Th1 Phenotype Research Articles

Combination of Ibrutinib with CD19 Chimeric Antigen Receptor (CAR) T Cells Suggests Greater Contribution to CAR T-Cell Efficacy Than Other Approved BTK Inhibitors in the Preclinical Setting

Background: CD19-directed CAR T-cell therapy has demonstrated remarkable clinical benefits for patients with B-cell malignancies, but impaired CAR T-cell fitness can result in lack of response or relapse. Rational combinations that enhance T-cell fitness may overcome primary and secondary resistance. Bruton tyrosine kinase inhibitors (BTKi), such as Ibrutinib (Ibr) have been shown to enhance CAR T-cell efficacy (Fraietta et al, Blood 2016;Liu et al, Cancer Sci 2020), but the precise mechanism remains unclear. In addition to BTK, Ibr potently inhibits IL-2-inducible tyrosine kinase (ITK), an important mediator of T-cell signaling. Ibr has been shown to enhance Th1 polarization through suppression of the Th2 differentiation pathway, thereby potentiating a cytotoxic effector response (Qin et al, J Immunother 2020). Consistent with this finding, the Th1 phenotype was more frequent in peripheral blood mononuclear cells of Ibr-exposed patients than in acalabrutinib (Acala)-exposed patients (Scarfò et al, Blood 2021). Therefore, we aimed to assess the potential impact of ITK inhibition (ITKi) on CD19 CAR T-cell function by comparing Ibr to other approved BTKi that display low or no ITKi activity (Estupiñán et al, Front Cell Dev Biol 2021). Methods: CD19-41bb CAR T cells (CAR19) were generated by lentiviral transduction of healthy donor T cells. BTKi additivity to CAR19 function was evaluated in a Luciferase (Luc)+ Ramos, BTKi resistant systemic tumor model in NSG mice co-treated with covalent BTKis Ibr, Acala, zanubrutinib (Zanu), and CAR T cells. Next, to evaluate the impact of ITKi on CAR19 fitness and cytotoxicity, repeated stimulation in an in vitro cytotoxicity assay was performed at low CAR19 effector-to-tumor ratio (1:20) to mimic higher tumor burden stress conditions in the presence of each BTKi, including non-covalent BTKi pirtobrutinib (Pirto) (500 nM). Tumor cells were replenished every 3 days and cytotoxicity was measured on day 7 by luciferase assay. For phenotypic characterization, CAR T cells were repeatedly stimulated by plate-bound anti-Whitlow linker every 3 days. Viability and proliferation were measured by cell counter. Flow cytometry (FACS) was performed on surface and intracellular T-cell phenotypic, activation, and exhaustion markers. Metabolic fitness (glycolysis and ATP production) was characterized using Seahorse (Van der Windt, 2012). Impact of Ibr on signal strength was evaluated in CD19-expressing Jurkat reporter cells (CAR-J19) with knock-in of mKate in frame with Nur77. Ibr was added to coculture of CAR-J19 and tumor cells and upregulation of Nur77 was measured by FACS. Downstream ITK signaling pathway was measured by Western blot. Results: Preliminary in vivo experiments demonstrated that co-administration of each BTKi with CD19 CAR T cells improved tumor control at day 26 (N=5 per group; P≤0.0004 vs CAR T cells alone). In serial killing experiments, addition of Ibr to the coculture increased proliferation (n=2; P<0.05), viability (n=2; P=0.01), and the cytotoxicity of CAR T cells against Ramos cells ( P=0.02 vs CAR19 alone) to a greater extent than other BTKis tested (Fig. 1A). Prolonged stimulation of CAR19 with Ibr led to downregulation of activation and inhibitory markers on CAR19 and maintenance of a naive T-cell phenotype (Fig. 1B) that was confirmed by increased metabolic fitness (n=5; P<0.05). Addition of Ibr to CAR-J19 resulted in a 72% reduction in CAR activation and reduced phosphorylation of ITK, PLCG, and Zap70. Additional data including the impact of ITK knockout on CAR-T cells will be presented. Conclusions: Our study expands on the hypothesized mechanism of action underlying the clinical benefit observed with Ibr in CD19 CAR T-cell-treated patients with chronic lymphocytic leukemia or mantle cell lymphoma (Liu et al, Front Immunol 2022; Wang et al, NEJM 2020). Our data suggest that Ibr enhances CAR T-cell fitness to a greater extent than both covalent and noncovalent BTKi that display reduced or no ITKi activity. Through ITKi, Ibr tempers CAR T-cell activation induced cell death, maintains a naive state and prevents premature exhaustion, thereby enabling more efficacious tumor cell killing over time. Together, these data support the further exploration of Ibr to enhance CD19 CAR T-cell efficacy.

Exploiting the Gut Microbiota to Boost Immunotherapy and Switch the Trajectory of Multiple Myeloma

Clonal proliferation of neoplastic plasma cells in the bone marrow (BM) and organ dysfunction characterizes multiple myeloma (MM), a treatable but incurable disease. Although asymptomatic smoldering MM (SMM) often precedes full blown MM, most patients affected by SMM are only offered active observation, which increases their frustration and anxiety. In transgenic Vk*MYC mice developing de novo MM, we found a direct link between gut microbiota composition and T helper 17 (Th17) lymphocytes, which migrated from the gut to the BM during asymptomatic MM (Early-MM), and favored the expansion of neoplastic plasma cells, thus fueling progression to symptomatic disease (Late-MM) [Calcinotto A. et al. Nat Commun (2018) 9(1):4832]. Similarly, in SMM patients, higher levels of BM Interleukin-17 (IL-17) predicted accelerated disease [Calcinotto A. et al. Nat Commun (2018) 9(1):4832]. Interestingly, Prevotella melaninogenica ( P.m.), a human commensal, limited MM aggressiveness in mice challenged with Vk*MYC-derived MM cells (t-Vk*MYC) by restraining Th17 cell expansion [Calcinotto et al. Nat Commun (2018) 9(1):4832]. Because the gut microbiota also contributes to the therapeutic efficacy of immune checkpoint blockade (ICB) and human neoplastic plasma cells express PD-L1, we hypothesized that modulation of the gut microbiota by P.m. could limit Th17 cell expansion in mice affected by Early-MM, thus fully exploiting the therapeutic potential of anti-PD-L1 against MM. Clinical trials combining anti-PD-1/PD-L1 antibodies, dexamethasone and immune modulatory drugs in MM were halted by US FDA because of unfavorable benefit-risk profile and life-threatening immune related adverse events (irAEs). Because IL-17 is involved in irAEs [Johnson et al. Cancer Immunol Res (2019) 7 (6): 860-865], we also hypothesized treatment with P.m. could restrain ICB-induced expansion of Th17 cells while unleashing CD8-mediated antitumor immunity. Early-MM Vk*MYC mice and t-Vk*MYC, the latter mimicking aggressive MM, were treated with P.m. and/or anti-PD-L1 and disease progression was monitored by paraprotein quantification in blood. Anemia provided clinical evidence of symptomatic disease. At sacrifice, gut, spleen and BM were analyzed by flow cytometry. In vitro assays were conducted to clarify the mechanism by which the gut microbiota influenced the induction of Th17 cells. To mimic cutaneous irAE, the skin of treated mice was exposed to imiquimod, which induces Th17-mediated inflammation [van der Fits et al. J Immunol (2009 )182(9): 5836-5845]. Body weight loss, skin inflammation, and inflammatory infiltrate into secondary lymphoid organs were taken as measure of imiquimod-induced toxicity. In Vk*MYC mice, all neoplastic plasma cells expressed PD-L1. Treatment of Early-MM Vk*MYC mice with anti-PD-L1 antibodies delayed progression to Late-MM, thus extending previous findings on the use of ICB in this model to anti-PD-L1 antibodies and Early-MM [Guillerey et al. J Clin Invest (2015 )125(5): 2077-2089]. Treatment with P.m. exerted a similar effect on disease progression, suggesting that a treatment as simple as taking a probiotic has an impact on MM evolution. When P.m. was combined with anti-PD-L1 antibodies, evolution to Late-MM was further delayed or even blocked. Single and combined treatments also reduced MM aggressiveness in t-Vk*MYC mice. Mechanistically, P.m. limited the expansion of gut-born Th17 cells and their accumulation in the BM of treated mice without dampening the antitumor cytotoxic response elicited by the ICB. Thus, only the combined treatment provided the most favorable CD8/Th17 and CD8/T regulatory cell ratios. Ex vivo, P.m.-conditioned BM DCs showed a limited propensity to produce Th17-polarizing cytokines. In vitro, stimulation of both human and mouse DCs with P.m. reduced polarization of naïve T cells toward a Th17 phenotype. Skin application of imiquimod in C57BL/6 mice induced psoriasis-like lesions and expansion of Th17 cells both in draining lymph nodes and spleen. The contextual administration of anti-PD-L1 antibodies worsened imiquimod-induced inflammation. An ongoing experiment will clarify if mice receiving P.m. and ICB experience reduced skin lesions. Taken together, our data support the development of microbiota-based strategies in combination with ICB to treat full-blown MM and to prevent progression of asymptomatic SMM to full-blown disease.

Deciphering the Dynamics of the B-NHL Single-Cell Landscape during BTK Inhibition

Introduction: The inhibition of Bruton's tyrosine kinase (BTK) as a therapeutic strategy has dramatically improved the management of patients with chronic lymphocytic leukemia (CLL) and other B-cell-Non Hodgkin lymphomas (B-NHL). However, therapy resistance and treatment toxicity persist as significant clinical challenges. It has been previously reported that BTK inhibition not only targets the overactivated BCR pathway in CLL and other B-NHL, but also coincides with profound alterations in the tumor microenvironment including immune cells. Therefore, it is vital to understand these tumor-immune cell interactions during BTK inhibition to enhance response and reduce toxicity, advancing personalized care for CLL and B-NHL patients in the future. Methods: We developed a high-parametric analysis pipeline based on full spectrum cytometry, a novel technology allowing for the simultaneous assessment of 40 markers at single-cell level. In order to comprehensively assess the phosphoproteome relevant to BTK inhibition we established a signaling panel covering >30 proteins including pBTK, pSyk, pPLCg, pS6 and pAMPKa. Additionally, we designed 3 panels with up to 40 markers characterizing T cell activation and exhaustion, cellular trafficking, inhibitory immune checkpoints and cytokine production. Using our scalable and reproducible bioinformatics pipeline, we analyzed a cohort of 160 samples, longitudinally collected from 57 patients (CLL, MCL, MZL) before BTKi treatment, directly after treatment initiation, at leukocyte peak count and during therapy up to 6 months after therapy initiation. Results: Given that T cells are a crucial arm in governing anti-lymphoma immune response, we sought to elucidate the changes associated with BTK inhibition in the T cell compartment of the patients at high resolution (figure A). We observed certain changes in the fraction of T cell subtypes, notably a decrease in central memory stem T cells and an increase in γδ T cells at leukocyte peak and during later stages of therapy. Our analysis uncovered substantial changes in the T cell marker expression over the course of BTK inhibition. This includes a reversal of exhausted phenotype marked by downregulation of PD1, CD39 and CD38 and a skewed differentiation of CD4 T cells towards Th1 phenotype illustrated by a pronounced upregulation of the transcription factor T-bet. In addition, we detected restored proliferative and cytotoxic capacity in the CD8 and CD4 T cell compartments characterized by upregulation of GRZB, CD226, CD28, CD25 and Ki67. By leveraging our platform, we successfully captured the signalosome of malignant B cells at a high resolution. As anticipated, we detected significant heterogeneity in the malignant B cell signatures across individual patients. Interestingly, even at the initial sampling timepoint following treatment start, we observed substantially altered B cell signatures encompassing both the phosphoproteome and inhibitory immune checkpoints (figure B). Our preliminary data suggest that the dynamic changes induced by BTK inhibition extend beyond the signaling nodes associated with the BCR pathway and also involve the NFkB and p53 pathways. Conclusion: In this study, we established a platform for deep and longitudinal interrogation of the tumor and immune cell compartments to capture the dynamic and patient-specific changes occurring during BTK inhibition. Our preliminary data obtained from a large patient cohort of 57 B-NHL patients reveals a profound remodeling of not only the BCR signaling pathway in tumor cells, but also of the T cell compartment. Our data will be useful for a better understanding of treatment response and toxicity in the context of BTK inhibition.

Mutual modulation of gut microbiota and the immune system in type 1 diabetes models

The transgenic 116C-NOD mouse strain exhibits a prevalent Th17 phenotype, and reduced type 1 diabetes (T1D) compared to non-obese diabetic (NOD) mice. A cohousing experiment between both models revealed lower T1D incidence in NOD mice cohoused with 116C-NOD, associated with gut microbiota changes, reduced intestinal permeability, shifts in T and B cell subsets, and a transition from Th1 to Th17 responses. Distinct gut bacterial signatures were linked to T1D in each group. Using a RAG-2−/− genetic background, we found that T cell alterations promoted segmented filamentous bacteria proliferation in young NOD and 116C-NOD, as well as in immunodeficient NOD.RAG-2−/− and 116C-NOD.RAG-2−/− mice across all ages. Bifidobacterium colonization depended on lymphocytes and thrived in a non-diabetogenic environment. Additionally, 116C-NOD B cells in 116C-NOD.RAG-2−/− mice enriched the gut microbiota in Adlercreutzia and reduced intestinal permeability. Collectively, these results indicate reciprocal modulation between gut microbiota and the immune system in rodent T1D models.

Sex-Related Effect of Aging in Gingival Gamma-Delta T Cells

Aging affects the number and function of gamma-delta (γδ) T cells in a tissue-specific manner, modifying the risk for inflammatory disease. These aging-related γδT-cell variations in gingival tissues that could increase the risk for inflammation and periodontal disease remain unknown. Here we sought to identify quantitative and qualitative variations in gingival γδT cells associated with aging that could have an impact in oral immunoinflammatory responses. For this, gingival tissues from young (4 mo) and aged (24 mo) male and female mice were collected and analyzed by flow cytometry. Cell suspensions were stimulated and stained with eFluor450 (cell viability), anti-CD45 (hematopoietic cells), anti-CD3 (lymphocytes), anti-TCRγδ (γδT cells), anti–IL-15rα (cell proliferation), and anti–Notch-3 (senescence marker). Detection of intracellular cytokines IL-17A and interferon γ (IFNγ) was performed. Gingival expression of specific γ- and δ-chains and cytokines was evaluated by quantitative reverse transcription polymerase chain reaction. A significantly higher number of IL-17A–producing γδT cells and IL-17A expression levels were observed in gingival tissues from aged females but not males. Similarly, the number of gingival Notch-3+ γδT cells increased with aging only in females. IL-15rα was not detected in gingival γδT cells. Chains γ1, 2, 4, 5, 6, and 7 as well as δ1, 2, 4, and 6 were detected. Detection levels of all γ chains except γ1 as well as δ1 and δ2 changed with aging in males, females, or both. Interestingly, number of IL-17A–producing conventional T cells similarly increased with aging only in females. Both sexes showed increased IFNγ+ conventional T-cell numbers with aging; however, it reached significance only in females. In conclusion, the number of gingival IL-17A–producing γδT cells and IL-17A expression increase naturally with aging specifically in females. This sexual dimorphism in gingival γδT and conventional Th17 cell numbers and phenotypes suggests distinct aging-related mechanisms of periodontitis in males and females.

T helper-1 activation via interleukin-16 is a key phenomenon in the acute phase of severe, first-episode major depressive disorder and suicidal behaviors

BackgroundImmune-inflammatory pathways in major depressive disorder are confined to the major dysmood disorder (MDMD) phenotype (Maes et al., 2022). No studies have addressed the immune profile of first episode MDMD (FE-MDMD). MethodsThis study investigated the immune profiles of 71 patients with the acute phase of first-episode major depressive disorder (FE-MDMD) and 40 healthy controls. We measured 48 cytokines/chemokines/growth factors, classical M1, alternative M2, T helper (Th)-1, Th-2, and Th-17 phenotypes, immune-inflammatory response system (IRS), compensatory immunoregulatory system (CIRS), and neuro-immunotoxicity profiles. ResultsFE-MDMD patients show significantly activated M1, M2, Th-1, IRS, CIRS, and neurotoxicity, but not Th-2 or Th-17, profiles compared to controls. FE-MDMD is accompanied by Th-1 polarization, while there are no changes in M1/M2 or IRS/CIRS ratios. The top single indicator of FE-MDMD was by far interleukin (IL)-16, followed at a distance by TRAIL, IL-2R, tumor necrosis factor (TNF)-β. The severity of depression and anxiety was strongly associated with IRS (positively) and Th-2 (inversely) profiles, whereas suicidal behavior was associated with M1 activation. Around 56–60% of the variance in depression, anxiety, and suicidal behavior scores was explained by IL-16, platelet-derived growth factor (PDGF) (both positively), and IL-1 receptor antagonist (inversely). Increased neurotoxicity is mainly driven by IL-16, TNF-α, TRAIL, IL-6, and chemokine (CCL2, CCL11, CXCL1, CXCL10) signaling. Antidepressant-treated patients show an increased IRS/CIRS ratio as compared with drug-naïve FE-MDMD patients. ConclusionsFE-MDMD is accompanied by positive regulation of the IRS mainly driven by Th-1 polarization and T cell activation (via binding of IL-16 to CD4), and TNF, chemokine, and growth factor signaling.

Ingested (oral) adrenocorticotropic hormone (ACTH) inhibits interleukin-17 in the central nervous system after adoptive transfer of T helper (Th)1/Th17 T cells in the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis

BackgroundExperimental autoimmune encephalomyelitis (EAE) is an inflammatory autoimmune disease of the central nervous system (CNS) that resembles multiple sclerosis (MS) and provides a useful animal model for the evaluation of mechanisms of action for potential immunomodulatory therapies. We have previously shown that oral adrenocorticotropic hormone (ACTH) decreased either interleukin (IL)-17 and/or interferon (IFN)γ in the CNS during EAE. ObjectiveWe wanted to examine whether oral ACTH showed a preferential effect on Th17 as opposed to Th1 phenotypes. Design/MethodsWe therefore examined whether oral ACTH could inhibit EAE in the C57BL/6 (B6) mouse strain after adoptive transfer of equal quantities of Th17 (CD4+IL-17+) and Th1 (CD4+IFN-γ+) T cells generated after in vitro skewing. B6 mice were injected with a 1:1 ratio of Th1:Th17 T cells and were gavaged daily with control scrambled peptide (s-MSH) or 10 μg ACTH. ResultsIngested (oral) ACTH attenuated ongoing clinical EAE disease and decreased the frequencies of Th17 cells in the spleen and in the CNS, but not Th1. ConclusionsThese findings suggest that there was preferential regulation of Th17 cells by oral ACTH compared to Th1 T cells in the CNS.

Vasoactive Intestinal Peptide Signaling in Transplant Recipients Modulates Gut Microbiome Diversity and Regulates Graft-Versus-Host Disease

Introduction: Allogeneic bone marrow transplantation (allo-BMT) presents a significant challenge in the form of graft-versus-host disease (GvHD), characterized by donor T cell-mediated attacks on recipient organs, leading to considerable morbidity and mortality. Vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, plays a crucial role in shaping T cell responses towards the Th2 phenotype while inhibiting proinflammatory cytokine production. Furthermore, VIP-producing enteric neurons contribute to enteric mucosal barrier homeostasis, maintaining optimal gut pH, and orchestrating the delicate balance between beneficial bacteria colonization and resistance against opportunistic pathogens. The potential impact of VIP production by host cells on GvHD regulation remains unclear. Our hypothesis proposes that the absence of VIP production by transplant recipients may exacerbate GvHD, either through increased T cell allo-reactivity, or via secondary modulation of the gut microbiome resulting from submucosal inflammation. Methods and Results: We investigated how endogenous production of VIP by host tissues on GvHD modulation in three murine allo-BMT models, where C57BL/6 wildtype (WT) and VIP-knockout (VIPKO) mice were subjected to 11Gy irradiation on day 0 and subsequently transplanted intravenously with 5x10 6 T cell depleted (TCD) bone marrow (BM) in combination with 0, 1x10 6, or 3x10 6 T cells from MHC mismatched B10.BR, Balb/c or FvB donors. In all three models, WT recipients had better survival than VIPKO recipients. Specifically, in B10BR B6, Balb/c B6 and FvB B6 model, the addition of 3 million donor T lymphocytes to the graft resulted in significantly more GvHD-related mortality in VIP-KO recipients compared with WT recipients, with 0% vs 82%, 18% vs 96%, and 0% vs 78% survival (p<0.01) among WT vs VIP-KO recipients. To elucidate the role of host cell-derived VIP production in T cell alloreactivity and GvHD exacerbation, we performed cytokine profile analysis using both serum and donor-derived lymphocytes from the spleen of transplant recipients. For serum cytokine analysis, VIP-KO recipients had significantly higher levels of IFNγ (p<0.05), TNFα (p<0.05) and IL-1beta (p<0.05) on day 7 post allo-BMT, and significantly higher levels of and IL-6 (p<0.01) and IL-10 (p<0.05) on day 14 post allo-BMT compared with WT recipients. For donor-derived lymphocytes cytokine analysis, we observed significantly increased Th1 and Th17 cytokines in VIP-KO recipient, with significantly higher frequencies of CD4+ IL2 (p<0.01) and CD4+ IFNγ (p<0.001) on day 4, significantly higher levels of CD4+ TNFα (p<0.05) and CD8+ TNFα (p<0.001) on day 19, and significantly increased CD8+ TNFα (p<0.05) on day 25 post allo-BMT compared to WT recipients. Immunofluorescence whole-mount imaging section staining demonstrated detectable VIP expression in enteric neurons innervating the entirety of the intestine in FvB VIP-GFP transplant recipients. Notably, VIP immunofluorescent signal was much higher in allo-BMT recipient intestines compared to syngeneic transplant recipient intestines, demonstrating immunomodulation of VIP-producing enteric neurons in response to local (GVHD-targeted organ) and systemic inflammation (Fig 1). To investigate the gut microbiome profile, we performed 16S ribosomal RNA sequencing and analyzed stool samples from three distinct groups: 1) naïve WT and VIP KO mice, 2) WT and VIPKO mice after co-housing, and 3) WT and VIPKO mice after co-housing followed by allo-BMT. Uniform Manifold Approximation and Projection high-dimensional cluster analysis revealed that VIP-KO mice exhibited a distinct gut microbiome compared to WT mice, regardless of their co-housing or allo-BMT conditioning (Fig 2A, 2B). The heatmap analysis demonstrated that VIP-KO mice had increased lactobacillus species (Fig 2C) and significantly decreased intestinal pH levels (p<0.01) compared to WT mice. Moreover, VIP-KO mice displayed significantly decreased gut microbiome diversity, indicating an inflammatory effect in the gut that influences changes in the microbiome composition. Conclusion: Overall, our findings highlight the crucial role of VIP in regulating immune responses and gut microbiome homeostasis, which could have implications for developing strategies to mitigate GvHD in allo-BMT recipients.

Leptin favors imbalance of antigen-specific CD4+ T-cells associated with severity of cat allergy.

Obesity can complicate IgE-mediated allergic diseases. In the present study, we aimed to investigate the ability of obesity-related concentrations of leptin to modulate the in vitro effector and regulatory Fel d1-specific CD4+ T-cell subsets in patients allergic to cat, considered the third most common cause of respiratory allergy in humans. For this study, plasma and peripheral blood mononuclear cells (PBMC) from 30 cat-allergic patients with mild, moderate and severe respiratory symptoms were obtained. The PBMC cultures were stimulated with Fel d1 antigen (10 µg/mL) in the presence or absence of obesity-related leptin dose (50 ηg/mL). After 6 days, the levels of cytokines and IgE in the supernatants were evaluated by multiplex and ELISA, respectively. The frequency of different non-follicular (CXCR5-) and follicular (CXCR5+) Fel d1-specific CD4+ T cell subsets was determined by flow cytometry. The plasma levels of leptin and IgE anti-cat titers were evaluated by ELISA and ImmunoCAP, respectively. Fel d1 induced both IgE production and release of cytokines related to Th2, Th9 and Th17 cell phenotypes. Feld1 was more efficient in increasing the frequency of TFHIL-21- cells positive for IL-4, IL-5 and IL-13 than TFHIL-21+ cell subsets. Leptin favored the expansion Th2-like and Th9-like cells and TFHIL-21- cells positive for IL-4, IL-5 and IL-13, but reduced the proportion of conventional (Treg/Tr-1) and follicular (TFR) regulatory CD4+ T-cell subsets expressing or not CD39 marker. Finally, many of the imbalances between Fel d1-specific CD4+ T-cells were also correlated with plasma leptin and anti-Fel d1 IgE titers. In summary, hyperleptinemia should negatively impact on the severity of cat allergies by favoring the expansion of pathogenic Fel d1-specific CD4+ T-cell phenotypes and damaging the functional status of regulatory CD4+ T-cell subsets.

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes.

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical characteristics, they are involved in numerous biological processes, such as modulation of the immune response, recognition of self and non-self antigens, carbohydrate-protein interactions, etc. The cellular content of sialic acid is regulated by sialidase, which catalyzes the removal of sialic acid residues. Several studies have shown that sialo-glycans are critical in monitoring immune surveillance by engaging with cis and trans inhibitory Siglec receptors on immune cells. Likewise, glyco-immune checkpoints in cancer are becoming crucial targets for developing immunotherapies. Additionally, dendritic cells (DCs) are envisioned as an important component in immunotherapies, especially in cancer research, due to their unique role as professional antigen-presenting cells (APC) and their capacity to trigger adaptive immune responses and generate immunologic memory. Nevertheless, the function of DCs is dependent on their full maturation. Immature DCs have an opposing function to mature DCs and a high sialic acid content, which further dampens their maturation level. This downregulates the ability of immature DCs to activate T-cells, leading to a compromised immune response. Consequently, removing sialic acid from the cell surface of human DCs induces their maturation, thus increasing the expression of MHC molecules and antigen presentation. In addition, it can restore the expression of co-stimulatory molecules and IL-12, resulting in DCs having a higher ability to polarize T-cells toward a Th1 phenotype and specifically activate cytotoxic T-cells to kill tumor cells. Therefore, sialic acid has emerged as a key modulator of DCs and is being used as a novel target to advance their therapeutic use. This study provides a unique approach to treat in vitro monocyte-derived DCs with sialidase, aimed at generating DC populations with different cell surface sialic acid phenotypes and tailored maturation and co-stimulatory profiles.

Toll-Like Receptor 4 Agonist Injection With Concurrent Radiotherapy in Patients With Metastatic Soft Tissue Sarcoma

Metastatic soft tissue sarcomas (STSs) have limited systemic therapy options, and immunomodulation has not yet meaningfully improved outcomes. Intratumoral (IT) injection of the toll-like receptor 4 (TLR4) agonist glycopyranosyl lipid A in stable-emulsion formulation (GLA-SE) has been studied as immunotherapy in other contexts. To evaluate the safety, efficacy, and immunomodulatory effects of IT GLA-SE with concurrent radiotherapy in patients with metastatic STS with injectable lesions. This phase 1 nonrandomized controlled trial of patients with STS was performed at a single academic sarcoma specialty center from November 17, 2014, to March 16, 2016. Data analysis was performed from August 2016 to September 2022. Two doses of IT GLA-SE (5 μg and 10 μg for 8 weekly doses) were tested for safety in combination with concurrent radiotherapy of the injected lesion. Primary end points were safety and tolerability. Secondary and exploratory end points included local response rates as well as measurement of antitumor immunity with immunohistochemistry and T-cell receptor (TCR) sequencing of tumor-infiltrating and circulating lymphocytes. Twelve patients (median [range] age, 65 [34-78] years; 8 [67%] female) were treated across the 2 dose cohorts. Intratumoral GLA-SE was well tolerated, with only 1 patient (8%) experiencing a grade 2 adverse event. All patients achieved local control of the injected lesion after 8 doses, with 1 patient having complete regression (mean regression, -25%; range, -100% to 4%). In patients with durable local response, there were detectable increases in tumor-infiltrating lymphocytes. In 1 patient (target lesion -39% at 259 days of follow-up), TCR sequencing revealed expansion of preexisting and de novo clonotypes, with convergence of numerous rearrangements coding for the same binding sequence (suggestive of clonal convergence to antitumor targets). Single-cell sequencing identified these same expanded TCR clones in peripheral blood after treatment; these T cells had markedly enhanced Tbet expression, suggesting TH1 phenotype. In this nonrandomized controlled trial, IT GLA-SE with concurrent radiotherapy was well tolerated and provided more durable local control than radiotherapy alone. Patients with durable local response demonstrated enhanced IT T-cell clonal expansion, with matched expansion of these clonotypes in the circulation. Additional studies evaluating synergism of IT GLA-SE and radiotherapy with systemic immune modulation are warranted. ClinicalTrials.gov Identifier: NCT02180698.

Reshaping Macrophage Polarization Potential Enhances Antitumor Immune Response to Radiation Therapy

Although radiation therapy (RT) remains a cornerstone in the treatment of breast cancer, many trials combining RT with immune checkpoint blockade (ICB) have failed to demonstrate benefit in solid tumors including breast cancer. Maximal efficacy of RT relies on the generation of antitumor immunity following treatment which largely consists of cytotoxic T cells and macrophages. Broad depletion of macrophages modestly enhances tumor responses to RT suggesting that they can shape RT-induced antitumor immunity. Although IL4 signaling through GATA-3 is known to polarize T cells into the protumor Th2 phenotype, such central drivers of macrophage polarization are not well established. Given that macrophages abundantly express IL4 receptor, we hypothesized that GATA-3 may direct the transition of macrophages to M2/alternative phase and that genetic ablation of GATA-3 in macrophages can enhance antitumor immunity by arresting macrophage transition to an M2-like pro-tumor state. We generated a macrophage specific GATA-3 KO mouse model (mG3KO) driven by the LysM-Cre promoter. Using a syngeneic orthotopic murine model of breast cancer (EO771), we evaluated the differential effect of RT (16Gy x 1) in WT and mG3KO mice. Multiparametric flow cytometry was performed to investigate the immune changes within the tumor microenvironment on day 3, day 5 and day 10 after RT. T cell depletion was performed using antibodies to CD4 and CD8 by intraperitoneal injections to understand the role of adaptive immunity in the response to RT in WT and mG3KO mice. We found that mG3KO mice bearing advanced EO771 tumors demonstrated significantly improved tumor regression compared to WT mice (p<0.001), which translated to increased overall survival. In vitro characterization of bone-marrow derived macrophages from mG3KO and WT mice suggest that macrophages with ablated GATA-3 expressed increased levels of iNOS and decreased levels of Arginase (Arg-1), consistent with an M1-like phenotype. Immune profiling of the tumors also revealed that mGATA-3 KO animals have significant enrichment of CD8+ T cells in the tumor milieu post RT and these CD8+ T cells express higher amounts of interferon gamma (p<0.001) and Granzyme B (p<0.0015) than their WT counterparts. Using neutralizing antibodies to deplete CD8+ T cells, we show that anti-tumor effects in the mG3KO mice were abolished, suggesting that mG3KO macrophages impact survival, at least, in part by enhancing cytotoxic CD8+T cells. Studies are currently ongoing to reveal the detailed mechanism of GATA-3 ablation in improving the efficacy of RT. Our data indicates that GATA-3 is a central regulator of macrophage polarization in response to RT. Further, directed ablation of GATA-3 appears to drive macrophages towards an M1-like phenotype, which enhances T cell recruitment to irradiated tumors. These data suggest that the antitumor efficacy of RT can be prolonged by targeting GATA-3-dependent signaling within myeloid cells.

T helper cell responses in adult diarrheal patients following natural infection with enterotoxigenic Escherichia coli are primarily of the Th17 type.

Infection with enterotoxigenic Escherichia coli (ETEC) gives rise to IgA antibodies against both the heat labile toxin (LT) and colonization factors (CFs), which are considered to synergistically protect against ETEC diarrhea. Since the development of ETEC-specific long lived plasma cells and memory B cells is likely to be dependent on T helper (Th) cells, we investigated if natural ETEC diarrhea elicits ETEC-specific Th cells and their relation to IgA responses. Th cell subsets were analyzed in adult Bangladeshi patients hospitalized due to ETEC diarrhea by flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) isolated from blood collected day 2, 7, 30 and 90 after hospitalization as well as in healthy controls. The LT- and CF-specific Th responses were determined by analysis of IL-17A and IFN-γ in antigen stimulated PBMC cultures using ELISA. ETEC-specific IgA secreted by circulating antibody secreting cells (plasmablasts) were analyzed by using the antibodies in lymphocyte supernatants (ALS) ELISA-based method and plasma IgA was also measured by ELISA. ETEC patients mounted significant ALS and plasma IgA responses against LTB and CFs on day 7 after hospitalization. ETEC patients had significantly elevated proportions of memory Th cells with a Th17 phenotype (CCR6+CXCR3-) in blood compared to controls, while frequencies of Th1 (CCR6-CXCR3+) or Th2 (CCR6-CXCR3-) cells were not increased. Antigen stimulation of PBMCs revealed IL-17A responses to LT, most clearly observed after stimulation with double mutant heat labile toxin (dmLT), but also with LT B subunit (LTB), and to CS6 in samples from patients with LT+ or CS6+ ETEC bacteria. Some individuals also mounted IFN-γ responses to dmLT and LTB. Levels of LTB specific IgA antibodies in ALS, but not plasma samples correlated with both IL-17A (r=0.5, p=0.02) and IFN-γ (r=0.6, p=0.01) responses to dmLT. Our results show that ETEC diarrhea induces T cell responses, which are predominantly of the Th17 type. The correlations between IL-17A and IFN-g and intestine-derived plasmablast responses support that Th responses may contribute to the development of protective IgA responses against ETEC infection. These observations provide important insights into T cell responses that need to be considered in the evaluation of advanced ETEC vaccine candidates.

Differential molecular programs of cutaneous anaplastic large cell lymphoma and CD30-positive transformed mycosis fungoides.

Discriminating between cutaneous anaplastic large cell lymphoma (cALCL) and CD30-positive transformed mycosis fungoides (CD30+ TMF) is challenging, particularly when they arise in the context of pre-existing mycosis fungoides. The development of molecular diagnostic tools was hampered by the rarity of both diseases and the limited understanding of their pathogenesis. In this study, we established a cohort comprising 25 cALCL cases and 25 CD30+ TMF cases, with transcriptomic data obtained from 31 samples. We compared the clinicopathological information and investigated the gene expression profiling between these two entities. Furthermore, we developed an immunohistochemistry (IHC) algorithm to differentiate these two entities clinically. Our investigation revealed distinct clinicopathological features and unique gene expression programs associated with cALCL and CD30+ TMF. cALCL and CD30+ TMF displayed marked differences in gene expression patterns. Notably, CD30+ TMF demonstrated enrichment of T cell receptor signaling pathways and an exhausted T cell phenotype, accompanied by infiltration of B cells, dendritic cells, and neurons. In contrast, cALCL cells expressed high levels of HLA class II genes, polarized towards a Th17 phenotype, and exhibited neutrophil infiltration. An IHC algorithm with BATF3 and TCF7 staining emerged as potential diagnostic markers for identifying these two entities. Our findings provide valuable insights into the differential molecular signatures associated with cALCL and CD30+ TMF, which contribute to their distinct clinicopathological behaviors. An appropriate IHC algorithm could be used as a potential diagnostic tool.

The Role of the Spleen and the Place of Splenectomy in Autoimmune Hemolytic Anemia-A Review of Current Knowledge.

Autoimmune hemolytic anemia (AIHA) is a rare, very heterogeneous, and sometimes life-threatening acquired hematologic disease characterized by increased red blood cell (RBC) destruction by autoantibodies (autoAbs), either with or without complement involvement. Recent studies have shown that the involvement of T- and B-cell dysregulation and an imbalance of T-helper 2 (Th2) and Th17 phenotypes play major roles in the pathogenesis of AIHA. AIHA can be primary (idiopathic) but is more often secondary, triggered by infections or drug use or as a part of other diseases. As the location of origin of autoAbs and the location of autoAb-mediated RBC clearance, as well as the location of extramedullary hematopoiesis, the spleen is crucially involved in all the steps of AIHA pathobiology. Splenectomy, which was the established second-line therapeutic option in corticosteroid-resistant AIHA patients for decades, has become less common due to increasing knowledge of immunopathogenesis and the introduction of targeted therapy. This article provides a comprehensive overview of current knowledge regarding the place of the spleen in the immunological background of AIHA and the rapidly growing spectrum of novel therapeutic approaches. Furthermore, this review emphasizes the still-existing expediency of laparoscopic splenectomy with appropriate perioperative thromboprophylaxis and the prevention of infection as a safe and reliable therapeutic option in the context of the limited availability of rituximab and other novel therapies.

In silico and in vitro potentials of crocin and amphotericin B on Leishmania major: Multiple synergistic mechanisms of actions.

A significant barrier to optimal antileishmanial treatment is low efficacy and the emergence of drug resistance. Multiple approaches were used to monitor and assess crocin (a central component of saffron) mixed with amphotericin B (AmpB) potential in silico and in vitro consequences. The binding behavior of crocin and iNOS was the purpose of molecular docking. The results showed that crocin coupled with AmpB demonstrated a safe combination, extremely antileishmanial, suppressed Leishmania arginase absorption, and increased parasite death. This natural flower component is a robust antioxidant, significantly promoting the expression of the Th1-connected cytokines (IL12p40, IFN-γ, and TNF- α), iNOS, and transcription factors (Elk-1, c-Fos, and STAT-1). In comparison, the expression of the Th2-associated phenotypes (IL-10, IL-4, and TGF-β) was significantly reduced. The leishmanicidal effect of this combination was also mediated through programmed cell death (PCD), as confirmed by the manifestation of phosphatidylserine and cell cycle detention at the sub-GO/G1 phase. In conclusion, crocin with AmpB synergistically exerted in vitro antileishmanial action, generated nitric oxide and reactive oxygen species, modulated Th1, and Th2 phenotypes and transfer factors, enhanced PCD profile and arrested the cell cycle of Leishmania major promastigotes. The main action of crocin and AmpB involved wide-ranging mechanistic insights for conducting other clinical settings as promising drug candidates for cutaneous leishmaniasis. Therefore, this combination could be esteemed as a basis for a potential bioactive component and a logical source for leishmanicidal drug development against CL in future advanced clinical settings.

Role of T and B lymphocyte cannabinoid type 1 and 2 receptors in major depression and suicidal behaviours.

Early flow cytometry studies revealed T cell activation in major depressive disorder (MDD). MDD is characterised by activation of the immune-inflammatory response system (IRS) and the compensatory immunoregulatory system (CIRS), including deficits in T regulatory (Treg) cells. This study examines the number of cannabinoid type 1 (CB1) and type 2 (CB2) receptor-bearing T/B lymphocytes in MDD, and the effects of in vitro cannabidiol (CBD) administration on CB1/CB2-bearing immunocytes. Using flow cytometry, we determined the percentage of CD20+CB2+, CD3+CB2+, CD4+CB2+, CD8+CB2+ and FoxP3+CB1+ cells in 19 healthy controls and 29 MDD patients in 5 conditions: baseline, stimulation with anti-CD3/CD28 with or without 0.1 µg/mL, 1.0 µg/mL, or 10.0 µg/mL CBD. CB2+ was significantly higher in CD20+ than CD3+ and CD4+ and CD 8+ cells. Stimulation with anti-CD3/CD8 increases the number of CB2-bearing CD3+, CD4+ and CD8+ cells, as well as CB1-bearing FoxP3+ cells. There was an inverse association between the number of reduced CD4+ CB2+ and IRS profiles, including M1 macrophage, T helper-(Th)-1 and Th-17 phenotypes. MDD is characterised by lowered basal FoxP3+ CB1+% and higher CD20+ CB2+%. 33.2% of the variance in the depression phenome (including severity of depression, anxiety and current suicidal behaviours) is explained by CD20+ CB2+ % (positively) and CD3+ CB2+% (inversely). All five immune cell populations were significantly increased by 10 µg/mL of CBD administration. Reductions in FoxP3+ CB1+% and CD3+ /CD4+ CB2+% contribute to deficits in immune homoeostasis in MDD, while increased CD20+CB2+% may contribute to the pathophysiology of MDD by activating T-independent humoral immunity.

Pre-existing CD4 T cell help boosts antibody responses but has limited impact on germinal center, antigen-specific B cell frequencies after influenza infection.

The influenza virus is a persistent burden on global health, with seasonal vaccines providing incomplete protection. CD4+ T cells help shape B cell and antibody responses; however, the selectivity of help and the effect on various antigen-specific B cell populations have not been fully elucidated. Here, we studied the specificity, selectivity, and influence of nucleoprotein (NP) CD4+ T cells on the magnitude and quality of hemagglutinin (HA) and NP-specific B cells and antibody responses. We identified immunodominant peptides and showed that peptide immunization was sufficient to induce CD4+ cells with Th1 and Tfh phenotypes. Surprisingly, while preexisting CD4+ T cells enhanced the influx of total germinal center (GC) B cells in the mediastinal lymph node after infection, this was not reflected by an increase in the frequency of antigen-specific cells within the GC. Furthermore, we demonstrated that NP-specific help was able to accelerate the kinetics and magnitude of the Ab response for NP but not for HA. Overall, our results showed that pre-existing CD4+ T cells provide strong cognate help during immunization or infection to enhance Ab production but not antigen-specific GC or memory B cells.

Elevated Interleukin-31 Levels in Serum, but Not CSF of Dogs with Steroid-Responsive Meningitis-Arteritis Suggest an Involvement in Its Pathogenesis.

Steroid-responsive meningitis-arteritis (SRMA) is a predominantly Th-2 immune-mediated disease, but the exact pathomechanism remains unclear. Interleukin-31 (IL-31) is predominantly produced by T cells with a Th-2 phenotype during proinflammatory conditions. We hypothesize that IL-31 might be involved in the pathogenesis of SRMA. IL-31 was measured in archived samples (49 serum and 52 CSF samples) of dogs with SRMA, meningoencephalitis of unknown origin (MUO), infectious meningoencephalitis, and atopic dermatitis, and of healthy control dogs using a competitive canine IL-31 ELISA. The mean serum IL-31 level in dogs with SRMA (n = 18) was mildly higher compared to dogs with atopic dermatitis (n = 3, p = 0.8135) and MUO (n = 15, p = 0.7618) and markedly higher than in healthy controls (n = 10, p = 0.1327) and dogs with infectious meningoencephalitis (n = 3, no statistics). Dogs with SRMA in the acute stage of the disease and without any pre-treatment had the highest IL-31 levels. The mean CSF IL-31 value for dogs with SRMA (n = 23) was quite similar to that for healthy controls (n = 8, p = 0.4454) and did not differ markedly from dogs with MUO (n = 19, p = 0.8724) and infectious meningoencephalitis. Based on this study, an involvement of IL-31 in the pathogenesis of the systemic Th-2 immune-mediated immune response in SRMA can be assumed as a further component leading to an aberrant immune reaction.

ApoE expression in macrophages communicates immunometabolic signaling that controls hyperlipidemia-driven hematopoiesis & inflammation via extracellular vesicles.

While apolipoprotein E (apoE) expression by myeloid cells is recognized to control inflammation, whether such benefits can be communicated via extracellular vesicles is not known. Through the study of extracellular vesicles produced by macrophages derived from the bone marrow of Wildtype (WT-BMDM-EV) and ApoE deficient (EKO-BMDM-EV) mice, we uncovered a critical role for apoE expression in regulating their cell signaling properties. WT-BMDM-EV communicated anti-inflammatory properties to recipient myeloid cells by increasing cellular levels of apoE and miR-146a-5p, that reduced NF-κB signalling. They also downregulated cellular levels of miR-142a-3p, resulting in increased levels of its target carnitine palmitoyl transferase 1A (CPT1A) which improved fatty acid oxidation (FAO) and oxidative phosphorylation (OxPHOS) in recipient cells. Such favorable metabolic polarization enhanced cell-surface MerTK levels and the phagocytic uptake of apoptotic cells. In contrast, EKO-BMDM-EV exerted opposite effects by reducing cellular levels of apoE and miR-146a-5p, which increased NF-κB-driven GLUT1-mediated glucose uptake, aerobic glycolysis, and oxidative stress. Furthermore, EKO-BMDM-EV increased cellular miR-142a-3p levels, which reduced CPT1A levels and impaired FAO and OxPHOS in recipient myeloid cells. When cultured with naïve CD4+ T lymphocytes, EKO-BMDM-EV drove their activation and proliferation, and fostered their transition to a Th1 phenotype. While infusions of WT-BMDM-EV into hyperlipidemic mice resolved inflammation, infusions of EKO-BMDM-EV increased hematopoiesis and drove inflammatory responses in myeloid cells and T lymphocytes. ApoE-dependent immunometabolic signaling by macrophage extracellular vesicles was dependent on transcriptional axes controlled by miR-146a-5p and miR-142a-3p that could be reproduced by infusing miR-146a mimics & miR-142a antagonists into hyperlipidemic apoE-deficient mice. Together, our findings unveil a novel property for apoE expression in macrophages that modulates the immunometabolic regulatory properties of their secreted extracellular vesicles.