The contribution of discoidin domain receptor 1 to the pathogenesis of diffuse large B cell lymphoma

Collagen is the ligand for the discoidin domain receptor-1 (DDR1), a receptor tyrosine kinase that is over-expressed in Hodgkin lymphoma. However, the role of DDR1 in diffuse large B cell lymphoma (DLBCL) is not known. I showed that DDR1 is over-expressed in a subset of DLBCL where it positively correlates with expression of its collagen ligands, and negatively correlates with expression of mitotic spindle genes. DDR1 correlated genes also overlapped with three aneuploidy signatures and DDR1 expression correlated significantly with autosomal aneuploidy index. RNAseq analysis revealed that over-expression of DDR1 in primary germinal centre B cells down-regulated expression of CENPE, an essential component of the mitotic spindle checkpoint that when inactivated leads to chromosome mis-segregation and aneuploidy. CENPE expression was also significantly reduced in primary DLBCL. Moreover, I showed that the constitutive activation of DDR1 in an in vitro lymphoma model led to aneuploidy. Finally, I showed that DDR1 can be inhibited by three small molecules and established the basis for in vivo model to test these inhibitors in DLBCL xenograft. My data provide evidence that DDR1 can induce aneuploidy in B cells, and as such identify a mechanism to potentially explain the link between chronic inflammation and lymphomagenesis

CD70 identifies alloreactive T cells and represents a potential target for prevention and treatment of acute GvHD

Graft-versus-host disease (GvHD) remains a major challenge following allogeneic hematopoietic stem cell transplantation (allo-HSCT) and further understanding of its immunopathology is crucial for developing new treatments. CD70 interacts with CD27 and is upregulated transiently on T cells following recent TCR engagement. Here we investigated the functional and clinical significance of CD70 expression on T cells during the early post-transplantation period. CD70 was expressed on a subset of highly activated memory T cells within the first 2 weeks post-transplant which then gradually declined in most patients. CD70+ T cells exhibited an open chromatin landscape and a transcriptional profile indicative of intense MYC-driven glycolysis and proliferation. CD4+ and CD8+ CD70+ T cell number increased by 9-fold and 4-fold respectively during acute GvHD (aGvHD) and displayed an oligoclonal TCR repertoire. These cells expressed CCR4 and CCR6 chemokine receptors and were markedly increased in aGvHD tissue samples. Furthermore, CD70+ T cells demonstrated alloreactive specificity in vitro and proliferative and inflammatory cytokine responses were markedly attenuated by CD70 blockade. These findings identify CD70 as a marker of highly activated alloreactive T cells and reveal the potential therapeutic importance of inhibiting CD27-CD70 co-stimulation in both the prophylaxis and treatment of aGvHD

Interplay in galectin expression predicts patient outcomes in a spatially restricted manner in PDAC

BACKGROUND: Galectins (Gal's) are a family of carbohydrate-binding proteins that are known to support the tumour microenvironment through their immunosuppressive activity and ability to promote metastasis. As such they are attractive therapeutic targets, but little is known about the cellular expression pattern of galectins within the tumour and its neighbouring stromal microenvironment. Here we investigated the cellular expression pattern of Gals within pancreatic ductal adenocarcinoma (PDAC).METHODS: Galectin gene and protein expression were analysed by scRNAseq (n=4) and immunofluorescence imaging (n=19) in fibroblasts and epithelial cells of pancreatic biopsies from PDAC patients. Galectin surface expression was also assessed on tumour adjacent normal fibroblasts and cancer associated primary fibroblasts from PDAC biopsies using flow cytometry.RESULTS: scRNAseq revealed higher Gal-1 expression in fibroblasts and higher Gal-3 and -4 expression in epithelial cells. Both podoplanin (PDPN+, stromal/fibroblast) cells and EpCAM+ epithelial cells expressed Gal-1 protein, with highest expression seen in the stromal compartment. By contrast, significantly more Gal-3 and -4 protein was expressed in ductal cells expressing either EpCAM or PDPN, when compared to the stroma. Ductal Gal-4 cellular expression negatively correlated with ductal Gal-1, but not Gal-3 expression. Higher ductal cellular expression of Gal-1 correlated with smaller tumour size and better patient survival. CONCLUSIONS: In summary, the intricate interplay and cell-specific expression patterns of galectins within the PDAC tissue, particularly the inverse correlation between Gal-1 and Gal-4 in ducts and its significant association with patient survival, highlights the complex molecular landscape underlying PDAC and provides valuable insights for future therapeutic interventions.</p

Robust SARS-CoV-2-specific and heterologous immune responses in vaccine-naïve residents of long-term care facilities who survive natural infection

We studied humoral and cellular immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 152 long-term care facility staff and 124 residents over a prospective 4-month period shortly after the first wave of infection in England. We show that residents of long-term care facilities developed high and stable levels of antibodies against spike protein and receptor-binding domain. Nucleocapsid-specific responses were also elevated but waned over time. Antibodies showed stable and equivalent levels of functional inhibition against spike-angiotensin-converting enzyme 2 binding in all age groups with comparable activity against viral variants of concern. SARS-CoV-2 seropositive donors showed high levels of antibodies to other beta-coronaviruses but serostatus did not impact humoral immunity to influenza or other respiratory syncytial viruses. SARS-CoV-2-specific cellular responses were similar across all ages but virus-specific populations showed elevated levels of activation in older donors. Thus, survivors of SARS-CoV-2 infection show a robust and stable immunity against the virus that does not negatively impact responses to other seasonal viruses

The role of the CXCR3 chemokine receptor in the innate immunity of common carp.

U karpia znanych jest obecnie 6 różnych chemokin z rodziny CXC. Dwie z nich zaliczane do grupy chemokin podobnych do ssaczej chemokiny CXCL8 (ang. CXCL8-like) to: CXCL8_L1, znana też jako CXCa, i CXCL8_L2. Kolejne dwie należą do grupy chemokin CXCb, indukowanych przez INF-γ (CXCb1 i CXCb2), natomiast pozostałe to CXC12 i CXC14. U karpia zsekwencjonowano także 4 receptory dla chemokin CXC: CXC1-4. Dobre poznanie zależności w oddziaływaniu między ligandem i receptorem dla chemokin CXC u ludzi, jak również wcześniejsze wyniki badań nad ekspresją genów receptora dla tej rodziny chemokin, sugerują, że chemokiny ryb należące do grupy CXCL8 mogą być potencjalnymi ligandami dla receptorów CXCR1 i CXCR2. Tym samym są zaangażowane w migrację neutrofili do miejsca zapalenia. Natomiast ligand dla receptora CXCR3 pozostaje u ryb w dalszym ciągu nieznany. W obecnej pracy badano rolę receptora CXCR3 w regulacji odporności wrodzonej karpia (Cyprinus carpio L.). W pracy mierzono profil ekspresji genów receptora CXCR3 w różnych tkankach/narządach oraz w populacjach leukocytów i komórek zaangażowanych w odporność, jak również sprawdzano, która chemokina z rodziny CXC jest u tego gatunku ligandem dla receptora CXCR3.Stwierdzono, że ekspresja genu receptora CXCR3 występuje we wszystkich przebadanych pod tym kątem tkankach/narządach, jednak najwyższą ekspresję mRNA dla tego receptora zanotowano w jelicie, skrzelach i nerce głowowej. Gen CXCR3 wykazywał także ekspresję w leukocytach, trombocytach, komórkach śródbłonka, przy czym najwyższą ekspresję genu CXCR3 zaobserwowano w dojrzałych makrofagach. Jednocześnie stwierdzono, że stymulacja komórek in vitro za pomocą lipopolisacharydu (LPS) i/lub interferonu gamma (INF-γ), nie ma wpływu na ekspresję genów receptora CXCR3. Z kolei, badania in vivo wykazały podniesioną ekspresję genu receptora CXCR3 na leukocytach w 96 godzinie odczynu zapalnego jamy otrzewnej wywołanego podaniem zymosanu, a więc w okresie nasilonego napływu do ogniska zapalnego makrofagów odpowiedzialnych za wyciszenie zapalenia.W celu wykazania, która z chemokin CXC (CXCL8, CXCa i CXCb1) jest ligandem receptora CXCR3 wykorzystano linie komórkowe: HEK T293 (ang. Human Embryonic Kidney Cells) i CLC (ang. Carp Leukocyte Cell Line) transfekowane receptorem CXCR3 karpia. W eksperymentach użyto 3 plazmidy z receptorem CXCR3: CXCR3pcDNA3 z białkiem znacznikowym m.Cherry, CXCR3pcDNA3 pozbawiony m.Cherry oraz CXCR3pBI-CMV1 z m.Cherry. Dodatkowo komórki transfekowano również plazmidem GECO (ang. green intensiometric genetically encoded Ca+ indicator), umożliwiającym obserwację zmian wewnątrzkomórkowego poziomu wapnia. Receptor CXCR3 należy bowiem do rodziny receptorów związanych z białkiem G, których aktywacja prowadzi do zmian poziomu wapnia wewnątrz komórki. Badania na komórkach HEK T293 nie dały wiarygodnych wyników. Z kolei, stymulacja transfekowanych komórek CLC sugeruje, że chemokina CXCb1, może być ligandem dla receptora CXCR3 u karpia. Podsumowując otrzymane wyniki, można stwierdzić, że występujący na dojrzałych makrofagach receptor CXCR3 zaangażowany jest w napływ tych komórek do miejsca zapalenia w późnej fazie tego procesu. Ponadto wydaje się, że istnieje zależność między chemokiną CXCb1, a receptorem CXCR3 u karpia. Jednocześnie komórki linii CLC stanowią lepszy model do badań nad ligandami dla receptorów u ryb, niż komórki HEK T293. Co więcej stwierdzono, że wykorzystanie GECO do badania zmian w wewnątrzkomórkowym poziomie wapnia, jest znacznie skuteczniejsze, niż używana dotychczas metoda z wykorzystaniem barwnika fluorescencyjnego Fluo-4.In common carp 6 different CXC chemokines has been discovered: two from CXCL8-like lineages: CXCL8_L1 (lineage 1, previous name CXCa) and CXCL8_L2 (lineage 2), two IFNγ-inducible CXCb genes (CXCb1 and 2) but also CXC12 and CXC14. Moreover, 4 CXC chemokine receptors: CXCR1-4 has been cloned in carp. Human CXC ligand-receptor relationships and previous gene expression data suggested that, also in fish CXCL8-like chemokines are potential ligands for CXCR1 and CXCR2 and are involved in directing neutrophil migration towards site of inflammation, while ligand for fish CXCR3 is still unknown. By this work we were investigated the role of CXCR3 chemokine receptor in regulation of carp immune response. We measured profile of CXCR3 gene expression in different tissues/organs and leukocytes and other cells involved in immune response. Moreover, we tested , which carp CXC chemokine is a ligand for CXCR3 receptor. We discovered that the expression of CXCR3 receptor appeared on every studied tissues/organs, whereas the highest one, was noticed on gut, gills and head kidney. It was also found that the expression of CXCR3 receptor appear on a different kind of the cells such as: leukocytes, trombocytes, epithelial cells or mature macrophages, where expression of CXCR3 receptor is the highest. At the same time we noticed that, in vitro cell stimulation with LPS and/or INF-γ do not influence significantly expression of CXCR3 gene .Interestingly, CXCR3 gene expression was significantly up-regulated at 96 h of zymosan-induced peritonitis, when high influx of mature macrophages, involved in inflammation resolution, appeared. To verified which CXC chemokine is CXCR3 ligand two different cell lines: human embryonic kidney HEK T293 cells and carp leukocyte CLC cell line were transfected with carp CXCR3. Moreover, both cell lines were co-transfected with GECO plasmid - green intensiometric genetically encoded Ca+ indicator – read out system for changes in intracellular calcium level. CXCR3 belongs to the G protein coupled receptors family, which activation leads changes in intracellular calcium level. To our experiments three plasmids with CXCR3 were prepared: CXCR3pcDNA3 with m.Cherry as the fluorescent tag, CXCR3pcDNA3 without m.Cherry and CXCR3pBI-CMV1 with m.Cherry. HEK 293 cells transfected with CXCR3 did not bring any significant results after chemokines stimulation. Although, our results indicated that some CLCs transfected with CXCR3 show the increase of intracellular calcium level in respond to CXC chemokines, however to confirm this observation some more experiments should be performed.Our results suggest that CXCR3 expressed on mature macrophages is involved in their migration in the late/resolution phase of inflammation. Moreover, most probably CXCb1 chemokine can be in carp the ligand for CXCR3 receptor. CLC seems to be better cell line to perform experiments on fish receptors, then HEK T293. We can also say that by using GECO, in comparison to Fluo-4, we obtain better and much more stable results

Inflammation vs depression

Depression is severe psychiatric disorder, which belongs to the group of the affective disorders. It can be characterized by several behavioral symptoms, e.g. sadness, sleep and appetite disorders and concentration problems. Depression is one of the most common complications in patients with chronic somatic illnesses e.g. with cardiological and genitor-urinary diseases. Disturbances of the biogenic amine metabolism (e.g. serotonin) are considered as main depression reasons. They can be explain by increased production of proinflammatory cytokines: IL-6, IL-1β, IFN-γ i TNF, and as a consequence stress axis activation and imbalance of neurotransmitter production. It has been shown in experimental animals that injection of proinflammatory cytokines or immunostimulants (e.g. lypopolisacharide) induces depression-like symptoms. Moreover, in patients treated with IL-2 and IFN-a, depression symptoms have been observed, and they disappeared when therapy was finished. Circulating cytokines can enter the brain in the region where brain-blood barrier is leaky, via vogues nerve or after cytokine binding to their receptors localized on brain-blood barrier endothelium. Also Alzheimer and Parkinson syndroms as well as tumors are linked now with chronic inflammations. Detailed study of cytokine function in those disorder can help to find new effective therapy.Depresja jest ciężką chorobą psychiczną należąca do grupy chorób afektywnych. Charakteryzuje ją szereg zmian behawioralnych takich jak smutek, zaburzenia snu i apetytu czy problemy z koncentracją. Depresja jest częstą komplikacją występującą u pacjentów z chronicznymi chorobami somatycznymi dotyczącym np. układu sercowo-naczyniowego, czy moczowo-płciowego. Podstawową przyczyną powstawania depresji są zaburzenia w metabolizmie amin biogennych w tym serotoniny. Obecnie sądzi się, że powodem tych zaburzeń może być między innymi wzmożona produkcja cytokin prozapalnych takich jak: IL-6, IL-1β, IFN-γ i TNF, w wyniku czego dochodzi do aktywacji osi stresu i zaburzenia równowagi w produkcji neurotransmiterów w mózgu. Udowodniono, że podanie zwierzętom doświadczalnym cytokin prozapalnych lub czynników wywołujących stan zapalny (np. lipopolisacharydu) wywołuje objawy podobne do depresji. Również u osób poddanych dłuższemu leczeniu z użyciem IL-2 i IFN-a, zaobserwowano objawy depresji, które ustępowały po zaprzestaniu terapii. Uważa się, że produkowane obwodowo cytokiny mogą docierać do mózgu poprzez narządy okołokomorowe, gdzie bariera krew-mózg jest słabsza, za pośrednictwem nerwu błędnego lub poprzez przyłączenie się cytokin do odpowiednich receptorów znajdujących się na komórkach śródbłonka bariery krew-mózg. Poza depresją istnieje szereg innych chorób będących konsekwencją chronicznego stanu zapalnego, do których zaliczamy między innymi chorobę Alzheimera, Parkinsona, oraz nowotwory. Poznanie dokładnej roli cytokin w ich wywoływaniu, daje nadzieję na znalezienie skutecznej terapii

NK cells in pancreatic cancer demonstrate impaired cytotoxicity and a regulatory IL-10 phenotype.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common tumor subtypes and remains associated with very poor survival. T cell infiltration into tumor tissue is associated with improved clinical outcome but little is known regarding the potential role of NK cells in disease control. Here we analyze the phenotype and function of NK cells in the blood and tumor tissue from patients with PDAC. Peripheral NK cells are present in normal numbers but display a CD16CD57 phenotype with marked downregulation of NKG2D. Importantly, these cells demonstrate reduced cytotoxic activity and low levels of IFN-γ expression but instead produce high levels of intracellular IL-10, an immunoregulatory cytokine found at increased levels in the blood of PDAC patients. In contrast, NK cells are largely excluded from tumor tissue where they display strong downregulation of both CD16 and CD57, a phenotype that was recapitulated in primary NK cells following co-culture with PDAC organoids. Moreover, expression of activatory proteins, including DNAM-1 and NKP30, was markedly suppressed and the DNAM-1 ligand PVR was strongly expressed on tumor cells. As such, and peripheral NK cells display differential features in patients with PDAC and indicate local and systemic mechanisms by which the tumor can evade immune control. These findings offer a number of potential options for NK-based immunotherapy in the management of patients with PDAC

Spatial determination and prognostic impact of the fibroblast transcriptome in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma has a poor clinical outcome and responses to immunotherapy are suboptimal. Stromal fibroblasts are a dominant but heterogenous population within the tumor microenvironment and therapeutic targeting of stromal subsets may have therapeutic utility. Here, we combine spatial transcriptomics and scRNA-Seq datasets to define the transcriptome of tumor-proximal and tumor-distal cancer-associated fibroblasts (CAFs) and link this to clinical outcome. Tumor-proximal fibroblasts comprise large populations of myofibroblasts, strongly expressed podoplanin, and were enriched for Wnt ligand signaling. In contrast, inflammatory CAFs were dominant within tumor-distal subsets and expressed complement components and the Wnt-inhibitor SFRP2. Poor clinical outcome was correlated with elevated HIF-1α and podoplanin expression whilst expression of inflammatory and complement genes was predictive of extended survival. These findings demonstrate the extreme transcriptional heterogeneity of CAFs and its determination by apposition to tumor. Selective targeting of tumor-proximal subsets, potentially combined with HIF-1α inhibition and immune stimulation, may offer a multi-modal therapeutic approach for this disease