The adoptive transfer of regulatory B lymphocytes prevents severe damage to lung tissues during respiratory infection with the influenza A/H1N1 virus

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Introduction

Every year, healthcare systems in all countries face the enormous challenge of preventing, treating, and eliminating the consequences of respiratory infection epidemics [1–3]. An important factor aggravating the course of respiratory infections is the development of uncontrolled inflammation in the lungs, which leads to significant damage and respiratory failure [4, 5]. Systemic inflammation and cytokine storm in the novel SARS-CoV-2 infection have attracted particular attention [6–9]. However, this problem has been noted before, for example, in severe damage to the lower respiratory tract during influenza virus infection (this was especially characteristic of variants of the pathogen with high epidemiological potential, such as H1N1). In these cases, the infection is accompanied by significant infiltration of lung tissue by neutrophils and an uncontrolled inflammatory process in the lungs [10, 11]. Significant neutrophil infiltration of the lower respiratory tract during influenza virus infection has also been demonstrated in animal models [12]. At the same time, uncontrolled production of pro-inflammatory factors significantly aggravates lung damage [13]. Thus, the shift in balance from a specific antiviral response towards excessive involvement of innate defense mechanisms can lead to significant damage to lung tissue [14] and significantly exacerbate its destruction [15]. As a result of such an uncontrolled immune response, lung tissue is damaged much more severely than under the direct effect of the virus. Based on this, it is of particular interest to study the mechanisms of regulation of excessive inflammation in lung tissue against the background of respiratory infections.

The natural mechanisms of immune response regulation are quite diverse. The key effectors in this process are two groups of immunocompetent cells: regulatory T lymphocytes (T_reg) [16] and regulatory B lymphocytes (B_reg) [17]. In recent years, much attention has been paid to studying the functional activity of T_reg, while the role of B_reg in the immune response to infectious diseases has been virtually unexplored. Nevertheless, it is B_reg that may be the key effector cells that regulate the local immune response in the mucous membranes, including the respiratory tract. This is due to their functional activity, predominant localization and origin.

The main source of B_reg is B-1 lymphocytes [18, 19], which are a unique subpopulation of B cells that, in adult organisms, predominantly inhabit body cavities (pleural, peritoneal) and mucous membranes (namely, lamina propria) of the digestive and respiratory tracts. B1 cells are the main source of normal IgM antibodies in the human body and at least half of the IgA antibodies in mucosal secretions, participating in the formation of the body's first line of defense against infection [20]. Furthermore, B1 lymphocytes are involved in maintaining homeostasis in non-lymphoid organs [21].

The role of B_reg in regulating inflammation in the respiratory tract has been demonstrated for a number of non-infectious pathologies. In particular, the involvement of B_reg in the regulation of inflammation in lung tissue has been demonstrated in a model of silicosis (pneumofibrosis resulting from prolonged inhalation of dust containing silicon dioxide), in which B_reg actively suppressed the inflammatory process via an interleukin (IL)-10-dependent pathway, as well as through the activation of T_reg [22]. It should be noted that CD1d^hiCD5⁺IL-10⁺-B_reg play a critical role [23]. B_reg participate in the suppression of allergic inflammation in the respiratory tract [24–26].

The predominant localization of the main precursors of B_reg — B-1 cells — in the mucous membranes and body cavities (including the pleural cavity), their participation in the formation of the first line of defense against infection, their ability to maintain the homeostasis of surrounding tissue on the one hand, and the proven role of B_reg in regulating inflammation in the respiratory tract on the other, suggest that B_reg are involved in suppressing excessive inflammation in the lungs during respiratory infections (in particular, influenza virus infection).

This hypothesis has not yet been confirmed in publications. Some review articles suggest a possible role for B_reg in preventing severe respiratory infections, but these suggestions have not been confirmed by experimental data obtained in models of influenza-induced lung damage. Despite the existence of a body of evidence suggesting a probable role for B_reg in protecting the lungs from severe damage during viral infections, there are no systematic studies in the literature specifically aimed at assessing the contribution of these cells to the regulation of inflammation in the lungs during influenza infection.

Based on the above, the aim of the present study is to evaluate the role of B_reg in regulating the inflammatory process in the lungs against the background of influenza virus infection (A/H1N1/WSN/1933).

Materials and methods

Laboratory animals

The experiments were conducted on female CBA mice weighing 18–20 g (obtained from the Andreevka breeding facility, and kept in the vivarium of the I. Mechnikov Research Institute of Vaccines and Sera during the study) and CBA/N mice weighing 18–20 g (line maintained in the vivarium of the I. Mechnikov Research Institute of Vaccines and Sera). Authors confirm compliance with institutional and national standards for the use of laboratory animals in accordance with «Consensus Author Guidelines for Animal Use» (IAVES, 23 July, 2010). The research protocol was approved by the Ethics Committee of the I. Mechnikov Research Institute for Vaccines and Sera (protocol No. 7/2025, May 16, 2025).

The animals had unlimited access to food and water. Infected and intact animals were kept in separate cages in separate rooms.

Viruses

A virulent strain of influenza A/H1N1/WSN/1933 virus was used for infection. For accumulation, the virus was introduced into the allantoic cavity of 10-day-old developing chicken embryos and incubated at 38°C for 48 hours. After incubation, the chicken embryos were cooled at 4°C for 16 hours and the virus-containing allantoic fluid was collected. The virus titer was determined using a hemagglutination reaction with 0.5% chicken erythrocytes and twofold dilutions of the virus-containing allantoic fluid.

The virus-containing allantoic fluid was clarified by low-speed centrifugation at 8000 rpm for 30 minutes in a Beckman Coulter J2-21 centrifuge, then layered onto 8 ml of a 20% sucrose solution in isotonic NaCl solution and precipitated by ultracentrifugation at 23,000 rpm at 4°C for 90 min in a Beckman Coulter Optima L-90K centrifuge. The resulting virus precipitate was resuspended in 25 mL of isotonic NaCl solution and centrifuged again at 23,000 rpm at 4°C for 90 min. The precipitated viral material was diluted in 5 mL of isotonic NaCl solution and its titer was determined by infecting developing 10-day-old chicken embryos with tenfold dilutions of the virus. The study used a stock suspension with a virus content of 5 × 10¹⁰ infectious units (IU) in isotonic NaCl solution.

Cells used for adoptive transfer

For adoptive transfer, Breg cells induced in vitro from CBA mouse peritoneal B cells were used. Since peritoneal B cells themselves can contribute to the immune response to influenza virus, two control groups were included: baseline peritoneal B cells isolated ex vivo and injected without prior incubation (B_PerC); peritoneal B cells incubated in vitro under conditions identical to the B_reg induction protocol, but without the addition of activators (controls, B_contr).

CBA mouse peritoneal cells were obtained by washing the peritoneal cavity with phosphate-buffered saline containing 1% bovine serum albumin (BSA, Invitrogen). The resulting suspension was washed three times in the same buffer by centrifugation at 300g and a cell suspension with a concentration of 10⁸/mL was prepared. B lymphocytes were isolated from the resulting suspension using the Regulatory B cell isolation kit (Miltenyi Biotec). In the first stage, a cell suspension enriched with B lymphocytes was obtained by negative selection: the cells were sequentially labeled with a Regulatory B-cell Biotin-Antibody Cocktail and anti-Biotin MicroBeads were added; all cells except B lymphocytes were separated on an LD column (Miltenyi Biotec). The isolated B lymphocytes were used for further transfer to CBA/N (B_PerC) mice or incubated in vitro for subsequent B_reg induction.

For in vitro cultivation, the isolated cells were resuspended in complete medium: RPMI-1640 with 10% fetal bovine serum (FBS; Capricorn) containing penicillin and streptomycin at a final concentration of 200 μg/mL (Invitrogen), 5 × 10^–5 M 2-mercaptoethanol (Gibco), and 2 mM L-glutamine (Gibco). The suspension was added to the wells of a 24-well plate (Nunc) at a concentration of 2.5 million cells per 1 mL of complete medium. To obtain B_contr, the cells were cultured in vitro in complete medium without the addition of activators for 24 hours at 37°C and 5% CO₂.

To induce B_reg, Escherichia coli lipopolysaccharide (Sigma) was added to the wells to a final concentration of 20 μg/mL and incubated for 24 hours. Five hours before the end of in vitro incubation, phorbol-12-myristate-13-acetate (Sigma) was added to the wells to a final concentration of 50 ng/mL, and ionomycin (Invitrogen) was added to a final concentration of 500 ng/mL. At the end of incubation, the cells were collected, washed twice with RPMI-1640 medium, and used for adoptive transfer to CBA/N mice.

Cytofluorometric assessment of cells

B_reg content in transmissible subpopulations was assessed by surface staining with anti-mouse CD19 antibodies labeled with PE-Vio770 (Miltenyi Biotec) and intracellular staining with IL-10-specific APC antibodies (Miltenyi Biotec) using the BD Fixation/Permeabilization with GolgiStop kit (BD). The results were evaluated by flow cytometry.

Cells (0.5 million) were weighed in Dulbecco's phosphate-buffered saline (DPBS, 1% FBS, 0.09% NaN₃) for staining and washed twice with 0.5 mL of the same buffer. To the resuspended cells, 30 μL of staining buffer and pre-titrated antibodies to surface markers were added: antibodies to CD19 labeled with PE-Vio770 (Miltenyi Biotec) and antibodies to CD3 labeled with FITC (BD Biosciences). After 30 minutes of incubation at 4°C, the cells were washed once. The stained cells were fixed by adding 0.5 mL of a 2% paraformaldehyde solution for subsequent assessment of B-lymphocyte content or, without fixing, additional intracellular staining against IL-10 was performed.

For intracellular staining, the BD Cytofix/Cytoperm Fixation/Permeabilization Kit GolgiStop (BD) was used according to the manufacturer's instructions. Cells were permeabilized using Fix/Perm buffer (200 μL/sample) for 20 min at 4°C, followed by two washes with 0.5 mL Perm/Wash buffer. The resuspended cells were added to 30 µL of Perm/Wash buffer and pre-titrated antibodies to IL-10 labeled with APC (Miltenyi Biotec) and incubated for 30 min at 4°C, followed by a single wash with 0.5 mL of Perm/Wash buffer. The cells were fixed by adding 0.5 mL of a 2% paraformaldehyde solution. The cells were analyzed by flow cytometry on a Canto II instrument (Becton Dickinson).

Mouse infection, adoptive cell transfer, and experimental design

CBA/N mice were infected intranasally with influenza A/H1N1/WSN/1933 virus at a dose of 10⁸ IU per mouse in 50 μL of sterile isotonic NaCl solution. Control mice were administered 50 μL of sterile isotonic NaCl solution intranasally in the same manner.

For adoptive transfer, CBA mouse cells were washed three times with RPMI-1640 medium containing 2% FBS after isolation (B_PerC) or in vitro incubation (B_contr, B_reg) and transferred to isotonic NaCl solution at a concentration of 20 million/mL.

The day after infection with A/H1N1/WSN/1933, mice were intravenously transferred 2 million CBA mouse cells in 100 μL of sterile isotonic NaCl solution or administered 100 μL of sterile isotonic NaCl solution without cells.

Obtaining suspensions of mononuclear cells from the spleen and lungs

To obtain suspensions of mononuclear cells from the lungs and spleen, mice were anesthetized by intraperitoneal injection of a mixture of 1.5 mg of tiletamine hydrochloride + zolazepam hydrochloride and 0.62 mg of xylazine in 0.5 mL of sterile isotonic NaCl solution.

After anesthesia, the mouse was fixed, dissected, the spleen was removed, the renal artery was additionally cut, and the lungs were washed of blood by injecting 10 mL of Versene solution (2 mM EDTA in DPBS) into the right ventricle of the heart. The washed lungs were excised and processed in accordance with subsequent manipulations. For histological studies, the lungs from one mouse were placed in 15 mL of 10% neutral buffered formalin for 24 hours. For subsequent PCR, the lungs were frozen. To obtain a single-cell suspension, the lungs were mechanically minced with scissors to a homogeneous state, weighed in 3 mL of RPMI-1640 medium containing 2% FBS (Capricorn), glutamine (Gibco), HEPES (Gibco), 3.77 mg/mL collagenase (Sigma), and 0.43 mg/mL DNase (Sigma), and incubated for 1.5 hours at 37°C and 5% CO₂. After incubation, the cells were homogenized by pipetting, filtered to remove undigested tissue particles and large cell agglomerates, and weighed in 45 mL of RPMI-1640 containing 2% FBS (Capricorn). The cells were precipitated and washed twice with 10 mL of the same medium at 300g. The B-lymphocyte content was assessed in the resulting cell suspension and used to identify antibody-forming cells (AFCs) by the ELISPOT method.

Spleen cells were obtained by mechanically homogenizing the spleen in a pestle homogenizer. The resulting suspension was filtered through sterile cotton filters to remove capsule residues and large cell agglomerates, precipitated at 300g for 7–10 min, and erythrocytes were removed by osmotic shock. After removing the erythrocytes, the cells were filtered, washed twice by centrifugation at 300g, evaluated for B-lymphocyte content, and used for ELISpot.

Cellular Enzyme-Linked Immunosorbent spot assay (ELISpot)

The content of AFCs to A/H1N1/WSN/1933 was assessed using the ELISPOT method with nitrocellulose-coated plates (MultiScreen-HA, Millipore). Nitrocellulose filters were sensitized by adding 100 μL of A/H1N1/WSN/1933 influenza virus suspension to the wells, previously inactivated by ultraviolet light for 5 min, in phosphate-buffered saline (PBS) at a concentration of 10 μg/mg. The antigen-coated plates were incubated at 37°C for 1 h, after which the unbound antigen was washed away with PBS. Non-specific sorption sites were blocked by adding a 2% BSA solution (Capricorn) to the PBS, followed by incubation at 37°C for 1 hour. Unbound BSA was washed away with PBS and 10⁵ spleen cells per well or 3 × 10⁵ lung cells per well were added to the wells. The cells were incubated on filters for 20 hours at 37°C and 5% CO₂. At the end of incubation, the cells were washed from the filters by pipetting into PBS containing 0.1% Tween-20 (FBS-Tween). Then, biotinylated antibodies against mouse IgM, IgA, or IgG (Life Technologies) were added to the wells in stages, followed by streptavidin conjugate with horseradish peroxidase (AbD Serotec). Each step was followed by incubation at 37°C for 1 hour and subsequent washing with FSB-Twin. The reaction was developed by incubation for 20 min in the dark after adding the substrate buffer (Tris-phosphate buffer pH 7.8 containing 1,4-chloronaphthol and H₂O₂). At the end of incubation, the reaction was stopped with distilled water. The spots (formed at the point of contact between the antibody-secreting cell and the membrane) were counted visually under a microscope after the filters had dried. The results were presented as the number of AFCs per 1 million B lymphocytes.

Preparation of histological specimens

Mouse lung samples were fixed in 10% neutral buffered formalin for 24 hours and subjected to standard histological processing. The process included dehydration in alcohols of increasing concentration, degreasing in xylene, and impregnation with paraffin. The resulting paraffin blocks were used to prepare 3–5 μm thick sections. The sections were mounted on glass slides (Thermo) and stained with hematoxylin and eosin. After deparaffinization and hydration, the sections were stained with Mayer's hematoxylin, followed by washing in running water. Contrast staining was performed with an aqueous solution of eosin, followed by dehydration, clearing in xylene, and mounting in a fixative. Morphological analysis of the preparations was performed using light microscopy on an Eclipse 80i microscope with a DS-Fi1 camera (Nikon) and NIS Basic Research software at ×100 magnification.

Determination of viral load in lung tissue using PCR

To determine the expression level of target genes from homogenized mouse lungs, RNA was extracted using the Ribo-Sorb kit (Sintol), then reverse transcription (RT) was performed to obtain complementary DNA (cDNA) using the RT-1 kit for reverse transcription (Sintol), after which the cDNA was used for real-time PCR (qPCR) in the presence of SYBR Green I (Sintol). The primer sequences were verified using GeneBank resources and the Primer Blast program of the NCBI (National Center for Biotechnological Information) bioinformatics database. To detect the nucleic acid of the A/H1N1/WSN/1933 virus, synthesized primers targeting the M segment of the influenza virus were used for quantitative PCR (Eurogen) with the following sequences: fluM-q-For — AAG ACC AAT CCT GTC ACC TCT GA; fluM-q-Rev — CAA AGC GTC TAC GCT GCA GTC C.

Statistical data processing

Statistical data processing was performed using standard software programs — Excel (Microsoft) and GraphPad Prism 8.4.3 (GraphPad Holdings). The 2-ΔΔCt method was used for statistical processing of data obtained by PCR. Results corresponding to threshold cycle (Ct) values > 40 were considered negative. One-way ANOVA was used to compare independent groups, and Tukey's test was used to assess differences between groups. The Mann–Whitney nonparametric test was used to process the results of the enzyme-linked immunosorbent assay. Differences were considered significant at p < 0.05.

Results

Experimental design

The experiments were conducted according to the following protocol. CBA/N mice were infected intranasally with influenza virus A/H1N1/WSN/1933. The day after infection, the mice were intravenously transfused with 2 million B cells from CBA mice or an equivalent volume of sterile isotonic NaCl solution without cells. Depending on the infection and the type of cells transfused, the following experimental groups were selected:

• intact — CBA/N mice, not infected;
• WSN — CBA/N mice, intranasally infected with influenza A/H1N1/WSN/1933 virus;
• WSN + B_PerC — CBA/N mice infected with influenza A/H1N1/WSN/1933 virus and receiving B cells from the abdominal cavity of CBA mice, not incubated in vitro (B_PerC);
• WSN + B_contr — CBA/N mice infected with influenza A/H1N1/WSN/1933 virus and receiving control B cells from CBA mice, incubated in vitro without the inclusion of inducers (B_contr);
• WSN + B_reg — CBA/N mice infected with influenza A/H1N1/WSN/1933 virus, receiving in vitro-induced B_reg from CBA mice (B_reg).

Three days after cell transfer (four days after infection with A/H1N1/WSN/1933), the mice were euthanized and their spleens and lungs were removed for further experiments.

Characteristics of cells used for adoptive transfer

Experiments with adaptive transfer of CBA mouse cells to CBA/N mice infected with influenza A/H1N1/WSN/1933 virus were repeated three times. The characteristics of the transferred cells were stable and are shown in Table 1. The admixture of T lymphocytes in all samples did not exceed 3%, and the content of IL-10⁺-T cells was < 0.1%.

 

Table 1. Content of B cells, IL-10+-B_reg, and AFCs to A/WSN/1933 B cells used for adoptive transfer

Transferable cells	Total B lymphocyte content, %	IL-10+-B lymphocyte content (М ± σ), %	AFC content per million cells (М ± σ)
			IgM	IgA	IgG
BPerC	> 98%	–*	0	0	0
Bcontr	> 96%	4.6 ± 0.6	776 ± 92	7 ± 3	538 ± 71
Breg	> 96%	38.9 ± 1.9	1160 ± 159	0	788 ± 103

Note. * The IL-10⁺-B lymphocyte content in isolated abdominal cavity B cells was not determined, since intracellular staining requires in vitro incubation in the presence of secretion blockers.

 

The specified cells were adoptively transferred to CBA/N mice, previously infected intranasally with influenza virus A/H1N1/WSN/1933 at a dose of 2 million per mouse.

Evaluation of histological sections of mouse lungs

Figure 1 shows the most characteristic images of histological sections characterizing the condition of lung tissue in the compared groups. Evaluation of histological preparations of mouse lungs showed significant tissue damage in CBA/N mice infected intranasally with influenza A/H1N1/WSN/1933 virus but not receiving additional CBA cells (Fig. 1, b). Significant edema and tissue infiltration are observed, and the tissue structure is not preserved. At the same time, lung damage in mice that received 2 million B_reg cells the day after infection (Fig. 1, e) is practically not expressed. The tissue structure is preserved, there is no edema, and the infiltration is focal and affects only a small part of the tissue. The histological picture observed in this case is similar to that in uninfected mice (Fig. 1, a). In CBA/N mice infected intranasally with influenza A/H1N1/WSN/1933 virus, which were treated with B_PerC (Fig. 1, c) and B_contr (Fig. 1, d), tissue damage was also significantly reduced compared to infected mice that did not receive CBA mouse cells. As shown in Fig. 1, с, and Fig. 1, d, the tissue structure is partially preserved, there is no edema, and diffuse infiltration is observed throughout the entire lung tissue. According to the data presented, the greatest protective effect against inflammatory damage to the lung tissue of mice infected intranasally with the influenza A/H1N1/WSN/1933 virus was provided by the adaptive transfer of B_reg containing a significant amount of 38.9 ± 1.9% IL-10⁺-B_reg.

 

Fig. 1. Histological picture of CBA/N mouse lung tissue, × 100.

a — intact; b — intranasally infected with influenza A/H1N1/WSN/1933 virus, not receiving adoptive transfer of CBA mouse cells; c — intranasally infected with influenza A/H1N1/WSN/1933 virus, receiving B_PerC; d — intranasally infected with influenza A/H1N1/WSN/1933 virus, received B_contr; e — intranasally infected with influenza A/H1N1/WSN/1933 virus, received B_Reg.

 

Assessment of the immune response to influenza virus A/H1N1/WSN/1933

Since abdominal B cells may participate in the immune response to the influenza virus, the identified “protective” effect against lung tissue damage in infected CBA/N mice recipients of CBA B cells may be due to both a reduction in lung inflammation under the action of regulatory B lymphocytes and the participation of transplanted B cells in the immune response to the virus. In this regard, the content of IgM, IgA and IgG AFCs to the influenza A/H1N1/WSN/1933 virus was evaluated in the spleen (systemic immune response) and lungs (local immune response) of infected mice and infected mice that received various experimental cell groups. The AFC content in intact mice was not evaluated because they were not used as recipients of B cells from CBA mice.

Fig. 1 shows that the lung tissues of mice from different groups are infiltrated unevenly, which may affect both the total number of cells in the organ and the content of individual subpopulations. In this regard, we evaluated the content of B cells in the suspension and recalculated their content for the entire organ. Table 2 shows the data on the content of B cells in single-cell suspensions of the spleen and lungs in mice of the compared groups. As can be seen from the data presented, the content of B cells varies between different groups, and therefore calculating the number of AFCs per 1 million of the entire cell suspension may lead to distortion of the results. In this regard, the data obtained on the number of AFCs individually for each sample were recalculated per 1 million B cells and, if necessary, for the entire organ.

 

Table 2. B-cell content in the spleen and lungs of mice in the compared groups, M ± σ

Group	Spleen		Lungs
	B cell content, %	total B cells per organ, mil	B cell content, %	total B cells per organ, mil
WSN	25.2 ± 2.6	13.1 ± 3.7	4.3 ± 0.7	1.1 ± 0.1
WSN + BPerC	21.3 ± 0.9	9.2 ± 0.4	2.9 ± 0.2	0.6 ± 0.1
WSN + Bcontr	20.3 ± 2.2	11.0 ± 0.4	3.4 ± 0.9	0.8 ± 0.3
WSN + Breg	21.1 ± 0.8	14.9 ± 1.2	3.2 ± 1.0	0.9 ± 0.4

 

The results of the assessment of the number of IgM-, IgA-, and IgG-AFCs to the influenza A/H1N1/WSN/1933 virus in the lungs and spleen are shown in Fig. 2 and Fig. 3, respectively. The data are presented as mean values with standard deviation. As can be seen from the data, the strongest increase in AFCs was observed in the spleen. At the same time, the greatest increase was noted for IgM- and IgG-AFCs (see Fig. 3). The number of AFCs in the lungs was insignificant. The transfer of CBA cells led to a significant increase in the number of IgM- and IgG-antibody producers in both the lungs (see Fig. 2, a, c) and the spleen (see Fig. 3, a, c) of infected mice. The most significant increase in IgM-AFCs in the spleen and lungs, as well as IgG-AFCs in the spleen, was observed when B_reg cells were transferred. It should be noted that in these cases, the level of AFCs in the group of mice that received B_reg was many times higher than not only the level in infected mice that did not receive CBA cells, but also the levels of AFCs achieved with B_PerC and B_contr transfer. In most cases, the level of IgA-AFCs to the influenza A/H1N1/WSN/1933 virus after transfer of CBA mouse cells did not differ significantly from the level detected in infected mice that did not receive cells. The exception is the level of IgA-AFCs in the spleen of infected mice that received B_PerC before transfer, which was not incubated in vitro, but even in this case, the AFC content per 1 million cells was insignificant.

 

Fig. 2. AFC content against influenza virus A/H1N1/WSN/1933 per 1 million B lymphocytes in the lungs of mice in the compared groups.

a — IgM-AFCs; b — IgA-AFCs; c — IgG-AFCs.

*Differences from the group of infected mice that did not receive CBA (WSN) B cells are significant (p < 0.05).

 

Fig. 3. AFC content against influenza virus A/H1N1/WSN/1933 per 1 million B lymphocytes in the spleen of mice in the compared groups. a — IgM-AFCs; b — IgA-AFCs; c — IgG-AFCs. *Differences from the group of infected mice that did not receive CBA (WSN) B cells are significant (p < 0.05).

Fig. 3. AFC content against influenza virus A/H1N1/WSN/1933 per 1 million B lymphocytes in the spleen of mice in the compared groups.

a — IgM-AFCs; b — IgA-AFCs; c — IgG-AFCs.

*Differences from the group of infected mice that did not receive CBA (WSN) B cells are significant (p < 0.05).

 

Assessment of viral load in lung tissue

Given the above data on the level of inflammation in lung tissue and the immune response to the A/H1N1/WSN/1933 influenza virus, it was necessary to assess the change in viral load in the lungs of infected mice in the compared groups. The viral load in lung samples from CBA/N mice that did not receive CBA mouse B cells varied widely, with a median value and interquartile range of 11.3 × 10⁷ (3.4 × 10⁷–53.0 × 10⁷). When B cells from CBA mice were transferred to infected mice, the viral load decreased by approximately one order of magnitude, but the differences with the group of infected mice that did not receive cells were not significant: B_PerC — 1.5 × 10⁷ (0.7 × 10⁷–7.8 × 10⁷; p > 0.41), B_contr — 6.2 × 10⁷ (2.0 × 10⁷–8.3 × 10⁷; p > 0.93), B_reg — 1.0 × 10⁷ (0.4 × 10⁷–6.0 × 10⁷; p > 0.39). Despite a stronger humoral response, there were no significant differences in viral load in the lungs of mice infected with influenza A/H1N1/WSN/1933, and the above-described differences in histological findings between the groups cannot be explained solely by more effective virus elimination.

Discussion

The role of B_reg in regulating inflammation in lung tissue during viral infections has not been studied extensively to date. One of the key challenges in researching this process is selecting an adequate model. The pandemic strain of influenza A/H1N1/WSN/1933 was used as a model virus in this study, since infection with the H1N1 virus has been shown to cause severe lung damage as a result of uncontrolled inflammation [10–13]. Intranasal infection of CBA mice with influenza A/H1N1/ WSN/1933 virus in doses comparable to those used in the described study did not lead to the development of lung damage (data not shown), which is most likely due to the low sensitivity of mice to the human influenza virus. At the same time, it is known that mouse peritoneal cells (most of which are B-1 lymphocytes) are capable of responding effectively to influenza A virus [27], and intraperitoneal infection of animals results in the formation of immune protection against subsequent intranasal infection. In addition, according to data obtained by X. Wang et al., B-1a lymphocytes (CD5⁺-B-1 cells) from the pleural cavity actively infiltrate lung tissue during influenza virus infection and differentiate into AFCs via the IL-17A-dependent pathway [28]. Based on this, a model of adoptive cell transfer from CBA mice to CBA/N mice was chosen as a model for studying the effect of B_reg on the development of inflammation in the lungs during influenza virus infection.

CBA/N mice carry the Xid mutation, which causes a non-lethal disruption in B-lymphocyte development in mice. An important feature of these mice, in addition to a generally reduced immune response, is the absence of B-1a cells, which, as noted above, may participate in the immune response to the influenza virus. In addition, due to impaired B-1 cell development, CBA/N mice have a reduced ability to differentiate B_reg. At the same time, the congenicity of the CBA and CBA/N mouse lines allows for the adoptive transfer of cells between them — the transferred cells are not perceived as foreign and can fully participate in the development of the immune response in the recipient's body [29]. As shown by experimental intranasal infection of CBA/N mice with influenza A/H1N1/WSN/1933 virus at a dose of 10⁸ IU per mouse, on the 4th day after infection, the mice develop severe lung tissue damage with tissue structure disruption and significant exudate.

Since adoptive transfer requires large numbers of cells (millions), B_reg were obtained by in vitro induction from CBA mouse peritoneal cells incubated in the presence of activators. Transfer of induced B_reg to infected mice resulted in a significant reduction in lung tissue damage (see Fig. 1). Unlike CBA/N mice, the CBA mice used for B_reg induction contain a large number of B1 lymphocytes, which themselves can participate in the response to the influenza virus. In this regard, it was necessary to ensure that the observed effect was due to the transfer of B_reg, and not simply B cells from the peritoneal cavity of CBA mice containing B1 cells. To this end, infected CBA/N mice were transferred with freshly isolated B cells from the peritoneal cavity of CBA mice (B_PerC) in parallel with B_reg. Furthermore, in vitro incubation itself can lead to cell activation, although the B_reg content in them increases insignificantly. To assess the effect of in vitro incubation of transferred cells on the detectable effects, infected CBA/N mice were also adoptively transferred with B cells from the peritoneal cavity of CBA mice, incubated in vitro under the same conditions as for B_reg induction, but without the addition of activators, i.e., only in complete medium (B_contr). Thus, all three variants of transplanted cells had a common origin and differed only in the degree of exposure to activating factors. Adoptive transfer was performed the day after infection, i.e., the cells were injected into mice already in the process of developing infection. The results showed that the transfer of B_PerC and B_contr also contributed to a reduction in inflammation in the lungs of infected mice, but the degree of lung structure damage and infiltration in these groups was still significantly more pronounced than in mice that received B_reg.

It should be noted that the result obtained could have been achieved not only due to the regulatory properties of the transferred B_reg, but also as a result of a stronger immune response of the transferred cells activated in vitro. The transferred B_reg contained more IgM- and IgG-AFCs to the influenza A/H1N1/WSN/1933 virus than B_contr, but these differences were insignificant — less than 1.5 times. Freshly isolated B_PerC from the abdominal cavity did not produce antibodies, as has been shown previously [30, 31]. At the same time, after transferring B cells from CBA mice to infected CBA/N mice, we observed a significant increase in the content of IgM- and IgG-AFC in recipients, with the most noticeable increase occurring when transferring B_reg: IgM in the spleen and lungs, IgG in the spleen. It should be noted that the increase in AFC in recipients significantly exceeded the number of AFC contained in the transferred cells. Thus, the transferred B_reg contained about 2,500 IgM-AFCs and 1,500 IgG-AFCs to the influenza A/H1N1/WSN/1933 virus, while when recalculated for the entire spleen after transfer, the number of IgM-AFCs in recipients increased from 2,600 (in infected CBA/N mice that did not receive B cells from CBA mice) to 31,500, and IgG-AFCs increased from 1,700 to 13,800. At the same time, during the transfer of B_PerC and B_contr, a disproportionate increase in the number of AFCs was also observed, although to a much lesser extent. The data obtained may indicate that the transferred cells can continue to activate and the number of AFCs in them may increase after transfer. This is indirectly confirmed by previously obtained data, according to which the number of IgM-AFCs in the B cells of the abdominal cavity of CBA mice gradually increases during in vitro incubation for 4 days [31]. On the other hand, the observed increase in the number of AFCs may also be due to a more effective induction of the immune response in CBA/N mice after adoptive transfer of CBA mouse B cells, including through the activation of recipient cells by the transferred cells. These assumptions require experimental verification.

Thus, the observed reduction in lung tissue damage in infected CBA/N mice after adoptive transfer of B_reg may be due to a stronger immune response, including in comparison with B_PerC and B_contr. In this case, the absence of lung damage could be due to more effective neutralization/elimination of the virus. Thus, a significant reduction in viral load in the lungs of infected CBA/N mice receiving B_reg could be expected compared to mice that did not receive CBA mouse B cells or received B_PerC or B_contr. However, the number of copies of influenza A/H1N1/WSN/1933 RNA in the lungs of infected mice in the compared groups did not differ significantly, which excludes a significantly more effective neutralization/elimination of the virus against the background of a stronger immune response during B_reg transfer.

Conclusion

The data obtained as a result of the presented work allow us to conclude that in vitro-induced B cells of CBA mice with a high content of IL-10⁺-B_reg during adoptive transfer to CBA/N mice intranasally infected with influenza virus A/H1N1/WSN/1933, prevent lung tissue damage against a background of persistent viral load. The observed effect is achieved both through a more effective immune response of the transferred cells and through the direct regulatory (anti-inflammatory) action of the transferred B_reg lymphocytes.

About the authors

Ilya N. Dyakov

I.I. Mechnikov Research Institute of Vaccines and Sera

Author for correspondence.
Email: dyakov.instmech@mail.ru
ORCID iD: 0000-0001-5384-9866

Cand. Sci. (Biol.), leading researcher, Head, Laboratory of biosynthesis of immunoglobulins, Immunology and allergology department, I. Mechnikov Research Institute of Vaccines and Sera; researcher, Laboratory of bacterial genetics, Department of medical microbiology, National Research Center for Epidemiology and Microbiology named after N.F. Gamaleya

Russian Federation, Moscow

Irina N. Chernyshova

I.I. Mechnikov Research Institute of Vaccines and Sera

Email: irina.n.chernyshova@gmail.com
ORCID iD: 0000-0001-5053-2433

Cand. Sci. (Med.), senior researcher, Laboratory of biosynthesis of immunoglobulins, Immunology and allergology department

Russian Federation, Moscow

Marina V. Gavrilova

I.I. Mechnikov Research Institute of Vaccines and Sera

Email: gavrilovamv@gmail.com
ORCID iD: 0000-0002-6936-2486

Cand. Sci. (Biol.), researcher, Laboratory of biosynthesis of immunoglobulins, Immunology and allergology department, I. Mechnikov Research Institute of Vaccines and Sera; researcher, Laboratory of bacterial genetics, Department of medical microbiology, National Research Center for Epidemiology and Microbiology named after N.F. Gamaleya

Russian Federation, Moscow

Kristina K. Bushkova

I.I. Mechnikov Research Institute of Vaccines and Sera

Email: christina_bushkova@mail.ru
ORCID iD: 0000-0002-4757-0751

researcher, Laboratory of biosynthesis of immunoglobulins, Immunology and allergology department

Russian Federation, Moscow

Artyom A. Rtishchev

I.I. Mechnikov Research Institute of Vaccines and Sera

Email: rtishchevartyom@gmail.com
ORCID iD: 0000-0002-4212-5093

researcher, Laboratory of genetics of RNA-containing viruses, Department of virology named after O.G. Andzhaparidze

Russian Federation, Moscow

Natalia E. Abayeva

I.I. Mechnikov Research Institute of Vaccines and Sera

Email: fabaeva.nata@list.ru
ORCID iD: 0000-0003-3984-959X

researcher, Laboratory of biosynthesis of immunoglobulins, Immunology and allergology department

Russian Federation, Moscow

Stanislav G. Markushin

I.I. Mechnikov Research Institute of Vaccines and Sera

Email: s.g.markushin@rambler.ru
ORCID iD: 0000-0003-0994-5337

Dr. Sci. (Med.), Head, Laboratory of genetics of RNA-containing viruses, Department of virology named after O.G. Andzhaparidze

Russian Federation, Moscow

Dmitry A. Khochenkov

N.N. Blokhin National Medical Research Center

Email: khochenkov@gmail.com
ORCID iD: 0000-0002-5694-3492

Cand. Sci. (Biol.), Head, Laboratory of biomarkers and mechanisms of tumor angiogenesis

Russian Federation, Moscow

Irina D. Bulgakova

I.I. Mechnikov Research Institute of Vaccines and Sera; Sechenov University

Email: bulgakova_i_d@staff.sechenov.ru
ORCID iD: 0000-0002-2629-9616

assistant, Microbiology, virology and immunology department named after Academician A.A. Vorobyev, Institute of Public Health named after F.F. Erisman, Sechenov University; junior researcher, Laboratory of molecular immunology, I. Mechnikov Research Institute of Vaccines and Sera

Russian Federation, Moscow; Moscow

Nadezhda A. Snegireva

I.I. Mechnikov Research Institute of Vaccines and Sera

Email: snegireva.nadezda@gmail.com
ORCID iD: 0000-0002-5399-3224

researcher, Laboratory of biosynthesis of immunoglobulins, Immunology and allergology department

Russian Federation, Moscow

Oxana A. Svitich

I.I. Mechnikov Research Institute of Vaccines and Sera

Email: svitichoa@yandex.ru
ORCID iD: 0000-0003-1757-8389

Dr. Sci. (Med.), Professor, Full Member of the Russian Academy of Sciences, Director

Russian Federation, Moscow

References

Supplementary files