Characterization of CD103+ CD8+ tissue-resident T cells in esophageal squamous cell carcinoma: may be tumor reactive and resurrected by anti-PD-1 blockade.

Though therapy that promotes anti-tumor response about CD8+ tumor-infiltrating lymphocytes (TILs) has shown great potential, clinical responses to CD8+ TILs immunotherapy vary considerably, largely because of different subpopulation of CD8+ TILs exhibiting different biological characters. To define the relationship between subpopulation of CD8+ TILs and the outcome of antitumor reaction, the phenotype and function of CD103+ CD8+ TILs in esophageal squamous cell carcinoma (ESCC) were investigated. CD103+ CD8+ TILs were presented in ESCC, which displayed phenotype of tissue-resident memory T cells and exhibited high expression of immune checkpoints (PD-1, TIM-3). CD103+ CD8+ TILs were positively associated with the overall survivals of ESCC patients. This population of cells elicited potent proliferation and cytotoxic cytokine secretion potential. In addition, CD103+ CD8+ TILs were elicited potent anti-tumor immunity after anti-PD-1 blockade and were not affected by chemotherapy. This study emphasized the feature of CD103+ CD8+ TILs in immune response and identified potentially new targets in ESCC patients.

Intrinsic Expression of Immune Checkpoint Molecule TIGIT Could Help Tumor Growth in vivo by Suppressing the Function of NK and CD8+ T Cells.

TIGIT, an immune checkpoint molecule widely expressed on NK cells, activated T cells and Tregs, has been involved in delivering inhibitory signals through the interaction with PVR. The blockade of TIGIT/PVR interaction is a promising approach in cancer immunotherapy. Here, we unexpectedly discovered the expression of TIGIT in murine tumor cells. To elucidate the mechanism of such intrinsic expression, TIGIT knockout murine colorectal CT26 and MC38 cell lines were generated by using CRISPR/Cas9 system. Although TIGIT knockout showed no effects on proliferation and colony formation of tumor cells in vitro, the tumor growth in mice was considerably inhibited. TIGIT knockout led to the increase of IFN-γ secretion by NK and CD8+ T cells. Further, in BABL/c nude mice, CD8+ T cells depleting mice and NK cells depleting nude mice, the promotion of tumor growth was significantly diminished, suggesting that both NK cells and CD8+ T cells were involved in the tumor promoting process mediated by intrinsic TIGIT. In addition, blocking TIGIT/PVR interaction by the antibody or recombinant PVR protein could elicit anti-tumor effects by facilitating the tumor infiltration and restoring the function of CD8+ T cells, and the antibody-mediate TIGIT blockade could inhibit MC38 tumor growth through blocking TIGIT expressed on tumor cells. We therefore propose a novel TIGIT/PVR interaction mode that tumor intrinsic TIGIT delivers inhibitory signals to CD8+ T cells and NK cells by engaging with PVR.

Novel epitopes identified from efflux pumps of Mycobacterium tuberculosis could induce cytotoxic T lymphocyte response.

Overcoming drug-resistance is one of the major challenges to control tuberculosis (TB). The up-regulation of efflux pumps is one common mechanism that leads to drug-resistance. Therefore, immunotherapy targeting these efflux pump antigens could be promising strategy to be combined with current chemotherapy. Considering that CD8+ cytotoxic T lymphocytes (CTLs) induced by antigenic peptides (epitopes) could elicit HLA-restricted anti-TB immune response, efflux pumps from classical ABC family (Mycobacterium tuberculosis, Mtb) were chosen as target antigens to identify CTL epitopes. HLA-A2 restricted candidate peptides from Rv2937, Rv2686c and Rv2687c of Mycobacterium tuberculosis were predicted, synthesized and tested. Five peptides could induce IFN-γ release and cytotoxic activity in PBMCs from HLA-A2(+) PPD(+) donors. Results from HLA-A2/K(b) transgenic mice immunization assay suggested that four peptides Rv2937-p168, Rv2937-p266, Rv2686c-p151, and Rv2686c-p181 could induce significant CTL response in vivo. These results suggested that these novel epitopes could be used as immunotherapy candidates to TB drug-resistance.

Blocking of the PD-1/PD-L1 Interaction by a D-Peptide Antagonist for Cancer Immunotherapy.

Blockade of the protein-protein interaction between the transmembrane protein programmed cell death protein 1 (PD-1) and its ligand PD-L1 has emerged as a promising immunotherapy for treating cancers. Using the technology of mirror-image phage display, we developed the first hydrolysis-resistant D-peptide antagonists to target the PD-1/PD-L1 pathway. The optimized compound (D) PPA-1 could bind PD-L1 at an affinity of 0.51 µM in vitro. A blockade assay at the cellular level and tumor-bearing mice experiments indicated that (D) PPA-1 could also effectively disrupt the PD-1/PD-L1 interaction in vivo. Thus D-peptide antagonists may provide novel low-molecular-weight drug candidates for cancer immunotherapy.

The immunogenicity of a novel cytotoxic T lymphocyte epitope from tumor antigen PL2L60 could be enhanced by 4-chlorophenylalanine substitution at position 1.

PIWIL2, a member of PIWI/AGO family, is expressed in germline stem cells and precancerous stem cells, but not in adult somatic cells. PIWIL2 plays an important role in tumor development. It is considered as a cancer­testis antigen (CT80). It has been reported that the spliced fragment of PIWIL2, PL2L60, was widely expressed in cancer cell lines. In this study, HLA-A2-restricted epitopes from PL2L60 were predicted by online tools. To improve the activity of the native epitope, a candidate peptide P281 with potent binding affinity was chosen to investigate the modification strategy. A series of aromatic amino acids were introduced to substitute the first residue of P281. Then, we tested the binding affinity and stability of the peptide analogs and their ability to elicit specific immune responses both in vitro and in vivo. Our results indicated that the cytotoxic T lymphocytes (CTLs) induced by [4-Cl-Phe1]P281 could elicit more potent activities than that of P281 and other analogs. The CTLs induced by this analog could lyze target cells in HLA-A2-restricted and antigen-specific manners. [4-Cl-Phe1]P281 also showed the best resistance against degradation in human serum. In conclusion, the introduction of the unnatural amino acid, 4-Cl-Phe, into the first position could enhance the activity of the native epitope to induce cytotoxic T lymphocytes. It might be a good strategy to modify other promising native epitopes. The novel epitopes identified in this study could be used as novel candidates to the immunotherapy of HLA-A2 positive patients with tumors expressing PL2L60.

In vitro and in vivo identification of a novel cytotoxic T lymphocyte epitope from Rv3425 of Mycobacterium tuberculosis.

The identification of novel cytotoxic T lymphocyte (CTL) epitopes is important to analysis of the involvement of CD8(+) T cells in Mycobacterium tuberculosis infection as well as to the development of peptide vaccines. In this study, a novel CTL epitope from region of difference 11 encoded antigen Rv3425 was identified. Epitopes were predicted by the reversal immunology approach. Rv3425-p118 (LIASNVAGV) was identified as having relatively strong binding affinity and stability towards the HLA-A*0201 molecule. Peripheral blood mononuclear cells pulsed by this peptide were able to release interferon-γ in healthy donors (HLA-A*02(+) purified protein derivative(+)). In cytotoxicity assays in vitro and in vivo, Rv3425-p118 induced CTLs to specifically lyse the target cells. Therefore, this epitope could provide a subunit component for designing vaccines against Mycobacterium tuberculosis.

Identification of novel T cell epitopes from efflux pumps of Mycobacterium tuberculosis.

Cytotoxic T lymphocytes (CTLs) play an important role in the immunity of Mycobacterium tuberculosis (Mtb) infection. In the present study, the identification of novel CTL epitopes from efflux pumps, Rv1258c and Rv1410c, was reported. Candidate native peptides and their analogues were predicted with prediction programs. Rv1410c-p510 (TLAPQVEPL) and Rv1410c-p510-1Y9V (YLAPQVEPV) showed potent binding affinity and stability towards HLA-A*0201 molecule. In enzyme-linked immunospot (ELISPOT) assay, the CTLs induced from peripheral blood mononuclear cells (PBMCs) by these peptides could release interferon-γ (IFN-γ) in at least one healthy donor (HLA-A*02(+), PPD(+)). In cytotoxicity assay in vitro and in vivo, the CTLs induced by Rv1410c-p510-1Y9V could specifically lyse peptide-loaded T2 cells. This is the first report to identify CTL epitopes from the efflux pumps of Mtb. The novel epitope identified could serve as candidate to the multivalent peptide vaccine against drug-resistant M. tuberculosis.

[Expression and significance of membrane skeleton protein 4.1 family in non-small cell lung cancer].

BACKGROUND AND OBJECTIVE: Protein 4.1, a component of cell membrane skeleton, plays a role in maintaining the shape and mechanical stability of erythrocytes. Recent researches showed that protein 4.1 may be associated with the development of tumors. This study was to investigate the expression and significance of membrane skeleton protein 4.1 family members (4.1B, 4.1R, 4.1N and 4.1G) in human non-small cell lung cancer (NSCLC). METHODS: The expression of proteins 4.1B, 4.1R, 4.1N and 4.1G in 147 specimens of NSCLC was detected by EnVision plus immunohistochemistry. The correlations of 4.1B, 4.1R, 4.1N and 4.1G expression to clinicopathologic features of NSCLC were analyzed by Wilcoxon rank sum test and Spearman rank correlation analysis. RESULTS: The protein levels of 4.1B, 4.1R and 4.1N were significantly lower in lung squamous cell carcinoma tissues than in adjacent normal tissues (P<0.01). The protein levels of 4.1B, 4.1R, 4.1N and 4.1G were significantly lower in lung adenocarcinoma tissues than in adjacent normal tissues (P<0.05). The protein levels of 4.1B and 4.1G were significantly lower in lung squamous cell carcinoma tissues than in lung adenocarcinoma tissues (P<0.05). Protein 4.1G expression in squamous cell carcinoma was positively correlated to tumor cell differentiation (rs=0.386,P<0.01). In adenocarcinoma, the expression of proteins 4.1B, 4.1N and 4.1G were positively correlated to tumor cell differentiation (rs=0.276, P<0.05; rs=0.248,P<0.05; rs=0.268, P <0.05). The expression of protein 4.1s in squamous cell carcinoma and adenocarcinoma were not related to lymph node metastasis, tumor size, patients'age and sex (P>0.05). CONCLUSIONS: Protein 4.1s are weakly expressed in NSCLC tissues than in adjacent normal tissues. The expression of proteins 4.1B, 4.1N and 4.1G are related to tumor cell differentiation.

Development and evaluation of a novel multiple-antigen ELISA for serodiagnosis of tuberculosis.

In this study, we describe the development and evaluation of a novel multiple-antigen ELISA for rapid diagnosis and screening of active tuberculosis (TB). The humoral immune responses of 136 active TB patients and 57 healthy subjects against antigens Rv3425, 38kDa and lipoarabinomannan (LAM) from Mycobacterium tuberculosis H37Rv were examined by ELISA. Three essential results were obtained. (i) Rv3425 antigen is a potential candidate for serodiagnosis of active TB. Of 136 active TB patients, Rv3425 antigen provided a sensitivity of 31.6%, lower than that of LAM antigen, but higher than that of 38kDa antigen, with an overall specificity of 100%. (ii) For 62 smear-negative pulmonary TB patients and 15 extra-pulmonary TB patients, the multiple-antigen test provided a sensitivity of 43.5% and 26.7%, respectively, representing an improvement over acid-fast bacilli (AFB) smear-based diagnosis. (iii) Compared with the single-antigen ELISA and the two available commercial kits, the multiple-antigen test offered the highest accuracy (71.0%). In conclusion, the multiple-antigen ELSIA test based on Rv3425, 38kDa, and LAM antigens is a potentially useful tool for the serodiagnosis and screening of active TB. Combinations of Rv3425 with other mycobacterial antigens may also be worthy of further investigation.

Identification of a new broad-spectrum CD8+ T cell epitope from over-expressed antigen COX-2 in esophageal carcinoma.

Cyclooxygenase-2 (COX-2) has been found to be over-expressed in esophageal carcinoma (EC) and it could be considered as a potential tumor-associated antigen (TAA). In the present study, six candidate peptides from COX-2 were firstly predicted and synthesized. Among them, P(479) had the highest affinity and stability toward both HLA-A *0201 and HLA-A *03 molecules and it could significantly promote the IFN-gamma release. The cytotoxic T lymphocytes (CTLs) induced by P(479) could specifically lyse COX-2-expressed EC cell lines, EC-1 (HLA-A3 supertype) and EC-9706 (HLA-A2 supertype). These results suggested that P(479) as a novel broad-spectrum T cell epitope would be very useful in immunotherapy against esophageal carcinoma.

Structure-activity relationships of the dimeric analogues of endomorphin-2 with different lengths of spacers.

In the present study, seven novel dimeric analogues of endomorphin-2 with longer spacers were designed and synthesized. Through dimerization, their affinity for delta-opioid receptor was mostly increased, especially the delta-opioid receptor preferred dimeric analogue, DEM(12). The results were confirmed by the in vitro bioassay. The structure-activity relationships were also discussed.

Entecavir up-regulates dendritic cell function in patients with chronic hepatitis B.

AIM: To investigate the in vitro effect of entecavir (ETV) on the function of dendritic cells (DCs) derived from chronic hepatitis B (CHB) patients. METHODS: Mononuclear cells were isolated from peripheral blood of patients with CHB. DCs were incubated with RPMI-1640 medium supplemented with fetal bovine serum, IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF). DCs were treated with or without ETV on the fourth day. Cell surface molecules, including CD1a, CD80, CD83 and HLA-DR, were assessed by flow cytometry. Concentrations of IL-6 and IL-12 in the supernatant were assayed by enzyme-linked immunosorbent assay (ELISA). The ability of the generated DCs to stimulate lymphocyte proliferation was observed. RESULTS: Compared with CHB control group, the expression levels of CD1a (29.07 +/- 3.20 vs 26.85 +/- 2.80), CD83 (25.66 +/- 3.19 vs 23.21 +/- 3.10), CD80 (28.00 +/- 2.76 vs 25.75 +/- 2.51) and HLA-DR (41.96 +/- 3.81 vs 32.20 +/- 3.04) in ETV-treated group were higher (P < 0.05). ETV-treated group secreted significantly more IL-12 (157.60 +/- 26.85 pg/mL vs 132.60 +/- 22.00 pg/mL (P < 0.05) and had a lower level of IL-6 in the culture supernatant (83.05 +/- 13.88 pg/mL vs 93.60 +/- 13.61 pg/mL, P < 0.05) than CHB control group. The ability of DCs to stimulate the proliferation of allogeneic lymphocytes was increased in ETV-treated group compared with CHB control group (1.53 +/- 0.09 vs 1.42 +/- 0.08, P < 0.05). CONCLUSION: Entecavir can enhance the biological activity of DCs derived from CHB patients.

Effects of lamivudine on the function of dendritic cells derived from patients with chronic hepatitis B virus infection.

AIM: To investigate if the nucleoside analogue lamivudine (LAM), a potent inhibitor of HBV replication, could restore the function of dendritic cells derived from patients with chronic hepatitis B (CHB) in an Asian population. METHODS: Dendritic cells (DCs) derived from mononuclearcytes of patients with chronic HBV infection were cultured in the presence of IL-4, granulocyte-macrophage colony-stimulating factors (GM-CSF) and gradient concentrations of LAM (0-2 mmol/L). Cell morphology was observed under light microscopy. Cell surface molecules, including HLA-DR, CD80, CD83, and CD1alpha, were analyzed with flow cytometry. The concentrations of IL-6 and IL-12 in the supernatant were assayed by ELISA. T cell proliferation was assayed by methyl thiazolyl tetrazolium (MTT). RESULTS: The expression of CD1alpha on DC treated with 0.5 mmol/L LAM (LAM-DC 0.5 mmol/L) was significantly higher than that of DC untreated with LAM (54.1 +/- 4.21 vs 33.57 +/- 3.14, P < 0.05), and so was the expression of CD83 (20.24 +/- 2.51 vs 12.83 +/- 2.12, P < 0.05) as well as the expression of HLA-DR (74.5 +/- 5.16 vs 52.8 +/- 2.51, P < 0.05). Compared with control group, LAM-DC group (0.5 mmol/L) secreted significantly more IL-12 (910 +/- 91.5 vs 268 +/- 34.3 pg/mL, P < 0.05), had lower levels of IL-6 in the culture supernatant (28 +/- 2.6 vs 55 +/- 7.36 pg/mL, P < 0.05), markedly enhanced the stimulatory capacity in the allogeneic mixed leukocyte reaction (MLR) (1.87 +/- 0.6 vs 1.24 +/- 0.51, P < 0.05). CONCLUSION: The lower expression of phenotypic molecules and impaired allogeneic mixed lymphocyte reaction function of dendritic cells derived from patients with HBV infection could be restored in vitro by incubation with LAM.

Effects of cytotoxic T lymphocytes on hepatoma cell line SMMC-7721 induced by different subsets of dendritic cells in vitro.

BACKGROUND: Dendritic cells (DCs) loaded with complex antigen are always used to induce cytotoxic T lymphocytes (CTLs) which have a specific anti-tumor activity. However, CTLs can assault autologous cells induced by DCs loaded with autologous antigen. This study aimed to explore how to weaken the autoimmune reaction induced by DC vaccine by combining mature DC (mDC) activating immunity and immature DC (imDC) leading to immune tolerance to make hepatocellular carcinoma (HCC) vaccine in vitro. METHODS: DC progenitors derived from human peripheral blood were assigned to two groups. One was cultured to mDC and pulsed with frozen-thawed antigen (FTA) of human HCC cell line SMMC-7721 cells (mDC group), and the other was cultured to imDC and pulsed with FTA of human liver cell line L-02 cells (imDC group). The morphology of DCs was monitored and cells phenotypes including HLA-DR, CD80, CD1alpha, CD83 were assayed by flowcytometry (FCM). The concentrations of interleukin-12 (IL-12) in the supernatant were assayed by ELISA. Methyl thiazolyl tetrazolium (MTT) was used to evaluate T cell proliferation induced by mDC and imDC and the killing rate of CTL induced by mDC and imDC respectively/together on SMMC-7721 and L-02 cells. RESULTS: Compared with the imDC group, the mDC group was characterized by the following: increased secretion of IL-12 (P<0.05); higher expression of HLA-DR, CD1alpha, CD80, CD83; and stronger activity in stimulating proliferation of isogenic T cells (P<0.05). CTL induced by the mDC group had a significant killing response to SMMC-7721 as well as a higher killing rate for L-02 (P>0.05). CTL induced by mDC and imDC together had a higher killing response to SMMC-7721, but a lower killing rate for L-02 (P<0.01). CONCLUSIONS: CTL induced by mDC and imDC together has a higher antigen-specific killing response in vitro than that induced by mDC alone. This may be of greater clinical value.

Association of peptic ulcer with increased expression of Lewis antigens, but not vacuolating cytotoxin activity or babA2 gene status, in Helicobacter pylori strains from China.

OBJECTIVE: Controversies exist regarding the virulence factors, such as vacA, babA2 and Lewis blood group antigens, of Western and Asian strains of Helicobacter pylori. The aim of the present study was to determine the significance of these potential virulence factors in the Chinese population. METHODS: Seventy-two strains of H. pylori isolated from patients in Zhengzhou, China, including 43 cases of peptic ulcer (PU) and 29 cases of chronic gastritis, were determined. Vacuolating cytotoxin assay was performed by HeLa cells. The expression of Le blood group antigens (Le(a), Le(b), Le(x) and Le(y)) was performed by enzyme-linked immunosorbent assay (ELISA). babA2 gene was identified by polymerase chain reaction. Frequencies were compared using two-tailed Fisher's exact test. Cytotoxin activities were compared using Spearman's rank correction test. RESULTS: Vacuolating cytotoxin activity was detected in 61 of the 72 strains (84.7%), but there was no significant difference in vacuolating cytotoxin activity (83.7% vs 86.2%, P = 0.821) or titer (4.4 +/- 3.8 vs 4.2 +/- 4.1, P = 0.876) between the PU and gastritis strains. Significantly more PU strains expressed two or more Lewis antigens (Le(x), Le(y), Le(a) or Le(b)) than strains from the chronic gastritis patients (90.7% vs 65.5%, P = 0.029). Of the 43 strains from PU patients, 17 (39.5%) were positive for babA2, compared with 11 (38.5%) of the 29 strains from gastritis patients (P = 0.924). There was no significant difference in the vacuolating cytotoxin activity or titer between strains expressing two or more Lewis antigens and less than two antigens (84.5% vs 85.7%, P = 1.000; 4.4 +/- 4.2 vs 4.3 +/- 3.2, P = 0.965). Of the 72 H. pylori strains, 28 were babA2 positive, of which 24 were cytotoxic, compared with 37 of 44 babA2-negative strains (P = 1.000). CONCLUSION: The present study suggests that PU is associated with increased Lewis antigen expression, but not vacuolating cytotoxin production or the presence of babA2, in the H. pylori strains in the Chinese population.

Endomorphins inhibit contractile responses of rat thoracic aorta rings induced by phenylephrine and angiotensin II in vitro.

AIM: To study the effects of opioid receptor agonists endomorphin-1 and -2 on contractile responses of rat thoracic aorta rings to phenylephrine (PE) and angiotensin II (Ang II), and their possible mechanism in vitro. METHODS: Isometric tension recording was progressed in thoracic aorta rings from Wistar rats. RESULTS: Pretreatment of morphine, endomorphin-1 and -2 (0.1, 1, and 10 micromol/L) could inhibit the contractile responses of the endothelium-intact aorta rings to PE (0.1 micromol/L) and Ang II ( 1 micromol/L) in a concentration-dependent manner (P < 0.01), but could not inhibit the contraction of rings without endothelium (P > 0.05). Naloxone (1 micromol/L) could partially antagonize the effects of endomorphine-1 and -2 (P < 0.01). N(omega)-nitro-L-arginine (L-NNA, 10 micromol/L) or endothelial rubbing could completely blocked the effects of morphine, endomorphine-1 and -2 (P < 0.01). CONCLUSION: Endomorphin-1 and -2 could inhibit PE- and Ang II-induced contractions of rat aorta rings, which was partially by naloxone-sensitive mechanism and related to the release of nitric oxide from vascular endothelium.