DC Populations Research Articles

Phylogenetic, growth performance and protein growth hormone of snakehead fish (Channa striata) from wild stock in Sumatra, Indonesia

Abstract Watershed isolation may influence morphological, genetic variations, and protein expression of snakehead fish (Channa striata). This research seeks to identify snakehead diversity from isolated Inland waters in Sumatra, i.e., Kampar River (SK), Merang River (SM), Cala Lake (DC), and Lampam Floodplain (RL). Channa striata samples were collected from inland waters and cultivated in Aquaculture laboratory. Cytochrome b gene was used for genetic characterization. Growth protein was analyzed using Enzyme-Linked Immunosorbent Assays (ELISA). We collected DNA from four samples at each site. The growth performance of cultivated snakehead fish was also observed. Five hundred post-juvenile snakehead fish from four populations were cultivated for 45-days with five repetitions. Each population had 12 snakehead fish tested for GH protein. Data were statistically tested using ANOVA, maximum likelihood phylogenetic tree analysis, linear regression, and Pearson correlation. The results showed four clusters of snakehead fish in the phylogenetic tree. The DC population had the highest significant value of the daily length growth rate (7.5±0.2 %). The GH protein-growth relationship was low (0.014-0.083). The SM snakehead fish population had the highest concentration of GH protein (1.77±0.11 pg μl−1) and had distinct cyt.b gene clusters from other populations, making it a potential candidate for future aquaculture strain development.

Molecular Mechanisms Underlying Response and Resistance to Glofitamab

The prognosis for patients with relapsed or refractory Diffuse Large B-cell Lymphoma (r/rDLBCL) is poor. Glofitamab (a CD20xCD3 T-cell-engaging bispecific antibody) demonstrated high and durable complete response rates and a manageable safety profile in patients with relapsed/refractory (R/R) Large B-cell lymphoma (LBCL). Despite its clinical efficacy and a rapid achievement of complete responses, some patients are refractory to glofitamab treatment, prompting us to investigate the mechanisms underlying response and resistance to glofitamab. To gain insight into these mechanisms, we performed molecular (single cell RNA and TCR sequencing data) and functional (ex-vivo assessment of cytotoxicity, cytokine and cytotoxic granules secretion, activation) characterisation of PBMCs from glofitamab-treated patients who achieved complete response (CR, n=18) or progressed (progressive disease, PD, n=13) by cycle 3, collected in a Phase I/II study (NCT03075696). All patients received glofitamab in a step-up-dose regimen of 2.5/10/30 mg following obinutuzumab pre-treatment (1000 mg) administered 7 days prior to the first glofitamab dose. The PBMC samples were analyzed at baseline (cycle 1 day 1 pre-dose) and on treatment (cycle 2 day 1 pre-dose). The assessment of PBMCs using single cell RNA sequencing did not reveal major differences in abundance of the main immune cell subsets between patients who achieved CR or PD. Paired analysis of on-treatment samples (compared to respective baseline) pointed to a tendency towards decreased frequency of CD8 T cells in patients with CRs and increased frequency of the same in patients with PDs (consisting mainly of an increase of proliferating CD8). Glofitamab treatment further increased the frequency of monocytes and DCs in patients with CRs, and of NKs in both CRs and PDs. Within the DC population, we observed a significant decrease of cDC1 population and increase of the AXL+SIGLEC6+ DC population with high capability of driving T cell activation and proliferation upon treatment in both CR and PD patients. Complementary to changes in cell proportions, we identified gene expression programs associated with response, of which some were shared between two or more cell types while others were cell type-specific. Using established gene expression programs of naiveness, cytotoxicity, and exhaustion, we found that CD8 T cells of CR patients maintain their naive phenotype on treatment, whereas the naiveness of CD8 T cells of PD patients decreases upon treatment. Subsequently, we observed a higher naiveness of CD8 T cells in patients with CRs compared to PDs on treatment. Moreover, CD4 T cells increase their naiveness upon treatment in patients with CRs and decrease in patients with PDs, resulting in higher overall naiveness of CD4 T cells on treatment in CR patients compared to PD patients. In line with the expected mode of action, glofitamab treatment led to an increase of the cytotoxicity and exhaustion scores in CD8 T cells, and of the exhaustion score in CD4 T cells, of both CR and PD patients, reflective of T cell engagement in both patient groups. However, CD8 T cells of PD patients display overall a higher cytotoxicity and exhaustion level compared to CR patients. TCR sequencing data showed a higher clonal diversity in CD4 T cells compared to CD8 T cellsand and enrichment of hyper-expanded clonotypes in the CD8 T cell compared to CD4 T cell compartment . However, we neither detected an increase in the proportion of clonal cells, nor a change in clonal diversity upon treatment, regardless of the patient response group. In addition, we did not find any association between the percent of TCR repertoire occupied by clones of a given size and response or treatment time point. In agreement with the molecular analysis, T cells from CR patients (collected at baseline and on treatment) displayed a tendency towards higher cytotoxicity, cytokine secretion and T cell activation compared to PD patients upon ex vivo re-stimulation with glofitamab, suggestive of a better functional state of T cells from patients with CR compared to PD. Taken together, our work provides a thorough characterization of peripheral blood immune cells from CR and PD patients treated with glofitamab, and provides evidence that T cells from CR patients maintain a fresher/naive and more functional state compared to PD patients, which display T cells in a more advanced differentiation state with lower functionality.

Type 2 Dendritic Cells Orchestrate a Local Immune Circuit to Confer Antimetastatic Immunity.

The progression of transformed primary tumors to metastatic colonization is a lethal determinant of disease outcome. Although circulating adaptive and innate lymphocyte effector responses are required for effective antimetastatic immunity, whether tissue-resident immune circuits confer initial immunity at sites of metastatic dissemination remains ill defined. Here we examine the nature of local immune cell responses during early metastatic seeding in the lung using intracardiac injection to mimic monodispersed metastatic spread. Using syngeneic murine melanoma and colon cancer models, we demonstrate that lung-resident conventional type 2 dendritic cells (DC2) orchestrate a local immune circuit to confer host antimetastatic immunity. Tissue-specific ablation of lung DC2, and not peripheral DC populations, led to increased metastatic burden in the presence of an intact T cell and NK cell compartment. We demonstrate that DC nucleic acid sensing and transcription factors IRF3 and IRF7 signaling are required for early metastatic control and that DC2 serve as a robust source of proinflammatory cytokines in the lung. Critically, DC2 direct the local production of IFN-γ by lung-resident NK cells, which limits the initial metastatic burden. Collectively, our results highlight, to our knowledge, a novel DC2-NK cell axis that colocalizes around pioneering metastatic cells to orchestrate an early innate immune response program to limit initial metastatic burden in the lung.

Cell-associated HIV cross-presentation by pDC is potentiated by non-cognate CD8+ T cell pre-activation

Antigens (Ag) from apoptotic HIV-infected cells can be cross-presented by human plasmacytoid dendritic cells (pDC) to CD8 + T cells 1 . We studied the mechanisms leading to this cross-presentation. For this, we generated HLA-A2-restricted HIV-Gag-specific CD8 + T cell clones. HLA-A2 negative, HIV-infected or uninfected, H9 cells were used as Ag donor cells. They were irradiated by UV-C to induce apoptosis. Direct viral presentation was prevented by using saquinavir. DC populations were immunomagnetically and FACS-sorted to >95% purity, from HLA-A2+ or -donor buffy coats. CD8 + T cells IFN-γ secretion or intracellular production were assessed by ELISA or flow cytometry, respectively. Apoptotic H9-HIV cell cross-presentation by pDC led to CD8 + T cells IFN-γ secretion, which was fully MHC-class I-restricted. Surprisingly, a lower level of non-MHC restricted intracellular IFN-γ production was also induced by HLA-A2-negative pDC incubated with HIV-infected cells. Both MHC-Class I-dependent and -independent IFN-γ intracellular productions were partly dependent on TLR-7 signalization, indicating a role for stimulation of TLR7 by HIV. Indeed, H9-HIV, and not H9 cells, induced maturation and production of cytokines by pDC. Among those cytokines, IFN-α or β induced intracellular IFN-γ production when added to the CD8 + T cell clones, in the absence of pDC or H9-HIV cells. To perform cognate activation of the CD8 + T cell clones without viral stimulation, we used CD3-and CD28-bound beads: IFN-γ secretion was stimulated, and was increased by adding these cytokines. Conversely, neutralizing antibodies against these cytokines abolished the non-MHC-restricted intracellular IFN-γ production triggered by pDC incubated with by HIV-infected cells. Compared to pDC, purified cDC1 or cDC2 required additional TLR-3 or -2/4 stimulation to cross-present antigens from H9-HIV cells. Indeed, H9-HIV apoptotic cells induced maturation of pDC, but not cDC. Our work demonstrates that pDC produce type I IFNs following HIV-infected cells detection, which pre-activate CD8+ T cells by making them produce, but not release, IFN-γ. Upon additional cognate interaction, IFN-γ. release is potentiated by this previous intracellular accumulation.

The nucleic acid sensor RIG-I as an inducer of anti-tumor response

Abstract The immune microenvironment plays a critical role in tumor progression. Recently, approaches to target Pattern Recognition Receptors (PRRs) have emerged as a promising strategy to enhance immune recognition of tumors. Among the PRRs, those which recognize DNA and RNA in the cells are unique in their ability to respond to both pathogenic and self-nucleic acids that are generated as a result of aberrant cell division/transcription. We previously showed that targeting the DNA sensor three prime exonuclease 1 (TREX1) enhanced immune responses in mouse tumor models. Here we show that the RNA sensor Retinoic acid Induced Gene 1 (RIG-I) acts as a robust immune activator across cancer types. According cBioportal data, expression of RIG-I seems to be a prognostic factor for progression free survival. To functional evaluate RIG-I response in tumor models, we used a hairpin RNA derived from H1N1 sequence. Interestingly, activation of RIG-I increased expression of TREX1 revealing a potential crosstalk between the two pathways. Both breast and colorectal tumor cell lines produce IFN-I after RIG-I activation. Consistently, RNAseq showed upregulation of IFN-I response genes and proinflammatory cytokines across different cell lines. Activation of RIG-I in the tumor cells decreased tumor burden with a concomitant broad cellular and molecular anti-tumor response. Thus, results showed an increase in NK and DC population, while the transcription profile was upregulation of innate immune response genes. Interestingly, tumor growth and NK results were consistent when tumor was implanted in immunocompromised mouse model. Taken together, our findings illustrate how tumor cell RIG-I is a potent inducer of cellular and molecular immune responses.

Abstract 2727: Heterodimeric IL-15 (hetIL-15) affects conventional dendritic cells and a distinct novel dendritic cell population in different mouse cancer models of breast and pancreatic cancer

Abstract Introduction: IL-15 is a cytokine inducing proliferation and cytotoxic function of lymphocytes, including CD8+ T and NKs cells. We have produced the native heterodimeric form of IL-15 (hetIL-15) which has advanced in clinical trials due to its anticancer activities in many different mouse cancer models. The objective of this study was to explore how hetIL-15 affects lymphoid and myeloid cell populations in the tumor microenvironment in different mouse cancer models of breast and pancreatic cancer. Study Design and Methods: We studied the therapeutic efficacy of intraperitoneal or locoregional administration of hetIL-15 in three different orthotopic mouse cancer models. We employed 4T1 and EO771 breast cancer models, which mimic human triple negative breast cancer and a KPC derived pancreatic cancer model. hetIL-15 effects on tumor infiltrating immune cells were evaluated by flow cytometry and immunohistochemistry. Results: hetIL-15 treatment showed a profound effect on the EO771 breast cancer model resulting in complete tumor regression in ~40% of the treated mice. Additionally, hetIL-15 caused tumor delay in 4T1 and KPC derived cancer models. Flow cytometry analysis revealed an increase of cytotoxic effector cells, and especially CD8+ T cells, with enhanced cytotoxic activity and proliferation in all cancer models tested. Immunohistochemistry verified the accumulation of cytotoxic effector cells intratumorally upon hetIL-15 treatment. Furthermore, flow analysis showed an increase of infiltrating conventional type 1 dendritic cells (cDC1s) which was found only in EO771 breast tumors. On the contrary, 4T1 breast and KPC derived pancreatic tumors, but not EO771 tumors, displayed an accumulation of infiltrating conventional type 2 dendritic cells (cDC2s). Importantly, the flow cytometry analysis revealed yet additional novel distinct dendritic cell population characterized by CD103intCD11b+ immunophenotype, which was mostly evident in tumor tissues treated with hetIL-15 in all three models. Phenotypic profiling of this novel DC population identified expression of several cDC1 specific markers, including CD103, IRF8 and XCR1. Both cDC1s and the novel DC population were inversely correlated with the tumor size in EO771 breast cancer model. Conclusions: hetIL-15 affects both T cells and conventional dendritic cells in syngeneic murine cancer models of breast and pancreatic cancer. We report that the treatment with hetIL-15 increases a novel distinct dendritic cell population (CD103intCD11b+) in all three models. Since we have previously reported that hetIL-15 induces long term immunological protection from tumor rechallenge, these findings suggest hetIL-15 as a promising therapeutic agent in treatment of triple negative breast and pancreatic cancer. Citation Format: Vasiliki Stravokefalou, Dimitris Stellas, Sevasti Karaliota, Bethany Nagy, Theresa Guerin, Serguei Kozlov, Barbara K. Felber, George N. Pavlakis. Heterodimeric IL-15 (hetIL-15) affects conventional dendritic cells and a distinct novel dendritic cell population in different mouse cancer models of breast and pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2727.

CcpA- and Shm2-Pulsed Myeloid Dendritic Cells Induce T-Cell Activation and Enhance the Neutrophilic Oxidative Burst Response to Aspergillus fumigatus.

Aspergillus fumigatus causes life-threatening opportunistic infections in immunocompromised patients. As therapeutic outcomes of invasive aspergillosis (IA) are often unsatisfactory, the development of targeted immunotherapy remains an important goal. Linking the innate and adaptive immune system, dendritic cells are pivotal in anti-Aspergillus defense and have generated interest as a potential immunotherapeutic approach in IA. While monocyte-derived dendritic cells (moDCs) require ex vivo differentiation, antigen-pulsed primary myeloid dendritic cells (mDCs) may present a more immediate platform for immunotherapy. To that end, we compared the response patterns and cellular interactions of human primary mDCs and moDCs pulsed with an A. fumigatus lysate and two A. fumigatus proteins (CcpA and Shm2) in a serum-free, GMP-compliant medium. CcpA and Shm2 triggered significant upregulation of maturation markers in mDCs and, to a lesser extent, moDCs. Furthermore, both A. fumigatus proteins elicited the release of an array of key pro-inflammatory cytokines including TNF-α, IL-1β, IL-6, IL-8, and CCL3 from both DC populations. Compared to moDCs, CcpA- and Shm2-pulsed mDCs exhibited greater expression of MHC class II antigens and stimulated stronger proliferation and IFN-γ secretion from autologous CD4+ and CD8+ T-cells. Moreover, supernatants of CcpA- and Shm2-pulsed mDCs significantly enhanced the oxidative burst in allogeneic neutrophils co-cultured with A. fumigatus germ tubes. Taken together, our in vitro data suggest that ex vivo CcpA- and Shm2-pulsed primary mDCs have the potential to be developed into an immunotherapeutic approach to tackle IA.

Benefits of exercise-based rehabilitation and Valsartan/Sacubitril association on cardiac function and physical capacites in patients with heart failure and reduced ejection fraction

Cardiac rehabilitation (CR) and Valsartan/Sacubitril (VS) association improved morbi-mortality and quality of live in patients (P) with Heart Failure and reduced Ejection Fraction (HFrEF), but few studies reported their impact on global systolic function and physical capacities. We evaluated the benefits of CR and VS in HFrEF P under optimal therapy (OT) (betablocker + conversion enzyme inhibitor or Angiotensin 2 receptor antagonist + Aldosterone antagonist + diuretics + Electric Resynchronization [ER] if necessary) on echocardiographic left ventricular (LV) EF and cardiorespiratory testing (CRT). VS was associated when OT was obtained (initial dosage 24/26 mg or 49/51 if tolerated). During follow-up, one P died, two were excluded for renal failure and nine were lost, leaving 26/38 P for final analysis (25 male, 62.5 ± 12 yo, 25 ± 7 kg/m2). 14 and 12 presented with ischemic (IC) and dilated (DC) cardiomyopathy. Peak oxygen uptake (VO2) and oxygen uptake at the first ventilatory threshold (VT1) were evaluated from CRT on a bicycle ergometer. All variables were serially measured at inclusion (T0), end of CR (28 ± 9 sessions,T1), as well six (T2) and twelve (T3) months later. LVEF in HFrEF P improved gradually from T0 to T2 (T0: 32.1 ± 7 vs. T1: 40.1 ± 8%, p = 0.006; vs. T2: 46.7 ± 9%, P < 0.0001; T1vsT2, p = 0.03) and then plateaued (T3: 49.6 ± 10%, T2vsT3, NS). Interestingly, LVEF kinetics differed between IC and DC P, values increasing up to T1 only in IC while constantly improving up to T3 in DC. Peak VO2 also gradually increased up to T2, differences with baseline being however significant only after six months (T0: 15.3 ± 5.3 vs. T1: 18.4 ± 6.6 ml/min/kg, NS; vs. T2: 20.7 ± 6.3 ml/min/kg, p = 0.01) before levelling-off (T3: 20.9 ± 5.8 ml/min/kg, T2vsT3 NS). Similar results were obtained for VT1 ( Table 1 , Fig. 1 ). CR associated to VS in P presented with HFrEF is able to enhance global systolic function and cardiorespiratory fitness parameters, improvement being particularly significant in the DC population.

Lactiplantibacillus plantarum 22A-3-induced TGF-β1 secretion from intestinal epithelial cells stimulated CD103+ DC and Foxp3+ Treg differentiation and amelioration of colitis in mice.

In the present study, we evaluated the anti-inflammatory properties of Lactiplantibacillus plantarum 22A-3 (LP22A3) and attempted to elucidate the underlying molecular mechanism. The oral administration of LP22A3 significantly inhibited body weight reduction and decreased colon shortening and colitis score in mice with dextran sulfate sodium (DSS)-induced colitis. It was demonstrated that the production of the active-form of TGF-β tended to increase in both the intestinal epithelial cells (IECs) of the ileum and serum but not in the colon of non-DSS-treated mice by LP22A3. IL-10 level in serum was also elevated by LP22A3-treatment. The mRNA expression of TGF-β, IL-10 and Foxp3 increased only in the small intestines of LP22A3-treated mice. Both the aldehyde dehydrogenase 1 family member A2 (Aldh1a2) mRNA expression and population of CD103+ dendritic cells (DCs) in the small intestine significantly increased in the LP22A3-treated group. LP22A3 induced TGF-β secretion from the IECs of the small intestine with retinoic acid production probably through TLR2, resulting in an increase in CD103+ DCs and the Foxp3+ Treg population. Both cells secrete a high level of anti-inflammatory cytokines, TGF-β and IL-10 contributing to the protective condition in the intestine and thus making it less susceptible to inflammation. This suggested that oral administration of LP22A-3 may be an alternative therapeutic strategy for IBD.

Chemical Composition and Antioxidant Activity of Essential Oils from Populations of Baccharis dracunculifolia DC. in Southern Brazil

HIGHLIGHTS Essential oils from populations of B. dracunculifolia were investigated. β-pinene and (E)-nerolidol were the main compounds in B. dracunculifolia populations. The difference in the chemical profile of the essential oils is quantitative only. There is a negative correlation between the antioxidant activity and spathulenol.

Abstract 5694: Complete regression of murine breast tumors by hetIL-15FC monotherapy correlates with infiltration by T, NK, cDC1 cells and a novel population of dendritic cells

Abstract Introduction: IL-15 cytokine stimulates the proliferation and cytotoxic function of CD8+ T and NK cells. Several preclinical models have supported the anti-tumor activity of IL-15. We have produced the native heterodimeric IL-15 (hetIL-15) of human, macaque or mouse and also fusions to the Fc antibody fragments (hetIL-15FC). Experimental procedures: We studied the therapeutic efficacy of hetIL-15 or hetIL-15FC immunotherapy in the murine EO771 orthotopic breast cancer model in syngeneic C57BL/6 mice. The effects of hetIL-15FC on immune cells were compared in tumors, lymph nodes and spleen by flow cytometry, immunohistochemistry (IHC) and gene expression profiling (Nanostring). We also assessed the metabolic profile of tumor infiltrating T cells. Results: hetIL-15 therapy delayed the primary tumor growth and increased overall survival. hetIL-15 or hetIL-15FC peritumoral administration resulted in tumor regression in 40% of the treated animals and resistance to tumor re-challenge. Therefore, monotherapy induced long term immunological memory able to block tumor growth. Both CD8+ T and NK cells were increased in hetIL-15FC treated tumors; phenotypic analysis showed they were activated and in an active proliferation state. Transcriptomic analysis confirmed the activated state of the T and NK cells, whereas metabolic flux analysis of the infiltrated CD8+T cells from treated mice confirmed a rise in oxygen consumption rate (OCR) with substantial increase of spare respiratory capacity (as a measure of mitochondrial reserve), which supports an activated/non exhausted phenotype of these hetIL-15 treated effector cells. In addition, peritumoral hetIL-15FC administration resulted in an increase of infiltrating, conventional type 1 dendritic cells (cDC1s), suggesting mechanisms of DC-lymphocyte interactions in the tumor. We also observed a new population of intra-tumoral DCs induced by hetIL-15, CD24+ F4/80high cDC2s (F4/80high DCs). The new DC population was negatively correlated with the tumor size of EO771 tumor bearing mice. Phenotypic profiling of F4/80high DCs identified expression of several cDC1 specific markers, including CD103 and IRF8 and t-SNE analysis clustered F4/80high DCs close to cDC1s and macrophages. Conclusion: Our results show complete eradication of EO771 tumors by hetIL-15FC treatment, correlating with different tumor infiltrating immune cell types. We propose that the anti-cancer activity of hetIL-15 in primary tumors is orchestrated by the interplay of CD8+T cells, cDC1s and a novel subset of antigen presenting cells with a distinct phenotypic profile. hetIL-15 supported a favorable metabolic profile of intra-tumoral effector lymphocytes, important for their function. Effective hetIL-15 treatment leads to development of long-term anti-tumor memory. Citation Format: Sevasti Karaliota, Dimitris Stellas, Vasiliki Stravokefalou, Bethany Nagy, Cristina Bergamaschi, Barbara K. Felber, George N. Pavlakis. Complete regression of murine breast tumors by hetIL-15FC monotherapy correlates with infiltration by T, NK, cDC1 cells and a novel population of dendritic cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5694.

350 Histone demethylase LSD1 is required for LC embryonic development but dispensable for LC maintenance and repopulation

Langerhans cells (LCs), the sole dendritic cell subpopulation in the epidermis, are potent regulators of immune surveillance and tolerance. Unlike conventional DCs, LCs follow unique patterns of development and maintenance under steady and inflamed states. Lysine-specific demethylase 1 (LSD1), known to mediate the demethylation of lysine amino acids on histone proteins, plays a key role in the maintenance and differentiation of hematopoietic stem cells. However, the role of LSD1 in LC ontogeny and homeostasis remains unknown. To address this, we generated Csf1rCreLSD1fl/fl conditional knockout (Csf1r.LSD1-KO) mice in which LSD1 was deficient in macrophage/monocytes and LC precursors from embryonic stage. We found a robust reduction of LC precursors in Csf1r.LSD1-KO mice at embryonic day 18.5 and postnatal day 0, suggesting that LSD1 is required for LC ontogeny at late embryonic stage. To investigate whether LSD1 is involved in LC maintenance and repopulation, we created CD11cCreLSD1fl/fl conditional knockout (CD11c.LSD1-KO) mice in which LSD1 was deficient in all DC populations including epidermal LCs after birth. Flow cytometry and immunofluorescent staining of skin showed that the frequency and number of LCs were unaltered in 2-week-old and adult CD11c.LSD1-KO mice compared to wild-type mice. Using an ultraviolet C (UVC)-induced skin damage model, we found that long-term LCs were able to repopulate to the inflamed epidermis of CD11c.LSD1-KO mice 2 weeks after UVC treatment. Overall, our data suggests that LSD1 is critical for LC embryonic development, but not required for the LC maintenance at steady state and repopulation under inflammatory conditions.

A novel lung DC(3) population potently induces Th17 responses to inhaled allergens

Abstract Allergic asthma is an inflammatory disease of the airways associated with T helper (Th)2-driven eosinophilia and Th17-directed neutrophilia. Although eosinophilic asthma generally responds well to inhaled glucocorticoids, neutrophilic asthma does not, and new therapies are needed for this disease subtype. A consensus DC subset in the lung that strongly promotes Th17 responses to inhaled allergens has remained elusive, as some groups have reported that CD103+ DCs (DC1) promote Th17 differentiation, whereas other groups have attributed this function to CD11bhi DCs (DC2). We hypothesized that this apparent discrepancy might be due to the heterogeneity of lung CD11b+ DCs and therefore used mass cytometry (CyTOF) to study these cells following allergic sensitization through the airway. We identified a novel CD11b+ APC that was recruited to the inflamed airways of mice following their inhalation of house dust extracts. These cells were bona fide DCs, as they were FLT3L-dependent, expressed Zbtb46, and formed dendrites. Bulk RNA-seq analysis of these DCs revealed that their transcriptome was similar to, yet distinct from, that of conventional DC2s. Although the novel DCs promoted only weak Th2 differentiation, they drove robust Th17 differentiation. Single cell RNA-sequencing of total lung CD11bhi DCs revealed seven distinct clusters, two of which co-expressed both Tgfb1 and Il1b. Pseudotime analysis suggested a progression from an immature form of these cells to a more mature form. Together, these findings identify a novel DC population (DC3) in the lung that potently stimulates Th17 responses to inhaled allergens.

Regression of murine breast tumors after hetIL-15 monotherapy correlates with a novel population of intratumoral dendritic cells, in addition to increased infiltration of T, NK and cDC1 cells

Abstract Background We studied mechanisms of therapeutic efficacy of the heterodimeric IL-15 (hetIL-15) of human, macaque or mouse in several mouse tumor models, including the murine EO771 orthotopic breast cancer model in syngeneic C57BL/6 mice. Methods The effects of hetIL-15 on immune cells were compared in tumors, lymph nodes and spleen by flow cytometry, immunohistochemistry (IHC), gene expression profiling (Nanostring) and metabolic profiling of tumor infiltrating T cells. Results hetIL-15 administration resulted in tumor regression in 40% of the treated animals and resistance to tumor rechallenge, inducing long term immunological memory. Activated NK and CD8+ T cells were increased in hetIL-15 treated tumors. hetIL-15 administration resulted in increased intratumoral infiltration of conventional Dendritic cells (cDC1) and also induced a new population of intratumoral DC, (F4/80high DCs), which were correlated negatively with the tumor size. Phenotypic profiling of F4/80high DCs identified expression of several cDC1 specific markers, including CD103, IRF8 and XCR1. tSNE analysis clustered F4/80high DCs close to cDC1s and macrophages. Conclusion Complete eradication of EO771 tumors by hetIL-15 treatment correlates with different tumor infiltrating immune cell types. We propose that the anti-cancer activity of hetIL-15 in primary tumors is orchestrated by the interplay of CD8+T cells, cDC1 and a novel subset of antigen presenting cells with a distinct phenotypic profile. Effective hetIL-15 treatment leads to development of long term anti-tumor memory. hetIL-15 supported a favorable metabolic profile of intratumoral effector lymphocytes, important for their function.

Inhibition of LSD1 in MDS progenitors restores differentiation of CD141Hi conventional dendritic cells.

The use of immunotherapy to treat patients with myelodysplastic syndromes (MDS) shows promise but is limited by our incomplete understanding of the immunologic milieu. In solid tumors, CD141Hi conventional dendritic cells (CD141Hi cDCs) are necessary for anti-tumor immunosurveillance and the response to immunotherapy. Here, we found that CD141Hi cDCs are reduced in MDS bone marrow and based on the premise established in solid tumors, we hypothesized that reduced numbers of CD141Hi cDCs are associated with inferior overall survival in MDS patients. We found that MDS patients with reduced numbers of CD141Hi cDCs, but not other DC populations, showed reduced overall survival. To examine the basis for reduction in CD141Hi cDCs, we found fewer numbers of progenitors committed to DC differentiation in the MDS bone marrow and these progenitors expressed lower levels of interferon regulatory factor-8 (IRF8), a master regulator of CD141Hi cDC differentiation. To rescue impaired CD141Hi cDC differentiation, we used pharmacologic inhibition of lysine-specific demethylase 1A (LSD1) to promote CD141Hi cDC differentiation by MDS progenitors. These data reveal a previously unrecognized element of the MDS immunologic milieu. Epigenetic regulation of CD141Hi cDC differentiation offers an intriguing opportunity for intervention and a potential adjunct to immunotherapy for patients with MDS.

Re-evaluation of human BDCA-2+ DC during acute sterile skin inflammation.

Plasmacytoid dendritic cells (pDCs) produce type I interferon (IFN-I) and are traditionally defined as being BDCA-2+CD123+. pDCs are not readily detectable in healthy human skin, but have been suggested to accumulate in wounds. Here, we describe a CD1a-bearing BDCA-2+CD123int DC subset that rapidly infiltrates human skin wounds and comprises a major DC population. Using single-cell RNA sequencing, we show that these cells are largely activated DCs acquiring features compatible with lymph node homing and antigen presentation, but unexpectedly express both BDCA-2 and CD123, potentially mimicking pDCs. Furthermore, a third BDCA-2-expressing population, Axl+Siglec-6+ DCs (ASDC), was also found to infiltrate human skin during wounding. These data demonstrate early skin infiltration of a previously unrecognized CD123intBDCA-2+CD1a+ DC subset during acute sterile inflammation, and prompt a re-evaluation of previously ascribed pDC involvement in skin disease.

Transcriptome landscape of myeloid cells in human skin reveals diversity, rare populations and putative DC progenitors

BackgroundThe heterogeneous functions of dermal myeloid cells in antigen presentation, and scavenging pathogens and cell debris places them centrally in cutaneous inflammation. Single cell transcriptomics can provide new understanding of the heterogeneity and function of yet incompletely understood human dermal myeloid cell subsets. ObjectiveInvestigate the transcriptome landscape of myeloid cells in healthy human skin. MethodsSingle cell RNA-sequencing was performed on skin biopsies from ten healthy donors and analyzed to identify myeloid cell populations. ResultsOne LIN− HLA-DR+ cluster with expression of myeloid-specific genes was identified as a cluster of myeloid cells. Upon reanalysis of this cluster, we identified three macrophage subsets, marked by high expression of CCR1, MARCO or TREM2; and six dendritic cell subsets, marked by high expression of CLEC9A, CXorf21, MCOLN2, LAMP3, KIAA0101 and Langerin, representing respectively cDC1, two subsets of cDC2, a novel DC type, a cluster of proliferating DC, and a Langerhans cell subset. GO term analysis indicated specialized functions for the discrete rare populations of myeloid cells: TREM2 Mφ in lipid metabolism and LAMP3 DC as a mature cDC. Proliferating DCs appeared to represent cDC2 progenitors. ConclusionThe transcriptional landscape of myeloid cell populations in human skin indicates several, novel populations with specialized functions, as well as a rare proliferating DC population that likely accounts for local regeneration or expansion of dermal DCs. We provide robust gene expression markers for each of these populations that should permit better understandings of their roles in various homeostatic and pathologic immune processes in the skin.

Inhibition of LSD1 in Myelodysplastic Syndrome Progenitors Restores Differentiation of CD141Hi Conventional Dendritic Cells

Background: Immunotherapeutic approaches for myelodysplastic syndrome (MDS) show promise, but progress is limited by our incomplete understanding of the immunologic milieu. In a recent Phase I trial, we found that MDS patients with higher numbers of CD141Hi conventional dendritic cells (cDCs) were more likely to respond to NY-ESO-1 vaccination. In solid tumor models, the CD141Hi cDC is critical for initiating anti-tumor immune responses but its impact in myeloid malignancies is unknown. In studies of primary human specimens and mouse models, we tested the hypothesis that MDS patients exhibit decreased quantity and quality of CD141Hi cDCs due to impaired myeloid differentiation. Methods: Bone marrow (BM) cells were collected from MDS patients (pre-treatment) and age matched healthy donors (HD; defined as absence of hematologic malignancy). We quantified DC populations, stem, progenitor cells and interferon regulatory factor-8 (IRF-8) expression using flow cytometry and RT-qPCR. Histone modifications were assessed by chromatin immunoprecipitation. To assess DC differentiation of progenitors, human CD34+ and mouse c-kit+ cells were expanded and differentiated in vitro. Results: We found fewer CD141Hi cDCs (p&lt;0.0001), CD1c+ cDCs (p&lt;0.005) and plasmacytoid DCs (CD123+ pDCs; p&lt;0.005) overall in BM samples from MDS patients (n=71) compared to HD (n=17). We stratified MDS patients based on the relative number of DCs and found that only those patients with highest number of CD141Hi cDCs had superior survival (p &lt; 0.05). No differences in survival were seen in patients stratified by the CD1c+ cDCs (p = 0.96) and CD123+ pDCs (p = 0.32) populations. We hypothesized that decreased numbers of CD141Hi cDCs and adverse survival in MDS patients resulted from impaired differentiation of DC progenitors. We showed that MDS patients (n=19) have fewer monocyte-DC progenitors (MDP) and common DC progenitors (CDP) compared to HD (n=11; p&lt;0.01). We then hypothesized that MDS progenitors express lower levels of IRF8, a master regulator of CD141Hi cDC differentiation. IRF8 expression was significantly lower in CDPs from MDS patients compared to HD (p&lt;0.05). Furthermore, MDS patients with lower levels of IRF8 (n=8) in their MDPs showed a trend towards production of fewer CDPs and significantly fewer CD141Hi cDCs compared to those with higher levels of IRF8 (n=10; p&lt;0.005). These results suggest that approaches to increase IRF8 expression could enhance CD141Hi cDC differentiation. We hypothesized that inhibition of lysine-specific histone demethylase 1A (LSD1), which increases IRF8 expression in myeloid leukemia cells, would induce CD141Hi cDC differentiation. Pharmacologic inhibition of LSD1 increased IRF8 expression (both mRNA and protein) in KG-1 cells, a model of human CD34+ cells, and in HD and MDS CD34+ progenitors (p&lt;0.05). LSD1 inhibition in KG-1 cells resulted in increased H3K27 acetylation (3861-fold change) and H3K4 dimethylation (922.4-fold change) compared to PBS (p&lt;0.05) at a region -70 kb to the IRF8 transcriptional start site, a putative regulatory element that demonstrated the highest level of LSD1 binding. These data indicate that LSD1 inhibition alters histone modifications at the IRF8 locus, resulting in increased expression of IRF8. Pharmacologic inhibition of LSD1 in HD CD34+ cells increased the number of mature CD141Hi cDCs in 92% of specimens (n = 12; 3.4 fold-change) compared to PBS. Similarly, LSD1 inhibition in MDS CD34+ cells increased the number of CD141Hi cDCs (n = 12) in 75% of patient specimens (16.8 fold-change) compared to PBS. IRF8 function is conserved between mice and humans. To test whether the effect of LSD1 inhibition on cDC differentiation was dependent on IRF8, we compared the effect of LSD1 inhibition on BM c-kit+ cells from Irf8 knock-out mice (Irf8-KO) and littermate controls (WT). LSD1 inhibition in WT c-kit+ cells resulted in increased numbers of CD141Hi cDCs in vitro (p&lt;0.05). By contrast, LSD1 inhibition of Irf8-KO c-kit+ cells did not result in differentiation of CD141Hi cDCs. These data suggest that LSD1 inhibition drives CD141Hi cDCs differentiation through IRF8. Conclusion: These data reveal a previously unrecognized determinant of the immune microenvironment in MDS. The opportunity for epigenetic regulation of CD141Hi cDC differentiation in MDS offers an opportunity for intervention and a potential adjunct to immunotherapy for patients. Disclosures Sait: Celgene: Consultancy. Griffiths:Boston Scientific: Consultancy; Boston Scientific: Consultancy; Genentech, Inc.: Research Funding; Persimmune: Consultancy; Persimmune: Consultancy; Abbvie, Inc.: Consultancy; Genentech, Inc.: Research Funding; Novartis Inc.: Consultancy; Celgene, Inc: Consultancy, Research Funding; New Link Genetics: Consultancy; New Link Genetics: Consultancy; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; Astex Phramaceuticals/Otsuka Pharmaceuticals: Consultancy, Research Funding; Novartis Inc.: Consultancy; Partner Therapeutics: Consultancy; Partner Therapeutics: Consultancy; Appelis Pharmaceuticals: Other: PI on a clinical trial; Onconova Therapeutics: Other: PI on a clinical trial; Appelis Pharmaceuticals: Other: PI on a clinical trial; Onconova Therapeutics: Other: PI on a clinical trial; Celgene, Inc: Consultancy, Research Funding; Abbvie, Inc.: Consultancy, PI on a clinical trial.

Abstract 1545: Altered myeloid and lymphoid composition of tumor microenvironment following anti-SEMA4D and immune checkpoint combination therapies

Abstract Anti-semaphorin 4D (SEMA4D, CD100) blocking antibody promotes immune infiltration, reduces immunosuppressive myeloid cells, and enhances T cell activity and tumor growth inhibition in combination with various immunotherapies in preclinical animal models. Clinical trials of immune checkpoint inhibitors (ICI) in combination with pepinemab (VX15/2503), humanized anti-SEMA4D antibody, are currently underway in several cancer indications. SEMA4D exerts multi-faceted effects within the tumor microenvironment by creating a barrier at the tumor-stroma margin to restrict immune cell infiltration and promote immunosuppressive activity of myeloid-derived cells. Blocking antibody to SEMA4D directly enhanced M1/M2 ratio and reduced both expression of chemokines that recruit MDSC and the ability of MDSC to suppress T cell activity. Antibody blockade simultaneously restored the ability of dendritic cells and cytotoxic T cells to infiltrate the TME, increasing ratio of Teffector to Tregulatory cells, in syngeneic tumor models. Importantly, anti-SEMA4D MAb enhanced the activity of co-administered immunotherapies, including antibodies to PD1, CTLA-4, and LAG3, epigenetic modulators, and additional novel immunomodulatory combinations in murine breast, colon, head and neck, and melanoma tumor models. New data will describe development and application of methods to assess immune phenotype and biomarkers in translational and clinical studies. These include flow cytometry and whole slide scans of multiplex IHC panels to examine MDSC, M1/M2 macrophage, monocytes, activated DC, B cells, exhausted, activated and stem-like populations of T cells in clinical samples. Expression of SEMA4D and its receptor PlexinB1 were also evaluated in spatial context and cellular co-localization. In summary, SEMA4D represents a novel target to regulate immune infiltration and mesenchymal suppression, sources of resistance to current immunotherapies. Pepinemab treatment was well tolerated in a Phase I oncology trial (NCT01313065) and is currently being evaluated as single agent or in ICI combinations in: (i) a Phase 1b/2a combination trial of pepinemab with avelumab in ICI naïve or ICI refractory NSCLC (CLASSICAL-Lung) (NCT03268057); (ii) a phase 1 combination trial of pepinemab with nivolumab or ipilimumab in melanoma patients who have progressed on any anti-PD-1/PD-L1 (NCT03425461); (iii) a neoadjuvant integrated biomarker trial in patients with metastatic colorectal, pancreatic (NCT03373188) and head and neck (NCT03690986) cancers treated with pepinemab in combination with nivolumab or ipilimumab; and (iv) a Phase 1/2 trial of pepinemab in children with solid tumors and children and young adults with osteosarcoma (NCT03320330). Clinical trials will evaluate safety, tolerability, efficacy, and biological endpoints, including immunophenotyping tumors and blood. Citation Format: Elizabeth E. Evans, Terrence L. Fisher, Holm Bussler, Crystal Mallow, Christine Reilly, Sebold Torno, Desa Rae Pastore, Maria Scrivens, Alan Howell, Leslie Balch, John E. Leonard, Clint Allen, Paul E. Clavijo, Gregory Lesinski, Christina Wu, Conor Steuer, Nabil F. Saba, Brian Olson, Siwen Hu-Lieskovan, Antoni Ribas, Emily G. Greengard, Ernest S. Smith, Maurice Zauderer. Altered myeloid and lymphoid composition of tumor microenvironment following anti-SEMA4D and immune checkpoint combination therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1545.

A 28-Year History of HIV-1 Drug Resistance and Transmission in Washington, DC.

Washington, DC consistently has one of the highest annual rates of new HIV-1 diagnoses in the United States over the last 10 years. To guide intervention and prevention strategies to combat DC HIV infection, it is helpful to understand HIV transmission dynamics in a historical context. Toward this aim, we conducted a retrospective study (years 1987–2015) of 3,349 HIV pol sequences (1,026 bp) from 1,995 individuals living in the DC area belonging to three different cohorts. We coupled HIV sequence data with clinical information (sex, risk factor, race/ethnicity, viral load, subtype, anti-retroviral regimen) to identify circulating drug resistant mutations (DRM) and transmission clusters and assess their persistence over time. Of the transmission clusters identified in the DC area, 78.0 and 31.7% involved MSM and heterosexuals, respectively. The longest spread of time for a single cluster was 5 years (2007–2012) using a distance-based network inference approach and 27 years (1987–2014) using a maximum likelihood phylogenetic approach. We found eight subtypes and nine recombinants. Genetic diversity increased steadily over time with a slight peak in 2009 and remained constant thereafter until 2015. Nucleotide diversity also increased over time while relative genetic diversity (BEAST) remained relatively steady over the last 28 years with slight increases since 2000 in subtypes B and C. Sequences from individuals on drug therapy contained the highest total number of DRMs (1,104–1,600) and unique DRMs (63–97) and the highest proportion (>20%) of resistant individuals. Heterosexuals (43.94%), MSM (40.13%), and unknown (44.26%) risk factors showed similar prevalence of DRMs, while injection drug users had a lower prevalence (33.33%). Finally, there was a 60% spike in the number of codons with DRMs between 2007 and 2010. Past patterns of HIV transmission and DRM accumulation over time described here will help to predict future efficacy of ART drugs based on DRMs persisting over time and identify risk groups of interest for prevention and intervention efforts within the DC population. Our results show how longitudinal data can help to understand the temporal dynamics of HIV-1 at the local level.