Abstract Pruritus is one of the cardinal symptoms found in patients with leukemic cutaneous T cell lymphoma (CTCL). The nature of the pruritus experienced by CTCL patients is complex, involving ...different pathways and cell mediators, thus making it poorly responsive to conventional anti-itch therapies. Recent reports highlight the role of interleukin 31 (IL-31) as a novel cytokine involved in the pathogenesis of pruritus in atopic dermatitis and CTCL. Here we provide both in vivo and in vitro evidence suggesting that histone deacetylase (HDAC) inhibitors may mitigate itch through lowering of levels of IL-31-expressing T cells. Furthermore, we demonstrate that chemokine receptor type-4 (CCR4)-bearing T cells are a main source of IL-31 in CTCL, and that neutralizing the IL-31 pathway through targeting of the CCR4-expressing T cells may represent a promising therapeutic strategy for symptomatic relief in CTCL.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
We have demonstrated previously that susceptibility of murine strains to the development of allergic airway responses is associated with a type 2 cytokine pattern. In the present study, we examine ...the in vivo role of IL-12 in the immune response to allergen exposure in susceptible (A/J) and resistant (C3H/HeJ, C3H) strains of mice. OVA sensitization and challenge induced significant increases in airway reactivity in A/J mice as compared with their PBS-challenged controls, while no increases in airway reactivity were observed in OVA-challenged C3H mice. OVA exposure of A/J mice resulted in marked increases in the Th2 cytokines, IL-4 and IL-10, in the bronchoalveolar lavage fluid, whereas increases in IFN-gamma were observed in C3H mice. Strikingly, anti-IL-12 mAb (1 mg/mouse) treatment resulted in threefold increases in airway reactivity in OVA-challenged resistant C3H mice, concomitant with significant increases in bronchoalveolar lavage levels of Th2 cytokines and decreases in IFN-gamma. IL-12 depletion of C3H mice also suppressed OVA-specific serum IgG2a levels and increased both serum OVA-specific IgG1 and IgE levels. Blockade of endogenous IL-12 levels in susceptible A/J mice resulted in further augmentation of type 2 immune responses. These results demonstrate that endogenous production of IL-12 is essential for resistance to Ag-induced airway hyperresponsiveness, and furthermore, that dysregulation of IL-12 production may lead to the development of deleterious type 2 immune responses to inhaled allergens.
Background Lenabasum is a cannabinoid type 2 receptor (CB2R) reverse agonist that demonstrates anti-inflammatory effects in vivo and in vitro in dermatomyositis (DM) and is currently being ...investigated for therapeutic potential. The purpose of our study is to investigate CB2R distribution as well as the effects of lenabasum in DM. Methods Immunohistochemistry staining (IHC) was utilized to examine immune cell and cytokine production changes in lesional DM skin biopsies from lenabasum and placebo-treated patients. CB2R expression in various immune cell populations within DM skin was investigated with image mass cytometry (IMC), whereas flow cytometry elucidated CB2R expression in DM peripheral blood mononuclear cells (PBMCs) as well as cytokine production by CB2R-expressing cell populations. Results After 12 weeks of lenabasum treatment, IHC staining showed that CD4+ T cells, CB2R, IL-31, IFN-gamma, and IFN-beta cytokines were downregulated. IFN-gamma and IFN-beta mRNA decreased in lesional DM skin but not in PBMCs. IMC findings revealed that CB2R was upregulated in DM lesional skin compared to HC skin and DM PBMCs (p<0.05). In DM skin, CB2R was upregulated on dendritic cells, B cells, T cells, and macrophages while dendritic cells had the greatest expression in both DM skin and PBMCs (p<0.05). These CB2R+ cells in the skin produce IL-31, IL-4, IFN-gamma, and IFN-beta. Conclusion Our findings of differential CB2R expression based on location and cell type suggest modes by which lenabasum may exert anti-inflammatory effects in DM and highlights dendritic cells as potential therapeutic targets. Keywords: CB2R, Dermatomyositis, Image mass cytometry, Lenabasum, Dendritic cells
Introduction The COVID-19 pandemic has been a great threat to facilities providing inpatient care to chronically/terminally ill patients or elderly people. This unprecedented situation has called for ...radical and previously untried solutions including restrictions or a complete ban on visiting inpatients, reductions in the number of staff, and measures to improve the sanitary regime. These far-reaching actions have had a direct impact on all actors involved in the system of social welfare facilities: employees, patients, and their families. Aim The analysis of the key needs of patients and their families and the potential differences in their hierarchy in the new situation. Material and methods A qualitative pilot study was conducted in a Polish inpatient hospice to identify the consequences of the changes outlined above and to help with the development of solutions adjusted to the current situation. The study, based on questionnaires containing open-ended questions, was conducted among patients and their families. The questionnaires were analysed using a medical-anthropological approach. Results The analysis provides a basis for identifying the key needs of both groups under study and highlighting the differences between them. The basic need of the patients were shown to be physical closeness. For families, it was a need to care for their relatives. Conclusions The differences in needs influenced the preferred forms of communication. Based on the results of the questionnaires, COVID-19 was also observed to play different roles and vary in importance among patients and their families. The findings of the pilot study are not exhaustive, and further in-depth research is required to explore this area of interest.
Full text
Available for:
IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, UL, UM, UPUK
Cutaneous T cell lymphomas (CTCLs) are a heterogenous group of lymphoproliferative disorders caused by clonally derived, skin-invasive T cells. Mycosis fungoides (MF) and Sezary syndrome (SS) are the ...most common types of CTCLs and are characterized by malignant CD4(+)/CLA(+)/CCR4(+) T cells that also lack the usual T cell surface markers CD7 and/or CD26. As MF/SS advances, the clonal dominance of the malignant cells results in the expression of predominantly Th2 cytokines, progressive immune dysregulation in patients, and further tumor cell growth. This review summarizes recent insights into the pathogenesis and immunobiology of MF/SS and how these have shaped current therapeutic approaches, in particular the growing emphasis on enhancement of host antitumor immune responses as the key to successful therapy.
Background: The genetic basis and the genome-wide abnormalities underlying most forms of cancer are being comprehensively annotated in many human malignancies. However, the contribution of epigenetic ...aberrations, particularly the post-translational modifications (PTM) of histone tails has not been investigated. This is largely due to lack of approaches to comprehensively interrogate the status of the numerous PTMs that define histone marks which control gene expression and complex biologic processes such as cancer. Sézary Syndrome (SS) is an aggressive form of cutaneous T-cell lymphoma (CTCL) characterized by poor outcomes and complex genetic alterations frequently targeting epigenetic regulators and chromatin remodelers. CTCLs represent the first FDA-approved disease for treatment using histone deacetylase inhibitors (HDACi) such as romidepsin, but the direct consequences of this treatment on histone PTMs remain unknown. Here we define the histone PTM signatures of CTCL/SS by using a novel unbiased strategy leveraging tandem mass spectrometry (MS/MS)-based quantitative proteomics to study the comprehensive combinatorial histone PTM code in a cohort of primary SS samples, and 4 CTCL/SS cell lines in comparison with normal reactive CD4+ T-lymphocytes from healthy individuals.
Methods: CD4+ T-cells were isolated using CD4 immunomagnetic beads from PBMC's of healthy volunteers (n=8) or patients with diagnosed SS (n=20). CD4+ cells were frozen and subsequently analyzed in batches. The CTCL/SS-derived cell lines (HH and MJ, HuT78 and H9) were also analyzed each with three biological replicates. Histones were acid extracted from isolated nuclei. Total bulk histones were propionylated and trypsinized prior to MS analysis. Nano-scale liquid chromatography followed by -tandem mass spectrometry (nano LC-MS/MS) data acquisition was performed in an Orbitrap FusionTM TribridTM MS in technical triplicates for each sample with a data-independent acquisition (DIA)-method using a 50 m/z quadrupole-isolation windows that steps across the 200-1500 m/z ranges. Data analysis was performed using EpiProfile for single and combinatorial histone PTM analysis and quantification. Orthogonal validation for selected modifications was performed using western blotting and single-cell mass cytometry by time of flight (CyTOF) analysis.
Results: Our quantitative epiproteomic strategy interrogated the relative ratios of a total of 228 histone PTM combinations, and 20 histone variants in a well-characterized cohort of SS patient samples, four CTCL cell lines and a cohort of normal primary CD4+ T-lymphocytes isolated from healthy individuals. With this approach, we were able to identify and quantify 23 unique histone peptides on histone H4, 105 on histone H3, 80 on histone H2A, 12 on histone H2B and 28 on histone H1 across every sample. Pearson correlation and principal component analysis of overall histone PTMs profiles across all samples provided accurate discrimination. We found a distinct pattern of histone PTMs in both SS patient samples and CTCL cell lines that were strikingly different from CD4+ T-cells obtained from healthy individuals. Notably, differences between cell lines and primary patient samples are more marked than those within individual SS primary patient samples, implying that cell lines may poorly recapitulate some aspects of in vivo biology. Unsupervised hierarchical clustering of the primary patient samples, cell lines and the normal CD4+ T-cells performed based on abundances of the identified histone PTMs revealed disease-specific ubiquitous marks as well as disease-specific unique marks for SS. Among the analyzed histone PTMs, H3K27ac, H3.3K27ac, H4K8ac, H4K20ac, H3K4me3, H3K18me1, H3K79me3 and H4K20me3 were distinct between healthy and CTCL CD4+ T-cells. Selected differential marks such as H4K20me3 were orthogonally corroborated by Western blotting and CyTOF mass cytometry analysis.
Conclusions: For the first time, we have defined the histone code of CTCL/SS using global mass spectrometry based quantitative proteomics with high specificity and sensitivity. The results of our MS-based epiproteomic profiling revealed disease-specific histone PTM signatures and opportunities to exploit tractable changes induced by HDACi treatment as clinically actionable molecular biomarkers and therapeutic targets.
No relevant conflicts of interest to declare.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Chimeric Antigen Receptor T cells (CART) have led to unprecedented clinical responses in relapsed or refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL), non-Hodgkin lymphomas (NHL), and ...multiple myeloma. However, despite these exciting results, most patients treated with CART therapy either do not respond or eventually relapse. Moreover, CART therapy has not yet been proven effective in several hematological malignancies, such as T cell lymphoma and leukemia (T-NHL/T-ALL) and acute myeloid leukemia (AML). Thus, there is a need to enhance currently available CART products and also to develop next-generation CART therapies to successfully treat additional neoplasms like T-NHL /T-ALL and AML.
To this goal, we studied the cysteine-rich scavenger receptor CD5, an attractive target for CART immunotherapy because of its dual role in malignant cells and normal T cells. In malignant cells, CD5 is expressed by ~90% of TCL cells, by ~15-20% of AML cells, and also by most cases of chronic lymphocytic leukemia and mantle cell lymphoma (MCL). Of note, promising results using a CD28-based anti-CD5 CART against T-NHL and T-ALL were reported at this meeting in 2019 (LaQuisa C. Hill #199). In T cells, CD5 is highly expressed and inhibits T cell receptor (TCR)-mediated activation through several mediators including SHP-1, CBL, CBL-B, and GRB2. Therefore, we hypothesized that the genetic deletion of CD5 in engineered T cells could potentially enhance their effector functions.
First, we designed and screened six 4-1BB-costimulated anti-CD5 lentiviral CAR constructs designed to have high, medium, and low affinity for CD5. We then selected the lead CAR5 construct (high affinity, heavy to light light chain orientation) based on its superior anti-tumor function in vivo in NOD-SCID IL2Rgnull (NSG) mice engrafted with T-cell leukemia (Jurkat). Then, to further improve CART5 activity, we optimized a CD5 short-guide RNA and deleted CD5 in CART5 cells using CRISPR-Cas9. CD5 gene deletion was reproducibly efficient (95-100% by flow cytometry and TIDE) during manufacturing (6 donors). Interestingly, the growth rate of wild type (WT) CART5 was comparable to CD5 KO CART5 and the expression of CD5 in WT CART5 was reduced. However, at the end of manufacturing, CD5 KO CART5 had increased central memory T cells (33.0% vs. 18.4%) and reduced expression of activation/exhaustion markers (PD-1 4.4% vs. 14.8%, LAG3 13.1% vs. 55.9%) compared to WT CART5, potentially indicating that CD5 KO reduces CART5-CART5 fratricide during manufacturing.
We then compared wild type (WT) CART5 to CD5 KO CART5 in vitro using several T-NHL/T-ALL, MCL, and AML models, including primary samples (Sezary cells, primary MCL cells, and CD5+ AML cells). Both WT and CD5 KO CART5 were highly effective in killing CD5+ malignant cells, but CD5 KO CART5 showed enhanced proliferation upon activation. In two xenograft models of T-cell leukemia (primary T-ALL and Jurkat), CD5 KO CART5 showed dramatically increased tumor control compared to WT (Fig.1A, median overall survival for WT= 62 days vs. CD5 KO=not reached, p = 0.006, Mantel-Cox). This enhanced anti-tumor effect was associated with increased expansion of CD5 KO CART5 in the peripheral blood (PB) compared to WT CART5.
To test the hypothesis that deletion of CD5 could increase the anti-tumor effect of CART targeting antigens other than CD5, we knocked out CD5 in anti-CD19 CART cells and tested their function in a CD19+ B-ALL xenograft model (NALM6). Remarkably, CD5 KO CART19 displayed significantly enhanced anti-leukemia activity and PB expansion compared to WT (Fig.1B,C, p<0.05, p = 0.001, Mantel-Cox).
Finally, we aimed to define the mechanisms by which CD5 KO enhances CART anti-tumor efficacy. We analyzed the phosphorylation of multiple targets in T cells after 15 minutes of CAR stimulation. Remarkably, CD5 KO CART5 cells had higher (>2fold) phosphorylation of several signaling proteins, including key regulators of T cell activation, migration, and survival compared to WT CART5 (Fig. 1D). To confirm that the CD5 pathway was indeed the mediator of this effect, we knocked out SHP-1 in CART19 cells using CRISPR/Cas9 and observed increased leukemia killing.
In conclusion, we demonstrate that CRISPR-Cas9 KO of CD5 enhances the anti-tumor activity of CAR T cells by enhancement of CAR-mediated activation and proliferation. These findings support the development of CD5 KO CART products in early-phase clinical trials.
Display omitted
Schuster:Novartis, Genentech, Inc./ F. Hoffmann-La Roche: Research Funding; AlloGene, AstraZeneca, BeiGene, Genentech, Inc./ F. Hoffmann-La Roche, Juno/Celgene, Loxo Oncology, Nordic Nanovector, Novartis, Tessa Therapeutics: Consultancy, Honoraria. Barta:Atara: Honoraria; Monsanto: Consultancy; Seattle Genetics: Honoraria, Research Funding; Janssen: Honoraria; Pfizer: Honoraria. June:Tmunity Therapeutics: Current equity holder in private company, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; Ziopharm Oncology: Current equity holder in private company, Membership on an entity’s Board of Directors or advisory committees; Novartis: Patents & Royalties, Research Funding; Bluesphere Bio: Membership on an entity’s Board of Directors or advisory committees; Cabaletta Bio: Current equity holder in private company, Membership on an entity’s Board of Directors or advisory committees; Carisma Therapeutics: Membership on an entity’s Board of Directors or advisory committees; Cellares: Membership on an entity’s Board of Directors or advisory committees; Celldex: Consultancy, Membership on an entity’s Board of Directors or advisory committees; DeCART Therapeutics: Membership on an entity’s Board of Directors or advisory committees; Immune Design: Membership on an entity’s Board of Directors or advisory committees; Kiadis Pharma: Current equity holder in private company. Ruella:Abclon, BMS, NanoString: Consultancy; UPenn/Novartis: Patents & Royalties; Abclon: Consultancy, Research Funding.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
IMPORTANCE: Sézary syndrome (SS) is an advanced form of cutaneous T-cell lymphoma with few long-term remissions observed. OBJECTIVE: To profile 3 patients with SS who have experienced long-term ...remission following the addition of low-dose total skin electron beam therapy (TSEBT) to systemic regimens of extracorporeal photopheresis, bexarotene, and interferon-γ. DESIGN, SETTING, AND PARTICIPANTS: This is a retrospective case series with additional investigations of patient-donated samples to assess therapeutic response. The study was conducted at the University of Pennsylvania Cutaneous Lymphoma Clinic and follows 3 patients with stage IVA1 CD4+ SS who presented to the clinic between November 1, 2009, and November 1, 2017, and who had a history of SS that was refractory to multimodality systemic therapy prior to receiving low-dose TSEBT. INTERVENTIONS: Patients were treated in a multimodality fashion with combined extracorporeal photopheresis, bexarotene, interferon-γ, and low-dose TSEBT. MAIN OUTCOMES AND MEASURES: To characterize treatment responses in these patients, the extent of skin disease was measured with the modified severity weighted assessment tool. Blood disease was measured with flow cytometric assessments of Sézary cell count, CD4:CD8 ratio, and high throughput sequencing of the T-cell receptors. To assess for restoration of immune function, we measured markers of immune exhaustion, including PD-1 (programmed cell death 1), TIGIT (T-cell immunoreceptor with immunoglobulin and ITIM domains), CTLA4 (cytotoxic T-lymphocyte-associated protein 4), TOX (thymocyte selection-associated high mobility group box protein), and Foxp3 (forkhead box P3) on circulating CD4 and CD8 T cells, along with production capacity of interferon-γ by lymphocytes following activation stimuli. RESULTS: Following administration of low-dose TSEBT and maintenance of the other therapies, remissions ranged from 24 to 30 months, with complete responses in 2 patients ongoing. Markers of immune exhaustion including PD-1, TIGIT, CTLA4, TOX, and Foxp3 were significantly reduced from baseline following TSEBT, along with enhanced production capacity of interferon-γ by lymphocytes following activation stimuli. High throughput sequencing demonstrated near-complete eradication of the circulating clone among 2 of 3 patients with stable levels in 1. CONCLUSIONS AND RELEVANCE: We describe 3 patients who achieved long-term clinical and molecular remissions following low-dose TSEBT as part of a multimodality regimen for treatment of SS. As long-term remissions in SS are uncommon, this approach demonstrates promise, and clinical trials should be considered.
PDF
