CI: 38.6–90.9%) in the CD40L treatment condition compared to 23% with the standard protocol. Also, TILs cultured in the presence of CD40L expanded to higher numbers than with the standard protocol (P = 0.02). Interestingly, most of the samples expanded with CD40L had a significant increase in the percentage of CD4+ T cells (P = 0.03), but not to the detriment of the absolute number of CD8+ T cells. Treatment with CD40L increased the percentage of effector memory-like T cells (P = 0.03) and of CD39- CD69- T cells (P < 0.05), which were recently associated with response to TIL therapy.3ConclusionsThis preliminary work demonstrates that the stimulation with CD40L at the initiation of TIL culture leads to enhanced TIL expansion and an increase in CD4+ T cells with an effector memory-like and stem-like phenotype. Our group and others have previously described cases of patients who had tumor regression after receiving TIL therapy that were predominantly CD4+ T cells, suggesting that expansion of the CD4+ TIL repertoire may enhance TIL therapy.4AcknowledgementsThis work has been supported in part by the Flow Cytometry, Genomics and Biostatistics and Bioinformatics Core Facilities at Moffitt Cancer Center, an NCI designated Comprehensive Cancer Center (P30-CA076292). We acknowledge Moffitt’s Melanoma Center of Excellence for the financial support.ReferencesCabrita R, Lauss M, Sanna A. Tertiary lymphoid structures improve immunotherapy and survival in melanoma. Nature 2020;577:561–565.Petitprez F, de Reynies A, Keung EZ. B cells are associated with survival and immunotherapy response in sarcoma. Nature 2020;577:556–560.Krishna S, Lowery FJ, Copeland AR. Stem-like CD8 T cells mediate response of adoptive cell immunotherapy against human cancer. Science 2020;370:1328–1334.Friedman KM, Prieto PA, Devillier LE. Tumor-specific CD4+ melanoma tumor-infiltrating lymphocytes. J Immunother 2012;35:400–408.Ethics ApprovalThe study was approved by Advarra IRB, approval number MCC20559.

scholarly journals First Evidence of Restoration of T and NK Cell Compartment after Venetoclax Treatment

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1860-1860 ◽  
Author(s):  
Iris de Weerdt ◽  
Tom Hofland ◽  
Johan Dobber ◽  
Julie Dubois ◽  
Eric Eldering ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Cytokine Production ◽  
Nk Cell ◽  
Advisory Committees ◽  
Ifn Γ

Abstract Introduction Chronic lymphocytic leukemia (CLL) is characterized by a profound immune suppression. In addition, CLL cells evade immune destruction by interacting with cells of the adaptive immune system, resulting in dysfunctional T cells. CD4+ T cells are skewed towards a TH2-profile and the number of regulatory T (Treg) cells, that diminish cellular immune responses, is increased in CLL patients. CD8+ T cells resemble exhausted T cells and have reduced cytotoxic, yet increased cytokine production capacity. The cytotoxic function of NK cells is impaired in CLL patients, but in contrast to CD8+ T cells their cytokine production is also compromised, presumably induced by CLL cells. These data are chiefly obtained from studies on peripheral blood (PB). Although the lymph node (LN) compartment has a central role in the pathobiology of CLL, very little is known about the composition of non-malignant lymphocytes in LN tissue. The Bcl-2 inhibitor venetoclax (Ven) is highly effective in CLL and, especially in combination with anti-CD20 monoclonal antibodies such as obinutuzumab (O), results in high rates of minimal residual disease (MRD) undetectable responses. However, the prospective effects of venetoclax on non-malignant lymphocytes in patient samples remain largely unexplored. Methods PB and LN biopsy specimens were collected at baseline from patients enrolled in the 1st-line FCR-unfit HOVON 139 / GIVE trial. Study treatment consisted of O (cycle 1-2), Ven+O (cycle 3-8) and Ven (cycle 9-14). Immune composition was analyzed by 7-color flow cytometry. Baseline PB samples were compared to paired LN samples. Moreover, PB samples of the first patients that completed 6 cycles of Ven monotherapy (cycle 14) were compared to baseline. Cytokine production and degranulation of T and NK cells was studied after stimulation of PBMCs with PMA/Ionomycin. Results Comparison of LN (n=28) vs PB (n=48) revealed a larger proportion of T cells in LN (13.2% vs 5.1% of the lymphocytes), at the expense of CLL cells, with a skewed CD4:CD8 ratio (5.2 in LN vs 1.8 in PB). Within the CD4+ T cells, significantly higher levels of both follicular T helper cells (15. 7% vs 5.2%) and Tregs (11.5% vs 6.9%) were found in LN (see Table). CD4+ T cells mostly consisted of naïve and memory T cells in both PB and LN. There were fewer CD8+ T cells and especially fewer effector CD8+ T cells in the LN in comparison to PB. CD8+ T cells in LN mostly had a naïve and memory phenotype. An increased percentage of LN-residing CD8+ T cells expressed the exhaustion marker PD-1 as compared to PB CD8+ T cells (30.4% in LN vs 12.4% in PB). We then compared PB baseline samples to PB obtained after cycle 14 (n=11). Ten patients achieved MRD undetectable levels (<10-4, determined by flow cytometry) and 1 patient was MRD intermediate (10-4-10-2). As expected, the treatment regimen led to complete elimination of CD19+ B cells. In contrast, absolute numbers of CD4+ and CD8+ T cells did not change during treatment. Differentiation status of CD4+ and CD8+ T cells remained similar. Interestingly, the proportion and absolute number of Tregs decreased after treatment (6.1% vs 0.9% of CD4+ T cells). After stimulation with PMA/Ionomycin, the percentage of IL-2 producing CD4+ T cells increased after treatment, leading to a higher IL-2:IL-4 ratio, that suggests normalization towards a TH1-profile. Fewer CD8+ T cells expressed PD-1 after treatment. The fraction of CD8+ T cells that produced IFN-γ (69.8% vs 56.2%) and TNF-α (58.4% vs 40.3%) decreased. Degranulation of CD8+ T cells did not change upon treatment. After treatment, the capacity of NK cells to degranulate increased. In addition, a larger proportion of NK cells produced IFN-γ, suggesting recovery of NK cell function after treatment. Conclusion In conclusion, our data strengthen the view that CLL cells reside in an immune suppressive environment in the LN. Moreover, we provide the first evidence that the Ven+O regimen does not harm non-malignant lymphocyte populations other than B cells. Both the improved cytokine production of NK cells and diminished cytokine production of CD8+ T cells may point to normalization of immune function. Collectively, the phenotypical and functional changes observed may reflect the eradication of the immunosuppressive CLL clone by Ven+O and subsequent recovery of the immune microenvironment in CLL patients. Disclosures Eldering: Celgene: Research Funding. Mobasher:F. Hoffmann-La Roche Ltd: Other: Ownership interests non-PLC; Genentech Inc: Employment. Levin:Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Kater:Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Anti-Asialo GM1 treatment during secondaryToxoplasma gondiiinfection is lethal and depletes T cells

2019 ◽  
Author(s):  
Daria L. Ivanova ◽  
Steve L. Denton ◽  
Jason P. Gigley
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
Secondary Infection ◽  
Parasite Burden ◽  
Effector Memory

AbstractUsing vaccine challenge model ofT. gondiiinfection, we found that treatments with two commonly used for NK cell depletion antibodies resulted in different survival outcomes during secondary infection. Anti-ASGM1 resulted in 100% death and greater parasite burden at the site of infection than anti-NK1.1. Anti-NK1.1 treatment resulted in increased parasite burdens, but animals did not die. Further we found that anti-ASGM1 treatment depleted T cells. CD8+ T cells were more susceptible that CD4+ T cells to the treatment. ASGM1 was expressed on a higher percentage of CD8+ T cells than CD4+ T cells and CD8+ T cells. InT. gondii-immunized animals ASGM1 was enriched on effector memory (Tem) and central memory (Tcm) CD8+ T cells. However, Tem were more susceptible to the treatment. After secondary infection, Tem, Tcm, effector (Tef) and naïve (Tn) CD8+ T cells were positive for ASGM1. Anti-ASGM1 treatment during reinfection resulted in greater depletion of activated IFNγ+, Granzyme B+, Tem and Tef than Tcm and Tn CD8+ T cells. Anti-ASGM1 also depleted IFNγ+ CD4+ T cells. Recombinant IFNγ supplementation prolonged survival of anti-ASGM1 treated mice, demonstrating that this antibody eliminated IFNγ-producing T and NK cells important for control of the parasite. These results highlight that anti-ASGM1 antibody is not an optimal choice for targeting only NK cells and more precise approaches should be used. This study uncovers ASGM1 as a marker of activated effector T cells and the potential importance of changes in sialylation in lipid rafts for T cell activation duringT. gondiiinfection.

scholarly journals Elimination of SHIV Infected Cells by Combinations of Bispecific HIVxCD3 DART® Molecules

2021 ◽  
Vol 12 ◽  
Author(s):  
Marina Tuyishime ◽  
Amir Dashti ◽  
Katelyn Faircloth ◽  
Shalini Jha ◽  
Jeffrey L. Nordstrom ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Viral Envelope ◽  
Host Immune System ◽  
Infected Cells ◽  
Anti Hiv ◽  
Hiv 1

Bispecific HIVxCD3 DART molecules that co-engage the viral envelope glycoprotein (Env) on HIV-1-infected cells and the CD3 receptor on CD3+ T cells are designed to mediate the cytolysis of HIV-1-infected, Env-expressing cells. Using a novel ex vivo system with cells from rhesus macaques (RMs) infected with a chimeric Simian-Human Immunodeficiency Virus (SHIV) CH505 and maintained on ART, we tested the ability of HIVxCD3 DART molecules to mediate elimination of in vitro-reactivated CD4+ T cells in the absence or presence of autologous CD8+ T cells. HIVxCD3 DART molecules with the anti-HIV-1 Env specificities of A32 or 7B2 (non-neutralizing antibodies) or PGT145 (broadly neutralizing antibody) were evaluated individually or combined. DART molecule-mediated antiviral activity increased significantly in the presence of autologous CD8+ T cells. In this ex vivo system, the PGT145 DART molecule was more active than the 7B2 DART molecule, which was more active than the A32 DART molecule. A triple combination of the DART molecules exceeded the activity of the individual PGT145 DART molecule. Modified quantitative virus outgrowth assays confirmed the ability of the DART molecules to redirect RM CD3+ T cells to eliminate SHIV-infected RM CD4+ T cells as demonstrated by the decreased propagation of in vitro infection by the infected cells pre-incubated with DART molecules in presence of effector CD8+ T cells. While mediating cytotoxic activity, DART molecules did not increase proinflammatory cytokine production. In summary, combination of HIVxCD3 DART molecules that have broadly-neutralizing and non-neutralizing anti-HIV-1 Env specificities can leverage the host immune system for treatment of HIV-1 infection but will require appropriate reactivation of the latent reservoir.

scholarly journals Characteristics of Malignant Pleural Effusion Resident CD8+ T Cells from a Heterogeneous Collection of Tumors

2020 ◽  
Vol 21 (17) ◽  
pp. 6178
Author(s):  
Rajeev Dhupar ◽  
Olugbenga T. Okusanya ◽  
Seth H. Eisenberg ◽  
Sara E. Monaco ◽  
Ayana T. Ruffin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Pleural Effusion ◽  
Cd8 T Cells ◽  
Malignant Pleural Effusion ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Great Promise ◽  
Primary Tumors ◽  
Indwelling Catheters

While T cell-based cancer immunotherapies have shown great promise, there remains a need to understand how individual metastatic tumor environments impart local T cell dysfunction. At advanced stages, cancers that metastasize to the pleural space can result in a malignant pleural effusion (MPE) that harbors abundant tumor and immune cells, often exceeding 108 leukocytes per liter. Unlike other metastatic sites, MPEs are readily and repeatedly accessible via indwelling catheters, providing an opportunity to study the interface between tumor dynamics and immunity. In the current study, we examined CD8+ T cells within MPEs collected from patients with heterogeneous primary tumors and at various stages in treatment to determine (1) if these cells possess anti-tumor activity following removal from the MPE, (2) factors in the MPE that may contribute to their dysfunction, and (3) the phenotypic changes in T cell populations that occur following ex vivo expansion. Co-cultures of CD8+ T cells with autologous CD45― tumor containing cells demonstrated cytotoxicity (p = 0.030) and IFNγ production (p = 0.003) that inversely correlated with percent of myeloid derived suppressor cells, lactate, and lactate dehydrogenase (LDH) within the MPE. Ex vivo expansion of CD8+ T cells resulted in progressive differentiation marked by distinct populations expressing decreased CD45RA, CCR7, CD127, and increased inhibitory receptors. These findings suggest that MPEs may be a source of tumor-reactive T cells and that the cellular and acellular components suppress optimal function.

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