The γδTCR combines innate immunity with adaptive immunity by utilizing spatially distinct regions for agonist selection and antigen responsiveness.

T lymphocytes expressing γδ T cell antigen receptors (TCRs) comprise evolutionarily conserved cells with paradoxical features. On the one hand, clonally expanded γδ T cells with unique specificities typify adaptive immunity. Conversely, large compartments of γδTCR+ intraepithelial lymphocytes (γδ IELs) exhibit limited TCR diversity and effect rapid, innate-like tissue surveillance. The development of several γδ IEL compartments depends on epithelial expression of genes encoding butyrophilin-like (Btnl (mouse) or BTNL (human)) members of the B7 superfamily of T cell co-stimulators. Here we found that responsiveness to Btnl or BTNL proteins was mediated by germline-encoded motifs within the cognate TCR variable γ-chains (Vγ chains) of mouse and human γδ IELs. This was in contrast to diverse antigen recognition by clonally restricted complementarity-determining regions CDR1-CDR3 of the same γδTCRs. Hence, the γδTCR intrinsically combines innate immunity and adaptive immunity by using spatially distinct regions to discriminate non-clonal agonist-selecting elements from clone-specific ligands. The broader implications for antigen-receptor biology are considered.

Empowering γδ T-cell functionality with vitamin C.

Vitamin C (ascorbic acid) is a potent antioxidant and a cofactor for various enzymes including histone demethylases and methylcytosine dioxygenases. Vitamin C also exerts direct cytotoxicity toward selected tumor cells including colorectal carcinoma. Moreover, vitamin C has been shown to impact immune cell differentiation at various levels including maturation and/or functionality of T cells and their progenitors, dendritic cells, B cells, and NK cells. γδ T cells have recently attracted great interest as effector cells for cell-based cancer immunotherapy, due to their HLA-independent recognition of a large variety of tumor cells. While γδ T cells can thus be also applied as an allogeneic off-the-shelf product, it is obvious that the effector function of γδ T cells needs to be optimized to ensure the best possible clinical efficacy. Here we review the immunomodulatory mechanisms of vitamin C with a special focus on how vitamin C enhances the effector function of γδ T cells. We also discuss future directions of how vitamin C can be used in the clinical setting to boost the efficacy of adoptive cell therapies.

Tumor resistance mechanisms and their consequences on γδ T cell activation.

Human γδ T lymphocytes are predominated by two major subsets, defined by the variable domain of the δ chain. Both, Vδ1 and Vδ2 T cells infiltrate in tumors and have been implicated in cancer immunosurveillance. Since the localization and distribution of tumor-infiltrating γδ T cell subsets and their impact on survival of cancer patients are not completely defined, this review summarizes the current knowledge about this issue. Different intrinsic tumor resistance mechanisms and immunosuppressive molecules of immune cells in the tumor microenvironment have been reported to negatively influence functional properties of γδ T cell subsets. Here, we focus on selected tumor resistance mechanisms including overexpression of cyclooxygenase (COX)-2 and indolamine-2,3-dioxygenase (IDO)-1/2, regulation by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/TRAIL-R4 pathway and the release of galectins. These inhibitory mechanisms play important roles in the cross-talk of γδ T cell subsets and tumor cells, thereby influencing cytotoxicity or proliferation of γδ T cells and limiting a successful γδ T cell-based immunotherapy. Possible future directions of a combined therapy of adoptively transferred γδ T cells together with γδ-targeting bispecific T cell engagers and COX-2 or IDO-1/2 inhibitors or targeting sialoglycan-Siglec pathways will be discussed and considered as attractive therapeutic options to overcome the immunosuppressive tumor microenvironment.

Peripheral T Cell Populations are Differentially Affected in Familial Mediterranean Fever, Chronic Granulomatous Disease, and Gout.

Both innate errors of immunity, such as familial Mediterranean fever (FMF) and chronic granulomatous disease (CGD), and the common inflammatory disease gout are characterized by episodes of sterile inflammatory attacks in the absence of an infection. While these disorders encompass distinct pathologies due to differentially affected metabolic pathways and inflammasome activation mechanisms, their common features are the excessive production of interleukin (IL)-1ß and innate immune cell hyperreactivity. On the other hand, the role of T cells and innate-like lymphocytes such as gamma delta (γδ) T cells in these pathologies is ill-defined. In order to widen our understanding of T cell involvement in CGD, FMF and gout pathology, we developed multicolour immunophenotyping panels for flow cytometry to characterize γδ T cells as well as CD4 and CD8 T cell populations in terms of their cytokine production, activation status, memory or naive phenotypes, exhaustion status, homing receptor expression, and cytotoxic activity. Our study is the first deep immunophenotyping analysis of T cell populations in CGD, FMF, and gout patients. We found that CGD affects the frequencies and activation status of T cells, while gout impairs the cytokine production capacity of Vδ2 T cells. FMF was characterized by decreased percentages of regulatory T cells in circulation and attenuated IFN-γ production capacity by Vδ2 T cells. Autoinflammatory syndromes and congenital defects of phagocyte differentially affect T cell compartments. Future studies are warranted to assess whether these phenotypical changes are relevant for disease pathology.

Frequency of B-Cell Subpopulations in Low Responders in Comparison with High Responders to Hepatitis B Vaccine Among Health Care Workers.

Vaccination is the most effective way to prevent Hepatitis B (HB) infection. The goal of vaccination is to induce immunological memory. Hence, determining the frequency of memory B-cell (MBC) subsets is an important indicator of vaccine efficacy. This study aimed to evaluate the frequency of different B-cell subpopulations and the expression of PD-1 on B-cell subsets in low responders (LR) and high responders (HR) to HB vaccine. According to our findings, the expression level of PD-1 was significantly higher on atypical MBC (atMBC) than that of naive B cell and classical MBC (cMBC) in LR and HR groups. Moreover, cMBCs had a significant higher PD-1 expression than naive B cells in LR group. No significant differences were found in the frequency of various B-cell subpopulations and the expression level of PD-1 on B-cell subsets between LR and HR groups. We observed a negative correlation between age and HBsAb titer and a positive correlation between age and PD-1 expression level on cMBC in LR group. It can be concluded that inadequate specific memory B-cell response, rather than total memory B-cell deficiency, may be implicated in low responsive rate to HB vaccine in healthy individuals.

Tumor cell lysis and synergistically enhanced antibody-dependent cell-mediated cytotoxicity by NKG2D engagement with a bispecific immunoligand targeting the HER2 antigen.

Natural killer group 2 member D (NKG2D) plays an important role in the regulation of natural killer (NK) cell cytotoxicity in cancer immune surveillance. With the aim of redirecting NK cell cytotoxicity against tumors, the NKG2D ligand UL-16 binding protein 2 (ULBP2) was fused to a single-chain fragment variable (scFv) targeting the human epidermal growth factor receptor 2 (HER2). The resulting bispecific immunoligand ULBP2:HER2-scFv triggered NK cell-mediated killing of HER2-positive breast cancer cells in an antigen-dependent manner and required concomitant interaction with NKG2D and HER2 as revealed in antigen blocking experiments. The immunoligand induced tumor cell lysis dose-dependently and was effective at nanomolar concentrations. Of note, ULBP2:HER2-scFv sensitized tumor cells for antibody-dependent cell-mediated cytotoxicity (ADCC). In particular, the immunoligand enhanced ADCC by cetuximab, a therapeutic antibody targeting the epidermal growth factor receptor (EGFR) synergistically. No significant improvements were obtained by combining cetuximab and anti-HER2 antibody trastuzumab. In conclusion, dual-dual targeting by combining IgG1 antibodies with antibody constructs targeting another tumor associated antigen and engaging NKG2D as a second NK cell trigger molecule may be promising. Thus, the immunoligand ULBP2:HER2-scFv may represent an attractive biological molecule to promote NK cell cytotoxicity against tumors and to boost ADCC.

Immune surveillance in glioblastoma: Role of the NKG2D system and novel cell-based therapeutic approaches.

Glioblastoma, formerly known as glioblastoma multiforme (GBM), is the most frequent and most aggressive brain tumour in adults. The brain is an immunopriviledged organ, and the blood-brain barrier shields the brain from immune surveillance. In this review, we discuss the composition of the immunosuppressive tumour micromilieu and potential immune escape mechanisms in GBM. In this respect, we focus on the role of the NKG2D receptor/ligand system. NKG2D ligands are frequently expressed on GBM tumour cells and can activate NKG2D-expressing killer cells including NK cells and γδ T cells. Soluble NKG2D ligands, however, contribute to tumour escape from immunological attack. We also discuss the current immunotherapeutic strategies to improve the survival of GBM patients. Such approaches include the modulation of the NKG2D receptor/ligand system, the application of checkpoint inhibitors, the adoptive transfer of ex vivo expanded and/or modified immune cells or the application of antibodies and antibody constructs to target cytotoxic effector cells in vivo. In view of the multitude of pursued strategies, there is hope for improved overall survival of GBM patients in the future.

Accelerated co-cultured dendritic cell (acDC) loaded with autologous apoptotic bodies might be a promising approach for antigen delivery.

Antigens derived from engulfed apoptotic bodies that are presented by dendritic cells can amplify Ag-specific T-cells. Accelerated co-cultured DC (acDC) strategy keeps lymphocytes in contact with differentiating DCs. Therefore, Ag-specific T-cell activation can occur during DC maturation. Our aim was to prepare DCs by acDC method and check the subsequent engulfment of the apoptotic body by acDC. We have proposed that this method could be feasible if we transfect the apoptotic bodies with the antigen. DCs were prepared using acDC method and their maturation markers were confirmed by flow cytometry. Ultraviolet was used for inducing apoptosis in the PBMCs and induction of apoptosis checked by propidium iodide and 7-aminoactinomycin D staining. Flow cytometry and immunohistochemistry were used for checking the uptake of apoptotic bodies by the DCs. The alloreactivity against apoptotic bodies was examined by enzyme-linked immunospot (ELISPOT) assay. Results showed that 40.4% of DCs could efficiently engulf the apoptotic bodies. The results indicated that acDC method is capable to isolate a high yield of DCs, and these cells could properly engulf the apoptotic bodies, more works should be performed to use this method for Ag discovery through delivering the Ag by apoptotic bodies into the DCs.

Correlation between IL-28 polymorphism and spontaneous clearance in HCV patients: systematic review and meta-analysis.

Hepatitis C virus (HCV) is a serious global health issue. Nearly 20% of HCV patients spontaneously clear the virus. While some studies have shown an association of spontaneous clearance (SC) of the virus with interleukin (IL) 28B single-nucleotide polymorphisms (SNPs), others did not show such a relationship. Thus, the purpose of the present study was to investigate the association of IL28B polymorphisms (12979860 SNP) with SC of HCV infection. Upon initial screening of the databases, a total of 545 articles were retrieved, of which 22 studies that met predefined eligibility criteria were entered into the meta-analysis. Odds ratios (ORs) with confidence intervals (95% CI), heterogeneity, publication bias, and sensitivity analysis were assessed. According to the meta-analysis results, a significant association was observed between the rs12979860 SNP and SC of HCV infection. The results indicated that the ORs of SC from hepatitis C virus infection were 2.75 times higher in those with cytokine gene polymorphisms (95% CI, 2.23 to 3.38). Moreover, it was found that the prevalence of rs12979860 CC was 0.33 with 95 CI 0.28-0.38 in genotype 1 and was 0.40 with 95 CI 0.34-0.47 in other genotypes. Our meta-analysis results suggest that IL28B rs12979860 CC is a strong predictor for SC of hepatitis C infection in PEG IFN-a/RBV-treated patients.

Degranulation of human cytotoxic lymphocytes is a major source of proteolytically active soluble CD26/DPP4.

Dipeptidyl peptidase 4 (DPP4, CD26) is a serine protease detected on several immune cells and on epithelial cells of various organs. Besides the membrane-bound enzyme, a catalytically active soluble form (sCD26/DPP4) is detected in several body fluids. Both variants cleave off dipeptides from the N-termini of various chemokines, neuropeptides, and hormones. CD26/DPP4 plays a fundamental role in the regulation of blood glucose levels by inactivating insulinotropic incretins and CD26/DPP4 inhibitors are thus routinely used in diabetes mellitus type 2 therapy to improve glucose tolerance. Such inhibitors might also prevent the CD26/DPP4-mediated inactivation of the T-cell chemoattractant CXCL10 released by certain tumors and thus improve anti-tumor immunity and immunotherapy. Despite its implication in the regulation of many (patho-)physiological processes and its consideration as a biomarker and therapeutic target, the cellular source of sCD26/DPP4 remains highly debated and mechanisms of its release are so far unknown. In line with recent reports that activated T lymphocytes could be a major source of sCD26/DPP4, we now demonstrate that CD26/DPP4 is stored in secretory granules of several major human cytotoxic lymphocyte populations and co-localizes with effector proteins such as granzymes, perforin, and granulysin. Upon stimulation, vesicular CD26/DPP4 is rapidly translocated to the cell surface in a Ca2+-dependent manner. Importantly, activation-induced degranulation leads to a massive release of proteolytically active sCD26/DPP4. Since activated effector lymphocytes serve as a major source of sCD26/DPP4, these results might explain the observed disease-associated alterations of sCD26/DPP4 serum levels and also indicate a so far unknown role of CD26/DPP4 in lymphocyte-mediated cytotoxicity.

Early HIV infection is associated with reduced proportions of gamma delta T subsets as well as high creatinine and urea levels.

Renal dysfunctions are major predictors of co-morbidities and mortality in HIV-infected individuals. Unconventional T cells have been shown to regulate kidney functions. However, there is dearth of information on the effect of HIV-associated nephropathies on γδ and DN T cells. It is also not clear whether γδ T cell perturbations observed during the early stages of HIV infection occur before immune activation. In this study, we investigated the relationship between creatinine and urea on the number of unconventional T cells in HIV-infected individuals at the early and chronic stages of infection. Persons in the chronic stage of infection were divided into treatment naïve and exposed groups. Treatment exposed individuals were further subdivided into groups with undetectable and detectable HIV-1RNA in their blood. Creatinine and urea levels were significantly higher among persons in the early HIV infection compared with the other groups. Proportions of γδ T, γδ + CD8, γδ + CD16 cells were also significantly reduced in the early stage of HIV infection (P < .01). Markers of immune activation, CD4 + HLA-DR and CD8 + HLA-DR, were also significantly reduced during early HIV infection (P < .01). Taken together, our findings suggest that high levels of renal markers as well as reduced proportions of gamma delta T cells are associated with the early stages of HIV infection. This event likely occurs before systemic immune activation reaches peak levels. This study provides evidence for the need for early HIV infection diagnosis and treatment.

The Ambiguous Role of γδ T Lymphocytes in Antitumor Immunity.

γδ T cells play a role in immune surveillance because they recognize stress-induced surface molecules and metabolic intermediates that are frequently dysregulated in transformed cells. Hence, γδ T cells have attracted much interest as effector cells in cell-based immunotherapy. Recently, however, it has been realized that γδ T cells can also promote tumorigenesis through various mechanisms including regulatory activity and IL-17 production. In this review we outline both the pathways involved in cancer cell recognition and killing by γδ T cells as well as current evidence for their protumorigenic activity in various models. Finally, we discuss strategies to improve the tumor reactivity of γδ T cells and to counteract their protumorigenic activities, which should open improved perspectives for their clinical application.

Pyrin and Hematopoietic Interferon-Inducible Nuclear Protein Domain Proteins: Innate Immune Sensors for Cytosolic and Nuclear DNA.

The innate immune system is the first line of defense against microbial pathogens. The response of innate immunity is initiated by molecules known as pattern recognition receptors (PRRs). Such responses are often triggered by nucleic acids that are delivered to the cytoplasm or nucleus of cells. The ability to recognize foreign nucleic acids in these two locations is an important defense mechanism of the human innate immune system. Several PRRs are located in the cytosol or nucleus and detect foreign DNAs. The pyrin and hematopoietic interferon-inducible nuclear (PYHIN) domain protein is a family of PRRs that includes interferon-inducible protein 16, absent in melanoma 2, PYHIN 1 (or interferon-inducible protein X, as it is also known), myeloid cell nuclear differentiation antigen, and pyrin domain only protein 3. These nuclear and cytosolic sensors play an essential part in host defense of intracellular pathogens. In addition, members of the PYHIN family are critical regulators of immune response, apoptosis, cell growth, differentiation, and transcription. In this review, we summarize important characteristics of these innate immune sensors and their roles in several diseases. A better understanding of the role of DNA sensors in the nucleus and cytoplasm will lead to the development of novel therapeutic approaches to control infections and associated diseases.

Predicting Humoral Alloimmunity from Differences in Donor and Recipient HLA Surface Electrostatic Potential.

In transplantation, development of humoral alloimmunity against donor HLA is a major cause of organ transplant failure, but our ability to assess the immunological risk associated with a potential donor-recipient HLA combination is limited. We hypothesized that the capacity of donor HLA to induce a specific alloantibody response depends on their structural and physicochemical dissimilarity compared with recipient HLA. To test this hypothesis, we first developed a novel computational scoring system that enables quantitative assessment of surface electrostatic potential differences between donor and recipient HLA molecules at the tertiary structure level [three-dimensional electrostatic mismatch score (EMS-3D)]. We then examined humoral alloimmune responses in healthy females subjected to a standardized injection of donor lymphocytes from their male partner. This analysis showed a strong association between the EMS-3D of donor HLA and donor-specific alloantibody development; this relationship was strongest for HLA-DQ alloantigens. In the clinical transplantation setting, the immunogenic potential of HLA-DRB1 and -DQ mismatches expressed on donor kidneys, as assessed by their EMS-3D, was an independent predictor of development of donor-specific alloantibody after graft failure. Collectively, these findings demonstrate the translational potential of our approach to improve immunological risk assessment and to decrease the burden of humoral alloimmunity in organ transplantation.

Vedolizumab is associated with changes in innate rather than adaptive immunity in patients with inflammatory bowel disease.

OBJECTIVE: Vedolizumab, a monoclonal antibody directed against the integrin heterodimer α4ß7, is approved for the treatment of Crohn's disease and ulcerative colitis. The efficacy of vedolizumab has been suggested to result from inhibition of intestinal T cell trafficking although human data to support this conclusion are scarce. We therefore performed a comprehensive analysis of vedolizumab-induced alterations in mucosal and systemic immunity in patients with inflammatory bowel disease (IBD), using anti-inflammatory therapy with the TNFα antibody infliximab as control. DESIGN: Immunophenotyping, immunohistochemistry, T cell receptor profiling and RNA sequencing were performed using blood and colonic biopsies from patients with IBD before and during treatment with vedolizumab (n=18) or, as control, the anti-TNFα antibody infliximab (n=20). Leucocyte trafficking in vivo was assessed using single photon emission computed tomography and endomicroscopy. RESULTS: Vedolizumab was not associated with alterations in the abundance or phenotype of lamina propria T cells and did not affect the mucosal T cell repertoire or leucocyte trafficking in vivo. Surprisingly, however, α4ß7 antibody treatment was associated with substantial effects on innate immunity including changes in macrophage populations and pronounced alterations in the expression of molecules involved in microbial sensing, chemoattraction and regulation of the innate effector response. These effects were specific to vedolizumab, not observed in response to the TNFα antibody infliximab, and associated with inhibition of intestinal inflammation. CONCLUSION: Our findings suggest that modulation of innate immunity contributes to the therapeutic efficacy of vedolizumab in IBD. TRIAL REGISTRATION NUMBER: NCT02694588.

Influence of forkhead box protein 3 polymorphisms (rs2232365, rs3761548) with the outcome of pregnancy: A meta-analysis.

Dysfunction of regulatory T cells (Tregs) may contribute to certain immune-related pregnancy complications. Forkhead box protein 3 (FOXP3) is the key transcription factor of Treg. We performed a systematic review and meta-analysis to evaluate the possible association between FOXP3 polymorphisms -924A/G (rs2232365) and -3279C/A (rs3761548) and immune-related pregnancy complications. After reviewing 78 fully published studies, 10 studies fulfilled previously defined eligibility criteria and were used for meta-analysis. Two single nucleotide polymorphisms showed a significant correlation with increased or reduced risk for immune-related pregnancy complications. For rs3761548, women with allele A were significantly at a higher risk than women carrying allele C (odds ratio = 1.29, 95% confidence interval: 1.20-1.38; p = 0.001). For rs2232365, women with GG or AG genotype were at a higher risk than women with genotype AA, thereby, allele G was significantly associated with a higher risk than allele A. Our meta-analysis supports the notion that immune-related pregnancy complications might be linked to genetic variations in the FOXP3 gene.

Hematopoietic stem cell involvement in BCR-ABL1-positive ALL as a potential mechanism of resistance to blinatumomab therapy.

The bispecific T-cell engager blinatumomab targeting CD19 can induce complete remission in relapsed or refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, some patients ultimately relapse with loss of CD19 antigen on leukemic cells, which has been established as a novel mechanism to escape CD19-specific immunotherapies. Here, we provide evidence that CD19-negative (CD19-) relapse after CD19-directed therapy in BCP-ALL may be a result of the selection of preexisting CD19- malignant progenitor cells. We present 2 BCR-ABL1 fusion-positive BCP-ALL patients with CD19- myeloid lineage relapse after blinatumomab therapy and show BCR-ABL1 positivity in their hematopoietic stem cell (HSC)/progenitor/myeloid compartments at initial diagnosis by fluorescence in situ hybridization after cell sorting. By using the same approach with 25 additional diagnostic samples from patients with BCR-ABL1-positive BCP-ALL, we identified HSC involvement in 40% of the patients. Patients (6 of 8) with major BCR-ABL1 transcript encoding P210BCR-ABL1 mainly showed HSC involvement, whereas in most of the patients (9 of 12) with minor BCR-ABL1 transcript encoding P190BCR-ABL1, only the CD19+ leukemia compartments were BCR-ABL1 positive (P = .02). Our data are of clinical importance, because they indicate that both CD19+ cells and CD19- precursors should be targeted to avoid CD19- relapses in patients with BCR-ABL1-positive ALL.

An update on immune dysregulation in obesity-related insulin resistance.

Obesity is associated with chronic low-grade inflammation of the adipose tissue (AT) that might develop into systemic inflammation, insulin resistance (IR) and an increased risk of type 2 diabetes mellitus (T2DM) in severe obese rodents and humans. In the lean state, small normal adipocytes and AT macrophages interact with each other to maintain metabolic homeostasis but during obesity, enlarged adipocytes secrete inflammatory mediators and express immune receptors to recruit immune cells and aggravate the inflammation. The better understanding of the obesity-related inflammatory milieu and the sequential events leading to IR could be helpful in designing new preventive and therapeutic strategies. The present review will discuss the cellular and molecular abnormalities participating in the pathogenesis of obesity in obese individuals as well as high-fat diet (HFD)-fed mice, a mouse model of obesity.

Mechanistic peculiarities of activation-induced mobilization of cytotoxic effector proteins in human T cells.

It is widely accepted that cytotoxic T and NK cells store effector proteins including granzymes, perforin and Fas ligand (FasL) in intracellular granules, often referred to as secretory lysosomes. Upon target cell encounter, these organelles are transported to the cytotoxic immunological synapse, where they fuse with the plasma membrane to release the soluble effector molecules and to expose transmembrane proteins including FasL on the cell surface. We previously described two distinct species of secretory vesicles in T and NK cells that differ in size, morphology and protein loading, most strikingly regarding FasL and granzyme B. We now show that the signal requirements for the mobilization of one or the other granule also differ substantially. We report that prestored FasL can be mobilized independent of extracellular Ca2+, whereas the surface exposure of lysosome-associated membrane proteins (Lamps; CD107a and CD63) and the release of granzyme B are calcium-dependent. The use of selective inhibitors of actin dynamics unequivocally points to different transport mechanisms for individual vesicles. While inhibitors of actin polymerization/dynamics inhibit the surface appearance of prestored FasL, they increase the activation-induced mobilization of CD107a, CD63 and granzyme B. In contrast, inhibition of the actin-based motor protein myosin 2a facilitates FasL-, but impairs CD107a-, CD63- and granzyme B mobilization. From our data, we conclude that distinct cytotoxic effector granules are differentially regulated with respect to signaling requirements and transport mechanisms. We suggest that a T cell might 'sense' which effector proteins it needs to mobilize in a given context, thereby increasing efficacy while minimizing collateral damage.