Elevated gene expression of Lymphocyte antigen 6K (LY6K) in cancer cells is associated with poor survival outcomes in multiple different cancer types including cervical, breast, ovarian, lung, and head and neck cancer. Since inhibition of LY6K expression inhibits cancer cell growth, we set out to explore whether pharmacological inhibition of LY6K could produce the same effect. We screened small molecule libraries for direct binding to recombinant LY6K protein in a surface plasmon resonance assay. We found that NSC243928 directly binds to the full-length and mature forms of LY6K and inhibits growth of HeLa cells that express LY6K. NSC243928 did not display binding with LY6D or LY6E. Our data demonstrate a first-time proof of principle study that pharmacological inhibition of LY6K using small molecules in cancer cells is a valid approach to developing targeted therapies against LY6K. This approach will be specifically relevant in hard-to-treat cancers where LY6K is highly expressed, such as cervical, pancreatic, ovarian, head and neck, lung, gastric, and triple-negative breast cancers.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an inhibitory immune checkpoint that can be expressed in tumor-infiltrating lymphocytes and colorectal cancer (CRC) cells. This immune checkpoint can attenuate anti-tumoral immune responses and facilitate tumor growth and metastasis. Although capecitabine is an effective chemotherapeutic agent for treating CRC, its effect on the tumoral CTLA-4 expression remains unclear. In the current research, we applied the GSE110224 and GSE25070 datasets to characterize CTLA-4 expression in CRC patients. Then, we analyzed CTLA-4 expression in CRC samples, HT-29, HCT-166, and SW480 cell lines using real-time PCR. Our bioinformatic results have highlighted the overexpression of CTLA-4 in the CRC tissues compared to the adjacent non-tumoral tissues. Our in vitro studies have indicated that SW480 cells can substantially overexpress CTLA-4 compared to HT-29 and HCT 116 cells. In addition, capecitabine can remarkably downregulate the expression of CTLA-4 in SW480 cells. Collectively, capecitabine can inhibit the expression of CTLA-4 in CRC cells and might bridge the immunotherapy approaches with chemotherapy.

The adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTL) shows promise in the treatment of cancer and infectious diseases. We utilize adeno-associated virus-(AAV-) based antigen gene-loaded dendritic cells (DCs) to stimulate such antigen-specific CTL. Yet further improvements in CTL stimulation and killing may result by gene delivery of various Th1-response interferons/cytokines, such as interferon gamma (IFN-gamma), as the delivered gene can continuously produce that interferon. However which immune cell type should optimally express IFN-gamma is unclear as the phenotypes of both DC and T cells are enhanced by it. Here, we used AAV to compare and contrast IFN-gamma gene delivery into DC or T cells, and versus the addition of exogenous IFN-gamma, for stimulating carcinoembryonic antigen-(CEA-) specific CTL. It was found that AAV/IFN-gamma delivery into T cells (autocrine) resulted in T cell populations with the highest CD8(+)/CD4(+) ratio, highest IFN-gamma(+)/IL-4(+) ratio, highest CD69(+),CD8(+) levels, and lowest CD4(+)/CD25(+) levels, all consistent with the strongest Th1 response. Most importantly, AAV/IFN-gamma transduction of T cells resulted in antigen-specific T cell populations with the highest killing capabilities, 49% above other treatments. These data strongly suggest that AAV/IFN-gamma autocrine gene delivery into T cells is worthy of further study towards maximizing the generation of antigen-specific anticancer CTL killers.

Heat shock proteins (HSPs) like glycoprotein (gp)96 (glucose-regulated protein 94 [grp94]) are able to induce specific cytotoxic T lymphocyte (CTL) responses against cells from which they originate. Here, we demonstrate that for CTL activation by gp96-chaperoned peptides, specific receptor-mediated uptake of gp96 by antigen-presenting cells (APCs) is required. Moreover, we show that in both humans and mice, only professional APCs like dendritic cells (DCs), macrophages, and B cells, but not T cells, are able to bind gp96. The binding is saturable and can be inhibited using unlabeled gp96 molecules. Receptor binding by APCs leads to a rapid internalization of gp96, which colocalizes with endocytosed major histocompatibility complex (MHC) class I and class II molecules in endosomal compartments. Incubation of gp96 molecules isolated from cells expressing an adenovirus type 5 E1B epitope with the DC line D1 results in the activation of E1B-specific CTLs. This CTL activation can be specifically inhibited by the addition of irrelevant gp96 molecules not associated with E1B peptides. Our results demonstrate that only receptor-mediated endocytosis of gp96 molecules leads to MHC class I-restricted re-presentation of gp96-associated peptides and CTL activation; non-receptor-mediated, nonspecific endocytosis is not able to do so. Thus, we provide evidence on the mechanisms by which gp96 is participating in the cross-presentation of antigens from cellular origin.

Lymphocytes circulate through lymph nodes (LN) in search for antigen in what is believed to be a continuous process. Here, we show that lymphocyte migration through lymph nodes and lymph occurred in a non-continuous, circadian manner. Lymphocyte homing to lymph nodes peaked at night onset, with cells leaving the tissue during the day. This resulted in strong oscillations in lymphocyte cellularity in lymph nodes and efferent lymphatic fluid. Using lineage-specific genetic ablation of circadian clock function, we demonstrated this to be dependent on rhythmic expression of promigratory factors on lymphocytes. Dendritic cell numbers peaked in phase with lymphocytes, with diurnal oscillations being present in disease severity after immunization to induce experimental autoimmune encephalomyelitis (EAE). These rhythms were abolished by genetic disruption of T cell clocks, demonstrating a circadian regulation of lymphocyte migration through lymph nodes with time-of-day of immunization being critical for adaptive immune responses weeks later.

CD8 cytotoxic T lymphocytes (CTLs) rely on rapid reorganization of the branched F-actin network to drive the polarized secretion of lytic granules, initiating target cell death during the adaptive immune response. Branched F-actin is generated by the nucleation factor actin-related protein 2/3 (Arp2/3) complex. Patients with mutations in the actin-related protein complex 1B (ARPC1B) subunit of Arp2/3 show combined immunodeficiency, with symptoms of immune dysregulation, including recurrent viral infections and reduced CD8+ T cell count. Here, we show that loss of ARPC1B led to loss of CTL cytotoxicity, with the defect arising at 2 different levels. First, ARPC1B is required for lamellipodia formation, cell migration, and actin reorganization across the immune synapse. Second, we found that ARPC1B is indispensable for the maintenance of TCR, CD8, and GLUT1 membrane proteins at the plasma membrane of CTLs, as recycling via the retromer and WASH complexes was impaired in the absence of ARPC1B. Loss of TCR, CD8, and GLUT1 gave rise to defects in T cell signaling and proliferation upon antigen stimulation of ARPC1B-deficient CTLs, leading to a progressive loss of CD8+ T cells. This triggered an activation-induced immunodeficiency of CTL activity in ARPC1B-deficient patients, which could explain the susceptibility to severe and prolonged viral infections.

Since their onset, display technologies have proven useful for the selection of antibodies against a variety of targets; however, most of the antibodies selected with the currently available platforms need to be further modified for their use in humans, and are restricted to accessible antigens. Furthermore, these platforms are not well suited for in vivo selections. We present here a novel cell based antibody display platform, which takes advantage of the functional capabilities of T lymphocytes. The display of antibodies on the surface of T lymphocytes, as a part of a chimeric-immune receptor (CIR) mediating signaling, may ideally link the antigen-antibody interaction to a demonstrable change in T cell phenotype, due to subsequent expression of the early T cell activation marker CD69. In this proof-of-concept, an in vitro selection was carried out using a human T cell line lentiviral-transduced to express a tumor-specific CIR on the surface, against a human tumor cell line expressing the carcinoembryonic antigen. Based on an effective interaction between the CIR and the tumor antigen, we demonstrated that combining CIR-mediated activation with FACS sorting of CD69(+) T cells, it is possible to isolate binders to tumor specific cell surface antigen, with an enrichment factor of at least 10(3)-fold after two rounds, resulting in a homogeneous population of T cells expressing tumor-specific CIRs.

Peste des petits ruminants (PPR) is a severe disease of goats and sheep that is widespread in Africa, the Middle East, and Asia. Several effective vaccines exist for the disease, based on attenuated strains of the virus (PPRV) that causes PPR. While the efficacy of these vaccines has been established by use in the field, the nature of the protective immune response has not been determined. In addition, while the vaccine derived from PPRV/Nigeria/75/1 (N75) is used in many countries, those developed in India have never been tested for their efficacy outside that country. We have studied the immune response in goats to vaccination with either N75 or the main Indian vaccine, which is based on isolate PPRV/India/Sungri/96 (S96). In addition, we compared the ability of these two vaccines, in parallel, to protect animals against challenge with pathogenic viruses from the four known genetic lineages of PPRV, representing viruses from different parts of Africa, as well as Asia. These studies showed that, while N75 elicited a stronger antibody response than S96, as measured by both enzyme-linked immunosorbent assay and virus neutralization, S96 resulted in more pronounced cellular immune responses, as measured by virus antigen-induced proliferation and interferon gamma production. While both vaccines induced comparable numbers of PPRV-specific CD8+ T cells, S96 induced a higher number of CD4+ T cells specifically responding to virus. Despite these quantitative and qualitative differences in the immune responses following vaccination, both vaccines gave complete clinical protection against challenge with all four lineages of PPRV.IMPORTANCE Despite the widespread use of live attenuated PPRV vaccines, this is the first systematic analysis of the immune response elicited in small ruminants. These data will help in the establishment of the immunological determinants of protection, an important step in the development of new vaccines, especially DIVA vaccines using alternative vaccination vectors. This study is also the first controlled test of the ability of the two major vaccines used against virulent PPRV strains from all genetic lineages of the virus, showing conclusively the complete cross-protective ability of these vaccines.

Conventional CD4+ T cells are differentiated into CD4+CD8αα+ intraepithelial lymphocytes (IELs) in the intestine; however, the roles of intestinal epithelial cells (IECs) are poorly understood. Here, we showed that IECs expressed MHC class II (MHC II) and programmed death-ligand 1 (PD-L1) induced by the microbiota and IFN-γ in the distal part of the small intestine, where CD4+ T cells were transformed into CD4+CD8αα+ IELs. Therefore, IEC-specific deletion of MHC II and PD-L1 hindered the development of CD4+CD8αα+ IELs. Intracellularly, PD-1 signals supported the acquisition of CD8αα by down-regulating the CD4-lineage transcription factor, T helper-inducing POZ/Krüppel-like factor (ThPOK), via the Src homology 2 domain-containing tyrosine phosphatase (SHP) pathway. Our results demonstrate that noncanonical antigen presentation with cosignals from IECs constitutes niche adaptation signals to develop tissue-resident CD4+CD8αα+ IELs.

Human leucocyte antigen (HLA) compatibility is the main factor determining the occurrence of graft-vs-host disease (GVHD) in patients. It has also been shown that minor histocompatibility antigen differences as well as genetic polymorphisms that are not sequenced by standard methodology for HLA typing can play a role. We used mixed lymphocyte cultures (MLCs) as a functional cellular test and investigated gene expression changes driven by HLA incompatibility in an effort to better understand the mechanisms involved in the disease. Gene expression profile of HLA matched and HLA mismatched MLC identified differentially regulated genes and pathways. We found that a great number of genes related to immune function were differentially regulated; these genes were also found to be associated with GVHD and graft rejection. The majority of differentially regulated genes were interferon-gamma (IFNγ)-inducible genes and IFNγ neutralisation in MLCs abrogated their induction. The microRNA-155, a recently identified target for acute GVHD (aGVHD), was also found to be significantly induced in HLA mismatched MLC but not in the matched setting and its induction was not diminished by blocking IFNγ. In this proof-of-principle study we show gene expression changes in mismatched MLC that represent alloreactive responses, correlate with markers involved in GVHD and can potentially be useful in the study of the biological processes involved in this disease.

The induction of long-lived effector CD8+ T cells is key to the development of efficient cancer vaccines. In this study, we demonstrated that a Toll-like receptor 2 (TLR2) agonist-fused antigen increased antigen presentation via TLR2 signaling and induced effector memory-like CD8+ T cells against cancer after immunization. The N-terminus of ovalbumin (OVA) was biologically fused with a bacterial lipid moiety TLR2 agonist to produce a recombinant lipidated ovalbumin (rlipo-OVA). We demonstrated that rlipo-OVA activated bone marrow-derived dendritic cells (BM-DCs) maturation and increased antigen presentation by major histocompatibility complex (MHC) class I via TLR2. After immunization, rlipo-OVA skewed the immune response towards T helper (Th) 1 and induced OVA-specific cytotoxic T lymphocyte (CTL) responses. Moreover, immunization with rlipo-OVA induced higher numbers of effector memory (CD44+CD62L-) CD8+ T cells compared with recombinant ovalbumin (rOVA) alone or rOVA mixed with the TLR2 agonist Pam3CSK4. Accordingly, the CD27+CD43+ effector memory CD8+ T cells expressed high levels of the long-lived CD127 marker. The administration of rlipo-OVA could inhibit tumor growth, but the anti-tumor effects were lost after the depletion of CD8 or CD127 cells in vivo. These findings suggested that the TLR2 agonist-fused antigen induced long-lived memory CD8+ T cells for efficient cancer therapy.

A coat of pericellular hyaluronan surrounds mature dendritic cells (DC) and contributes to cell-cell interactions. We asked whether 4-methylumbelliferone (4MU), an oral inhibitor of HA synthesis, could inhibit antigen presentation. We find that 4MU treatment reduces pericellular hyaluronan, destabilizes interactions between DC and T-cells, and prevents T-cell proliferation in vitro and in vivo. These effects were observed only when 4MU was added prior to initial antigen presentation but not later, consistent with 4MU-mediated inhibition of de novo antigenic responses. Building on these findings, we find that 4MU delays rejection of allogeneic pancreatic islet transplant and allogeneic cardiac transplants in mice and suppresses allogeneic T-cell activation in human mixed lymphocyte reactions. We conclude that 4MU, an approved drug, may have benefit as an adjunctive agent to delay transplantation rejection.

Assembly of the CARD11/CARMA1-BCL10-MALT1 (CBM) signaling complex upon T or B cell antigen receptor (TCR or BCR) engagement drives lymphocyte activation. Recruitment of pre-assembled BCL10-MALT1 complexes to CARD11 fosters activation of the MALT1 protease and canonical NF-κB signaling. Structural data and in vitro assays have suggested that CARD11 acts as a seed that nucleates the assembly of BCL10 filaments, but the relevance of these findings for CBM complex assembly in cells remains unresolved. To uncouple cellular CARD11 recruitment of BCL10 and BCL10 filament assembly, we generated a BCL10-CARD11 fusion protein that links the C-terminus of BCL10 to the N-terminus of CARD11. When stably expressed in CARD11 KO Jurkat T cells, the BCL10-CARD11 fusion induced constitutive MALT1 activation. Furthermore, in CARD11 KO BJAB B cells, BCL10-CARD11 promoted constitutive NF-κB activation to a similar extent as CARD11 containing oncogenic driver mutations. Using structure-guided destructive mutations in the CARD11-BCL10 (CARD11 R35A) or BCL10-BCL10 (BCL10 R42E) interfaces, we demonstrate that chronic activation by the BCL10-CARD11 fusion protein was independent of the CARD11 CARD. However, activation strictly relied upon the ability of the BCL10 CARD to form oligomers. Thus, by combining distinct CARD mutations in the context of constitutively active BCL10-CARD11 fusion proteins, we provide evidence that BCL10-MALT1 recruitment to CARD11 and BCL10 oligomerization are interconnected processes, which bridge the CARD11 seed to downstream pathways in lymphocytes.

Background: Immunotherapy is one promising therapeutic strategy against glioma, an aggressive form of brain cancer. Previous studies have demonstrated that multiple tumor antigens exist and can be used to induce tumor specific T cell responses. Furthermore, recently it was shown that TLR4-primed mesenchymal stem cells (MSCs), also known as MSC1, mostly elaborate pro-inflammatory mediators. Compared to MSCs, MSC-derived microvesicles (MVs) have advantageous properties that present them as stable, long lasting effectors with no risk of immune rejection. Therefore, peripheral blood monocyte derived dendritic cells (MoDCs) have been used to load tumor antigens and stimulate T cell mediated responses in the presence of MSC1-derived MVs in vitro. Methods: The B92 tumor cell line was heated to 43°C for 90 min prior to preparation of tumor cell lysates. MVs were purified by differential ultracentrifugation after isolation, stimulation of proliferation and treatment of MSCs. Autologous T cells isolated from non-adherent cells were harvested during the procedure to generate MoDCs and then incubated with heat stressed tumor cell lysate pulsed DCs in the presence of MSC1-derived MVs. T cells were then co-cultured with tumor cells in 96-well plates at a final volume of 200 μl CM at an effector: target ratio of 100:1 to determine their specific cytotoxic activity. Results: Flow cytometric analysis, T cell mediated cytotoxicity showed that heat stressed tumor antigen pulsed MoDCs and MSC1-derived MVs primed T cells elicited non-significantly enhanced cytotoxic activity toward B92 tumor cells (P≥0.05). Conclusion: These findings may offer new insights into tumor antigen presenting technology involving dendritic cells and MSC1-derived MVs. Further exploration of the potential of such nanoscale particles in immunotherapy and in novel cancer vaccine settings appears warranted.

New diagnostic tests for the etiology of childhood pneumonia are needed. We evaluated the antibody-in-lymphocyte supernatant (ALS) assay to detect immunoglobulin (Ig) G secretion from ex vivo peripheral blood mononuclear cell (PBMC) culture, as a potential diagnostic test for pneumococcal pneumonia. We enrolled 348 children with pneumonia admitted to Patan Hospital, Kathmandu, Nepal between December 2015 and September 2016. PBMCs sampled from participants were incubated for 48 h before harvesting of cell culture supernatant (ALS). We used a fluorescence-based multiplexed immunoassay to measure the concentration of IgG in ALS against five conserved pneumococcal protein antigens. Of children with pneumonia, 68 had a confirmed etiological diagnosis: 12 children had pneumococcal pneumonia (defined as blood or pleural fluid culture-confirmed; or plasma CRP concentration ≥60 mg/l and nasopharyngeal carriage of serotype 1 pneumococci), and 56 children had non-pneumococcal pneumonia. Children with non-pneumococcal pneumonia had either a bacterial pathogen isolated from blood (six children); or C-reactive protein <60 mg/l, absence of radiographic consolidation and detection of a pathogenic virus by multiplex PCR (respiratory syncytial virus, influenza viruses, or parainfluenza viruses; 23 children). Concentrations of ALS IgG to all five pneumococcal proteins were significantly higher in children with pneumococcal pneumonia than in children with non-pneumococcal pneumonia. The concentration of IgG in ALS to the best-performing antigen discriminated between children with pneumococcal and non-pneumococcal pneumonia with a sensitivity of 1.0 (95% CI 0.73-1.0), specificity of 0.66 (95% CI 0.52-0.78) and area under the receiver-operating characteristic curve (AUROCC) 0.85 (95% CI 0.75-0.94). Children with pneumococcal pneumonia were older than children with non-pneumococcal pneumonia (median 5.6 and 2.0 years, respectively, p < 0.001). When the analysis was limited to children ≥2 years of age, assay of IgG ALS to pneumococcal proteins was unable to discriminate between children with pneumococcal pneumonia and non-pneumococcal pneumonia (AUROCC 0.67, 95% CI 0.47-0.88). This method detected spontaneous secretion of IgG to pneumococcal protein antigens from cultured PBMCs. However, when stratified by age group, assay of IgG in ALS to pneumococcal proteins showed limited utility as a test to discriminate between pneumococcal and non-pneumococcal pneumonia in children.

Therapeutic HIV vaccines may prove helpful to intensify antiretroviral treatment (ART) efficacy and may be an integral part of future cure strategies.

Vaccines remain the most effective tool to prevent infectious diseases. Here, we introduce an in vitro booster vaccination approach that relies on antigen-dependent activation of human memory B cells in culture. This stimulation induces antigen-specific B cell proliferation, differentiation of B cells into plasma cells, and robust antibody secretion after a few days of culture. We validated this strategy using cells from healthy donors to retrieve human antibodies against tetanus toxoid and influenza hemagglutinin (HA) from H1N1 and newly emergent subtypes such as H5N1 and H7N9. Anti-HA antibodies were cross-reactive against multiple subtypes, and some showed neutralizing activity. Although these antibodies may have arisen as a result of previous influenza infection, we also obtained gp120-reactive antibodies from non-HIV-infected donors, indicating that we can generate antibodies without prior antigenic exposure. Overall, our novel approach can be used to rapidly produce therapeutic antibodies and has the potential to assess the immunogenicity of candidate antigens, which could be exploited in future vaccine development.

A current focus in cancer treatment is to broaden responses to immunotherapy. One reason these therapies may prove inadequate is that T lymphocytes fail to recognize the tumor due to differences in immunogenic epitopes presented by the cancer cells under inflammatory or non-inflammatory conditions. The antigen processing machinery of the cell, the proteasome, cleaves proteins into peptide epitopes for presentation on MHC complexes. Immunoproteasomes in inflammatory melanomas, and in antigen presenting cells of the immune system, are enzymatically different to standard proteasomes expressed by tumors with no inflammation. This corresponds to alterations in protein cleavage between proteasome subtypes, and a disparate repertoire of MHC-presented epitopes.

PD-1 is an immunoinhibitory receptor expressed by activated T cells, B cells, and myeloid cells. Mice deficient in PD-1 exhibit a breakdown of peripheral tolerance and demonstrate multiple autoimmune features. We report here that the ligand of PD-1 (PD-L1) is a member of the B7 gene family. Engagement of PD-1 by PD-L1 leads to the inhibition of T cell receptor-mediated lymphocyte proliferation and cytokine secretion. In addition, PD-1 signaling can inhibit at least suboptimal levels of CD28-mediated costimulation. PD-L1 is expressed by antigen-presenting cells, including human peripheral blood monocytes stimulated with interferon gamma, and activated human and murine dendritic cells. In addition, PD-L1 is expressed in nonlymphoid tissues such as heart and lung. The relative levels of inhibitory PD-L1 and costimulatory B7-1/B7-2 signals on antigen-presenting cells may determine the extent of T cell activation and consequently the threshold between tolerance and autoimmunity. PD-L1 expression on nonlymphoid tissues and its potential interaction with PD-1 may subsequently determine the extent of immune responses at sites of inflammation.

Equisetum giganteum is a plant used in traditional medicine as diuretic. From our knowledge this is the first time this plant is tested in an in vivo model of acute inflammation. To evaluate the effect of aqueous extract of giant horsetail (AEGH) as immunomodulatory therapy, antigen-induced arthritis (AIA) was generated in mice with methylated bovine serum albumin (mBSA). Inflammation was evaluated by articular nociception, leukocytes migration and lymphocyte proliferation. AEGH reduced nociception at 3, 6 and 24 h (P < 0.01), decreased leukocyte migration (P < 0.015), and inhibited lymphocyte proliferation stimulated with Concanavalin A and Lipopolysaccharide (P < 0.05). In conclusion, AEGH has an anti-inflammatory potential in acute model of inflammation, as well as immunomodulatory effect on both B and T lymphocytes, with an action independent of cytotoxicity.