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Background Staphylococcal enterotoxin-B (SEB) is one of the most potent bacterial superantigens that exerts profound toxic effects by inducing cytokine storm. When SEB is inhaled, it can cause Acute Respiratory Distress Syndrome (ARDS), which is often fatal and currently there are no effective treatment modalities. Experimental approach We used mouse model of SEB-mediated ARDS to test the efficacy of Δ9-tetrahydrocannabinol (THC). These mice were monitored for lung inflammation, alterations in gut and lung microbiota and production of short-chain fatty acids (SCFA). Gene dysregulation of lung epithelial cells was studied by transcriptome arrays. Fecal microbiota transplantation (FMT) was performed to confirm the role of microbiota in suppressing ARDS. Key results While SEB triggered ARDS and 100% mortality in mice, THC protected the mice from fatality effects. Pyrosequencing analysis revealed that THC caused significant and similar alterations in microbiota in the lungs and gut of mice exposed to SEB. THC significantly increased the abundance of beneficial bacterial species, Ruminococcus gnavus, but decreased pathogenic microbiota, Akkermansia muciniphila. FMT confirmed that THC-mediated reversal of microbial dysbiosis played crucial role in attenuation of SEB-mediated ARDS. THC treatment also led to increase in SCFA, of which propionic acid was found to inhibit the inflammatory response. Transcriptome array showed that THC up-regulated several genes like lysozyme-1&2, β-defensin-2, claudin, zonula-1, occludin-1, Mucin2 and Muc5b while downregulating β-defensin-1. Conclusions Current study demonstrates for the first time that THC attenuates SEB-mediated ARDS and toxicity by altering the microbiota in the lungs and the gut as well as promoting anti-microbial and anti-inflammatory pathways.

Endometriosis affects women of reproductive age via unclear immunological mechanism(s). Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of myeloid cells with potent immunosuppressive and angiogenic properties. Here, we found MDSCs significantly increased in the peripheral blood of patients with endometriosis and in the peritoneal cavity of a mouse model of surgically induced endometriosis. Majority of MDSCs were granulocytic, produced ROS, and arginase, and suppressed T-cell proliferation. Depletion of MDSCs by antiGr-1 antibody dramatically suppressed development of endometrial lesions in mice. The chemokines CXCL1, 2, and 5 were expressed at sites of lesion while MDSCs expressed CXCR-2. These CXC-chemokines promoted MDSC migration toward endometriotic implants both in vitro and in vivo. Also, CXCR2-deficient mice show significantly decreased MDSC induction, endometrial lesions, and angiogenesis. Importantly, adoptive transfer of MDSCs into CXCR2-KO mice restored endometriotic growth and angiogenesis. Together, this study demonstrates that MDSCs play a role in the pathogenesis of endometriosis and identifies a novel CXC-chemokine and receptor for the recruitment of MDSCs, thereby providing a potential target for endometriosis treatment.

Acute Respiratory Distress Syndrome (ARDS) is triggered by a variety of agents, including Staphylococcal Enterotoxin B (SEB). Interestingly, a significant proportion of patients with COVID-19, also develop ARDS. In the absence of effective treatments, ARDS results in almost 40% mortality. Previous studies from our laboratory demonstrated that resveratrol (RES), a stilbenoid, with potent anti-inflammatory properties can attenuate SEB-induced ARDS. In the current study, we investigated the role of RES-induced alterations in the gut and lung microbiota in the regulation of ARDS. Our studies revealed that SEB administration induced inflammatory cytokines, ARDS, and 100% mortality in C3H/HeJ mice. Additionally, SEB caused a significant increase in pathogenic Proteobacteria phylum and Propionibacterium acnes species in the lungs. In contrast, RES treatment attenuated SEB-mediated ARDS and mortality in mice, and significantly increased probiotic Actinobacteria phylum, Tenericutes phylum, and Lactobacillus reuteri species in both the colon and lungs. Colonic Microbiota Transplantation (CMT) from SEB-injected mice that were treated with RES as well as the transfer of L. reuteri into recipient mice inhibited the production of SEB-mediated induction of pro-inflammatory cytokines such as IFN-γ and IL-17 but increased that of anti-inflammatory IL-10. Additionally, such CMT and L. reuteri recipient mice exposed to SEB, showed a decrease in lung-infiltrating mononuclear cells, cytotoxic CD8+ T cells, NKT cells, Th1 cells, and Th17 cells, but an increase in the population of regulatory T cells (Tregs) and Th3 cells, and increase in the survival of mice from SEB-mediated ARDS. Together, the current study demonstrates that ARDS induced by SEB triggers dysbiosis in the lungs and gut and that attenuation of ARDS by RES may be mediated, at least in part, by alterations in microbiota in the lungs and the gut, especially through the induction of beneficial bacteria such as L. reuteri.

MicroRNAs (miRNAs) are highly conserved, noncoding small RNAs that regulate gene expression, and consequently several important functions including early embryo development, cell cycle, programmed cell death, cell differentiation, and metabolism. While there are no effective treatments available against hepatocellular carcinoma (HCC), some Chinese herbal medicines have been shown to regulate growth, differentiation, invasion, and metastasis of HCC. Many studies have shown that Chinese herbal medicines regulate the expression of miRNAs and this may be associated with their ability to control the development of HCC. In this article, the effects of Chinese herbal medicines on the expression of miRNAs and their functions in the regulation of HCC have been reviewed and discussed. miRNAs such as miRNA-221 and miRNA-222 mediated by Chinese herbal medicines may be good biomarkers and therapeutic targets for HCC.

Cancer immunotherapy is a promising and effective treatment modality for patients with cancers. Cytokine, anticytokine, and antibody therapies appear to be effective in treating various forms of cancer. The human papillomavirus vaccine is protective for cervical cancer, and this discovery has paved the way to the development of cancer vaccines for other forms of virus-associated cancers such as liver cancer and Merkel cell carcinoma. Clinical trials have demonstrated that adoptive cell therapy using tumor-infiltrating lymphocytes can induce tumor regression in approximately 75% of metastatic melanoma patients, suggesting the possibility of using similar technique to effectively treat breast, lung, and renal cancers in the near future. Besides, genetically engineered T cells transduced with genes encoding specific T cell receptors and chimeric antigen receptors have been shown effective in the treatment of cancer patients. These studies suggest that combination therapies are superior choices in cancer immunotherapy for patients.

MicroRNAs (miRNAs) are single-stranded 19-25 nucleotide-long RNAs and have an important role in post-transcriptional gene silencing. It has been demonstrated that miRNAs are dysregulated in patients with multiple sclerosis (MS). For instance, miR-21, miR-142-3p, miR-146a, miR-146b, miR-155 and miR-326 were up-regulated in both peripheral blood mononuclear cells (PBMCs) and brain white matter lesions from MS patients and mouse model as well. These up-regulated miRNAs may be used as a signature for MS and play critical roles in MS pathogenesis. Moreover, miR-15a, miR-19a, miR-22, miR-210 and miR-223 were up-regulated in both regulatory T cells (Tregs) and other samples such as plasma, blood cells, PBMCs and brain white matter tissues from MS patients, suggesting that these up-regulated miRNAs and Tregs may also play a role in MS pathogenesis. Contrarily, other miRNAs such as miR-15a, miR-15b, miR-181c and miR-328 were down-regulated in MS. Drugs such as interferon-β and glatiramer acetate for MS treatment may regulate miRNA expression and thus have benefits for MS patients. The dysregulated miRNAs such as miR-155 and miR-326 may be used as diagnostic markers and therapeutic targets for MS.

55 Background: Immune regulation may play an important role in cancer development. The immune dysregulation and underlying mechanisms in patients with liver cancer have not fully understood. Methods: Peripheral blood mononuclear cells (PBMC) were isolated from patients with liver cancer. Deep-sequencing, FACS analysis, ELISA and real-time PCR were used to analyze the immune dysregulation and underlying mechanisms in patients with liver cancer. Results: Our analysis discovered that the percentages of immune cell populations in PBMC from patients with liver cancer were significantly different from those in normal controls. Specifically, the percents of B cells and regulatory T cells were high in PBMC from patients with liver cancer. Expression of 161 genes such as EGF, IRF3, CD177, MAP2K2 and MMP9 was found to be significantly inhibited, but 66 genes including CD19, CD70, FOXP1 and IL-32 were significantly up-regulated in PBMC from patients with liver cancer. Further analysis showed that 190 long non-coding RNAs (lncRNAs) were significantly down-regulated; whereas150 lncRNAs were significantly up-regulated in PBMC from patients with liver cancer. Dysregulated lncRNAs were involved in the control of immune cell signaling, cell division and differentiation. Conclusions: The results suggest that the immune dysregulation may play a critical role in the pathogenesis of liver cancer. It also has a great implication in the development of immune therapeutic methods for patients with liver cancer.

Multiple sclerosis (MS) is not only an autoimmune disease in which autoreactive immune cells against myelin damage axons and nerves in the central nervous system, but also a neurodegenerative disease, in which progressive loss of structure and function of neurons occurs. The mechanisms of MS pathogenesis have not been fully understood. It has been reported that miRNAs may play a critical role in MS pathogenesis. In this review, we have extensively discussed the alterations in the expression of miRNAs detected in patients with MS. The dysregulated miRNAs have been shown to be associated with the pathogenesis of MS. We suggest that such dysregulated miRNAs may potentially be used as biomarkers in the diagnosis of MS, to discover new therapeutic targets for MS treatment, and to predict prognostic markers in responses to MS treatment.

Recent studies have indicated that adoptive immunotherapy with autologous antitumor tumor-infiltrating lymphocytes (TILs) following nonmyeloablative chemotherapy mediates tumor regression in ∼50% of treated patients with metastatic melanoma, and that tumor regression is correlated with the degree of persistence of adoptively transferred T cells in peripheral blood. These findings, which suggested that the proliferative potential of transferred T cells may play a role in clinical responses, led to the current studies in which telomere length as well as phenotypic markers expressed on the administered TILs were examined. TILs that were associated with objective clinical responses following adoptive transfer possessed a mean telomere length of 6.3 kb, whereas TILs that were not associated with significant clinical responses were significantly shorter, averaging 4.9 kb (p < 0.01). Furthermore, individual TIL-derived T cell clonotypes that persisted in vivo following adoptive cell transfer possessed telomeres that were longer than telomeres of T cell clonotypes that failed to persist (6.2 vs 4.5 kb, respectively; p < 0.001). Expression of the costimulatory molecule CD28 also appeared to be associated with long telomeres and T cell persistence. These results, indicating that the telomere length of transferred lymphocytes correlated with in vivo T cell persistence following adoptive transfer, and coupled with the previous observation that T cell persistence was associated with clinical responses in this adoptive immunotherapy trial, suggest that telomere length and the proliferative potential of the transferred T cells may play a significant role in mediating response to adoptive immunotherapy.

The authors recently reported that adoptive immunotherapy with autologous tumor-reactive tumor infiltrating lymphocytes (TILs) immediately following a conditioning nonmyeloablative chemotherapy regimen resulted in an enhanced clinical response rate in patients with metastatic melanoma. These observations led to the current studies, which are focused on a detailed analysis of the T-cell antigen reactivity as well as the in vivo persistence of T cells in melanoma patient 2098, who experienced a complete regression of all metastatic lesions in lungs and soft tissues following therapy. Screening of an autologous tumor cell cDNA library using transferred TILs resulted in the identification of novel mutated growth arrest-specific gene 7 (GAS7) and glyceral-dehyde-3-phosphate dehydrogenase (GAPDH) gene transcripts. Direct sequence analysis of the expressed T-cell receptor beta chain variable regions showed that the transferred TILs contained multiple T-cell clonotypes, at least six of which persisted in peripheral blood for a month or more following transfer. The persistent T cells recognized both the mutated GAS7 and GAPDH. These persistent tumor-reactive T-cell clones were detected in tumor cell samples obtained from the patient following adoptive cell transfer and appeared to be represented at higher levels in the tumor sample obtained 1 month following transfer than in the peripheral blood obtained at the same time. Overall, these results indicate that multiple tumor-reactive T cells can persist in the peripheral blood and at the tumor site for prolonged times following adoptive transfer and thus may be responsible for the complete tumor regression in this patient.

In recent clinical trials in patients with metastatic melanoma, adoptive transfer of tumor-reactive lymphocytes mediated the regression of metastatic tumor deposits. To better understand the role of individual T cell clones in mediating tumor regression, a 5′ RACE technique was used to determine the distribution of TCR β-chain V region sequences expressed in the transferred cells as well as in tumor samples and circulating lymphocytes from melanoma patients following adoptive cell transfer. We found that dominant T cell clones were present in the in vitro-expanded and transferred tumor-infiltrating lymphocyte samples and certain T cell clones including the dominant T cell clones persisted at relatively high levels in the peripheral blood of the patients that demonstrated clinical responses to adoptive immunotherapy. However, these dominant clones were either undetected or present at a very low level in the resected tumor samples used for tumor-infiltrating lymphocyte generation. These data demonstrated that there was selective growth and survival, both in vitro and in vivo, of individual T cell clones from a relatively small number of T cells in the original tumor samples. These results suggest that the persistent T cell clones played an active role in mediating tumor regression and that 5′ RACE analysis may provide an important tool for the analysis of the role of individual T cell clones in mediating tumor regression. A similar analysis may also be useful for monitoring autoimmune responses.

145 Background: Clinical trials have shown that adoptive cell transfer therapy is a promising method for cancer treatment. In the current study, we aim to generate and characterize anti-tumor tumor-infiltrating lymphocytes from patients with breast cancer for adoptive cell transfer therapy. Methods: In vitro culture method was used to generate anti-tumor, tumor-infiltrating lymphocytes from patients with breast cancer. FACS analysis, ELISA, and Elispot assay were used to characterize tumor-infiltrating lymphocytes. Autologous anti-tumor tumor-infiltrating lymphocytes from patients with breast cancer were used in adoptive cell transfer therapy. Results: FACS analysis indicated that tumor-infiltrating lymphocytes were present in the tumor tissues, but not detectable in the normal breast tissues from patients with breast cancer. Tumor-infiltrating lymphocytes could be generated in vitro from fresh tumor specimens of patients with breast cancer. Both CD4 T cells and CD8 T cells were detected in tumor-infiltrating lymphocytes. Autologous tumor cells could also generate in vitro from fresh tumor tissue samples of patients with breast cancer. Among 22 samples screened, 6 samples (25%) of tumor-infiltrating lymphocytes are tumor-reactive. Anti-tumor, tumor-infiltrating lymphocytes could recognize autologous tumor cells and allogenic tumor cells. After a large scale T cell expansion, anti-tumor reactivity was maintained in tumor-infiltrating lymphocytes. All of tumor-infiltrating lymphocytes were NK cells in some samples from patients with breast cancer, and these NK cells could recognize autologous tumor cells and a panel of allogenic tumor cells. T cell cloning assay demonstrated that some of the tumor-reactive, tumor-infiltrating lymphocytes were CD4 T cells. Conclusions: The results suggest that anti-tumor, tumor-infiltrating lymphocytes may be generated from patients with breast cancer, which may be used in clinical applications of adoptive cell transfer therapy for patients with breast cancer. The clinical trial of adoptive cell transfer therapy using autologous anti-tumor tumor-infiltrating lymphocytes for patients with breast is under way.

Sepsis is a major cause of death worldwide. It triggers systemic inflammation, the role of which remains unclear. In the current study, we investigated the induction of microRNA (miRNA) during sepsis and their role in the regulation of inflammation. Patients, on days 1 and 5 following sepsis diagnosis, had reduced T cells but elevated monocytes. Plasma levels of IL-6, IL-8, IL-10 and MCP-1 dramatically increased in sepsis patients on day 1. T cells from sepsis patients differentiated primarily into Th2 cells, whereas regulatory T cells decreased. Analysis of 1163 miRNAs from PBMCs revealed that miR-182, miR-143, miR-145, miR-146a, miR-150, and miR-155 were dysregulated in sepsis patients. miR-146a downregulation correlated with increased IL-6 expression and monocyte proliferation. Bioinformatics analysis uncovered the immunological associations of dysregulated miRNAs with clinical disease. The current study demonstrates that miRNA dysregulation correlates with clinical manifestations and inflammation, and therefore remains a potential therapeutic target against sepsis.

RNA helicase II/Gu (RH II/Gu) is a nucleolar antigen originally identified using an autoimmune serum from a patient with watermelon stomach. A later report showed that anti-RH II/Gu autoantibodies were also present at low frequency in connective tissue disease (CTD) patients who did not show any symptoms suggestive of a watermelon stomach lesion. In an attempt to understand the relationship between watermelon stomach, also called gastric antral vascular ectasia (GAVE), and autoimmune disorder, we identified the antigenic sites recognized by these autoantibodies. Serum Gu uniquely recognized epitopes at amino acids 646-748 of RH II/Gu and all four CTD patient sera recognized antigenic sites within amino acids 1-173. Anti-RH II/Gu serum produced by immunizing rabbit with recombinant human RH II/Gu protein bound to the same antigenic sites recognized by the CTD patient sera, but it did not recognize the serum Gu epitopes. Results are also presented showing the use of these anti-RH II/Gu antibodies in the analysis of the evolutionary conservation of RH II/Gu in human, monkey and mouse.

Objective To determine the impact of endometriotic lesion removal on local and systemic inflammation. Design Multiplex cytokine analysis on samples from endometriosis patients before surgery, 2 weeks after surgery, and 3 months after surgery. Setting Academic teaching hospital and university. Patient(s) A total of 43 endometriosis patients before and after excision of lesions by means of laparoscopic surgery, and 25 normal women. Intervention(s) None. Main Outcome Measure(s) Plasma, eutopic and ectopic tissue, and peritoneal fluid cytokine levels. Result(s) Compared with presurgery plasma samples, levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL) 2, IL-8, and IL-10 decreased significantly by 2 weeks after surgery in endometriosis patients. Interestingly, levels began to rise at 3 months after surgery in most cases. In tissue, levels of GM-CSF and IL-15 were lower in eutopic tissue, while levels of basic fibroblast growth factor, interferon-inducible protein 10, IL-1 receptor antagonist, granulocyte colony–stimulating factor, macrophage inflammatory protein 1β, IL-7, and IL-5 were higher in eutopic than in ectopic tissue. In peritoneal fluid, levels of IL-5 and IL-12 were higher in early versus advanced stages of endometriosis. Compared with normal women, plasma from endometriosis patients had higher levels of inflammatory cytokines. Conclusion(s) Endometriotic lesion removal significantly alters the inflammatory profile both locally and systemically in women with endometriosis. Our findings indicate that ectopic lesions are the major drivers of systemic inflammation in endometriosis. The transitory nature of the change may reflect the recurrence of the condition and the influence of systemic factors in its onset.

CD44 is a cell-surface glycoprotein involved in many cellular functions including lymphocyte activation, recirculation and homing, hematopoiesis and tumor metastasis, suggesting that CD44 may play an important role in breast cancer development. In this study, we examined whether CD44 exon 2 polymorphisms are associated with increased susceptibility to breast cancer. Direct nucleotide sequencing analysis showed that multiple single nucleotide polymorphisms were present in the CD44 exon 2 coding region in female patients with breast cancer. There was no significant difference in the frequency of any one single nucleotide polymorphism in the CD44 exon 2 coding region between patients with breast cancer and normal donors. However, CD44 polymorphisms in the CD44 exon 2 coding region were identified in approximately 40% of patients with breast cancer, which was significantly higher than in normal donors (odds ratio, 9.34; 95% confidence interval = 2.58-33.82; P < 0.0001). The Wilcoxon-Mann-Whitney test analysis showed that the patients with the CD44 polymorphisms in CD44 exon 2 coding sequence had breast cancer at earlier ages, 49 ± 3 versus 62 ± 2 years (P < 0.0005), and larger tumor burdens (4.9 ± 1.22 vs. 1.6 ± 0.15 mm, P < 0.01) at the time of diagnosis. Interestingly, African-American female patients having the CD44 polymorphisms in CD44 exon 2 coding sequence were diagnosed with breast cancer at very young age (41 ± 2 years). Our results show that CD44 exon 2 polymorphisms are associated with breast cancer development, and such analysis may be effectively used in the evaluation of risk, prediction of cancer, prevention, diagnosis, and epidemiological studies of breast cancer.

Clinical symptoms in MOG-induced EAE mice significantly exacerbated following chondroitin sulfate A (CS-A) injection, whereas administration of a degraded product, CSPG-DS, caused dramatic inhibition of EAE development. Also, administration of CSPG-DS but not CS-A, after the onset of clinical symptoms of EAE, was able to suppress the disease. Further studies demonstrated that CS-A up-regulated STAT4 expression and thus, induced IFN-gamma production and Th1 CD4 T cell differentiation. CS-A also up-regulated STAT3 and IL-23 expression and thus increased IL-17 producing T cells. CSPG-DS treatment both in vivo and in vitro decreased TNFalpha production from splenocytes. In vitro and in vivo studies indicated that CSPG-DS treatment in EAE mice significantly blocked migration of lymphocytes, whereas CS-A treatment increased lymphocyte infiltration in the brain.

Adoptive cell therapy (ACT) with tumor reactive lymphocytes in patients with refractory melanoma can result in tumor regression and prolonged survival. Generating tumor reactive lymphocyte cultures is technically difficult and resource intensive; these limitations have restricted the widespread application of ACT. Tumor infiltrating lymphocytes (TIL) from melanoma contain tumor antigen reactive cells. The “standard” method for producing TIL cultures for clinical administration requires extended in vitro expansion in interleukin-2, then identification of tumor reactive cells by immunological assays. We show here that limitations in reagents and methods during screening under-represent the actual reactivity of TIL cultures. Furthermore, the extended culture times necessitated by the screening assays resulted in telomere shortening and reduced expression of CD27 and CD28 in the TIL cultures, properties that our prior studies showed are correlated within vivo persistence and clinical response. We have thus developed an alternative “young” TIL method that demonstrated superior in vitro attributes compared with standard TIL. This approach utilizes the entire resected tumor to rapidly expand TIL for administration without in vitro testing for tumor recognition. Our observations suggest that younger TIL can have an undetermined but high level of antigen reactivity, and other advantageous attributes such as long telomeres and high levels of CD27 and CD28. We suggest that minimally cultured, unselected lymphocytes represent an alternative strategy for generating TIL cultures suitable for use in ACT therapy that, if effective in vivo, may facilitate the widespread application of this approach to a broader population of patients with melanoma.

Transfer of autologous tumor-specific tumor infiltrating lymphocytes (TILs) in adoptive immunotherapy can mediate the regression of tumor in patients with metastatic melanoma. In this procedure, TILs from resected tumors are expanded in vitro, then administered to patients and further stimulated to proliferate in vivo by the administration of high dose IL-2. After in vitro expansion, TILs are often dominated by a few specific clonotypes, and recently it was reported that the persistence in vivo of one or more of these clonotypes correlated with positive therapeutic response. We and others have previously shown that repeated in vitro stimulation and clonal expansion of normal human T lymphocytes results in progressive decrease in telomerase activity and shortening of telomeres, ultimately resulting in replicative senescence. In the studies reported here, we therefore compared telomerase activity and telomere length in persistent and nonpersistent TIL clonotypes before transfer in vivo, and found a correlation between telomere length and clonal persistence. We also observed that TILs proliferate extensively in vivo in the days after transfer, but fail to induce substantial telomerase activity, and undergo rapid decreases in telomere length within days after transfer. Thus, in vivo loss of telomeres by clonotypes that have the shortest telomeres at the time of administration may drive these clones to replicative senescence, whereas cells with longer telomeres are able to persist and mediate antitumor effects. These findings are relevant both to predicting effectiveness of adoptive immunotherapy and in deriving strategies for improving effectiveness by sustaining telomere length.

Meeting abstracts Cytokine-induced killer (CIK) cells have been extensively used in treatment of cancer patients in China. Clinical trials demonstrated that there were markedly decreased tumor nodules in size in a patient with advanced pancreatic adenocarcinoma following four cycles of CIK cell

The lack of persistence of transferred autologous mature lymphocytes in humans has been a major limitation to the application of effective cell transfer therapies. The results of a pilot clinical trial in 13 patients with metastatic melanoma suggested that conditioning with nonmyeloablative chemotherapy before adoptive transfer of activated tumor-reactive T cells enhances tumor regression and increases the overall rates of objective clinical responses. The present report examines the relationship between T cell persistence and tumor regression through analysis of the TCR β-chain V region gene products expressed in samples obtained from 25 patients treated with this protocol. Sequence analysis demonstrated that there was a significant correlation between tumor regression and the degree of persistence in peripheral blood of adoptively transferred T cell clones, suggesting that inadequate T cell persistence may represent a major factor limiting responses to adoptive immunotherapy.