Cell-attracting Chemokine Research Articles

T cell-attracting CCL18 chemokine is a dominant rejection signal during limb transplantation

SummaryLimb transplantation is a life-changing procedure for amputees. However, limb recipients have a 6-fold greater rejection rate than solid organ transplant recipients, related in part to greater immunogenicity of the skin. Here, we report a detailed immunological and molecular characterization of individuals who underwent bilateral limb transplantation at our institution. Circulating Th17 cells are increased in limb transplant recipients over time. Molecular characterization of 770 genes in skin biopsies reveals upregulation of T cell effector immune molecules and chemokines, particularly CCL18. Skin antigen-presenting cells primarily express the chemokine CCL18, which binds to the CCR8 receptor. CCL18 treatment recruits more allo-T cells to the skin xenograft in a humanized skin transplantation model, leading to signs of accelerated graft rejection. Blockade of CCR8 remarkedly decreases CCL18-induced allo-T cell infiltration. Our results suggest that targeting the CCL18:CCR8 pathway could be a promising immunosuppressive approach in transplantation.

Knockout of the KH-Type Splicing Regulatory Protein Drives Glomerulonephritis in MRL-Faslpr Mice.

KH-type splicing regulatory protein (KSRP) is an RNA-binding protein that promotes mRNA decay and thereby negatively regulates cytokine expression at the post-transcriptional level. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by dysregulated cytokine expression causing multiple organ manifestations; MRL-Faslpr mice are an established mouse model to study lupus disease pathogenesis. To investigate the impact of KSRP on lupus disease progression, we generated KSRP-deficient MRL-Faslpr mice (MRL-Faslpr/KSRP−/− mice). In line with the predicted role of KSRP as a negative regulator of cytokine expression, lupus nephritis was augmented in MRL-Faslpr/KSRP−/− mice. Increased infiltration of immune cells, especially of IFN-γ producing T cells and macrophages, driven by enhanced expression of T cell-attracting chemokines and adhesion molecules, seems to be responsible for worsened kidney morphology. Reduced expression of the anti-inflammatory interleukin-1 receptor antagonist may be another reason for severe inflammation. The increase of FoxP3+ T cells detected in the kidney seems unable to dampen the massive kidney inflammation. Interestingly, lymphadenopathy was reduced in MRL-Faslpr/KSRP−/− mice. Altogether, KSRP appears to have a complex role in immune regulation; however, it is clearly able to ameliorate lupus nephritis.

CXCL13 is expressed in a subpopulation of neuroendocrine cells in the murine trachea and lung

The conducting airways are lined by distinct cell types, comprising basal, secretory, ciliated, and rare cells, including ionocytes, solitary cholinergic chemosensory cells, and solitary and clustered (neuroepithelial bodies) neuroendocrine cells. Airway neuroendocrine cells are in clinical focus since they can give rise to small cell lung cancer. They have been implicated in diverse functions including mechanosensation, chemosensation, and regeneration, and were recently identified as regulators of type 2 immune responses via the release of the neuropeptide calcitonin gene-related peptide (CGRP). We here assessed the expression of the chemokine CXCL13 (B cell attracting chemokine) by these cells by RT-PCR, in silico analysis of publicly available sequencing data sets, immunohistochemistry, and immuno-electron microscopy. We identify a phenotype of neuroendocrine cells in the naïve mouse, producing the chemokine CXCL13 predominantly in solitary neuroendocrine cells of the tracheal epithelium (approx. 70% CXCL13+) and, to a lesser extent, in the solitary neuroendocrine cells and neuroepithelial bodies of the intrapulmonary bronchial epithelium (< 10% CXCL13+). In silico analysis of published sequencing data of murine tracheal epithelial cells was consistent with the results obtained by immunohistochemistry as it revealed that neuroendocrine cells are the major source of Cxcl13-mRNA, which was expressed by 68–79% of neuroendocrine cells. An unbiased scRNA-seq data analysis of overall gene expression did not yield subclusters of neuroendocrine cells. Our observation demonstrates phenotypic heterogeneity of airway neuroendocrine cells and points towards a putative immunoregulatory role of these cells in bronchial-associated lymphoid tissue formation and B cell homeostasis.

P08.02 Harnessing T cells to target brain metastasis

Abstract BACKGROUND Up to 60% of melanoma patients develop brain metastases (BrM). These patients have a poor prognosis and limited treatment options. Immune checkpoint inhibitors (ICI) targeting Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) and Programmed cell death protein-1 (PD-1) have revolutionized the treatment of melanoma and their efficacy has been also demonstrated in melanoma BrM. Our group previously demonstrated that ICI (combined α-PD-1 and α-CTLA-4) enhances chemokine-dependent infiltration of cytotoxic T lymphocytes (CTLs) into melanoma BrMs in preclinical models, accompanied by upregulated expression of T cell attracting chemokines in tumours. Notably, CTLs infiltrating BrM expressed only some of the chemokine receptors (CRs) interacting with ICI-induced chemokines in BrM, providing a rationale to over-express the “missing” CRs in T cells to enhance their homing to tumours in the context of adoptive T cell therapy (ACT). MATERIALS AND METHODS OT-I cells were isolated from OT-I mice and differentiated ex vivo into effector (TEF) and memory (TCM) CD8+ T cells. Tumour infiltrating lymphocytes (TILs) from B16 tumour-bearing mice treated with ICI were isolated using magnetic beads, activated and expanded ex vivo. Expression of CRs and activation markers in ex vivo cultured T cells were quantified by qPCR and/or flow cytometry. The migration of human blood CD8+ T cells towards chemokines of interest were measured in ex vivo migration assays. RESULTS The same CRs that were missing on BrM-infiltrating CTLs in vivo models were also absent from OT-I TEF (CCR7low/CD44high/CD62Llow) and TCM (CCR7high/CD44low/CD62Lhigh) cells, as well as from TILs expanded ex vivo for use in ACT. Furthermore, we observed no increase in migration of human T cells towards chemokines interacting with the “missing” CRs in comparison to the baseline migration, suggesting that these CRs are also absent from human T cells. CONCLUSION Ex vivo expanded T cells that are used in ACT are missing several CRs that are interacting with chemokines upregulated in BrM. We hypothesise that the use of genetically engineered T cells expressing the “missing” CRs in ACT has the potential to enhance ACT efficacy in combination with ICI.

Abstract 1489: Deposition of genetically engineered T cell attracting antigen presenting cells in the glioma microenvironment with low intensity pulsed ultrasound-based blood-brain barrier opening triggers therapeutic responses in preclinical glioma models

Abstract Introduction: In contrast to brain metastases, gliomas have few antigen-presenting cells (APCs) in the tumor microenvironment, which may limit T cell effector responses. We hypothesized that: (1) an APC could be modified to express a T cell attracting chemokine that could enhance localized immune activation, and (2) that ultrasound-based blood-brain barrier (BBB) opening would enhance delivery of these APCs at the site of tumor antigens. Methods: Murine F4/80 and CD11c expressing APCs were isolated from the bone marrow of C57BL/6 mice and modified via lentiviral transduction to encode the cDNA of immune cell chemokines CXCL9 or CXCL10 and methionine-deficient green fluorescent protein (co-expression) for cell tagging. C57BL/6 mice were implanted with GL261 cells and treated with 1 x 106 CXCL9 or CXCL10 over-expressing APCs intracranially (i.c.), or intravenously (i.v.) with or without ultrasound-based BBB opening. To determine how the CXCL10 expressing APCs were altering the tumor microenvironment, immune cells were isolated from the glioma implanted hemisphere of all treatment groups and analyzed by multicolor flow cytometry. Results: Transduced APCs produced 3,392 pg/ml and 2,987 pg/ml of CXCL9 or CXCL10, respectively. Mice tolerated the treatments well without any adverse events, behavioral changes, or neurological toxicity. Survival analysis of mice treated with CXCL9 expressing APCs using i.c., or i.v. with and without BBB opening ultrasound showed no significant survival benefit compared with PBS-treated control mice. Mice treated i.c. with CXCL10-expressing APCs had significantly improved survival relative to the PBS control group (p&amp;lt;0.05), demonstrating that this cellular therapy required access to the tumor microenvironment in order to be effective. Notably, the most therapeutically efficacious administration route was when the CXCL10-expressing APCs were administered i.v. before ultrasound-based BBB opening even relative to the i.c. administered group (p&amp;lt;0.05). Intravenous administration of CXCL10 APCs combined with ultrasound-based BBB opening also significantly increased the percentage of T cells in the glioma microenvironment relative to all other treatment groups. Conclusions: The delivery of CXCL10-secreting APCs to the glioma microenvironment with ultrasound-based BBB opening was superior to delivery with direct i.c. injection. BBB disruption may have triggered more diffuse dispersal of the APCs throughout the tumor microenvironment or positioned these cells in closer proximity to the T cells emigrating from the vascular space into the localized glioma microenvironment. Citation Format: Aria Sabbagh, Kevin Beccaria, Xiaoyang Ling, Anantha Marisetty, Martina Ott, Ling-Yuan Kong, Dexing Fang, Jun Wei, Carole Desseaux, Guillaume Bouchoux, Michael Canney, Alexandre Carpentier, Amy B. Heimberger. Deposition of genetically engineered T cell attracting antigen presenting cells in the glioma microenvironment with low intensity pulsed ultrasound-based blood-brain barrier opening triggers therapeutic responses in preclinical glioma models [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 1489.

A Comparison of Ex Vivo Expanded Human Regulatory T Cells Using Allogeneic Stimulated B Cells or Monocyte-Derived Dendritic Cells.

Alloreactive regulatory T cells (arTregs) are more potent than polyclonal Tregs at suppressing immune responses to transplant antigens. Human arTregs can be expanded with allogeneic CD40L-stimulated B cells (sBcs) or stimulated-matured monocyte-derived dendritic cells (sDCs). Here, we compared the expansion efficiency and properties of arTregs stimulated ex vivo using these two types of antigen-presenting cells. Compared to sBcs, sDCs stimulated Tregs to expand two times more in number. The superior expansion-inducing capacity of sDCs correlated with their higher expression of CD80, CD86, and T cell-attracting chemokines. sBc- and sDC-arTregs expressed comparable levels of FOXP3, HELIOS, CD25, CD27, and CD62L, demethylated FOXP3 enhancer and in vitro suppressive function. sBc- and sDCs-arTregs had similar gene expression profiles that were distinct from primary Tregs. sBc- and sDC-arTregs exhibited similar low frequencies of IFN-γ, IL-4, and IL-17A-producing cells, and the cytokine-producing arTregs expressed high levels of FOXP3. Almost all sBc- and sDC-arTregs expressed CXCR3, which may enable them traffic to inflammatory sites. Thus, sDCs-arTregs that expand more readily, are phenotypically similar to sBc-arTregs, supporting sDCs as a viable alternative for arTreg production for clinical evaluation.

Association of triple negative breast cancer (TNBC) with high thermogenesis with worse survival and unfavorable tumor microenvironment (TME).

e12546 Background: Norepinephrine released during cold stress by housing 4T1 TNBC mice at 22°C increases tumor growth with a pro-tumorigenic TME [lower CD8+ T-cells, higher myeloid derived suppressor cells (MDSC) and regulatory T-cells (Treg)] compared with mice housed at 30°C. Norepinephrine activates thermogenesis in adipose tissue. Worse survival has been reported among females with malignancies living in cold versus warm climate. These findings led us to hypothesize that TNBC with enhanced thermogenesis is associated with worse outcome and unfavorable TME. Methods: mRNA expression data for 298 and 143 TNBC patients was accessed from METABRIC and GSE96058 breast cancer cohorts, respectively. TNBC were classified into “high” (top tertile) and “low” thermogenesis (bottom two-third) using Gene Set Variation Analysis (GSVA) with KEGG thermogenesis pathway. Computer algorithms xCell and Gene Set Enrichment Analysis (GSEA) were used. Interferon (IFN)-γ signature was calculated using GSVA. Gene expression data was used to quantify adipocytes, angiogenesis, chemokines and cytolytic activity (CYT). R software was used for analysis. False discovery rate (FDR) &lt; 0.25 for GSEA and p &lt; 0.05 was considered statistically significant. Results: High-thermogenesis TNBC was associated with a worse median disease specific survival 14.7 years vs. not reached [HR 1.49 (95% CI 1.02-2.18)], p &lt; 0.05 in METABRIC and a trend towards worse overall survival in both METABRIC and GSE96058. Higher fraction of adipocytes (p &lt; 0.001) and angiogenesis markers including CD31, vWF, Vascular endothelial cadherin, claudin 5, JAM2 and sphingosine-1-phosphate related gene S1PR1 (p &lt; 0.01) were observed explaining the worse survival in high-thermogenesis TNBC. This group was significantly enriched in fatty acid metabolism and adipogenesis pathways (FDR &lt; 0.25) on GSEA. On the other hand, low-thermogenesis TNBC was enriched in mitotic spindle, E2F targets, G2M checkpoint, MYC targets consistent with higher Ki67 (p &lt; 0.05) in this group; and also in IFN-α and IFN-γ response (FDR &lt; 0.25). High-thermogenesis TNBC was associated with lower IFN-γ and granzyme B expression (p &lt; 0.05). Favorable cytotoxic-T cell attracting chemokines CCL5, CXCL9, CXCL10 and CXCL11 were lower; MDSC-, Treg-favoring chemokine CXCL12 was higher (p &lt; 0.05). There were lower M1 and more M2 macrophages (p &lt; 0.001) with lower CYT. There was higher expression of dual-specificity phosphatase-1 ( DUSP1)(p &lt; 0.05), which is upregulated following glucocorticoid receptor activation. Conclusions: We investigated the clinical relevance of preclinical findings and observed that high-thermogenesis TNBC is associated with worse survival and unfavorable TME. Further research is warranted to validate if high thermogenesis could reflect tumors under thermal stress and develop novel strategies to abrogate stress and improve outcomes.

The imitation game: How glioblastoma outmaneuvers immune attack

Immune evasion and resistance to immunotherapy mark major roadblocks in treating glioblastoma, the deadliest form of brain cancer. In this issue of Cell, Gangoso etal. demonstrate that the immune microenvironment drives glioblastoma cells to hijack myeloid-characteristic transcriptional and epigenetic circuits as a mode of immune evasion.

B-T Cell Interactions in GRAFT-Versus-Host Disease

Cytotoxic T cells (CTLs) and B cells engage distinct interactions in GVHD patients' blood and tissues, detectable in regular flow-cytometry screenings, by size and by double positive CD19-CD8 antibody markers (Deola, BMT 2017). B-CTL couplets are formed by alpha-betaTCR+ CD8+ CTLs preferentially targeting CD27+ CD19+ cells displaying an activated CD80 and CD86 phenotype. Interactions may last from 5 minutes to roughly 1 hour, and release a pattern of T cell attracting chemokines, as IP10, MIG, ITAC, which are also known GVHD biomarkers. To further unravel the mechanism of this cell interaction, we built an in-vitro model where human PBMCs cells are expanded with cognate peptides and IL2 for 1-2 weeks, then immune-selected for CD8 antigen by Miltenyi microbeads negative-selection and incubated (2-18 hours) with fresh autologous CD19-B cells, immune-selected with the same method. The interactions are studied under confocal microscope video-imaging (Zeiss LSM 880+Imaris 3D analysis software) and in flow-cytometry (SymphonyA5 BD) after deep phenotype antibody staining. The intensity of interaction, measured by fluorescence interference on cell membranes, revealed an active engagement of CD19 and CD8 antigens. CD19 antigen penetrates deeper in contacting T cells, than CD8 on B cells, and consistently with this finding, after the interactions there is an antigen exchange between cells with CD19 antigen actively transferred in CD8 cells (p value =&amp;lt;0.001), but not the contrary. We already proved that this type of B-T interaction is not antigen specific in CTL-to-B direction (Deola et a, JI 2008) but to exclude cross-presentation from B to CTLs and to unravel the role of CD8, we interfered by antibody blocking of MHC class I pathway on B cells and CD8 on CTLs. B-T cell interactions are not abolished after MHC-I or CD8 blocking, the intensity of coupling is unchanged after MHC-I block, and is higher after blocking CD8 (p value=&amp;lt;0.001). In particular, by blocking CD8 molecule, T cells target preferentially CD19+/CD27- cells rather than CD19+/27+ cells. Interestingly, B cell engagement follows 2 repetitive patterns of interaction: a high intensity interaction that visually corresponds to tight coupling cells with high CD19 penetration in T cells, and a low-intensity continuous interaction, visually measurable by cells "sniffing" each other. Both patterns correspond to diverse Calcium flux activation on T cells and B cells, suggesting functional different pathways triggered by the 2 type of interactions. Deep phenotype flow cytometry analyses after coupling reveals distinct programs triggered by the contact in both B cells and T cells. While after the interaction CTLs double their pool of perforin bearing effectors and their fraction of CD45RA-/CD27+ memory CTLs, CD19 preferentially undergo a deletion of IgD- CD27- (DN) cells (13,85%+/-1,1 and 22,95%+/-4,5 CD95/Fas+, respectively in B cells alone and B+CTLs, n=2) and a rescue of affinity mature CD27+ IgD- cells (39.8%+/-25,47 and 21,2%+/- 29% CD95/Fas+ in the same groups) CTLs are the ultimate line of "tissue attack" in GVHD and several diseases, as autoimmune diseases, cancer, viral diseases, sharing a common pathological program definable as "immune rejection". B cells are key players in immune rejection, but a link between these 2 types of cells is still unclear. Our findings enforce the hypothesis of a program of peripheral tolerance/activation triggered directly between B cells and activated CTLs in the context of inflammation and of GVHD. Disclosures No relevant conflicts of interest to declare.

Streptococcus pneumoniae aminopeptidase N regulates dendritic cells that attenuates type-2 airway inflammation in murine allergic asthma.

Epidemiological and experimental studies suggest that microbial exposure in early childhood is linked with reduced risk to suffer asthma. Thus microbial components with immunoregulatory capabilities might serve as a preventive strategy for allergic asthma. Recently, it was identified that Streptococcus pneumoniae aminopeptidase N (PepN) could suppress T cell effector function. We sought to investigate the effect of PepN on murine allergic asthma and elucidate the underlying mechanism. The effects of intranasal administration of PepN during or before sensitization were examined in ovalbumin (OVA)-induced murine allergic asthma. The roles of CD11b+ dendritic cells in PepN treated OVA-induced allergic asthma were evaluated by flow cytometry, cytokines detection and adoptive transfer. Moreover, the numbers of lung type 2 innate lymphoid cells (ILC2s) were also detected. Administration of PepN during or before sensitization attenuated type-2 airway inflammation (eosinophilia, mucus hypersecretion, Th2 cytokines production and IgE production) in allergic asthma mice. PepN reduced lung accumulation of CD11b+ dendritic cells, which was accompanied by diminished dendritic cell-attracting chemokine CCL20 production as well as CCL17 and CCL22, which are Th2-cell chemokines predominantly produced by CD11b+ dendritic cells. Adoptive transfer of BM-derived CD11b+ dendritic cells abolished the inhibitory effect of PepN on OVA-induced type-2 airway inflammation. The numbers of lung ILC2s were decreased in asthmatic mice receiving PepN. PepN alleviated type-2 inflammation in OVA-induced allergic asthma mice, which was mediated by regulation of lung CD11b+ dendritic cells. Our study provides a novel strategy for the prevention of allergic asthma.

Oral Administration of Live and Dead Cells of Lactobacillus sakei proBio65 Alleviated Atopic Dermatitis in Children and Adolescents: a Randomized, Double-Blind, and Placebo-Controlled Study.

Several studies suggest that probiotics might be useful in the management of atopic dermatitis (AD). However, the efficacy and comparison between both the administration of viable and non-viable probiotics on alleviation of AD is not well studied. Therefore, the purpose of this study was to evaluate the effect of L. sakei proBio65 live and dead cells when administered (1 × 1010 cells/day) for 12weeks to children and adolescents (aged 3 to 18) with atopic dermatitis. In this randomized double-blind, placebo-controlled study, ninety patients were recruited and randomly allocated to either the L. sakei proBio65 live cells, L. sakei proBio65 dead cells, or placebo groups. Assessment of efficacy was based on the change in SCORing Atopic Dermatitis (SCORAD) score, Investigators Global Assessment (IGA) score, serum inflammatory markers such as the serum eosinophil (count), IgE, eosinophil cationic protein (ECP), CCL17 (thymus and activation-regulated chemokine [TARC]), and CCL27 (cutaneous T cell-attracting chemokine [CTACK]), and changes in skin condition (moisture and sebum) at baseline, week 6 and week 12. The SCORAD total score decreased in the live cells (p =0.0015) and dead cell group (p =0.0017) from the baseline after 12weeks, whereas there were no significant changes in the placebo group when compared with baseline. The skin sebum content increased in both the live cell (p <0.0001) and the dead cell group (p <0.0001), suggesting potential improvements in skin barrier functions. Current data suggested a positive improvement in alleviation of AD symptoms upon oral administration of L. sakei proBio65 in both viable and non-viable forms.

Tumor inhibition or tumor promotion? The duplicity of CXCR3 in cancer.

Tumor tissue includes cancer cells and normal stromal cells such as vascular endothelial cells, connective tissue cells (cancer associated fibroblast, mesenchymal stem cell), and immune cells (tumor-infiltrating lymphocytes or TIL, dendritic cells, eosinophils, basophils, mast cells, tumor-associated macrophages or TAM, myeloid-derived suppressor cells or MDSC). Anti-tumor activity is mainly mediated by infiltration of NK cells, Th1 and CD8+ Tcells, and correlates with expression of NK cell and Tcell attracting chemokines. Nevertheless, cancer cells hijack tissue homeostasis through secretion of cytokines and chemokines that mediate not only the induction of an inflamed status that supports cancer cell survival and growth, but also the recruitment and/or activation of immune suppressive cells. CXCL9, CXCL10, and CXCL11 are known for their tumor-inhibiting properties, but their overexpression in several hematologic and solid tumors correlates with disease severity, suggesting a role in tumor promotion. The dichotomous nature of CXCR3 ligands activity mainly depends on several molecular mechanisms induced by cancer cells themselves able to divert immune responses and to alter the whole local environment. A deep understanding of the nature of such phenomenon may provide a rationale to build up a CXCR3/ligand axis targeting strategy. In this review, we will discuss the role of CXCR3 in cancer progression and in regulation of anti-tumor immune response and immunotherapy.

The role of thymus and activation-regulated chemokine as a marker of severity of atopic dermatitis

The role of thymus and activation-regulated chemokine as a marker of severity of atopic dermatitis

Effect of Low-Dose Radiation Therapy on Abscopal Responses to Hypofractionated Radiation Therapy and Anti-PD1 in Mice and Patients With Non-Small Cell Lung Cancer

Hypofractionated radiation therapy (HFRT) can induce antitumor T cell responses, particularly in combination with immune checkpoint inhibitors (ICI), but abscopal effects are often precluded by insufficient T cell infiltration of distant, nonirradiated tumors. Additional noncytotoxic, low-dose radiation therapy (LDRT) of distant tumors may enhance the abscopal response, but clinical evidence and preclinical studies for this scenario are lacking. We investigated whether triple treatment consisting of HFRT, ICI, and LDRT could achieve better systemic antitumor response in bilateral mouse tumor models and in patients with stage IV non-small cell lung cancer. Our analyses of bilateral mouse tumor models show that HFRT treatment of the primary tumor combined with LDRT treatment of the abscopal tumor and anti-PD1 therapy enhances the abscopal response compared with HFRT/anti-PD1, HFRT/LDRT, or LDRT/anti-PD1 double treatments; complete cure was observed in more than half of the mice treated with triple therapy. The enhanced abscopal effect was associated with increased infiltration of CD8+ effector T cells and upregulated expression of T cell-attracting chemokines. Of 9 patients with metastatic non-small cell lung cancer who were treated with this triple therapy, 3 and 2 patients showed partial responses and stable disease, respectively. Among 9 relatively large (175.7 ± 42.3 cm3) LDRT lesions, 6 lesions decreased by 28% in size, on average. Our study demonstrates preclinically that LDRT of established metastases significantly enhances the abscopal response to HFRT plus ICI. It also shows that additional LDRT was well tolerated by patients and that this treatment profile is effective and worth further study.

Deep abscopal response to radiotherapy and anti-PD-1 in an oligometastatic melanoma patient with unfavorable pretreatment immune signature

Radiotherapy can elicit abscopal effects in non-irradiated metastases, particularly under immune checkpoint blockade (ICB). We report on two elderly patients with oligometastatic melanoma treated with anti-PD-1 and stereotactic body radiation therapy (SBRT). Before treatment, patient 1 showed strong tumor infiltration with exhausted CD8+ T cells and high expression of T cell-attracting chemokines. This patient rapidly mounted a complete response, now ongoing for more than 4.5 years. Patient 2 exhibited low CD8+ T cell infiltration and high expression of immunosuppressive arginase. After the first SBRT, his non-irradiated metastases did not regress and new metastases occurred although neoepitope-specific and differentiation antigen-specific CD8+ T cells were detected in the blood. A second SBRT after 10 months on anti-PD-1 induced a radiologic complete response correlating with an increase in activated PD-1-expressing CD8 T cells. Apart from a new lung lesion, which was also irradiated, this deep abscopal response lasted for more than 2.5 years. However, thereafter, his disease progressed and the activated PD-1-expressing CD8 T cells dropped. Our data suggest that oligometastatic patients, where a large proportion of the tumor mass can be irradiated, are good candidates to improve ICB responses by RT, even in the case of an unfavorable pretreatment immune signature, after progression on anti-PD-1, and despite advanced age. Besides repeated irradiation, T cell epitope-based immunotherapies (e.g., vaccination) may prolong antitumor responses even in patients with unfavorable pretreatment immune signature.

Immunomodulating Effects Depend on Prolactin Levels in Patients with Hyperprolactinemia.

Prolactin is known to have immune modulatory effects acting through the prolactin receptor, which is present on a variety of immune cells. Certain chemokines contribute to form the type of T helper (Th) preponderance in the immune response. The objective of this work was to assess if hyperprolactinemia not related to pregnancy is associated with changes in circulating levels of chemokines and other immunological markers. In this cross sectional study, 35 patients with hyperprolactinemia (5 men), and 102 healthy blood donors (19 men) were included. Serum levels of Th1- Th2- and Th17-associated chemokines, C-reactive protein, immunoglobulins, and the B cell attracting chemokine CXCL13 were assessed. The hyperprolactinemic group had significantly higher levels of Th2 associated CCL22 (p=0.022), Th17 associated CXCL1 (p=0.001), B cell attracting CXCL13 (p=0.003), and C-reactive protein (p<0.001) compared to controls, and these proteins were also positively correlated with prolactin levels. While differences in CCL22, CXCL1, CXCL13, and C-reactive protein were present in patients with low or moderate hyperprolactinemia, no differences were observed at high (>3600 mU/l) prolactin levels. To evaluate a possible dose-associated response to prolactin, an in vitro model was used, showing prolactin-induced increase in T-helper cell activation at moderate levels, while activation decreased at higher levels. Hyperprolactinemia seems to have several immunomodulatory effects and was associated with increased levels of chemokines associated with Th2 and Th17 responses and B cell attraction. However, patients with greatly increased prolactin had normal levels of chemokines, and in vitro, high levels of prolactin decreased T-helper cell activation.

IL-17 inhibits CXCL9/10-mediated recruitment of CD8+ cytotoxic T cells and regulatory T cells to colorectal tumors

BackgroundThe IL-17 family cytokines are potent drivers of colorectal cancer (CRC) development. We and others have shown that IL-17 mainly signals to tumor cells to promote CRC, but the underlying mechanism remains unclear. IL-17 also dampens Th1-armed anti-tumor immunity, in part by attracting myeloid cells to tumor. Whether IL-17 controls the activity of adaptive immune cells in a more direct manner, however, is unknown.MethodsUsing mouse models of sporadic or inducible colorectal cancers, we ablated IL-17RA in the whole body or specifically in colorectal tumor cells. We also performed adoptive bone marrow reconstitution to knockout CXCR3 in hematopoietic cells. Histological and immunological experimental methods were used to reveal the link among IL-17, chemokine production, and CRC development.ResultsLoss of IL-17 signaling in mouse CRC resulted in marked increase in the recruitment of CD8+ cytotoxic T lymphocytes (CTLs) and regulatory T cells (Tregs), starting from early stage CRC lesions. This is accompanied by the increased expression of anti-inflammatory cytokines IL-10 and TGF-β. IL-17 signaling also inhibits the production of T cell attracting chemokines CXCL9 and CXCL10 by tumor cells. Conversely, the inability of hematopoietic cells to respond to CXCL9/10 resulted in decreased tumor infiltration by CTLs and Tregs, decreased levels of IL-10 and TGF-β, and increased numbers of tumor lesions. Blockade of IL-17 signaling resulted in increased expression of immune checkpoint markers. On the other hand, treatment of mouse CRC with anti-CTLA-4 antibody led to increased expression of pro-tumor IL-17.ConclusionIL-17 signals to colorectal tumor cells and inhibits their production of CXCL9/10 chemokines. By doing so, IL-17 inhibits the infiltration of CD8+ CTLs and Tregs to CRC, thus promoting CRC development. Cancer immunotherapy may be benefited by the use of anti-IL-17 agents as adjuvant therapies, which serve to block both IL-17-mediated tumor promotion and T cell exclusion.

Vδ1+ T Cells: Adoptive Cell Therapy for the Treatment of Haematological Malignancies in Allogeneic Settings

The treatment of haematological malignancies with adoptive cell therapy such as leukaemia is limited to conventional ab T cells transduced at great cost with chimeric antigen receptors (CAR) recognising the B-cell associated antigen CD19. Although greatly successful especially in paediatric cases, this therapy remains with its challenges such as associated toxicities, relapse rate as well as high production costs and production failure rates due to its autologous nature, indicated by the high risk of αβ T cells inducing sever graft versus host disease (GvHD). In contrast to αβ T cells, γδ T cells are not restricted to, or recognise major histocompatibility complex (MHC) peptide complexes. Especially Vδ1 T cells, enriched in human tissues but also found in small numbers in the human blood have been shown to elicit potent anti-tumour responses recognising stress ligands up-regulated on malignant cells, whilst sparing healthy tissue. A study looking at 39 malignancies, solid tumours as well as haematological malignancies, identified the presence of γδ T cells as the single most positive correlative factor for overall survival. Even more striking, expanding γδ T cells and more specifically Vδ1 T cells correlate with strong graft versus leukaemia and reduced incident rates of GvHD after αβ T cell depleted, haploidentical stem cell transplantation. These correlative findings have been confirmed in several retrospective data-sets but more recently in a prospective study looking at multiple haematological indications such as AML, ALL and MM. We have developed a 2½ week, good manufacturing practice (GMP) compliant and scalable (≥ 1x109) process to consistently generate αβ T cell depleted, γδ T cell lymphocyte cultures (93.5% ± 3.7 of live), strongly enriched for Vδ1 T cells (67.9%±2.2% of live). Vδ1 T cells show good recoverability and proliferation in response to cytokines after cryopreservation, kill a variety of malignant leukaemia and myeloma cells at low effector to target ratios (2:1) and show a favourable innate phenotype expressing high levels of the co-stimulatory molecule CD27, CD69 and the natural cytotoxicity receptors NKG2D, DNAM-1 and NKp30. Expression of negative modulators such as PD-1, CTLA4 and TIGIT remain low or non-detectable. Vδ1 T cells produce large levels of IFN-γ, TNF-α but little to no IL-17 on activation. Furthermore, large amounts of the pro-inflammatory chemokines CCL2 and CLL5, as well as the antigen-presenting cell attracting chemokine CCL4 additionally to the colony stimulating factors M-CSF and GM-CSF, support the idea of Vδ1 T cells orchestrating secondary adaptive immune responses after activation. Importantly, and in accordance with their MHC-independent biology, Vδ1 T cells do not kill healthy lymphocytes or primary tissue-derived stromal cells such as fibroblasts. Vδ1 T cells derived from healthy blood and generated under GMP conditions constitute a potential 'off-the-shelf', fully allogeneic cell therapy for the treatment of multiple haematological malignancies. Disclosures Simoes: GammaDelta Therapeutics Ltd.: Employment. Kawalkowska:GammaDelta Therapeutics Ltd.: Employment. Recaldin:GammaDelta Therapeutics Ltd.: Employment. Foxler:GammaDelta Therapeutics Ltd.: Employment. Nussbaumer:GammaDelta Therapeutics Ltd.: Employment, Equity Ownership, Patents &amp; Royalties. Koslowski:GammaDelta Therapeutics Ltd.: Employment, Equity Ownership.

Evaluation of Potential Serum Biomarkers of Disease Activity in Diverse Forms of Vasculitis.

We evaluated potential circulating biomarkers of disease activity in giant cell arteritis (GCA), Takayasu arteritis (TA), polyarteritis nodosa (PAN), and eosinophilic granulomatosis with polyangiitis (EGPA). A panel of 22 serum proteins was tested in patients enrolled in the Vasculitis Clinical Research Consortium Longitudinal Studies of GCA, TA, PAN, or EGPA. Mixed models were used for most analyses. A J48 classification tree method was used to find the most relevant markers to differentiate between active and inactive GCA. Tests were done on 418 samples from 152 patients (60 GCA, 29 TA, 26 PAN, 37 EGPA), during both active vasculitis and remission. In GCA, these showed significant (p < 0.05) differences between disease states: B cell-attracting chemokine 1 (BCA)-1/CXC motif ligand 13 (CXCL13), erythrocyte sedimentation rate (ESR), interferon-γ-induced protein 10/CXC motif chemokine 10, soluble interleukin 2 receptor α (sIL-2Rα), and tissue inhibitor of metalloproteinase-1 (TIMP-1). In EGPA, these showed significant increases during active disease: granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF, interleukin (IL)-6, IL-15, and sIL-2Rα. BCA-1/CXCL13 also showed such increases, but only after adjustment for treatment. In PAN, ESR and matrix metalloprotease (MMP)-3 showed significant differences between disease states. Differences in biomarker levels between diseases were significant for 11 markers and were more striking (all p < 0.01) than differences related to disease activity. A combination of lower values of TIMP-1, IL-6, interferon-γ, and MMP-3 correctly classified 87% of samples with inactive GCA. We identified novel biomarkers of disease activity in GCA and EGPA. Differences of biomarker levels between diseases, independent of disease activity, were more apparent than differences related to disease activity. Further studies are needed to determine whether these serum proteins have potential for clinical use in distinguishing active disease from remission or in predicting longer-term outcomes.

Cytokine Changes in Maternal Peripheral Blood Correlate With Time-to-Delivery in Pregnancies Complicated by Premature Prelabor Rupture of the Membranes.

Premature prelabor rupture of the membranes (PPROM) causes one-third of preterm births worldwide and is most likely caused by subclinical intrauterine infection and/or inflammation. We proposed that women with systemic inflammation at the time of PPROM would have shorter latency. Peripheral blood samples were collected from 20 singleton pregnant women with PPROM between 23 ± 1 and 33 ± 6 weeks. The first sample was drawn within 48 hours of admission, followed by weekly blood draws until delivery. Pregnancies complicated with acute chorioamnionitis, preeclampsia, intrauterine growth restriction, obesity, substance abuse, and chronic maternal disease were excluded. Twenty uncomplicated, gestational age-matched pregnancies served as controls. Plasma concentration of 39 cytokines was measured longitudinally using Luminex assays to investigate their value as predictive biomarkers of latency. Women with PPROM exhibited significantly lower plasma concentration of interferon-γ-inducible protein 10-Chemokine (c-x-c motif; IP10/CXCL10), Chemokine (c-x-c motif) Ligand 9 (MIG/CXCL9), Platelet-derived growth factor BB (PDGFbb), and cutaneous T cell-attracting chemokine, also known as CCL27/CCL27 than controls at admission but significantly elevated interleukin (IL)1RA, tumor necrosis factor α, monocyte chemotactic protein-1/CCL2 at delivery compared to admission. Women with PPROM who delivered within 7 days had significantly lower plasma concentration of anti-inflammatory cytokine IL1RA than those with latency periods >7 days. The IL1RA and endotoxin activity in conjunction with clinical parameters results in excellent prediction of latency to delivery (area under the receiver-operating characteristic curve = 0.91). We concluded that higher levels of anti-inflammatory cytokines in women with PPROM were associated with increased latency until delivery, likely due to counterbalancing of proinflammatory load. When used in conjunction with other predictive characteristics of time until delivery, cytokines may further assist clinical decision-making and optimize pregnancy outcomes in women with PPROM.