IL-4+TGF-β in presence of pbCD3/sCD28 activation induce generation of CD4⁺IL-9⁺ T cells.

<p><b>A)</b> CD4⁺CD25⁻ T cells (1.0×10⁶/ml in 24 well plates) were activated with plate bound-anti-CD3 mAb (pbCD3)/soluble-anti-CD28 mAb (sCD28) in presence or absence of IL-4 or TGF-β or IL-4+TGF-β for 96hrs and analyzed by flow cytometry for IL-9 expression. IL-4+TGF-β in combination induced significantly higher percentage of CD4⁺ T cells positive for IL-9, as compared to IL-4, TGF-β, or neither (<i>n = 14</i>). Data is expressed as the mean±SD. <b>B)</b> CD4⁺CD25⁻ T cells, CD4⁺CD25⁻CD45RA⁺ T cells (naïve T cells), CD4⁺CD25⁻CD45RO⁺ T cells (resting memory T cells) (2.0×10⁵/ml in 96 well plates) were activated with pbCD3/sCD28 in presence of IL-4+TGF-β in a 96 well plate for 96hrs. Cells were surface stained for CD4 PerCP-Cy5.5 and intracellular stained for IL-9 PE. IL-4+TGF-β in combination induced IL-9 expression by both naïve and memory T cells, but memory T cells expressed high levels of IL-9. Data are representative of six independent experiments (six different donors).</p

Cytokine and transcription factor profile of memory CD4⁺CD25⁻CD45RO⁺ T cells activated with IL-4+TGF-β.

<p><b>A)</b> Log-transformed quantities of cytokines (pg/ml) are shown. CD4⁺CD25⁻CD45RO⁺ T cells (1.0×10⁶/ml in 24 well plates) were activated with pbCD3/sCD28 in presence or absence of IL-4+TGF-β. Supernatants were collected at 96hrs post activation and IFNγ, IL-2, IL-5, IL-9, IL-10, IL-13, and IL-17 were quantified by ELISA. IL-4+TGF-β treated CD4⁺ T cells produced significantly high IL-2 and IL-9, but significantly low IFNγ, IL-13, and IL-17, as compared to CD4⁺CD25⁻CD45RO⁺ T cells not treated with IL-4+TGF-β (<i>n = 3</i>). Data is expressed as the mean±SD. <b>B)</b> Log-transformed ratios of mRNA copies to GAPDH mRNA copies for GATA3, RORC, IL-9, and Tbet are shown. CD4⁺CD25⁻CD45RO⁺ T cells (1.0×10⁶/ml in 24 well plates) were activated with pbCD3/sCD28 in presence of IL-4+TGF-β. Cells were harvested and single cell sorted. IL-9 transcripts were quantified by qt-RT-PCR. 10,000 cells comprising of total cell population (TC) was also taken and the gene expression was averaged for single cell for reference. Cells positive for IL-9 transcripts were further quantitated for GATA3, RORC, and Tbet (<i>n</i> = 3). As IL-9 is expressed in only 10% of all CD4⁺ T cells activated with pbCD3/sCD28 in presence of IL-4+TGF-β, average IL-9 mRNA copies of TC are always lower than that of a single IL-9⁺ cell. CD4⁺IL-9⁺ T cells expressed GATA3 and RORC, but not Tbet. <b>C)</b> CD4⁺CD25⁻CD45RO⁺ T cells (1.0×10⁶/ml in 24 well plates) were activated with pbCD3/sCD28 in presence of IL-4+TGF-β. Cells were surface stained for CD4 and intracellular stained for IL-9 and FOXP3. Cells were gated for CD4 and then IL-9⁺ or/and FOXP3⁺ cells were analyzed. 25% of CD4⁺IL-9⁺ T cells were also FOXP3⁺. Data are representative of seven independent experiments. <b>D)</b> CD4⁺CD25⁻CD45RO⁺ T cells (1.0×10⁶/ml in 24 well plates) were activated with pbCD3/sCD28 in presence or absence of IL-4 or TGF-β or IL-4 plus TGF-β for 96hrs and analyzed by flow cytometry for FOXP3 expression. IL-4 significantly inhibited TGF-β induced FOXP3 expression (<i>n = 3</i>). Data is expressed as the mean±SD.</p

IL-1β amplifies IL-4+TGF-β induced IL-9 production by memory CD4⁺CD25⁻CD45RO⁺ T cells.

<p>Resting memory CD4⁺CD25⁻CD45RO⁺ T cells (2.0×10⁵/ml in 96 well plates) activated with pbCD3/sCD28 alone or with IL-4+TGF-β, in the presence or absence of IL-1β, IL-2, IL-6, IL-12, and IL-21 for 96hrs and supernatants were collected. Influence of IL-1β, IL-2, IL-6, IL-12, and IL-21 on IL-9 production of IL-4+TGF-β treated CD4⁺CD25⁻CD45RO⁺ T cells was examined. IL-1β, IL-12 or IL-21 significantly elevated IL-4+TGF-β induced IL-9 production, but IL-1β had significantly higher influence compared to IL-12 or IL-21 (<i>n = 3</i>). Data is expressed as the mean±SD.</p

CD4⁺CD25⁻ T cells activated with IL-4+TGF-β express more IL-9 than Th1, Th2, Th17, or iTregs.

<p>CD4⁺CD25⁻ T cells (1.0×10⁶/ml in 24 well plates) were activated with pbCD3/sCD28 in presence or absence of IL-4+TGF-β or Th1-, Th2-, Th17-, iTreg-polarizing condition for 96hrs. Cells were harvested, gated on CD4⁺ T cells, and were analyzed for IL-9⁺ cells by flow cytometry or were used to quantitate IL-9 transcripts by real-time PCR. Data is expressed as the mean±SD. (A) 2% of Th2 cells, 4% of iTregs, or 10% of cells treated with IL-4+TGF-β in combination were IL-9⁺, whereas Th1-, Th17-, or Th0-cells had negligible number of IL-9⁺ cells (<i>n = 7</i>); (B) Log-transformed ratios of IL-9 mRNA copies to 18S rRNA are shown. Cells treated with IL-4+TGF-β in combination had significantly higher levels of IL-9 mRNA as compared to polarized Th1-, Th2-, Th17-, iTreg-, or Th0-cells (<i>n</i> = 9).</p

Virus-Like Particles of SARS-CoV‑2 as Virus Surrogates: Morphology, Immunogenicity, and Internalization in Neuronal Cells

The engineering of virus-like particles (VLPs) is a viable strategy for the development of vaccines and for the identification of therapeutic targets without using live viruses. Here, we report the generation and characterization of quadruple-antigen SARS-CoV-2 VLPs. VLPs were generated by transient transfection of two expression cassettes in adherent HEK293T cellsone cassette containing Mpro for processing of three structural proteins (M, E, and N), and the second cassette expressing the Spike protein. Further characterization revealed that the VLPs retain close morphological and antigenic similarity with the native virus and also bind strongly to the SARS-CoV-2 receptor hACE-2 in an in vitro binding assay. Interestingly, the VLPs were found to internalize into U87-MG cells through cholesterol-rich domains in a dynamin-dependent process. Finally, our results showed that mice immunized with VLPs induce robust humoral and cellular immune responses mediated by enhanced levels of IL-4, IL-17, and IFNγ. Taken together, our results demonstrate that VLPs mimic the native virus and induce a strong immune response, indicating the possible use of these particles as an alternative vaccine candidate against SARS-CoV-2. VLPs can also be effective in mapping the initial stages of virus entry and screening inhibitors