Targeting alternative splicing as a new cancer immunotherapy-phosphorylation of serine arginine-rich splicing factor (SRSF1) by SR protein kinase 1 (SRPK1) regulates alternative splicing of PD1 to generate a soluble antagonistic isoform that prevents T cell exhaustion.

BACKGROUND: Regulation of alternative splicing is a new therapeutic approach in cancer. The programmed cell death receptor 1 (PD-1) is an immunoinhibitory receptor expressed on immune cells that binds to its ligands, PD-L1 and PD-L2 expressed by cancer cells forming a dominant immune checkpoint pathway in the tumour microenvironment. Targeting this pathway using blocking antibodies (nivolumab and pembrolizumab) is the mainstay of anti-cancer immunotherapies, restoring the function of exhausted T cells. PD-1 is alternatively spliced to form isoforms that are either transmembrane signalling receptors (flPD1) that mediate T cell death by binding to the ligand, PD-L1 or an alternatively spliced, soluble, variant that lacks the transmembrane domain. METHODS: We used PCR and western blotting on primary peripheral blood mononuclear cells (PBMCs) and Jurkat T cells, IL-2 ELISA, flow cytometry, co-culture of melanoma and cholangiocarcinoma cells, and bioinformatics analysis and molecular cloning to examine the mechanism of splicing of PD1 and its consequence. RESULTS: The soluble form of PD-1, generated by skipping exon 3 (∆Ex3PD1), was endogenously expressed in PBMCs and T cells and prevents cancer cell-mediated T cell repression. Multiple binding sites of SRSF1 are adjacent to PD-1 exon 3 splicing sites. Overexpression of phosphomimic SRSF1 resulted in preferential expression of flPD1. Inhibition of SRSF1 phosphorylation both by SRPK1 shRNA knockdown and by a selective inhibitor, SPHINX31, resulted in a switch in splicing to ∆Ex3PD1. Cholangiocarcinoma cell-mediated repression of T cell IL-2 expression was reversed by SPHINX31 (equivalent to pembrolizumab). CONCLUSIONS: These results indicate that switching of the splicing decision from flPD1 to ∆Ex3PD1 by targeting SRPK1 could represent a potential novel mechanism of immune checkpoint inhibition in cancer.

Activated tissue resident memory T-cells (CD8+CD103+CD39+) uniquely predict survival in left sided "immune-hot" colorectal cancers.

Introduction: Characterization of the tumour immune infiltrate (notably CD8+ T-cells) has strong predictive survival value for cancer patients. Quantification of CD8 T-cells alone cannot determine antigenic experience, as not all infiltrating T-cells recognize tumour antigens. Activated tumour-specific tissue resident memory CD8 T-cells (TRM) can be defined by the co-express of CD103, CD39 and CD8. We investigated the hypothesis that the abundance and localization of TRM provides a higher-resolution route to patient stratification. Methods: A comprehensive series of 1000 colorectal cancer (CRC) were arrayed on a tissue microarray, with representative cores from three tumour locations and the adjacent normal mucosa. Using multiplex immunohistochemistry we quantified and determined the localization of TRM. Results: Across all patients, activated TRM were an independent predictor of survival, and superior to CD8 alone. Patients with the best survival had immune-hot tumours heavily infiltrated throughout with activated TRM. Interestingly, differences between right- and left-sided tumours were apparent. In left-sided CRC, only the presence of activated TRM (and not CD8 alone) was prognostically significant. Patients with low numbers of activated TRM cells had a poor prognosis even with high CD8 T-cell infiltration. In contrast, in right-sided CRC, high CD8 T-cell infiltration with low numbers of activated TRM was a good prognosis. Conclusion: The presence of high intra-tumoural CD8 T-cells alone is not a predictor of survival in left-sided CRC and potentially risks under treatment of patients. Measuring both high tumour-associated TRM and total CD8 T-cells in left-sided disease has the potential to minimize current under-treatment of patients. The challenge will be to design immunotherapies, for left-sided CRC patients with high CD8 T-cells and low activate TRM,that result in effective immune responses and thereby improve patient survival.

A review of the racial heterogeneity of breast cancer stem cells.

Breast Cancer Stem Cells has become the toast of many breast cancer investigators in the past two decades owing to their crucial roles in tumourigenesis, progression, differentiation, survival and chemoresistance. Despite the growing list of research data in this field, racial or ethnic comparison studies on these stem cells remain scanty. This study is a comparative racial analysis of putative breast cancer stem cells. Research articles on the clinicopathological significance of breast cancer stem cells within a period of 17 years (2003-2020) were reviewed across 5 major races (African/Black American, Asian, Caucasian/White, Hispanic/Latino, and American). The associations between the stem cells markers (CD44+/CD24-/low, BMI1, ALDH1, CD133, and GD2) and clinicopathological and clinical outcomes were analysed. A total of 40 studies were included in this study with 50% Asian, 25% Caucasian, 10% African, 5% American and 2.5% Hispanic/Latino, and 7.5% other mixed races. CD44+/CD24-/low has been associated with TNBC/Basal like phenotype across all races. It is generally associated with poor clinicopathological features such as age, tumour size, lymph node metastasis and lymphovascular invasion. In Asians, CD44+/CD24-/low was associated with DFS and OS but not in Caucasians. ALDH1 was the most studied breast CSC marker (40% of all studies on breast cancer stem cell markers) also associated with poor clinicopathological features including size, age, stage, lymph node metastasis and Nottingham Prognostic Index. ALDH1 was also associated with DFS and OS in Asians but not Caucasians. Racial variations exist in breast cancer stem cell pattern and functions but ill-defined due to multiple factors. Further research is required to better understand the role of breast CSC.

Structural assessment of SARS-CoV2 accessory protein ORF7a predicts LFA-1 and Mac-1 binding potential.

ORF7a is an accessory protein common to SARS-CoV1 and the recently discovered SARS-CoV2, which is causing the COVID-19 pandemic. The ORF7a protein has a structural homology with ICAM-1 which binds to the T lymphocyte integrin receptor LFA-1. As COVID-19 has a strong immune component as part of the disease, we sought to determine whether SARS-CoV2 would have a similar structural interaction with LFA-1. Using molecular docking simulations, we found that SARS-CoV2 ORF7a has the key structural determinants required to bind LFA-1 but also the related leukocyte integrin Mac-1, which is also known to be expressed by macrophages. Our study shows that SARS-CoV2 ORF7a protein has a conserved Ig immunoglobulin-like fold containing an integrin binding site that provides a mechanistic hypothesis for SARS-CoV2's interaction with the human immune system. This suggests that experimental investigation of ORF7a-mediated effects on immune cells such as T lymphocytes and macrophages (leukocytes) could help understand the disease further and develop effective treatments.

Connexin 43 is an independent predictor of patient outcome in breast cancer patients.

PURPOSE: Gap junctions are specialized membrane structures that form channels between adjacent cells allowing cell communication. Gap junctions and specifically Connexin 43 (Cx43) are down-regulated in cancer; however, there are contrasting reports on how this effects breast cancer patient survival. This paper is the first large-scale tissue microarray analysis of Cx43 expression in breast cancer patients with an associated clinical long-term follow-up. METHODS: Using a validated TMA of 1118 primary breast cancers, coupled to a comprehensive database of clinicopathological variables, the expression levels and subcellular localisation of Cx43 was assessed by immunohistochemistry. Its impact in terms of survival, distant metastasis-free survival, and clinicopathological variables was determined. RESULTS: Patients whose tumors expressed high levels of Cx43 had significantly better survival (p < 0.001) than patients with low levels. High Cx43 expression within tumors was associated with an 18-month survival advantage. Loss of Cx43 expression was associated with markers of poor prognosis, namely large tumor size, high grade, high proliferation status, high pleomorphism, high mitosis, poor Nottingham Prognostic Index (NPI), and triple negative tumors. Cx43 expression was independent of tumor size, grade, stage and ER-status in predicting poor survival on multivariate analysis (p = 0.004). CONCLUSION: Connexin 43 (Cx43) is an independent predictor of breast cancer survival and distant metastasis-free survival. High expression of Cx43 was seen in only 13% of tumors, suggesting that drugs to increase Cx43 expression may result in prolonged patients survival.

Macrophage-derived interleukin-1beta promotes human breast cancer cell migration and lymphatic adhesion in vitro.

Lymphovascular invasion (LVI), encompassing blood and lymphatic vessel invasion, is an important event in tumourigenesis. Macrophages within the tumour microenvironment are linked to the presence of LVI and angiogenesis. This study investigates the role of macrophage-derived, caspase-1-dependent interleukin-1beta (IL-1ß) in an in vitro model of LVI. IL-1ß significantly augmented the adhesion and transmigration of breast cancer cell lines MCF7 and MDA-MB-231 across endothelial cell barriers. MDA-MB-231 and MCF7 showed a higher percentage of adhesion to lymphatic endothelial cells than blood endothelial cells following endothelial cell IL-1ß stimulation (P < 0.001 and P < 0.0001, respectively). Supernatants from activated macrophages increased the adhesion of tumour cells to lymphatic and blood endothelium. Secretion of IL-1ß was caspase-1 dependent, and treatment with caspase-1 inhibitor reduced IL-1ß production by 73% and concomitantly reduced tumour cell adhesion to levels obtained with resting macrophages. Transmigration of MDA-MB-231 cells across blood and lymphatic endothelial monolayers was significantly increased following IL-1ß stimulation. Furthermore, supernatants from activated macrophages increased transmigration of MDA-MB-231 cells across endothelial monolayers, which was abolished by caspase-1 inhibition. IL-1ß stimulation of tumour cells significantly increased their migratory ability and a significant increase in migration was observed when MDA-MB-231 cells were stimulated with macrophage conditioned media (two of three donors). Results demonstrate that macrophage production of IL-1ß plays an important role in the migration of breast cancer cells and their adhesion to, and transmigration across, blood and lymphatic endothelial cells. Results suggest that IL-1ß may play a role in the adhesion to lymphatic endothelial cells in particular.

Immuno-silent polymer capsules encapsulating nanoparticles for bioimaging applications.

PEGylated polymer capsules encapsulating LaVO4:Tb3+, GdVO4:Tb3+, Gd2O3:Tb3+, GdF3:Tb3+, YVO4:Tb3+ and iron oxide nanoparticles are promising new fluorescence, magnetic and magnetofluorescence imaging agents. Recently, we have reported their in vitro and in vivo level toxicity profiles which show the non-toxic nature of these polymer capsules encapsulating nanoparticles. However, prior to clinical use, it is essential to ensure that these agents are unlikely to activate immune responses. Herein, we investigated the immunocompatibility of polymer capsules with dendritic cells (DCs), macrophages (MOs), and major antigen presenting cell (APC) subsets required for the activation of innate and adaptive immunity. The capsules were efficiently internalized by both DCs and MOs in vitro. Importantly, despite the presence of intracellular capsules, there was no significant impact on the viability of the cells. We studied the impact of different capsules on the cytokine profiles of the DCs and MOs, which is known to be important for the polarization of T-cell immunity. None of the capsules elicited a change in cytokine secretion from the DCs. Furthermore, the capsules did not alter the polarization of either M1 or M2 MO subsets as determined by the balance of IL-12 and IL-10 secretion. These data support the notion that PEGylated polymer capsules loaded with nanoparticles have the potential to remain immunologically silent as they do not activate APCs nor do they hinder the response of DCs or MOs to pathogen activating signals. These systems, therefore, exhibit promising characteristics for bioimaging applications.

Stromal fibroblasts support dendritic cells to maintain IL-23/Th17 responses after exposure to ionizing radiation.

Dendritic cell function is modulated by stromal cells, including fibroblasts. Although poorly understood, the signals delivered through this crosstalk substantially alter dendritic cell biology. This is well illustrated with release of TNF-α/IL-1ß from activated dendritic cells, promoting PGE2 secretion from stromal fibroblasts. This instructs dendritic cells to up-regulate IL-23, a key Th17-polarizing cytokine. We previously showed that ionizing radiation inhibited IL-23 production by human dendritic cells in vitro. In the present study, we investigated the hypothesis that dendritic cell-fibroblast crosstalk overcomes the suppressive effect of ionizing radiation to support appropriately polarized Th17 responses. Radiation (1-6 Gy) markedly suppressed IL-23 secretion by activated dendritic cells (P < 0.0001) without adversely impacting their viability and consequently, inhibited the generation of Th17 responses. Cytokine suppression by ionizing radiation was selective, as there was no effect on IL-1ß, -6, -10, and -27 or TNF-α and only a modest (11%) decrease in IL-12p70 secretion. Coculture with fibroblasts augmented IL-23 secretion by irradiated dendritic cells and increased Th17 responses. Importantly, in contrast to dendritic cells, irradiated fibroblasts maintained their capacity to respond to TNF-α/IL-1ß and produce PGE2, thus providing the key intermediary signals for successful dendritic cell-fibroblasts crosstalk. In summary, stromal fibroblasts support Th17-polarizing cytokine production by dendritic cells that would otherwise be suppressed in an irradiated microenvironment. This has potential ramifications for understanding the immune response to local radiotherapy. These findings underscore the need to account for the impact of microenvironmental factors, including stromal cells, in understanding the control of immunity.

Impact of p38 mitogen-activated protein kinase inhibition on immunostimulatory properties of human 6-sulfo LacNAc dendritic cells.

p38 Mitogen-activated protein kinase (MAPK) plays a crucial role in the induction and regulation of innate and adaptive immunity. Furthermore, p38 MAPK can promote tumor invasion, metastasis, and angiogenesis. Based on these properties, p38 MAPK inhibitors emerged as interesting candidates for the treatment of immune-mediated disorders and cancer. However, the majority of p38 MAPK inhibitor-based clinical trials failed due to poor efficacy or toxicity. Further studies investigating the influence of p38 MAPK inhibitors on immunomodulatory capabilities of human immune cells may improve their therapeutic potential. Here, we explored the impact of the p38 MAPK inhibitor SB203580 on the pro-inflammatory properties of native human 6-sulfo LacNAc dendritic cells (slanDCs). SB203580 did not modulate maturation of slanDCs and their capacity to promote T-cell proliferation. However, SB203580 significantly reduced the production of pro-inflammatory cytokines by activated slanDCs. Moreover, inhibition of p38 MAPK impaired the ability of slanDCs to differentiate naïve CD4(+) T cells into T helper 1 cells and to stimulate interferon-γ secretion by natural killer cells. These results provide evidence that SB203580 significantly inhibits various important immunostimulatory properties of slanDCs. This may have implications for the design of p38 MAPK inhibitor-based treatment strategies for immune-mediated disorders and cancer.

Expression of arginase I in myeloid cells limits control of residual disease after radiation therapy of tumors in mice.

An accumulating body of evidence demonstrates that radiation therapy can generate adaptive immune responses that contribute to tumor control. However, in the absence of additional immune therapy, the adaptive immune response is insufficient to prevent tumor recurrence or affect distant disease. It has been shown in multiple models that tumor-infiltrating myeloid cells exhibit alternative activation phenotypes and are able to suppress adaptive immune responses, and recent data suggests that the myeloid response in tumors treated with cytotoxic therapy limits tumor control. We hypothesized that tumor myeloid cells inhibit the adaptive immune response after radiation therapy through expression of the enzyme arginase I. Using a myeloid cell-specific deletion of arginase I in mice, we demonstrate an improved tumor control after radiation therapy. However, tumors still recurred despite the conditional knockdown of arginase I. Since multiple alternative factors may combine to inhibit adaptive immunity, we propose that targeting macrophage differentiation may be a more effective strategy than targeting individual suppressive pathways.

Novel function for the p38-MK2 signaling pathway in circulating CD1c+ (BDCA-1+) myeloid dendritic cells from healthy donors and advanced cancer patients; inhibition of p38 enhances IL-12 whilst suppressing IL-10.

There is growing interest in myeloid (my) dendritic cells (DC) as an alternative to monocyte-derived DC (moDC) for immunotherapy. However, in contrast to moDC, little is known regarding the effect of malignancy on the function, abundance or use of intracellular signaling pathways in myDC. Understanding the molecular detail of circulating myDC is therefore important for future use in advanced cancer. Advanced cancer patients had similar numbers of circulating myDC to cancer-free patients and healthy individuals, and secreted similar levels of IL-1ß, IL-6, IL-10, IL-12 and IL-23. However, myDC from some patients failed to secrete the Th1-cytokine IL-12. Surprisingly, inhibiting p38 (p38i) signaling (using BIRB0796 or SB203580) markedly increased IL-12 secretion by myDC. This is in complete contrast to what is established for moDC where inhibiting p38 ablates IL-12. Interestingly, this was specific to IL-12, since IL-10 was suppressed by p38i in both DC types. The opposing effect of p38i on IL-12 was evident at the transcriptional level and in both DC types was mediated through the p38-MK2 pathway but did not involve differential phosphorylation of the distal Rsk kinase. Importantly, where patient myDC did not secrete IL-12 (or after treatment with suppressive melanoma lysate), p38i restored IL-12 to normal levels. In contrast to p38, inhibiting the other MAPK pathways had similar consequences in both DC types. We show for the first time the differential use of a major intracellular signaling pathway by myDC. Importantly, there are sufficient circulating myDC in advanced cancer patients to consider development of adoptive immunotherapy.

CD55 costimulation induces differentiation of a discrete T regulatory type 1 cell population with a stable phenotype.

Unlike other helper T cells, the costimulatory ligands responsible for T regulatory type 1 (Tr1) cell differentiation remain undefined. Understanding the molecular interactions driving peripheral Tr1 differentiation is important because Tr1s potently regulate immune responses by IL-10 production. In this study, we show that costimulation of human naive CD4(+) cells through CD97/CD55 interaction drives Tr1 activation, expansion, and function. T cell activation and expansion was equipotent with CD55 or CD28 costimulation; however, CD55 costimulation resulted in two IL-10-secreting populations. Most IL-10 was secreted by the minor Tr1 population (IL-10(high)IFN-γ(-)IL-4(-), <5% cells) that expresses Tr1 markers CD49b, LAG-3, and CD226. This Tr1 phenotype was not restimulated by CD28. However, on CD55 restimulation, Tr1s proliferated and maintained their differentiated IL-10(high) phenotype. The Tr1s significantly suppressed effector T cell function in an IL-10-dependent manner. The remaining (>95%) cells adopted a Th1-like IFN-γ(+) phenotype. However, in contrast to CD28-derived Th1s, CD55-derived Th1s demonstrated increased plasticity with the ability to coexpress IL-10 when restimulated through CD55 or CD28. These data identify CD55 as a novel costimulator of human Tr1s and support a role for alternative costimulatory pathways in determining the fate of the growing number of T helper populations. This study demonstrates that CD55 acts as a potent costimulator and activator of human naive CD4(+) cells, resulting in the differentiation of a discrete Tr1 population that inhibits T cell function in an IL-10-dependent manner and maintains the Tr1 phenotype upon restimulation.

The peripheral myeloid expansion driven by murine cancer progression is reversed by radiation therapy of the tumor.

Expansion of myeloid-lineage leukocytes in tumor-bearing mice has been proposed as a cause of systemic immunosuppression. We demonstrate that radiation therapy of tumors leads to a decline in myeloid cell numbers in the blood and a decrease in spleen size. The frequency of myeloid cells does not decline to the level seen in tumor-free mice: we demonstrate that metastatic disease can prevent myeloid cell numbers from returning to baseline, and that tumor recurrence from residual disease correlates with re-expansion of myeloid lineage cells. Radiation therapy results in increased proliferation of T cells in the spleen and while T cell responses to foreign antigens are not altered by tumor burden or myeloid cell expansion, responses to tumor-associated antigens are increased after radiation therapy. These data demonstrate that myeloid cell numbers are directly linked to primary tumor burden, that this population contracts following radiation therapy, and that radiation therapy may open a therapeutic window for immunotherapy of residual disease.

The ataxia telangiectasia mutated kinase pathway regulates IL-23 expression by human dendritic cells.

Little is known of the regulation of IL-23 secretion in dendritic cells (DC) despite its importance for human Th17 responses. In this study, we show for first time, to our knowledge, that the ataxia telangiectasia mutated (ATM) pathway, involved in DNA damage sensing, acts as an IL-23 repressor. Inhibition of ATM with the highly selective antagonist KU55933 markedly increased IL-23 secretion in human monocyte-derived DC and freshly isolated myeloid DC. In contrast, inhibiting the closely related mammalian target of rapamycin had no effect on IL-23. Priming naive CD4(+) T cells with ATM-inhibited DC increased Th17 responses over and above those obtained with mature DC. Although ATM blockade increased the abundance of p19, p35, and p40 mRNA, IL-12p70 secretion was unaffected. To further examine a role for ATM in IL-23 regulation, we exposed DC to low doses of ionizing radiation. Exposure of DC to x-rays resulted in ATM phosphorylation and a corresponding depression of IL-23. Importantly, ATM inhibition with KU55933 prevented radiation-induced ATM phosphorylation and abrogated the capacity of x-rays to suppress IL-23. To explore how ATM repressed IL-23, we examined a role for endoplasmic reticulum stress responses by measuring generation of the spliced form of X-box protein-1, a key endoplasmic reticulum stress transcription factor. Inhibition of ATM increased the abundance of X-box protein-1 mRNA, and this was followed 3 h later by increased peak p19 transcription and IL-23 release. In summary, ATM activation or inhibition, respectively, inhibited or augmented IL-23 release. This novel role of the ATM pathway represents a new therapeutic target in autoimmunity and vaccine development.

Gene expression changes in human islets exposed to type 1 diabetic serum.

A major obstacle to the success of islet cell transplantation as a standard treatment for labile type 1 diabetes mellitus is the immediate loss of up to 70% of the transplanted islet mass. Activation of the complement cascade and coagulation factors has been implicated in initiating the destruction of the islet graft. In this study, we analyzed the gene expression changes in islet cells following exposure to type 1 diabetes mellitus serum (T1DM). Isolated human pancreatic islet cells were cultured for 2 d to stabilize islet cell gene expression. Cultured islets were divided into three groups for treatment as follows: group 1 was treated with autologous donor serum, while groups two and three were treated with sera from ABO-matched allogeneic donors or autoantibody positive type 1 diabetic patient, respectively. Complement was detected using anti-C3 FITC and CH50 assay. Islet gene expression was analyzed using Illumina micro-array technology. Results were confirmed using real-time PCR. Immunofluorescent imaging demonstrated complement deposition only in the T1DM condition. Gene array and class prediction analysis generated a list of 50 genes that were able to predict the effect of T1DM serum on islets. Quantitative PCR corroborated microarray results. Both techniques demonstrated upregulation of MMP9 (243%), IL-1ß (255%), IL-11 (220%), IL-12A (132%), RAD (343%) and a concomitant downregulation of IL-1RN (64%) in islets treated with T1DM serum. Islets treated with T1DM serum overexpressed genes associated with angiogenesis while decreasing transcription of genes that protect islets from inflammatory cytokines and reactive oxygen species.

Novel approach for interleukin-23 up-regulation in human dendritic cells and the impact on T helper type 17 generation.

Interleukin-23 (IL-23) is important for T helper type 17 (Th17) responses and strategies to regulate IL-23 in human dendritic cells (DC) are limited. This study describes a novel means to control IL-23 secretion by conditioning DC with a phosphatidyl inositol 3-kinase inhibitor Wortmannin (WM). Treatment of monocyte-derived DC with WM increased Toll-like receptor (TLR) -dependent IL-23 secretion 10-fold and IL-12p70 twofold, but IL-27 was unaffected. The effect of WM was restricted to TLR3/4 pathways, did not occur through TLR2, TLR7/8 or Dectin-1, and was characterized by increased p19, p35 and p40 transcription. These responses were not solely dependent on phosphatidyl inositol 3-kinase as the alternative inhibitor LY294002 did not modulate IL-23 production. The normal patterns of activation of mitogen-activated protein kinase pathways were unaffected by WM-conditioning but IL-23 secretion required p38, ERK and JNK pathways. Importantly, this effect was manifest in populations of blood DC. Conditioning freshly isolated myeloid DC with WM before TLR3 or TLR4 triggering resulted in high levels of IL-23 secretion and an absence of IL-12p70. These WM-conditioned myeloid DC were highly effective at priming Th17 responses from naive CD4(+) T cells. Our findings provide a novel means to generate IL-23-rich environments and Th17 responses and suggest as yet unidentified regulatory factors, identification of which will provide new approaches to control IL-23-dependent immunity in infectious disease, autoimmunity and malignancy.

Role of mitogen-activated protein kinase and PI3K pathways in the regulation of IL-12-family cytokines in dendritic cells and the generation of T H-responses.

Mitogen-activated protein kinases (MAPK) are targets for the immune-modulation of dendritic cells (DC). However, our knowledge of their role in the regulation of IL-12-family cytokines is limited. This study investigated the roles of p38, JNK, p44/42 and PI3K pathways in IL-12/23/27 production by human DC, and their impact on naïve T(H)-responses. We first identified TOP and UBC as robust DC housekeeping genes. Peak transcription of p35 and p40 occurred by 12h, p19 and p28 by 8h and EBI3 by 12-24h. Using selective antagonists, we showed that p38 was a positive regulator of IL-12, 23 and 27, JNK positively regulated IL-12 and IL-27, and inhibition of MEK1/2 had no marked effect. In contrast, the PI3K pathway markedly attenuated IL-23 responses and, to a lesser extent, IL-12, but not IL-27. To identify the role of these soluble factors, we co-stimulated naïve CD4+ T-cells in the presence of DC supernatant. The presence of mature DC supernatant induced not only strong IFNγ responses, but also IL-10 and IL-17A. Inhibition of p38 ablated T(H1), and IL-10 and IL-17A responses, whilst modestly enhancing IL-5 secretion. In contrast, inhibition of MEK1/2 abolished IL-17A production, whilst leaving other responses unaffected, whereas inhibition of JNK or PI3K had no discernable effect. In summary, we describe the expression of IL-12-family cytokines from DC and propose a modified model for their regulation. This study further clarifies the potential for therapeutic modulation through these mediators.

Recent advances in bacillus Calmette-Guerin immunotherapy in bladder cancer.

The concept of using Mycobacterium for cancer treatment goes back to the 19th Century. Today, bacillus Calmette-Guerin (BCG) vaccine is a well-established treatment for human bladder cancer that is arguably superior to intravesical chemotherapy for superficial disease and is commonly used as the first-line adjuvant treatment. Much has been learnt about the effects of BCG on bladder cancer and the immune system, but deeper understanding is required in order to improve its efficacy further, to be able to reliably predict responders and ultimately to adapt this most successful form of cancer immunotherapy for the treatment of other malignancies. This article summarizes the current understanding of BCG cancer immunotherapy mechanisms and discusses possible future developments.

Assessment of islet quality following international shipping of more than 10,000 km.

Islet transplantation is an attractive therapy for type 1 diabetes, although some issues remain. One of them is the severe donor shortage in some countries. In this study, we investigated the possibility of international islet shipping beyond 10,000 km to supply islets to countries with donor shortages. Human islets were isolated from six cadaver donors and cultured until shipment. Islets were packed in either gas-permeable bags or in non-gas-permeable bags and shipped from Baylor Research Institute (Dallas, TX, USA) to Fukuoka University (Fukuoka, Japan). Pre- and postshipment islet number, purity, viability, and stimulation index (by glucose stimulation test) were assessed. Shipped 1,500 IE islets were transplanted into streptozotocin-induced diabetic nude mice for in vivo assay. The distance of our shipment was 11,148.4 km, and the mean duration of the shipments was 48.2 ± 8.2 h. The islet number recovery rate (postshipment/preshipment) was significantly higher in gas-permeable bags (56.4 ± 10.1% vs. 20.5 ± 20.6%, p < 0.01). Islet purity was significantly reduced during shipment in non-gas-permeable bags (from 47.7 ± 18.6% to 40.2 ± 28.2 in gas-permeable bags vs. from 50.4 ± 6.4% to 25.9 ± 15.6% in non-gas-permeable bags, p < 0.05). Islet viability and stimulation index did not change significantly between pre- and postshipping, in either gas-permeable bags or in non-gas-permeable bags. One of three diabetic nude mice (33.3%) converted to normoglycemia. It is feasible to ship human islet cells internationally in gas-permeable bags. This strategy would promote basic and preclinical research for countries with donor shortages, even though the research centers are remote (over 10,000 km from the islet isolation center).

Evidence for Induced Expression of HLA Class II on Human Islets: Possible Mechanism for HLA Sensitization in Transplant Recipients.

Recent reports have shown that islet transplant recipients develop antibodies against donor human leukocyte antigen (HLA) class I and II. Because human islets do not express HLA class II under normal conditions, mechanisms underlying induction of the anti-class II response are unclear. We hypothesized that under inflammatory conditions, islets will have induced expression of HLA class II leading to sensitization. Isolated human islets were divided into two groups. Group 1 was cultured at 37 degrees C as control; group 2 was cultured similarly in presence of tumor necrosis factor alpha and interferon gamma. After treatment, islets were analyzed for expression of HLA class II using real-time polymerase chain reaction, immunofluorescence and flow cytometry. Furthermore, serum from an islet transplant recipient who developed anti-class II antibody was tested by flow cytometry for immunoglobulin (Ig) binding to cytokine-stimulated islets. Real-time polymerase chain reaction analysis for gene transcripts of class II transactivator, HLA-DRagr;, and HLA-DRbeta1 showed maximum 9.38-, 18.95-, and 46.5-fold increase, respectively in group 2 when compared with control at 24 hr. Cytokine treatment increased HLA class II expression markedly on both alpha and beta cells in islets as evidenced by fluorescent imaging and flow cytometric analysis. When patient serum was analyzed by flow cytometry, both IgM and IgG binding was observed in cytokine-treated, HLA class II matched islet cells alone. We conclude that inflammation leads to induced expression of HLA class II on transplanted islet cells potentially causing antidonor sensitization and adversely impacting islet transplant outcomes.