Enthalpy-Driven Swelling of Photonic Block Polymer Films

Nonvolatile, soft photonic films that reflect UV/vis light were prepared by enthalpy-driven swelling of lamellar-forming polystyrene-<i>b</i>-poly­(2-vinyl­pyridine) (PS–P2VP) block copolymer thin films with a neat protic solvent. These films are very sensitive to further swelling with the addition of a small amount of acid. Transmission electron microscopy and ultrasmall-angle X-ray scattering of the films before and after the addition of the neat protic solvent revealed selective swelling of the P2VP phase while maintaining the lamellar morphology due to the presence of the layered glassy PS domains. P2VP is swollen due to the hydrogen bonding between a hydroxy group of a protic solvent and the pyridyl group of P2VP. The interdomain distance of the neat PS–P2VP film as measured by U-SAXS increased by about 200% and the films acted as a 1D photonic crystal reflecting UV light. Moreover, by exposing the neat PS–P2VP films to a mixture of a protic solvent and a small amount of sulfonic acid that can protonate the pyridyl groups of the P2VP block, the degree of swelling, therefore the interdomain distance and the wavelength of the reflection light, became significantly larger, resulting in color variations across the visible spectrum and suggesting that such a nonvolatile material system can be a sensor of the acid concentration in the millimolar regime for anhydrous solutions

Image_1_Immunological imprint on peripheral blood in kidney transplant recipients after two doses of SARS-CoV-2 mRNA vaccination in Japan.TIFF

The immunological imprint after two doses of severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2) mRNA vaccination for patients after kidney transplantation (KTx) remain unclear. This study included KTx recipients and volunteer healthy controls (HCs) who received two doses of SARS-CoV-2 mRNA vaccine (Pfizer BioNTech) from January 2021 to December 2021. We analyzed safety within 21 days after each vaccination dose and compared the immune response in peripheral blood mononuclear cells (PBMCs) between the two groups. No graft rejection was observed throughout this study. Adverse events were generally observed within 5 days. The KTx group exhibited a significantly lower degree of symptoms between doses 1 and 2 (P +CD8+ T cells and CD38+CD19+ B cells (P = 0.042 and P = 0.031, respectively). In addition, PD1+CD8+ T cells—but not PD1+CD4+ T cells—increased significantly in the HC group (P = 0.027). In the KTx group, however, activated HLA-DR+, CD38+, and PD1+ cells remained at baseline levels. Immunoglobulin (Ig)G against SARS-CoV-2 was detected in only four KTx recipients (13.3%) after dose 2 (P +CD8+ T cells and ΔCD38+CD19+ B cells were significantly associated with IgG formation (both P = 0.02). SARS-CoV-2 mRNA vaccine generates impaired cellular and humoral immunity for KTx recipients. Results indicate the need for modified vaccination strategies in immunocompromised KTx recipients.</p

Photonic Block Copolymer Films Swollen with an Ionic Liquid

Nonvolatile solvent swollen 1D periodic films were fabricated from lamellae-forming block copolymers with medium molecular weight by infiltrating an ionic liquid. A mixture of imidazole and imidazolium bis­(trifluoromethanesulfonyl)­imide as a room temperature ionic liquid was added after spin-coating of thin films of polystyrene-<i>b</i>-poly­(2-vinylpyridine) (PS–P2VP) block copolymers having an approximately 50/50 composition to create photonic films reflecting in the visible regime. Under normal conditions of temperature and humidity, the films maintained their photonic properties for more than 100 days without perceptible change, stemming from the nonvolatility of the ionic liquid. Transmission electron microscopy revealed the selective swelling of the P2VP nanodomains by the IL and ultrasmall angle X-ray scattering measurements provided quantitative nanostructure information on the periodicities of the films. The wavelength of reflected light from photonic films was tunable by using different molecular weight block copolymers as well as by employing blends of two block copolymers. The experimental wavelength of the reflected light, detected by a fiber-optic spectrophotometer, agreed with values estimated from the Bragg condition and was able to be controlled from about 380 to 620 nm

Generation of Large Numbers of Antigen-Expressing Human Dendritic Cells Using CD14-ML Technology

<div><p>We previously reported a method to expand human monocytes through lentivirus-mediated introduction of cMYC and BMI1, and we named the monocyte-derived proliferating cells, CD14-ML. CD14-ML differentiated into functional DC (CD14-ML-DC) upon addition of IL-4, resulting in the generation of a large number of DC. One drawback of this method was the extensive donor-dependent variation in proliferation efficiency. In the current study, we found that introduction of BCL2 or LYL1 along with cMYC and BMI1 was beneficial. Using the improved method, we obtained CD14-ML from all samples, regardless of whether the donors were healthy individuals or cancer patients. <i>In vitro</i> stimulation of peripheral blood T cells with CD14-ML-DC that were loaded with cancer antigen-derived peptides led to the establishment of CD4⁺ and CD8⁺ T cell lines that recognized the peptides. Since CD14-ML was propagated for more than 1 month, we could readily conduct genetic modification experiments. To generate CD14-ML-DC that expressed antigenic proteins, we introduced lentiviral antigen-expression vectors and subjected the cells to 2 weeks of culture for drug-selection and expansion. The resulting antigen-expressing CD14-ML-DC successfully induced CD8⁺ T cell lines that were reactive to CMVpp65 or MART1/MelanA, suggesting an application in vaccination therapy. Thus, this improved method enables the generation of a sufficient number of DC for vaccination therapy from a small amount of peripheral blood from cancer patients. Information on T cell epitopes is not necessary in vaccination with cancer antigen-expressing CD14-ML-DC; therefore, all patients, irrespective of HLA type, will benefit from anti-cancer therapy based on this technology.</p></div

Induction of CD8⁺ T cell lines that are reactive to cancer antigens by CD14-ML-DC.

<p>(A) Protocol for the induction of cancer antigen-specific CD8⁺ T cells by CD14-ML-DC. In order to generate CD14-ML-DC, we added IL-4 to CD14-ML. After 3 days, we added OK432. CD14-ML-DC were pulsed with peptides for 3 h, X-ray-irradiated (45 Gy), and subsequently mixed with autologous CD8⁺ T cells. Cells were cultured with rIL-7 (10 ng/ml) in AIM-V with 5% human decomplemented plasma. On days 7 and 14, the T cells were restimulated with the autologous peptide-pulsed CD14-ML-DC and on days 9 and 16, and were supplemented with rIL-2 (20 IU/ml). CD14-ML-DC were prepared each time, and we only added IL-4 (did not add OK432). IFN-γ ELISPOT assay and flow cytometry were performed after 6 or 7 days from the third round of peptide stimulation. (B, C) Peripheral blood CD8⁺ T cells were obtained from a HLA-A*24:02-positive healthy donor (healthy donor 1) and were co-cultured with 4 peptides (CDCA1_56-64, KIF20A_66-75, LY6K_177-186 and IMP-3_508–516)-loaded autologous CD14-ML-DC. (B) On day 21, the number of IFN-γ producing CD8⁺ T cells were analyzed by ELISPOT assay (Day 21). The results of the T cells before stimulation culture are also shown (Day 0). The HIV-peptide was used as a control peptide. (C) On day 21, the T cells were recovered and stained with anti-CD8 mAb and the HLA-A*24:02/CDCA1_56-64 or HLA-A*24:02/LY6K_177-186 tetramer. The numbers in the figure indicate the percentage of the CD8⁺ T cells that were positively stained with the tetramer of the HLA-peptide complex (Day 21). The results of the T cells before stimulation culture are also shown (day 0). (D, E) A similar experiment as in (B, C) was done with the cells obtained from a HLA-A*02:01-positive donor (healthy donor 2). We used 4 peptides (CDCA1_351-359, KIF20A_809-817, MART1_26-35 and IMP3_515-523) for the stimulation of the T cells. (D) The number of IFN-γ producing CD8⁺ T cells was analyzed by ELISPOT assay. (E) The T cells were recovered and stained with an anti-CD8 mAb and a HLA-A*02:01/MART1_26-35 dextramer, HLA-A*02:01/CDCA1_351-359 tetramer or HLA-A*02:01/IMP3_515-523 tetramer. The numbers in the figure indicate the percentage of the CD8⁺ T cells that were positively stained with the dextramer or tetramer of HLA-peptide complex.</p

Induction of CD8⁺ T cell lines that are reactive to antigens by CD14-ML-DC that express antigenic proteins.

<p>(A) The lentivirus constructs for CMVpp65 (EF-CMV-IP) and MART1 (EF-MART1-IP) are shown. CD14-ML were transduced with the lentivirus vector and cultured in the presence of puromycin (5 μg/ml) to select and expand the cell population carrying the transgene, resulting in the generation of CD14-ML/CMV and CD14-ML/MART1. (B) Expression of CMVpp65 by CD14-ML/CMV was analyzed by flow cytometric analysis. The staining profiles of the specific mAb (black lines) and isotype-matched control mAb (gray area) are shown. (C) CD14-ML-DC/CMV derived from a HLA-A*24:02-positive healthy donor were cultured with autologous CD8⁺ T cells. On day 9, the number of T cells reactive to the CMVpp65_341-349 peptide was analyzed by ELISPOT assay. The HIV-peptide was used as a control peptide. The results of the T cells before stimulation culture are shown (Day 0). (D) On day 9, the T cells were recovered and stained with an anti-CD8 mAb and a tetramer of HLA-A*24:02/CMVpp65_341-349 complex. The numbers in the figure indicate the percentage of the CD8⁺ T cells positively stained with the tetramer of the HLA-peptide complex. The results of the T cells before stimulation culture are also shown (Day 0). (E) CD8⁺ T cells obtained from an HLA-A*24:02-negative healthy donor were co-cultured with autologous CD14-ML-DC/CMV. On day 9, the number of IFN-γ producing CD8⁺ T cells was analyzed by ELISPOT assay, using CD14-ML and CD14-ML/CMV as stimulators. The results of the T cells before stimulation culture are also shown (Day 0). (F) Expression of MART1 by CD14-ML/MART1 was analyzed by flow cytometric analysis. The staining profiles of the specific mAbs (black lines) and isotype-matched control mAbs (gray area) are shown. (G) CD8⁺ T cells obtained from an HLA-A*02:01-positive healthy donor were co-cultured with autologous CD14-ML-DC/MART1 cells. On day 21, the frequency of CD8⁺ T cells reactive to MART1_26-35 was analyzed by ELISPOT assay. The HIV-peptide was used as a control peptide. The results of the T cells before stimulation culture are also shown (Day 0). (H) On day 21, the T cells were recovered and stained with an anti-CD8 mAb and HLA-A*02:01/MART1_26-35 dextramer. The numbers in the figure indicate the percentage of the CD8⁺ T cells that were positively stained with the dextramer of the HLA-peptide complex. The results of the T cells before stimulation culture are also shown (Day 0). (I) CD8⁺ T cells obtained from an HLA-A*02:01-negative healthy donor were co-cultured with autologous CD14-ML-DC/MART1. On day 21, the frequency of CD8⁺ T cells reactive to MART1 was analyzed by ELISPOT assay, using CD14-ML-DC and CD14-ML-DC/MART1 as stimulators. The results of the T cells before stimulation culture are also shown (Day 0).</p

Induction of CD4⁺ T cell lines that are reactive to cancer antigens by CD14-ML-DC obtained from HNC patients.

<p>CD4⁺ T cells isolated from PBMCs of HNC patients were stimulated with CD14-ML-DC that were pulsed with a mixture of 6 kinds of peptides (CDCA1_39-64, CDCA1_55-78, KIF20A_60-84, KIF20A_809-833, LY6K_119-142 and LY6K_172-191). After more than three rounds of stimulation, the number of CD4⁺ T cells that reacted to each peptide was analyzed by ELISPOT assay. CD14-ML-DC were used as stimulators in the assay, because only a few amount of blood samples could be obtained from cancer patients. The results for cancer patient 1 (A) and cancer patient 2 (B) are shown.</p

Cell surface molecules of CD14-ML-DC and the production of IL-12p70 by CD14-ML-DC.

<p>(A) CD14-ML-DC, before and after treatment with OK432, were analyzed for the expression of CD40, CD80, CD83, CD86, HLA class I and HLA class II. As a control, results of the analysis of OK432-stimulated mo-DC (monocyte-derived DC) are also shown. The staining profiles of the specific mAbs (black lines) and isotype-matched control mAbs (gray area) are shown. (B) CD14-ML-DC and mo-DC were cultured in 96-well flat-bottomed culture plates (1×10⁵ cells/200 μl medium/well) in the presence OK432 (10 μg/ml). After 60 h, the concentration of IL-12p70 in the culture supernatant was measured by ELISA. The data are representative of 2 experiments.</p

Morphology of CD14-ML and CD14-ML-DC generated by the current procedure.

<p>(A) Phase-contrast images of live cells (left) and cytospin samples stained with May–Grünwald Giemsa (right) of the human monocytes and monocyte-derived myeloid cell lines (CD14-ML) are shown. (B) Morphology of OK432-stimulated mo-DC and CD14-ML-DC are shown. mo-DC and CD14-ML-DC were stimulated with OK432 for 2 days and subjected to microscopic analysis. The data are representative of 2 experiments.</p

Induction of CD4⁺ T cell lines that are reactive to cancer antigens by CD14-ML-DC.

<p>(A) Protocol for the induction of cancer antigen-specific CD4⁺ T cells by CD14-ML-DC. In order to generate CD14-ML-DC, we added IL-4 to CD14-ML. After 3 days, we added OK432. CD14-ML-DC were pulsed with a mixture of 6 peptides (CDCA1_39-64, CDCA1_55-78, KIF20A_60-84, KIF20A_809-833, LY6K_119-142 and LY6K_172-191) for 3 h, X-ray-irradiated (45 Gy), and subsequently mixed with autologous CD4⁺ T cells in AIM-V with 5% human decomplemented plasma. On day 7, the T cells were restimulated with the autologous peptide-pulsed CD14-ML-DC and supplemented with rIL-7 (5 ng/ml). After two days, these cultures were supplemented with rIL-2 (10 IU/ml). CD14-ML-DC were added with only IL-4 (did not add OK432). On day 14, the stimulated CD4⁺ T cells in each well were analyzed for specificity in IFN-γ ELISPOT assays. The T cells showing a specific response to the cognate peptide were transferred to 24-well plates and restimulated with the autologous peptide-pulsed CD14-ML-DC, and subsequently supplemented with rhIL-7 (5 ng/ml) and rhIL-2 (20 IU/ml). On day 21, the T cells were restimulated with the autologous peptide-pulsed CD14-ML-DC and supplemented with rhIL-7 and rhIL-2. IFN-γ ELISPOT assays were performed after 6 or 7 days from the fourth round of peptide stimulation. (B, C) After the stimulation (more than three times), the number of CD4⁺ T cells reacting to each peptide was analyzed with an IFN-γ ELISPOT assay (Day 28). The results of the T cells before stimulation culture are shown (Day 0). Dimethyl sulfoxide was used as a control. The results for the healthy donor 3 (B) and donor 2 (C) are shown.</p