CD83 expression regulates antibody production in response to influenza A virus infection

Madin–Darby Canine Kidney (MDCK) cell lines used for this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown on Minimum Essential Medium (MEM) with 10% fetal bovine serum (FBS), 100 µg/ml streptomycin and 100 U/ml penicillin. The influenza A virus, A/WSN/1933 (H1N1), was obtained from Professor Man-Seong Park (Korea University, Seoul, Korea). The virus was amplified using specific-pathogen-free (SPF) embryonated eggs followed by infecting the MDCK cell lines. MDCK cells (2 × 10⁵/well) were cultured in 6-well plates using MEM media containing 10% FBS at 37 °C overnight in a CO₂ incubator. After the overnight culture, the cells were washed with PBS, and each well was infected with the influenza A virus at MOI 0.01 in MEM media containing 1 µg/ml l-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin and then incubated at 37 °C in CO₂ incubator. After 1 h incubation, the supernatants were removed and then cultured for 3 days in MEM media containing 0.3% BSA at 37 °C in a CO₂ incubator. The virus culture supernatants were collected and centrifuged at 2,000 rpm for 10 min at 4 °C to remove the cell debris. The quantification of the amplified viruses was performed by plaque assay. MDCK cells (6 × 10⁵/well) were cultured in 6-well plates. The cells were cultured, washed with PBS, and infected with the amplified influenza A virus culture supernatants after a ten-fold serial dilution. After 1 h incubation, the supernatants were removed and overlaid with 2 ml of DMEM/F12 media containing 2 mM glutamine, 4% BSA, 10 mM HEPES, 2.5% sodium bicarbonate, 50 mg/ml DEAE dextran, 1 µg/ml l-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin, 100 U/ml penicillin, 100 μg/ml streptomycin and 0.6% immunodiffusion-grade agar. After 72 h incubation, the plates were stained with crystal violet (0.1% crystal violet in 20% methanol) for 1 h. The plaques were counted to determine the virus titers. The entire process for the virus preparation and cell culture procedure was conducted in a biosafety level 2 laboratory.

Eight-week-old female C57BL/6J mice were purchased from NARA Biotech, Inc. (Seoul, Korea), and C57BL/6J CD83 KO mice (B6.129S4-Cd83^tm1Tft/J, JAX stock #017703) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were maintained at the Animal Center of Hallym University under specific pathogen-free conditions (20–25 °C, 40–45% humidity, 12 h light/dark cycle, and food and water access, ad libitum). Mice experiments were performed in a biosafety level 2 facility in the Hallym Clinical and Translational Science Institute.

A/WSN/1933 virus inactivation was performed by 254 nm UV light exposure with 1,500 mW/s/cm² UV for 15 min from a height of 5 cm. The virus inactivation was verified by a plaque assay with MDCK cells [27]. The live A/WSN/1933 virus or UV-inactivated A/WSN/1933 virus was intraperitoneally infected at a dose of 5 × 10⁶ pfu per mouse and CD83 expression in the peritoneal cavity, bone marrow and spleen cells was observed at 1 h, 3 h, 6 h, and 24 h after infection. Changes in lymphocyte population were observed at 5, 7 and 14 days after live A/WSN/1933 virus infection. The production of virus-specific antibody was measured at 14 days after live A/WSN/1933 virus infection.

C57BL/6J wild type or C57BL/6J CD83 KO mice were infected intraperitoneally with 5 × 10⁶ pfu of A/WSN/1933 virus. After the virus infection, cells of the peritoneal cavity, splenocytes, and cells of the bone marrow were harvested in RPMI 1640 media containing 5% FBS. The cells were centrifuged at 1,200 rpm and 4 °C for 5 min, and the pellets were treated with RBC lysis buffer (20 mM Tris HCl and 140 mM NH₄Cl) for 5 min. After washing with RPMI media, total cell numbers of peritoneal cavity, spleen and bone marrow were counted and analyses of cell populations were performed as described previously (26). The cells were washed with FACS buffer (1% FBS in PBS) and transferred into Falcon tubes and then treated with 10 µg/ml anti-FcγRII/III antibody (Catalogue No: 553142, BD Biosciences, San Jose, CA, USA). To analyze lymphoid population, PerCP Cy5.5-conjugated anti-CD3 antibody (Catalog No: 551163, BD Biosciences), Pacific blue ef450-conjugated anti-CD4 antibody (Catalog No: 558107, BD Biosciences), FITC-conjugated anti-CD8 antibody (Catalog No: 553031, BD Biosciences), APC-Cy7-conjugated anti-CD19 antibody (Catalog No: 557655, BD Biosciences), PE-conjugated anti-CD23 antibody (Catalog No: 12-0232-81, eBioscience, San Diego, CA, USA), and APC-conjugated anti-CD83 antibody (Catalog No: 558208, BD Biosciences) were used. We pre-gated peritoneal cells, bone marrow cells, splenocytes (FSC^lowSSC^low) for the enriched lymphoid population and then analyzed specific cell population with a FACSCanto™ II Flow Cytometry (BD Biosciences).

For the detection of A/WSN/1933 virus-specific IgG, the subclasses of IgG, ninety-six well Immuno plates (Nunc™, Roskilde, Denmark) were coated with the A/WSN/1933 virus. After overnight incubation at 4 °C, the plates were washed three times with PBST (0.1% Tween-20 in PBS) and blocked with 1% BSA for 1 h and then loaded with three-fold dilutions of sera and peritoneal supernatants in PBST. After incubation at room temperature for 2 h, the plates were washed three times with PBST and goat anti-mouse IgG/IgG1/IgG2a/IgG2b/IgG3 antibodies labeled with Horseradish peroxidase (Southern Biotechnology Associates, Inc. Birmingham, AL, USA) with dilutions 1:500 were used to bind total IgG and each subclasses antibody. The plates were incubated for 1 h at room temperature, and then, TMB substrate solution was added (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA). The absorbance was measured by a Spectra Max 250 microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 450 nm.

Results are shown as the mean ± standard deviation. The statistical significance of the differences between the two samples was evaluated using Student’s t-test with P < 0.05 as the threshold for statistical significance.

Mice experimental procedures were undertaken following the guidelines of Laboratory Animals of the National Veterinary Research and Quarantine Service of Korea. Animal use and related experimental procedures were approved by the Institutional Animal Care and Use Committee of Hallym University (Permit Number: Hallym R2/2018-25). Mice were anesthetized with 3% to 5% isoflurane (Pharmaceutical, Seoul, Korea) inhalation to minimize any pain. The mice were sacrificed by CO₂ inhalation after the experiments and euthanized by CO₂ inhalation if they lost 25% of their baseline adult body weight or if they revealed evidence of debilitation, pain or distress such as a hunched posture, rough hair coat, reduced food consumption, emaciation, inactivity, ambulation difficulty, and respiratory problems, and all efforts were made to limit suffering.

B cells perform a vital function in combating infection through the production of antibodies and cytokines and antigen presentation to CD4⁺ and CD8⁺ T cells [28]. Because CD83 has an important role in the development and function of B cells and T cells [17], we investigated the role of CD83 in B cell function during influenza A virus infection in mice. To determine the effect of influenza A virus on CD83 expression in peritoneal cells, C57BL/6J wild type mice were intraperitoneally injected with 5 × 10⁶ plaque forming units (pfu)/mouse of live or UV-inactivated A/WSN/1933 virus. FACS analysis results showed that the expression of CD83 on CD19⁺ peritoneal B cells was drastically increased at 3 h and then restored to basal level at 24 h after infection with live A/WSN/1933 virus (Fig. 1b). The expression of CD83 on CD19⁺ B cells was also increased at 3 h in the mice infected with UV-inactivated WSN virus; however, the levels of CD83 expression at 6 h were substantially lower than those of live virus-infected mice (Fig. 1a). CD83 expression on CD19⁺ B cells in mice infected with UV-inactivated virus was also restored to basal level at 24 h. The expression of CD83 on CD19⁺ B cells from bone marrow and the spleen was not affected by live or UV-inactivated A/WSN/1933 virus-infection (Fig. 1b). However, CD3⁺ T cells from the peritoneum cavity, bone marrow, and spleen showed no difference in expression of CD83 upon infection with either live or UV-inactivated virus (Fig. 1c). These results show that infection of influenza A virus leads to a transient but distinct increase of CD83 expression in the surface of peritoneal B cells. As overall CD83 expression patterns induced by live or UV-inactivated virus are similar, we then performed further experiments with live A/WSN/1933 virus using PBS as a control.

Previously, we have reported that intraperitoneal inoculation with the A/WSN/1933 virus induced a substantial but transient B cell depletion at 12 h post-infection, which was partially recovered at 7 days with a gradual restoration to normal levels by 14 days [26]. To investigate the effect of CD83 expression in the lymphocyte population and its restoration pattern in the peritoneal fluid, bone marrow, and spleen, wild type mice and CD83 KO mice were intraperitoneally injected with the live A/WSN/1933 virus at a dose of 5 × 10⁶ pfu/mouse. Peritoneal cells, bone marrow cells, and splenocytes were harvested at 5, 7, and 14 days post-infection, and cell populations were analyzed by flow cytometry. In the wild type mice, depletion of CD19⁺ B cells was observed at 5 days post-infection in the peritoneal cavity (Fig. 2a) and bone marrow (Fig. 2b), followed by a partial recovery at 7 days and restoration to near-normal levels at 14 days post-infection as previously described [26]. In CD83 KO mice, transient depletion and then recovery to the normal level were observed in CD19⁺ B cells of the peritoneal cavity similar to wild type mice (Fig. 2a). However, the CD19⁺ B cells in bone marrow exhibited an incomplete recovery in the CD83 KO mice until 14 days post-infection, suggesting a delay in the restoration process (Fig. 2b). On the other hand, the peritoneal cells exhibited an increment in CD3⁺ T cells for both the wild type and CD83 KO mice after infection although the amounts of T cells at 7 and 15 days post-infection were higher in the wild type mice than in the CD83 KO mice (Fig. 2a). Furthermore, the basal levels of CD3⁺ T cells in the peritoneal cavity of the CD83 KO mice were much lower (about 50%) compared to those of wild type mice (Fig. 2a). The T cell numbers of the bone marrow declined at day 5 and slowly recovered at day 7 and 14 in the wild type mice and CD83 KO mice (Fig. 2b). In the spleen, the CD19⁺ B cells and CD3⁺ T cells were decreased at 5 days post-infection, followed by a partial recovery at 7 days and restoration to near-normal levels at 14 days post-infection with no prominent difference between the wild type mice and CD83 KO mice (Fig. 2c). Collectively, these results show the requirement of CD83 for the homeostasis of T cells in the peritoneal cavity fluids and for the restoration of B cells in the bone marrow following influenza A virus infection.

B cell populations are composed of subclasses B-1 and B-2 cells. B-1 cells, unlike the classical B-2 cells, are characterized by a low level of CD23 expression and participates in the antibody response during vaccination and infection [29]. Therefore, we analyzed the B-1 and B-2 cells in the peritoneal cavity fluids after intraperitoneal infection of live A/WSN/1933 virus. The depletion of both the B-1 and B-2 cells was observed at 5 days post-infection, followed by partial recovery at 7 days in both wild type mice and CD83 KO mice (Fig. 3a). While B-2 cells had a full restoration of their cell numbers at day 14, B-1 cells were unable to recover to a normal level. However, there was no significant difference between the wild type and CD83 KO mice (Fig. 3a). Next, we checked the T cell populations and found that after the virus inoculation, the CD8⁺ T cell number underwent continuous expansion as observed at day 5, 7, and 14 in both the wild type (threefold) and CD83 KO (fivefold) mice (Fig. 3b). In contrast, CD4⁺ T cells exhibited an altered behavior between the wild type and CD83 KO mice. As observed earlier, the T cell population was smaller in the CD83 KO mice [30]. In the wild type mice, the CD4⁺ T cell number was reduced at day 5 post-infection and later was restored to the normal level by day 14. However, the CD4⁺ T cell number mildly increased after infection in the CD83 KO mice; however, the total number of cells in the CD83 KO mice was still much lower compared to the wild type mice (Fig. 3b). Overall, these results suggest that CD83 loss could lead to differential properties, especially in the T cell population in the peritoneal cavity.

Upon infection, some of the naïve B cells are converted to plasma cells that produce antibodies against the antigenic components of invading pathogens, which has an essential role in the clearance of pathogens [31]. Antibody production by B cells could be CD4⁺ T cell-dependent or independent [32]. Because the loss of CD83 affected the B cell population and CD4⁺ T cell population after infection, we investigated the effect of CD83 expression on antibody production in response to virus infection by measuring the level of virus-specific IgG and its subclasses in the peritoneal cavity fluids and serum at 5, 7 and 14 days post-infection. There were no detectable virus-specific IgG in the PBS control mice. A negligible amount of the virus-specific IgG was produced in the peritoneal cavity fluids and serum at day 5 and 7 post-infection, which later increased to a considerable value by day 14 in the wild type mice. Notably, IgG2b and IgG3 were the major IgG isotypes in the peritoneal cavity fluids (Fig. 4a), whereas IgG2b was the most prominent in the serum (Fig. 4b). Conversely, the CD83 KO mice failed to produce virus-specific antibodies comparable to the wild type mice (Fig. 4). Although IgG levels were increased at day 14 in the CD83 KO mice, the amount was significantly lower compared to the wild type mice both in the peritoneal cavity fluids (Fig. 4a) and serum (Fig. 4b). Taken together, these results suggest that CD83 has an important role in B cell response to influenza A virus infection with a direct influence on the production of virus-specific antibodies.