HDACi induce cancer cell apoptosis and are in use for T-cell lymphoma. Prior studies have indicated that HDACi are immune-suppressive. In this study, we address this issue by examining the effects of ...two different HDACi on human monocyte derived dendritic cell (hmDC) biology.
hmDC were activated by TLR stimuli (LPS or poly:IC). HDACi effect on TLR-induced hmDC activation was assessed by up regulation of co-stimulatory molecules CD80, CD86 and CD83 along with secretion of IL-12p70, IL-10 and IL1β. To address the mechanism for drug-induced altered cytokine secretion mRNA for IL-12p70, IL-10 and IL1β were assessed followed by mir-155 mRNA and SOCS-1 protein. The functional outcome was assessed in an allogeneic MLR.
At doses sub-optimal to induce myeloma cell death, both drugs significantly inhibited TLR-induced hmDC increase in CD80 and CD83, but not CD86. TLR-induced secretion of IL-10 and IL12p70 was reduced while IL-1β secretion was increased. This occurred regardless of drug sequencing. HDACi did not significantly alter expression of IL-12p70, IL-10 or IL-1β mRNA despite changes in protein levels. To reveal the mechanism for the HDACi-induced hmDC IL-12p70 defect, the upstream molecules miR-155 and SOCS-1 were examined. Interestingly, panobinostat but not romidepsin induced mir-155 down-regulation and SOCS-1 protein increase. Subsequently, allogeneic T-cells co-cultured with HDACi/TLR-ligand-treated hmDC secreted lower levels of cytokines (decreased secretion of IFN-γ, IL-2, TNF, IL-4 and IL-17) compared to those allogeneic T cells co-cultured with control hmDC.
In this study, we show that whilst HDACi impair DC maturation and modulate subsequent T cell responses. Our findings suggest that panobinostat induces the IL-12p70 effect via the mir-155/SOCS-1 pathway, whereas romidepsin induces the effect via a mir-155 independent pathway. This data suggests that HDACi affect the hmDC maturation response to pathogen, and that these drugs may alter the ability of treated patients to raise an effective immune response to pathogens or, indeed, tumour antigens.
No relevant conflicts of interest to declare.
This review specifically examines the role of regulatory T cells (Tregs) in cancer in both mice and the clinic. Due to the rapid refinement of the definition of Tregs and their heterogeneity, ...emphasis is given to research findings over the past three years. For clarity, this review is broadly divided into three short sections that outline the basic biology of Tregs – (1) Treg lineage and development, (2) Treg subsets, and (3) mechanisms of Treg-mediated immune suppression; followed by two more comprehensive sections that cover; (4) clinical observations of Tregs and cancer, and (5) modifications of Treg biology as cancer immunotherapies. The latter two sections discuss the measurement of function and frequency of Treg in model systems and clinical trials and possible ways to interfere with Treg-mediated immune suppression with the focus on recent pre-clinical and clinical findings.
CD57+ NK cells are a subset of CD56lo/CD16+ NK cells which have been alternatively described as terminally differentiated or memory NK cells; however their physiological role remains unclear. In this ...study, we explored this unique NK subset and define their role in the context of MM.
CD57+ NK cell frequency was initially assessed by FACS. Thus, newly diagnosed untreated (NEW) Multiple Myeloma (MM) patients had a significantly increased frequency of CD57+ NK cells in the PB (p=0.0029) and BM (p=0.0095) compared with age matched normal donor controls. Refractory relapsed (RR) MM patients also had significantly increased CD57+ cells in the BM (p=0.008) (Figure 1). There was no change in CD57+ NK cell numbers (in either patient cohort) following three cycles of lenalidomide+dexamethasone (len+dex) therapy. Normal donor CD57+ NK cells had significantly less natural cytotoxicity function (compared with CD57- NK cells) against K562 (p=0.04) or U266 (p=0.0265) (Figure 2A). The same trend was observed in MM patient CD57+ NK cells; however this did not reach statistical significance (Figure 2B). In contrast, the ADCC activity for CD57+ and CD57- NK cells was the same whether the effector cells were from normal donors (Figure 2A) or MM patients (Figure 2B). To explain the reason for this difference in natural cytotoxicity function between the two NK cell subsets, the balance of NK activating (KAR) and inhibitory receptors (KIR) was examined (Figure 3). CD57+NK cells expressed significantly lower levels of the NK activating receptor Nkp46 than CD57-NK cells, in the PB of normal donors (p=0.001), NEW MM patients at presentation (p=0.008) or NEW MM patients after three cycles of len-dex therapy (p=0.0237) (Figure 3). In contrast, both CD57+ and CD57- NK cell subsets in RR MM patients had the same levels of Nkp46, this did not change with len-dex therapy. To further explore reasons for the CD57+ NK cell functional state, expression of NK effector molecules (perforin and granzyme) and NK effector transcription factors (TF) T-bet and Eomesdermin (Eomes) was assessed. Surprisingly, PB CD57+ NK cells had significantly higher perforin expression than CD57- NK cells in the PB of normal donors (p=0.0089) and NEW (p=0.0815) MM patients, and the BM of NEW MM patients (p=0.0487). Similarly, granzyme B was significantly elevated in CD57+ compared to CD57- NK cells in normal donors (p=0.013), NEW MM patients in the PB (p=0.0024) and BM (p=0.0007). There was no statistical difference in the expression of Eomes or T-bet in either NK subset, whether the cells were from normal donors or MM patients. Nonetheless, a trend in higher Eomes expression was observed in CD57+ NK cells from MM patients compared to age matched controls. Lastly, CD57+ (compared to CD57-) NK cells have a significantly lower proliferation rate in a NK homeostatic proliferation assay.
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Studies to date indicate CD57+ NK cells are terminally differentiated NK cells; however to date their role remains undefined. We observed an increased frequency of CD57+ NK in the PB and BM of newly diagnosed untreated MM patients. These CD57+ NK cells have the same functional and phenotypic features in normal donors and MM patients, whether at diagnosis or in RR patients. The strikingly reduced natural cytotoxicity in CD57+ NK cells, coupled with reduced Nkp46 expression, indicates these cells are not important for surveillance of viral infected or tumour cells. Rather CD57+ NK express elevated effector proteins and the effector TF Eomes and are ‘primed' for killing of target cells via ADCC. Unlike CD57- NK cells, CD57+ NK cells do not undergo homeostatic proliferation; whilst this is consistent with terminally differentiated cells, it is not clear how these cells persist in the periphery of MM patients. Nonetheless, CD57+ NK cells are elevated in MM patients at presentation, whilst this likely contributes to the overall decreased natural cytotoxicity observed in MM patients, it also provides an opportunity to activate NK cells via CD16 (FcγRIII) to induce ADCC against MM.
Harrison:Celgene Corp: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Janssen Cilag: Honoraria, Research Funding; Virolytics: Consultancy.
To investigate variations in the management and outcomes of peritonsillar abscesses, and to develop a trainee collaborative network in the UK.
Data were collected prospectively on suspected ...peritonsillar abscess cases presenting over a 2-month period at 42 participating secondary care centres, covering a population of 16 million. The primary outcome was an adverse event at 30 days, defined as re-presentation or re-drainage.
Eighteen per cent of the 325 cases experienced an adverse event. Follow-up data were valid for 90 per cent of cases. Regression analyses showed a significant reduction in adverse events in the 12 per cent of patients who were discharged within 12 hours, and there was no significant increase in adverse events for the 70 per cent receiving corticosteroids.
Out-patient management of peritonsillar abscess is not commonly practised in the UK. Corticosteroid usage is common and appears safe. This study demonstrates that trainees working in collaboration can effectively deliver prospective multicentre cohort studies in the UK.
OBJECTIVEThe immune system can halt cancer progression by suppressing outgrowth of clinically occult micrometastases in a state of cancer-immune equilibrium. Cutaneous melanoma provides a unique ...opportunity to study the immune contexture of such lesions, as miniscule skin metastases are accessible to clinical inspection and diagnostic biopsy. METHODSHere, we analysed by multiplex immunofluorescence microscopy samples from a melanoma patient presenting with an overt and an occult in-transit metastasis (ITM), the latter of which appeared as a small erythematous papule. RESULTSMicroarchitecture and immune composition in the two lesions were vastly different. CD4+ and CD8+ T cells accumulated around the margin of the overt SOX10+ Melan A+ ITM but were largely excluded from the tumor centre. By contrast, the occult micrometastasis contained only few SOX10+ Melan A- melanoma cells which were scattered within a dense infiltrate of T cells, including a prominent population of CD103+ CD8+ T cells resembling tissue-resident memory T (TRM) cells. Notably, almost every single melanoma cell in the micrometastasis was in close proximity to these TRM-like cells. CONCLUSIONSuch results support the emerging concept that CD103+ CD8+ TRM cells are key mediators of cancer surveillance and imply an important function of these cells in controlling clinically occult micrometastases in humans.
Abstract
In a phase 2 trial of panobinostat in 129 patients with relapsed or refractory Hodgkin lymphoma, exploratory analyses of chemokines and cytokines were prospectively performed in 109 patients ...to determine their association with clinical outcomes. Patients were categorized into two groups (reductions > median and reductions ≤ median) based on percentage change from baseline of log10 transformed measurements. Thymus and activation-regulated chemokine (TARC) was most strongly associated with clinical outcome. Early reduction of TARC was observed in responding patients, with the greatest reduction at cycle 1, day 15 (C1D15). Of 93 patients with C1D15 samples, there were three complete and 25 partial responses. The group with TARC reductions > median at C1D15 had more responders (18 39% vs. 10 21%), longer progression-free survival (10.6 vs. 4.9 months), shorter time to response and longer overall survival than the group with reductions ≤ median. This study is registered at www.ClinicalTrials.gov, NCT00742027.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Abstract 1862
Multiple Myeloma (MM) and its treatment are associated with impaired humoral and cellular immunity (CI). Lenalidomide (Len) is thought to mediate its anti-MM effect in part via ...stimulation of in vivo T cell, NK cell and NKT cell activity. However, as we have previously demonstrated (Hsu et al, Blood, 2011;117:1605), the immunostimulatory effects of Len are substantially abrogated by co-administration of high doses of Dexamethasone (Dex). It is unknown whether the efficacy of the Len/Dex induction regimen is maintained in newly diagnosed MM if the dose of Dex is lowered to preserve CI. In a prospective study (PMCC HREC #05/56), we examined both anti-MM responses and CI using a regimen of low dose Len and Dex induction followed by consolidation with autologous stem cell transplant (ASCT) in previously untreated patients with MM.
Twenty patients were enrolled. Induction therapy was with four 28 day (d) cycles of Len 15 mg d1 − 21 and Dex 20 mg weekly followed by hematopoietic stem cell (HSC) mobilization with cyclophosphamide 2 – 4 gm/m2 and G-CSF 10mcg/kg/day, and a melphalan 140 – 200mg/m2 conditioned ASCT. Maintenance with Rev 25mg d1-21 of a 28d cycle was commenced on d21-35 post ASCT until progression. Interim assessments of response were made post-induction, post-ASCT and on 31/05/2011 using IMWG uniform response criteria. Assessments of CI were undertaken at enrolment and again at the end of 4 cycles of induction by flow cytometric analysis of peripheral blood lymphocyte subsets (CD19+, CD3+, CD4+, CD8+, CD4+/CD25+/CD127+ Treg, CD3−/ CD16+/CD56+ NK cells), T cell proliferation to allogeneic stimulators (ratio 1:1) in a 7d mixed lymphocyte reaction (MLR) and analysis of NK function by cytotoxicity against K562 targets. All data were compared to age-matched controls, and analysed for statistical significance using a student T-test.
All patients have completed induction and ASCT. Median age=57.5 yrs, (range 44 – 70); male= 15; IgG = 10, IgA = 6 and light chain = 4. Stage at enrolment ISS1 = 9, ISS2 = 9 and ISS3 = 2. Median harvested CD34+ cells=10.8×106 /kg (range 4.9 – 40.6). The median number of CD34+ cells infused = 4.8×106 / kg (range 2.9 – 11.5) and median time to recovery of neutrophils > 0.5 ×109/L and platelets > 20 ×109/L was 12d (range 9 – 18) and 11d (range 8 – 25) respectively. The post-induction, overall response rate was 85%, very good partial response (VGPR)=20%, partial response (PR)= 65%, stable disease =5%, and 10% were refractory. Post-autograft responses improved to complete response (CR)=10%, VGPR= 40% and PR=35%. At a median follow-up of 17 months (range 5 – 29 months), the best response achieved to date is 35% CR, 25% VGPR and 30% PR (Fig 1). Immunology: CI data is available on 19 patients. Compared-to age matched controls, patients at enrolment had reduced CD3+ (p≤0.05), CD4+ (p≤0.01) (Fig 2)and Treg (P≤0.01) numbers and reduced NK cell function (p<0.05); however CD8+ T cell, NK cell and B cell numbers were normal. Following four cycles of Len/Dex therapy, CD3+, CD4+ and Treg numbers all increased, but remained below those of normal controls. Conversely, circulating B lymphocyte numbers fell substantially (P≤0.01) and NK cell function remained significantly impaired (p<0.05) following induction therapy. However, functional analysis of CD3+ (Fig 3), CD4+ and CD8+ proliferation in an MLR at enrolment and following induction therapy was identical to that of controls. Display omitted Display omitted Display omitted
This novel treatment regimen of low dose Len and low dose Dex followed by ASCT in untreated patients with MM is associated with high response rates and successful HSC collection. The depth of response progressively improved following ASCT. Cellular immunology in MM patients at diagnosis shows specific reductions in CD4+ and Treg cell numbers, and NK cell function that is not rescued by Len therapy when co-administered with Dex, even at low doses, which induced a further reduction in CD19+ cells. However despite these deficiencies, preserved proliferative capacity of both CD4+ and CD8+ T-cells is seen at diagnosis and following this induction therapy, suggesting that with the low dose Len-Dex regimen, the immune environment is conducive to novel strategies that are aimed at inducing adaptive anti-MM immune responses.
Harrison:Celgene: Honoraria, Research Funding. Off Label Use: Low dose lenalidomide in newly diagnosed myeloma. Neeson:Celgene: Research Funding. Prince:Celgene: Honoraria, Research Funding.
Abstract 4180▪▪This icon denotes a clinically relevant abstract
High risk acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) may be amenable to immunotherapy with adoptive transfer of ...chimeric antigen receptor (CAR) T cells. However, persistence and generation of central memory (CM) are essential for ongoing clinical responses (Morgan et al Science 2006). Our prior work showed that the carbohydrate antigen LewisY (LeY) is expressed in 46% of AML patients (n=33) to varying degrees, making it a suitable and novel target for CAR therapy. In our phase I study, we have assessed the trafficking, persistence and functional capacity of CAR-T cells transduced with an anti-LeY chimeric receptor gene in high-risk patients with LeY positive AML or MDS.
The CAR comprised extracellular humanized scFv recognizing the LeY Ag, linked to an extracellular CD8 hinge region, a transmembrane and cytoplasmic CD28 and CD3 zeta signalling domain. The humanized anti- LeY scFv-CD28-ζ receptor vector was produced as described previously (Westwood J et al PNAS 2005). T cells, harvested from the patient, were transduced with LeY scFv-CD28-ζ pSAMEN retroviral vector. Bulk transduced T cells (LeY-T) were re-infused into the patient after lymphodepleting fludarabine chemotherapy. AML immunophenotyping and cytogenetics were used to monitor minimal residual disease (MRD). PB and BM samples were collected prior to and post CAR-T adoptive transfer. An optimized PCR assay (sensitivity 1:1e5) for the presence of the LeY transgene (TG) was performed on genomic DNA extracted from each sample. To measure both CAR-T cell functional polarization and persistence of CAR expression, PB or BM cells were co-cultured with a cell line expressing LeY (OVCAR-3), in the presence or absence of MHC class I or II blocking antibodies. Culture supernatant was collected at 24 hours and assessed by Luminex assay for Th1 (IFN-g, IL-2), Th2 (IL-4, IL-10), pro-inflammatory (IL-6, IL-17 and TNF) cytokines and TGF-b.
Five AML patients have been enrolled to date. Four patients have had sufficient CAR-T cells generated to provide doses of 0.5 × 109 −1.3 × 109T cells (transduced T cells: 8 – 30% of total T-cells) with a viability of 96.1– 98.5%. 2 patients (01 and 04) had progressive disease 1 and 2 months after infusion. 2 patients (02 and 05) remain in morphologic remission with stable cytogenetic MRD, 3 and 14 months post infusion.
Patients 01, 02 and 04 all had LeY TG in PB and BM samples at all time points measured, up to day 49 in patient 01 (Figure 1) and up to 10 months in patient 02. In patient 04 LeY TG was also detected in a skin biopsy (day +6) of leukemic skin infiltrate. Display omitted
Co-culture supernatant cytokine analysis showed LeY-T cells from patient 01, 02 and 04 secreted high levels of IFN-g (805.5 ± 198.9 pg/ml) and low levels of IL-2 (3.14 ± 0.65 pg/ml) prior to adoptive transfer. In contrast, post adoptive transfer, co-culture with OVCAR-3 cells, and MHC class I and II blocking antibody, induced these PB LeY-T cells to secrete IL-4 (24.13 ± 12.47pg/ml) and IL-10 (9.35 ± 0.44 pg/ml); no IFN-g or IL-2 was detected. This Th2 polarization of CAR-T cells was shown in PB samples taken at days 5 to 28 (patient 01), day 28 to 8 months (patient 02) and day 1 to day 28 (patient 04) post-adoptive transfer (Figure 2). In addition, when Th1/Th2 cytokines were measured in patient 02 PB and BM plasma, Th1 cytokines were detected immediately post-adoptive transfer (IFN-g=39 pg/ml, IL-2=8.6 pg/ml at peak); however these decreased to basal levels by day 3 (IFN-g=1.8 pg/ml) and day 2 (IL-2=2.6 pg/ml). The same trend in serum cytokines was observed for patient 01. Display omitted
Therefore, our studies reveal that, after adoptive transfer, LeY CAR-T cells persist in vivo, maintain their expression of the CAR, but show rapid polarization from a Th1 to a Th2 phenotype.
No relevant conflicts of interest to declare.
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