Th1 Phenotype Research Articles

Dopamine receptor D2 on CD4+ T cells is protective against neuroinflammation and neurodegeneration in a mouse model of Parkinson's disease

Parkinson's disease (PD) is a chronic neurodegenerative disease. Recently, neuroinflammation driven by CD4+ T cells has been involved in PD pathophysiology. Human and murine lymphocytes express all the five subtypes of dopamine receptors (DRs), DRD1 to DRD5. However, roles of DRs particularly DRD2 expressed on CD4+ T cells in PD remain elucidated. Global Drd1- or Drd2-knockout (Drd1−/− or Drd2−/−) mice or CD4+ T cell-specific Drd2-knockout (Drd2fl/fl/CD4Cre) mice were intraperitoneally injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to induce PD with the different mutants. On the 7th day following MPTP injection, mice were assessed for dopaminergic neurodegeneration, locomotor impairments, microglial activation, as well as CD4+ T-cell differentiation and function. Furthermore, in vitro CD4+ T cells were exposed to DRD2 agonist and antagonist and then differentiation and function of the cells were determined. MPTP induced dopaminergic neuronal loss in the nigrostriatal system, motor coordinative and behavioral impairments, microglial activation, and CD4+ T-cell polarization to pro-inflammatory T-helper (Th)1 and Th17 phenotypes. Importantly, either Drd2−/− or Drd2fl/fl/CD4Cre mice manifested more severe dopaminergic neurodegeneration, motor deficits, microglial activation, and CD4+ T-cell bias towards Th1 and Th17 phenotypes in response to MPTP, but Drd1−/− did not further alter MPTP intoxication. DRD2 agonist sumanirole inhibited shift of CD4+ T cells obtained from MPTP-intoxicated mice to Th1 and Th17 phenotypes and DRD2 antagonist L-741,626 reversed sumanirole effects. These findings suggest that DRD2 expressed on CD4+ T cells is protective against neuroinflammation and neurodegeneration in PD. Thus, developing a therapeutic strategy of stimulating DRD2 may be promising for mitigation of PD.

Role of Toll-Like Receptor 2 in Regulation of T-Helper Immune Response in Chronic Obstructive Pulmonary Disease.

Objective According to modern views, the differences in the clinical course of chronic obstructive pulmonary disease (COPD) are associated with certain types of T-helper (Th) immune response. Recent data have shown that toll-like receptor 2 (TLR2) is involved in the development of Th immune response. However, TLR2-mediated regulation of Th subpopulation balance in COPD needs to be elucidated. The aim of our work is to determine the mechanisms of TLR2-mediated regulation of Th immune response in COPD of varying severity. Methods The study included 323 smokers/ex-smokers with stable COPD (GOLD I, GOLD II, and GOLD III) and 97 healthy nonsmokers (control group). Serum levels of Th1 (TNF-α and IFN-γ), Th2 (IL-4), Th17 (IL-6 and IL-17A), Treg (IL-10) cytokines, and the percentage of peripheral blood Th cells expressing TLR2 (CD4+CD282+) were assessed by flow cytometry. Serum concentrations of IL-21 (Th17) and TGF-β1 (Treg) were measured using the ELISA method. The predominant Th cytokine profile in serum was determined by calculating the ratios between levels of Th1 and Th17 cytokines. Spearman's correlation test was performed. Results Patients with COPD GOLD II and III with Th1 and Th17 cytokine profiles exhibited an increase in the percentage of CD4+CD282+ cells compared to the control group. In COPD GOLD I–III, positive correlations between CD4+CD282+ cell frequency and Th17 cytokine levels (IL-6, IL-17A, and IL-21) were found. In COPD GOLD I, IL-10 concentration was negatively correlated with the percentages of studied cells; in COPD GOLD II, a positive correlation between these parameters was noted. Conclusions Enhanced TLR2 expression on CD4+ cells shifts cytokine profile toward Th17 phenotype that plays a crucial role in COPD progression. The level of TLR2 expression on peripheral blood CD4+ cells may be considered as a biomarker for diagnosing and predicting the progression of COPD.

Clonally expanded, GPR15-expressing pathogenic effector TH2 cells are associated with eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is an allergic disorder characterized by the recruitment of eosinophils to the esophagus, resulting in chronic inflammation. We sought to understand the cellular populations present in tissue biopsies of patients with EoE and to determine how these populations are altered between active disease and remission. To this end, we analyzed cells obtained from esophageal biopsies, duodenal biopsies, and peripheral blood of patients with EoE diagnosed with active disease or remission with single-cell RNA and T cell receptor (TCR) sequencing. Pathogenic effector TH2 (peTH2) cells present in the esophageal biopsies of patients with active disease expressed distinct gene signatures associated with the synthesis of eicosanoids. The esophageal tissue-resident peTH2 population also exhibited clonal expansion, suggesting antigen-specific activation. Peripheral CRTH2+CD161- and CRTH2+CD161+ memory CD4+ T cells were enriched for either a conventional TH2 phenotype or a peTH2 phenotype, respectively. These cells also exhibited substantial clonal expansion and convergence of TCR sequences, suggesting that they are expanded in response to a defined set of antigens. The esophagus-homing receptor GPR15 was up-regulated by peripheral peTH2 clonotypes that were also detected in the esophagus. Finally, GPR15+ peTH2 cells were enriched among milk-reactive CD4+ T cells in patients with milk-triggered disease, suggesting that these cells are an expanded, food antigen-specific population with enhanced esophagus homing potential.

Immunisation of ferrets and mice with recombinant SARS-CoV-2 spike protein formulated with Advax-SM adjuvant protects against COVID-19 infection

The development of a safe and effective vaccine is a key requirement to overcoming the COVID-19 pandemic. Recombinant proteins represent the most reliable and safe vaccine approach but generally require a suitable adjuvant for robust and durable immunity. We used the SARS-CoV-2 genomic sequence and in silico structural modelling to design a recombinant spike protein vaccine (Covax-19™). A synthetic gene encoding the spike extracellular domain (ECD) was inserted into a baculovirus backbone to express the protein in insect cell cultures. The spike ECD was formulated with Advax-SM adjuvant and first tested for immunogenicity in C57BL/6 and BALB/c mice. Covax-19 vaccine induced high spike protein binding antibody levels that neutralised the original lineage B.1.319 virus from which the vaccine spike protein was derived, as well as the variant B.1.1.7 lineage virus. Covax-19 vaccine also induced a high frequency of spike-specific CD4 + and CD8 + memory T-cells with a dominant Th1 phenotype associated with the ability to kill spike-labelled target cells in vivo. Ferrets immunised with Covax-19 vaccine intramuscularly twice 2 weeks apart made spike receptor binding domain (RBD) IgG and were protected against an intranasal challenge with SARS-CoV-2 virus given two weeks after the last immunisation. Notably, ferrets that received the two higher doses of Covax-19 vaccine had no detectable virus in their lungs or in nasal washes at day 3 post-challenge, suggesting that in addition to lung protection, Covax-19 vaccine may have the potential to reduce virus transmission. This data supports advancement of Covax-19 vaccine into human clinical trials.

Tocilizumab in a patient affected by chronic active antibody mediated rejection; histological improvement, reduction of proteinuria and renal function stabilization

Introduction: Chronic active antibody-mediated rejection (cAMR) is a significant and rapid destructive form of allograft rejection and it is related to donor specific antibodies (DSA). Interleukin 6 (IL-6) plays an important role in mediating the allograft rejection by promoting CD4+ T cells differentiation to Th17 phenotype while inhibiting Treg. Tocilizumab is a humanized monoclonal antibody directed to IL-6 receptor (IL6-R). The aim of the study is to demonstrate the efficacy of tocilizumab as rescue therapy for cAMR. Case Presentation: A 50-year-old man with Alport syndrome and with positive DSA against B7 e B55 underwent a second kidney transplant (HLA 2 mismatch). He received thymoglobulin and three plasma exchanges as induction therapy. Proteinuria (1-1.3 g/24 h) and decline in kidney function (serum creatinine; 1.5 mg/dL) appeared at 9 months. Kidney biopsy showed endocapillary proliferation, mononuclear cells infiltration, glomerular basal membrane duplication and tubulitis suggestive of cAMR. The patient has been treated with tocilizumab (6 mg/kg/mon) for six months. Reduction of proteinuria (0.6 g/24 h) and mild improvement of kidney function (serum creatinine; 1.3 mg/dL) were observed after tocilizumab treatment. A second biopsy revealed a significant decrease of glomerulitis and peritubular capillaritis. A significant reduction in DSA was detected. Conclusion: Inhibition of the IL-6 receptor by tocilizumab may represent a novel and cheering approach to treat cAMR.

Transgenic expression of a T cell epitope in Strongyloides ratti reveals that helminth-specific CD4+ T cells constitute both Th2 and Treg populations.

Helminths are distinct from microbial pathogens in both size and complexity, and are the likely evolutionary driving force for type 2 immunity. CD4+ helper T cells can both coordinate worm clearance and prevent immunopathology, but issues of T cell antigen specificity in the context of helminth-induced Th2 and T regulatory cell (Treg) responses have not been addressed. Herein, we generated a novel transgenic line of the gastrointestinal nematode Strongyloides ratti expressing the immunodominant CD4+ T cell epitope 2W1S as a fusion protein with green fluorescent protein (GFP) and FLAG peptide in order to track and study helminth-specific CD4+ T cells. C57BL/6 mice infected with this stable transgenic line (termed Hulk) underwent a dose-dependent expansion of activated CD44hiCD11ahi 2W1S-specific CD4+ T cells, preferentially in the lung parenchyma. Transcriptional profiling of 2W1S-specific CD4+ T cells isolated from mice infected with either Hulk or the enteric bacterial pathogen Salmonella expressing 2W1S revealed that pathogen context exerted a dominant influence over CD4+ T cell phenotype. Interestingly, Hulk-elicited 2W1S-specific CD4+ T cells exhibited both Th2 and Treg phenotypes and expressed high levels of the EGFR ligand amphiregulin, which differed greatly from the phenotype of 2W1S-specific CD4+ T cells elicited by 2W1S-expressing Salmonella. While immunization with 2W1S peptide did not enhance clearance of Hulk infection, immunization did increase total amphiregulin production as well as the number of amphiregulin-expressing CD3+ cells in the lung following Hulk infection. Altogether, this new model system elucidates effector as well as immunosuppressive and wound reparative roles of helminth-specific CD4+ T cells. This report establishes a new resource for studying the nature and function of helminth-specific T cells.

STING Agonist Combined to a Protein-Based Cancer Vaccine Potentiates Peripheral and Intra-Tumoral T Cell Immunity.

Combining different immunotherapy approaches is currently building the future of immunotherapy, with the view to maximize anti-tumoral efficacy for larger patient population. The KISIMA™ platform allows the development of protein-based cancer vaccines able to induce tumor-specific T cell response resulting in anti-tumoral efficacy in various mouse models. Intra-tumoral administration of stimulator of interferon gene agonists (STINGa) was shown to induce a potent inflammatory response leading to the development of tumor-specific immunity. Here, we explored the efficacy and mechanisms of action of subcutaneous STINGa treatment combined with therapeutic vaccination in various mouse tumor models. This combinatory treatment highly enhanced frequency and effector function of both peripheral and intra-tumoral antigen-specific CD8 T cells, promoting potent IFNγ and TNFα production along with increased cytotoxicity. Moreover, combination therapy favorably modulated the tumor microenvironment by dampening immune-suppressive cells and increasing CD4 T cell infiltration together with their polarization toward Th1 phenotype. Combination with STINGa treatment improved the effect of therapeutic vaccination, resulting in a prolonged control and slower growth of B16-OVA and TC-1 tumors. Altogether, the results presented here highlight the potential of combining STINGa with a therapeutic protein vaccine for cancer treatment.

Abstract 1719: OR2805, an anti-CD163 antibody derived from an elite responder to checkpoint inhibitor therapy relieves immunosuppression caused by tumor associated macrophages

Abstract Background: OR2805 is a fully human IgG1 antibody that binds to CD163, an immune-suppressive receptor highly expressed on tumor associated macrophages (TAMs). High numbers of CD163-expressing TAMs generally predict an unfavorable prognosis in solid tumors. CD163-expressing TAMs contribute to an immune-suppressive tumor microenvironment (TME) and inhibit an anti-tumor T-cell response by engaging immune checkpoints, producing immune-suppressive cytokines, and promoting T-cell skewing towards a pro-cancer Th2 phenotype. Relieving the immune suppression of CD163-expressing TAMs in the TME to improve T-cell-mediated responses is a rational adjunct to immune checkpoint inhibitor (CPI) therapy. Methods: OR2805 was discovered using OncoResponse's discovery platform by cell-based functional and phenotypic assays. B cells derived from CPI elite responders were cultured at clonal density, and IgG antibodies in supernatants were evaluated for binding to myeloid-derived suppressor cells. Variable-regions from positive hits were sequenced, cloned, and expressed as recombinant IgG1. OR2805 was identified as one of the top hits that relieved myeloid cell-mediated immune suppression. Co-cultures of immunosuppressive primary human M2 macrophages and autologous T cells were used to interrogate OR2805-dependent immunomodulatory responses in vitro. The anti-tumor activity of OR2805 in vivo was evaluated in a humanized mouse model. Results: OR2805 binds to CD163 expressed on TAMs and M2-like macrophages. OR2805 does not bind to other hematopoietic cells nor to a panel of human primary non-immune cells. OR2805 treatment reduces expression of cell-surface markers associated with tumor-promoting M2c-like macrophages. In co-culture assays, OR2805 relieves the suppressive effect of M2 macrophages and results in increased T-cell activation and proliferation, upregulation of T-cell activation markers, and enhanced T-cell-mediated tumor cell killing. Administration of OR2805 in humanized NSG-SGM3 mouse tumor models resulted in approximately 55% and 75% reduction in A549 tumor growth and NCI-H1975 tumor growth, respectively. In this model, OR2805 treatment significantly increased the proportions of human CD8+ T cells and human CD11b+ myeloid cells, as well as significantly enhanced expression of activation markers by human CD8+ T cells. Conclusions: OR2805 reduces M2 macrophage-mediated immunosuppression and enhances anti-tumor immune responses. OR2805 treatment induces anti-tumor activity in lung cancer xenograft models in humanized mice. These data support further development of OR2805 as an anti-cancer therapy, both as a monotherapy and an addition to current CPI therapy. Citation Format: Peter Probst, Randi Simmons, Valerie Wall, Meghan Zuck, Myriam Bouchlaka, Sam Lam, Raymond Fox, Darbie Whitman, Tom Graddis, Kamal D. Puri. OR2805, an anti-CD163 antibody derived from an elite responder to checkpoint inhibitor therapy relieves immunosuppression caused by tumor associated macrophages [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 1719.

Exploring the Cord Blood T Cell Compartment with Mass Cytometry Reveals the Existence of Memory CD4, CD8 and Regulatory T Cell Pools in the Fetal Period

It has been long perceived that cord blood (CB) T cells are exclusively composed of antigen-inexperienced ‘naïve’ T cells under Th2 trophic and/or Th1 suppressive conditions, and that this is important for the maintenance of tolerance during pregnancy. However, a recent study reported the existence of a small fraction of memory CD4+ T cells with inflammatory cytokine profile within the sterile CB environment. This is in contrast to the common dogma challenging the presence of inflammatory T cells in the CB environment. To produce a more in-depth picture of the T cell compartment in CB, we assembled a panel of 40 antibodies to characterize the phenotypic and functional complexity of CB T cells. We interrogated the immune landscape of 12 CB donors vs 12 healthy adult peripheral blood (PB) donors to obtain single-cell profiles of the T-cell compartment. We used t-SNE algorithm to visualize the high dimensional data and the nearest-neighbor based clustering algorithm, PhenoGraph, to identify the potential clusters in high dimensional space. We observed significant segregation between PB and CB T cells. As expected, the majority of CD4+ and CD8+ T cells in CB were composed of naïve cells, while PB CD4+ and CD8+ T cells were characterized by a dominantly memory T cell phenotype. Within the CB CD4+ T cell compartment, in addition to the CD127+CD25- naïve T cell subset, we observed two more populations, including CD127+CD25int memory T cells (median 1.92%; range 0.69%-4.14%) and CD127lowCD25hi regulatory T-cells (Tregs) (median 1.9%; range 1.36%-2.98%). Similarly, a small subset of memory T cells, defined as CD45RA-CCR7+/-CD95+ (median 3.23%; range:1.53%-7.45%) was present in the CD8 T cell compartment in CB.Each T-cell presents a diversity of surface phenotypes defined by distinct combinations of marker expression. Thus, we sought an overview of the similarities and differences between CB and PB T-cell subsets. Analysis of CB Tregs revealed a repertoire composed mainly of naïve and central memory subsets, the latter expressed CD95, a marker of activation and memory, suggesting the generation of memory Tregs during fetal life. Both CB and PB Tregs were characterized by expression of PD-1, CD39, TIGIT, ICOS, CD161, CCR4, DNAM-1, CD49f and CD26. Expression of TIGIT, PD-1, CD39, CD49f, ICOS and CCR4 was exclusively confined to the memory Treg subset. In contrast to PB Tregs, CB Tregs lacked CCR10 and CXCR5 expression, suggesting differential migratory properties for CB and PB Tregs. We next focused on the differences between the naïve and memory CD4+ T cell subsets in CB. Chemokine receptors can be used to classify T-cell subsets with distinct migratory capacities and effector function. Naïve CD4 T cells do not express chemokine receptors, while memory T cells express different combinations of chemokine receptors that give clues to their lineage commitment. We discovered CCR6, CCR4, CXCR3, CD161, DNAM-1, CD49f, KLRG1, Beta7, PD-1, and TIGIT as the set of combinatorial markers to identify the memory CD4+ T cell pool in CB. Notably, a significant proportion of memory CD4+ T cells in CB expressed CCR4, supporting the notion that the CB environment drives a Th2 rather than a Th1 or Th17 functional phenotype. Moreover, in CB, expression of CCR6, CXCR3 and CD161 was confined to the memory CD4 T cell pool, suggesting that Th1, Th2 and Th17 lineage commitment starts in the fetal period, although the exact mechanism leading to memory T cell formation is not known.Taken together, interrogation of the T cell compartment in CB and PB in high dimensional space reveals well-defined and distinct memory signatures. Acquisition of memory phenotype instructs expression of unique sets of markers for each memory subset and varies among CD4, CD8 and Treg. Our data indicate the presence of a seemingly full-blown immune response, resulting in the generation of committed memory T cells and changes in the immune landscape of CB, although the initiating factor for this is yet to be determined. Mass cytometry proves to be a valuable tool in the characterization and discovery of novel T cell subsets, even if present at low frequencies. Studies to track the differentiation and diversification of T cells in CB transplant recipients and to link the impact of the T cell repertoire on clinical outcomes are under way. DisclosuresNo relevant conflicts of interest to declare.

Myeloid-Derived Suppressor Cell Subtypes Are Responsible for the Th2 Phenotype of T Cells in CLL

In chronic lymphocytic leukemia (CLL), monoclonal B cells expand and accumulate in the blood, bone marrow, spleen and secondary lymphoid organs in response to incompletely defined cellular and molecular interactions involving multiple cell types within the tumor microenvironment (TME). Bidirectional interactions of CLL cells with components of the TME - mesenchymal stromal cells, myeloid cells, T cells, and matrix - are critical for leukemic cell survival and proliferation. Here, we focused on understanding how two non-leukemic cell types within the TME, T lymphocytes and myeloid-derived suppressor cells (MDSCs), interact to create a TME supportive of a CLL clone.In CLL, it has been suggested alterations in CD4+ and CD8+ T cell ratios and functions are responsible for a non-inflammatory and immunosuppressive cytokine TME, with expansions of Th2s and Tregs. Conversely, higher numbers of Th17 and CD8+ cells are associated with longer survival. MDSCs are not well studied in CLL, although existing reports suggest that their expansion suppresses T cell proliferation in vitro. Here we studied the effects of MDSCs and CLL B cells on T cell numbers and function.First, we identified a correlation between blood T cells and MDSCs in 56 CLL patients. Absolute numbers of MDSCs correlated with total numbers of CD3+, CD4+ and CD8+ cells (P= 0.002, < 0.001, < 0.001 and Spearman r = 0.44, 0.646, = 0.61, respectively). However, the correlation of MDSC subtypes with CD4+ and CD8+ cells differed: monocyte-like MDSCs (mMDSCs) associated with CD4+ cells (P < 0.001, Spearman r = 0.73) whereas granulocyte-like MDSCs (gMDSCs) associated with CD8+ cells (P= 0.008; Spearman r = 0.45).Moreover, in a subset of 18 CLL patients tested, MDSC numbers directly correlated with the levels of Th2 (P= 0.049, Spearman r = 0.470), Th17 (P= 0.040, Spearman r = 0.488), Tc17 (P= 0.018, Spearman r = 0.550) and Tc22 (P= 0.010, Spearman r = 0.587) cells. When dividing MDSCs into subsets, gMDSCs exhibited a similar correlation except for the absence of Th2 and the presence of a correlation with Th22 (P= 0.033, Spearman r = 0.505). Conversely, mMDSCs positively associated with Th1 (P= 0.024, Spearman r = 0.549) and Tc1 (P= 0.019, Spearman r = 0.569) cells.We next addressed the functional effect of MDSCs on naïve CD4+ cell differentiation using samples from 16 CLL patients and 10 healthy, age-matched controls (HCs). CD3+CD45RO-CD62L+ CD4+ lymphocytes were FACS-sorted and stimulated in vitro with anti-CD3/CD28 beads plus IL-2, and the percentages of CD4+ T cells producing various cytokines were quantified by intracellular flow cytometry. These results were compared with those obtained in the presence of mMDSCs (HLA-DRlo/CD11b+/CD33+/CD14+), gMDSCs (HLA-DRlo/CD11b+/CD33+/CD15+), monocytes (HLA-DRhi/CD11b+/CD14+) or CLL B lymphocytes (CD19+). On day 7, CLL T cells, cultured in the absence of other cells, generated a higher percentage of cytokine-producing cells than those from HCs (22% vs. 8%; P < 0.001). This difference was due to an increase in all cytokine-producing cells except classical Th17s producing IL-17A. When CLL T cells were co-cultured with gMDSCs, a significant expansion of Th2 (IL-4+) (P= 0.044) and a trend in growth of IL-17F+ Th17cells (P= 0.101) was found. In contrast, co-culture of CLL T cells with mMDSCs led to a reduction in Th22 (IL-22+) (P= 0.005) and Treg (FoxP3+) (P= 0.006) cells and to a trend towards expansion of Th2 (IL-4+) cells. Co-cultures of CLL T cells with monocytes, similarly to mMDSCs, led to an expansion of Th2 (IL-4+) cells (P= 0.01) and a reduction in Treg (FoxP3+) cells (P= 0.002). Finally, co-culturing naïve CD4+ lymphocytes with CLL B cells had a different and unexpected effect, promoting the expansion of Th1 (IFNγ+) cells (P= 0.033) and Th22 (IL-22+) T cells (P= 0.052).In summary, the absolute number of blood T cells, CD4+ and CD8+ subsets, and MDSCs are linked, although associations of T cell subsets differ between mMDSCs and gMDSCs. MDSCs and their subsets influence naïve T cell differentiation differently, in general leading to expansion of Th2 (IL-4+) cells. In contrast, CLL B cells had a different effect on T cell polarization, preferentially expanding Th1 (IFNγ+) cells. Thus, it appears that myeloid cells and especially MDSCs are responsible for polarization of the T cell compartment to Th2 which is said to be dominant in CLL patients. DisclosuresKolitz:Gilead, Magellan, and Novartis: Honoraria; Gilead, Novartis, and Seattle Genetics: Other: Travel Support; Boehringer Ingelheim, Cantex, Erytech, and Millennium: Research Funding; Celgene, Jazz: Equity Ownership; Gilead, Magellan, Novartis, Pharmacyclics, and Seattle Genetics: Consultancy. Barrientos:Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Janssen: Consultancy; Gilead: Consultancy, Research Funding. Stamatopoulos:Novartis SA: Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding.

A ROR1 Bispecific T Cell Engager for the Treatment of Chronic Lymphocytic Leukaemia Demonstrates Enhanced Function Following Ibrutinib Treatment

ROR1 is expressed on a range of solid and haematological malignancies, including chronic lymphocytic leukaemia, mantle cell lymphoma and a subset of acute lymphoblastic lymphoma, making it an attractive therapeutic target. In keeping with this, ROR1 CAR T cells and a ROR1 antibody, are being investigated as novel treatments [NCT02222688, NCT02776917 & NCT02706392]. BiTEs are small, 55kDa antibody based therapeutics composed of two single chain variable fragments (scFv) linked in tandem by a flexible linker. One scFv binds to tumour antigens and the other to the CD3 complex, bringing target cells and T cells in close proximity, facilitating formation of cytotoxic synapses and resulting in target cell death. We have generated a fully humanised ROR1 BiTE which unlike conventional antibodies recruits and utilises the inherent cytotoxic potential of polyclonal resident T cells and does not require ex vivo cell manipulation. We therefore investigated the potential of a ROR1 BiTE for the treatment of CLL.We first assessed the efficacy of our ROR1 BiTE against SKW6.4, Jeko1 and Kasumi-2 haematological cell lines which are ROR1 positive, confirming significant cytotoxicity even at low concentrations of 1ng/ml (Figure 1A). To demonstrate in vivo potential, mice were injected with SKW6.4 cells and disease established before treatment with T cells and 5 days of ROR1 BiTE or control PBS therapy. BiTE treated animals had improved survival compared to the control cohort (50 days vs 18 days), despite only a single infusion of T cells (Figure 1B).We next assessed the potential of the BiTE to target primary CLL cells: allogeneic healthy donor T cells were co-cultured with CLL cells (3:1 effector:target ratio) in the presence of ROR1 BiTE with resultant decrease in CLL viability at 24 hours (Mean CLL survival 32%, Figure 1C). In contrast, we saw minimal cytotoxicity in co-cultures of autologous patient T cells and CLL cells with ROR1 BiTE under identical conditions (Mean CLL survival 92%, Figure 1D). We hypothesised this was secondary to the previously described multifactorial T cell dysfunction in CLL.Ibrutinib, the first in class Bruton's tyrosine kinase inhibitor, demonstrates impressive efficacy in front line and relapsed/refractory CLL patients including those with high risk 17p deletion. In addition, it modulates the tumour micro-environment, skews T cells to a Th1 phenotype, reduces PD-1 expression and reverses pseudo-exhaustion. In view of this, we isolated T cells from patients receiving Ibrutinib and cultured them with autologous CLL cells and demonstrated significantly enhanced cytotoxicity compared to patients not exposed to Ibrutinib (Mean CLL survival 43% vs 85%, p=0.012) (Figure 1E). This cohort included patients who had failed multiple prior lines of therapy and/or 17p deleted cases. Ibrutinib treatment was also associated with markedly enhanced secretion of a range of pro-inflammatory mediators including IFNγ and TNFα (Figure 1F). To ensure this effect was not due to Ibrutinib increasing sensitivity of CLL cells to a ROR1 BiTE, we analysed matched samples from 2 patients pre and post treatment. Post Ibrutinib T cells demonstrated enhanced killing, irrespective of the CLL cells exposure to Ibrutinib, confirming enhanced T cell function as the driving force of improved BiTE mediated cytotoxicity (Figure 1G).Taken together, we demonstrate the potential of a ROR1 BiTE to target haematological malignancies and highlight for CLL, the clinical context in which BiTEs are used is critical for effective responses. Baseline T cell dysfunction in untreated or relapsed CLL patients will likely limit efficacy, whilst Ibrutinib treatment allows for improved T cell effector function. In keeping with this CD19 CAR T cells demonstrate superior expansion, function and persistence when generated from CLL patients on Ibrutinib, confirming a global positive immunomodulatory role. Other small molecule inhibitors may improve T cell function by reducing tumour bulk but Ibrutinib inhibits other tyrosine kinases including ITK, which may play a role in T cell modulation. Despite Ibrutinib monotherapy being effective, patients often have a low level disease burden and minimal residual disease is rarely achieved. BiTE treatment for high risk patients in this situation represents a rational option to deepen responses. Overall, our data supports the assessment of our ROR1 BiTE in the post Ibrutinib setting for CLL. [Display omitted] DisclosuresGohil:UCL Business: Patents & Royalties. Nathwani:UCL Business: Patents & Royalties. Della Peruta:UCL Business: Patents & Royalties.

Successful treatment with benralizumab in a patient with eosinophilic granulomatosis with polyangiitis refractory to mepolizumab.

IntroductionEosinophilic granulomatosis with polyangiitis (EGPA) is characterized by necrotizing eosinophilic granulomatous inflammation that frequently involves the respiratory tract (90% of cases). Asthma in EGPA is systematically severe and often refractory to common treatment, it is corticosteroid resistant and can often anticipate the onset of systemic vasculitis by many years. A release of cytokines necessary for the activation, maturation and survival of eosinophils, such as IL-4, IL-5 and IL-13 occurs in the activated Th-2 phenotype. In particular, IL-5 level is high in active EGPA and its inhibition has become a key therapeutic target. Oral glucocorticoids (OCS) are effective treatment options but unfortunately, frequent relapses occur in many patients and they lead to frequent side effects. As for now, there are currently no official recommendations on doses and treatment schedules in the management of EGPA.Case presentationIn this article, we describe the case of a man with EGPA, severe asthma and chronic rhinosinusitis with nasal polyps (CRSwNP), with poor asthma and CRSwNP control despite OCS and mepolizumab treatment. Respiratory and vasculitis symptoms improved markedly after therapeutic switch to benralizumab. During the treatment, in addition to clinical effects, we witnessed a depletion of blood eosinophils, as well as an improvement in both pulmonary function tests, CT scan and skin lesions present initially.ConclusionsWhile there are many studies confirming the efficacy of benralizumab in EGPA, the most interesting aspect of our report is that efficacy was confirmed in a patient previously unresponsive to mepolizumab, known to be effective in EGPA.

Derangement of cell cycle markers in peripheral blood mononuclear cells of asthmatic patients as a reliable biomarker for asthma control

In asthma, most of the identified biomarkers pertain to the Th2 phenotype and no known biomarkers have been verified for severe asthmatics. Therefore, identifying biomarkers using the integrative phenotype-genotype approach in severe asthma is needed. The study aims to identify novel biomarkers as genes or pathways representing the core drivers in asthma development, progression to the severe form, resistance to therapy, and tissue remodeling regardless of the sample cells or tissues examined. Comprehensive reanalysis of publicly available transcriptomic data that later was validated in vitro, and locally recruited patients were used to decipher the molecular basis of asthma. Our in-silicoanalysis revealed a total of 10 genes (GPRC5A, SFN, ABCA1, KRT8, TOP2A, SERPINE1, ANLN, MKI67, NEK2, and RRM2) related to cell cycle and proliferation to be deranged in the severe asthmatic bronchial epithelium and fibroblasts compared to their healthy counterparts. In vitro, RT qPCR results showed that (SERPINE1 and RRM2) were upregulated in severe asthmatic bronchial epithelium and fibroblasts, (SFN, ABCA1, TOP2A, SERPINE1, MKI67, and NEK2) were upregulated in asthmatic bronchial epithelium while (GPRC5A and KRT8) were upregulated only in asthmatic bronchial fibroblasts. Furthermore, MKI76, RRM2, and TOP2A were upregulated in Th2 high epithelium while GPRC5A, SFN, ABCA1 were upregulated in the blood of asthmatic patients. SFN, ABCA1 were higher, while MKI67 was lower in severe asthmatic with wheeze compared to nonasthmatics with wheezes. SERPINE1 and GPRC5A were downregulated in the blood of eosinophilic asthmatics, while RRM2 was upregulated in an acute attack of asthma. Validation of the gene expression in PBMC of locally recruited asthma patients showed that SERPINE1, GPRC5A, SFN, ABCA1, MKI67, and RRM2 were downregulated in severe uncontrolled asthma. We have identified a set of biologically crucial genes to the homeostasis of the lung and in asthma development and progression. This study can help us further understand the complex interplay between the transcriptomic data and the external factors which may deviate our understanding of asthma heterogeneity.

Immune changes beyond Th2 pathways during rapid multifood immunotherapy enabled with omalizumab.

Multifood oral immunotherapy (mOIT) with adjunctive anti-IgE (omalizumab, XOLAIR® ) treatment affords safe, effective, and rapid desensitization to multiple foods, although the specific immune mechanisms mediating this desensitization remain to be fully elucidated. Participants in our phase 2 mOIT trial (NCT02643862) received omalizumab from baseline to week 16 and mOIT from week 8 to week 36. We compared the immune profile of PBMCs and plasma taken at baseline, week 8, and week 36 using high-dimensional mass cytometry, component-resolved diagnostics, the indirect basophil activation test, and Luminex. We found (i) decreased frequency of IL-4+ peanut-reactive CD4+ T cells and a marked downregulation of GPR15 expression and CXCR3 frequency among γδ and CD8+ T-cell subsets at week 8 during the initial, omalizumab-alone induction phase; (ii) significant upregulation of the skin-homing receptor CCR4 in peanut-reactive CD4+ T and Th2 effector memory (EM) cells and of cutaneous lymphocyte-associated antigen (CLA) in peanut-reactive CD8+ T and CD8+ EM cells; (iii) downregulation of CD86 expression among antigen-presenting cell subsets; and (iv) reduction in pro-inflammatory cytokines, notably IL-17, at week 36 post-OIT. We also observed significant attenuation of the Th2 phenotype post-OIT, defined by downregulation of IL-4 peanut-reactive T cells and OX40 in Th2EM cells, increased allergen component-specific IgG4/IgE ratio, and decreased allergen-driven activation of indirectly sensitized basophils. This exploratory study provides novel comprehensive insight into the immune underpinnings of desensitization through omalizumab-facilitated mOIT. Moreover, this study provides encouraging results to support the complex immune changes that can be induced by OIT.

Peripheral blood basophils are the main source for early interleukin-4 secretion upon in vitro stimulation with Culicoides allergen in allergic horses

Interleukin-4 (IL-4) is a key cytokine secreted by type 2 T helper (Th2) cells that orchestrates immune responses during allergic reactions. Human and mouse studies additionally suggest that basophils have a unique role in the regulation of allergic diseases by providing initial IL-4 to drive T cell development towards the Th2 phenotype. Equine Culicoides hypersensitivity (CH) is a seasonal immunoglobulin E (IgE)-mediated allergic dermatitis in horses in response to salivary allergens from Culicoides (Cul) midges. Here, we analyzed IL-4 production in peripheral blood mononuclear cells (PBMC) of CH affected (n = 8) and healthy horses (n = 8) living together in an environment with natural Cul exposure. During Cul exposure when allergic horses had clinical allergy, IL-4 secretion from PBMC after stimulation with Cul extract was similar between healthy and CH affected horses. In contrast, allergic horses had higher IL-4 secretion from PBMC than healthy horses during months without allergen exposure. In addition, allergic horses had increased percentages of IL-4+ cells after Cul stimulation compared to healthy horses, while both groups had similar percentages of IL-4+ cells following IgE crosslinking. The IL-4+ cells were subsequently characterized using different cell surface markers as basophils, while very few allergen-specific CD4+ cells were detected in PBMC after Cul extract stimulation. Similarly, IgE crosslinking by anti-IgE triggered basophils to produce IL-4 in all horses. PMA/ionomycin consistently induced high percentages of IL-4+ Th2 cells in both groups confirming that T cells of all horses studied were capable of IL-4 production. In conclusion, peripheral blood basophils produced high amounts of IL-4 in allergic horses after stimulation with Cul allergens, and allergic horses also maintained higher basophil percentages throughout the year than healthy horses. These new findings suggest that peripheral blood basophils may play a yet underestimated role in innate IL-4 production upon allergen activation in horses with CH. Basophil-derived IL-4 might be a crucial early signal for immune induction, modulating of immune responses towards Th2 immunity and IgE production.

Treg-Resistant Cytotoxic CD4+ T Cells Dictate T Helper Cells in Their Vicinity: TH17 Skewing and Modulation of Proliferation.

Cytotoxic CD4+ T cells (CD4 CTL) are terminally differentiated T helper cells that contribute to autoimmune diseases, such as multiple sclerosis. We developed a novel triple co-culture transwell assay to study mutual interactions between CD4 CTL, conventional TH cells, and regulatory T cells (Tregs) simultaneously. We show that, while CD4 CTL are resistant to suppression by Tregs in vitro, the conditioned medium of CD4 CTL accentuates the suppressive phenotype of Tregs by upregulating IL-10, Granzyme B, CTLA-4, and PD-1. We demonstrate that CD4 CTL conditioned medium skews memory TH cells to a TH17 phenotype, suggesting that the CD4 CTL induce bystander polarization. In our triple co-culture assay, the CD4 CTL secretome promotes the proliferation of TH cells, even in the presence of Tregs. However, when cell−cell contact is established between CD4 CTL and TH cells, the proliferation of TH cells is no longer increased and Treg-mediated suppression is restored. Taken together, our results suggest that when TH cells acquire cytotoxic properties, these Treg-resistant CD4 CTL affect the proliferation and phenotype of conventional TH cells in their vicinity. By creating such a pro-inflammatory microenvironment, CD4 CTL may favor their own persistence and expansion, and that of other potentially pathogenic TH cells, thereby contributing to pathogenic responses in autoimmune disorders.

CpG oligodeoxynucleotides attenuate ROR\u03b3t-mediated Th17 response by restoring histone deacetylase-2 in cigarette smoke-exposure asthma

BackgroundCigarette smoke (CS) exposure increases corticosteroid insensitive asthma related to increased Th17 phenotype, and new treatment strategies are needed for CS-associated asthma. Histone deacetylase 2 (HDAC2), found in the airway epithelium, is critical for ameliorating glucocorticoids insensitivity. We recently demonstrated the anti-inflammatory effects of CpG oligodeoxynucleotides (CpG-ODNs) on CS-exposure asthma. However, the effects of CpG-ODNs on HDAC2 expression and enzymatic activity remain unclear. This study aimed to assess whether CpG-ODNs protect against excessive Th17 immune responses in CS-induced asthma through HDAC2-dependent mechanisms and compared their effects with those of corticosteroids.MethodsThe effects of CpG-ODNs alone and in combination with budesonide (BUD) on airway inflammation and Th2/Th17-related airway immune responses were determined using an in vivo model of CS-induced asthma and in cultured bronchial epithelial (HBE) cells administered ovalbumin (OVA) and/or cigarette smoke extract (CSE). HDAC2 and retinoid-related orphan nuclear receptor γt (RORγt) expression were also assessed in mouse lung specimens and HBE cells.ResultsCpG-ODNs and BUD synergistically attenuated CS exposure asthmatic responses in vivo by modulating the influx of eosinophils and neutrophils, airway remodeling, Th2/Th17 associated cytokine and chemokine production, and airway hyperresponsiveness and blocking RORγt-mediated Th17 inflammation through induced HDAC2 expression/activity. In vitro, CpG-ODNs synergized with BUD to inhibit Th17 cytokine production in OVA- and CSE-challenged HBE cells while suppressing RORγt and increasing epithelial HDAC2 expression/activity.ConclusionsCpG-ODNs reversed CS-induced HDAC2 downregulation and enhanced the sensitivity of CS-exposed asthmatic mice and CSE-induced HBE cells to glucocorticoid treatment. This effect may be associated with HDAC2 restoration via RORγt/IL-17 pathway regulation, suggesting that CpG-ODNs are potential corticosteroid-sparing agents for use in CS-induced asthma with Th17-biased immune conditions.

Silk Fibroin Scaffold Architecture Regulates Inflammatory Responses and Engraftment of Bone Marrow-Mononuclear Cells.

Despite being one of the most clinically trialed cell therapies, bone marrow-mononuclear cell (BM-MNC) infusion has largely failed to fulfill its clinical promise. Implanting biomimetic scaffolds at sites of injury prior to BM-MNC infusion is a promising approach to enhance BM-MNC engraftment and therapeutic function. Here, it is demonstrated that scaffold architecture can be leveraged to regulate the immune responses that drive BM-MNC engraftment. Silk scaffolds with thin fibers and low porosity (LP) impairs immune activation in vitro compared with thicker fiber, high porosity (HP) scaffolds. Using the authors' established in vivo bioluminescent BM-MNC tracking model, they showed that BM-MNCs home to and engraft in greater numbers in HP scaffolds over 14 days. Histological analysis reveals thicker fibrous capsule formation, with enhanced collagen deposition in HP compared to LP scaffolds consistent with substantially more native CD68+ macrophages and CD4+ T cells, driven by their elevated pro-inflammatory M1 and Th1 phenotypes, respectively. These results suggest that implant architecture impacts local inflammation that drives differential engraftment and remodeling behavior of infused BM-MNC. These findings inform the future design of biomimetic scaffolds that may better enhance the clinical effectiveness of BM-MNC infusion therapy.

Immunomodulation of T Helper Cells by Tumor Microenvironment in Oral Cancer Is Associated With CCR8 Expression and Rapid Membrane Vitamin D Signaling Pathway.

The immune system plays a key role in the protective response against oral cancer; however, the tumor microenvironment (TME) impairs this anti-cancer response by modulating T helper (Th) responses and promoting an anti-inflammatory environment. Regulatory T cells (Tregs) and Th2 effector cells (Teff) are associated with poor prognosis in oral squamous cell carcinoma (OSCC). However, the main immunomodulatory mechanisms associated with the enrichment of these subsets in OSCC remain unknown. We characterized Th-like lineages in Tregs and Teff and evaluated immunomodulatory changes induced by the TME in OSCC. Our phenotypic data revealed a higher distribution of tumour-infiltrating CCR8+ and Th2-like Treg in OSCC compared with non-malignant samples, whereas the percentages of Th1 cells were reduced in cancer. We then analyzed the direct effect of the TME by exposing T cell subsets to cancer secretomes and observed the OSCC secretome induced CCR8 expression and reduced cytokine production from both subsets. Transcriptomic analysis showed that the co-culture with OSCC secretome induced several gene changes associated with the vitamin D (VitD) signaling pathway in T cells. In addition, proteomic analysis identified the presence of several proteins associated with prostaglandin E2 (PGE2) production by rapid membrane VitD signaling and a reduced presence of the VitD binding protein. Thus, we analyzed the effect of VitD and PGE2 and observed that VitD promotes a regulatory Th2-like response with CCR8 expression whilst PGE2 also modulated CCR8 but inhibited cytokine production in combination with VitD. Finally, we evaluated the presence of CCR8 ligand in OSCC and observed increased chemokine CCL18, which was also able to upregulate CCR8 in activated Th cells. Overall, our data showed the immunomodulatory changes induced by the TME involving CCR8 expression and regulatory Th2 phenotypes, which are associated with PGE2 mediated VitD signaling pathway and CCL18 expression in OSCC.

Plasmacytoid Dendritic Cells Regulate Th17 Activation in Diabetic Wound CD4+ T-cells

Abstract Introduction: CD4+ T-cell activation is vital for normal wound repair but the factors that control T-cell activation in wounds in vivo are not clear. Our group and others have found increased Th17 activation in diabetic wounds resulting in increased IL-17a and pathologic inflammation that prevents tissue repair. Plasmacytoid dendritic cells (pDC) are antigen presenting cells that are present in early diabetic wound tissue and may play a key role in modulating CD4+ T-cell phenotype. Hence, we hypothesized that diabetic pDCs may influence wound CD4+ T-cells towards Th17 T-cell expansion. Methods: Wild type C57BL/6 mice were fed normal chow diet (13.5% kcal fat; LabDiet) or high fat diet chow (60% kcal fat; Research Diets) for 12–14 weeks to generate the diet-induced obesity (DIO) model of glucose intolerance/insulin resistance. These mice were subsequently wounded, and wound plasmacytoid dendritic cells harvested on day 1 and day 3. These cells were co-cultured with naïve CD4+ T-cells for 3 days, after which T-cell phenotype was determined by flow cytometry. Additionally, pDC in wounds 1-day post wounding were examined by quantitative PCR for cytokine production. Results: Following exposure to DIO pDCs, wild type activated CD4+ T-cells were activated towards a Th17 phenotype via significant increases in TGFb CD4+ T-cell activation and may act to increase inflammation in diabetic wounds.