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Am J Respir Cell Mol Biol
Am J Respir Cell Mol Biol
ajrcmb
American Journal of Respiratory Cell and Molecular Biology
1044-1549
1535-4989
American Thoracic Society

38696270
2024-0004OC
10.1165/rcmb.2024-0004OC
Original Research
Infection and Lung Inflammation
Monocyte Production of C1q Potentiates CD8+ T-Cell Function Following Respiratory Viral Infection
Eddens Taylor 1 *
Parks Olivia B. 6 7 *
Lou Dequan 2
Fan Li 2
Sojati Jorna 6 7
Ramsey Manda Jo 3
Schmitt Lori 4
Salgado Claudia M. 4
https://orcid.org/0000-0001-5698-3702
Reyes-Mugica Miguel 4
Evans Alysa 5
Zou Henry M. 2
Oury Tim D. 4
Byersdorfer Craig 3
Chen Kong 2
Williams John V. 6 8
1 Division of Allergy/Immunology, Department of Pediatrics,
2 Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine,
3 Division of Blood and Marrow Transplant and Cellular Therapies, Department of Pediatrics,
4 Department of Pathology,
5 Program in Microbiology and Immunology, and
6 Division of Infectious Diseases, Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania;
7 University of Pittsburgh Medical Scientist Training Program, Pittsburgh, Pennsylvania; and
8 Institute for Infection, Inflammation, and Immunity in Children, Pittsburgh, Pennsylvania
Correspondence and requests for reprints should be addressed to John V. Williams, M.D., Division of Infectious Diseases, Department of Pediatrics, University of Pittsburgh School of Medicine, 4401 Penn Avenue, Pittsburgh, PA 15224. E-mail: jvw@chp.edu.
* Co–first authors.

2 5 2024
1 9 2024
2 5 2024
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4 1 2024
2 5 2024
Copyright © 2024 by the American Thoracic Society
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0. For commercial usage and reprints, please e-mail Diane Gern.

Respiratory viral infections remain a leading cause of morbidity and mortality. Using a murine model of human metapneumovirus, we identified recruitment of a C1q-expressing inflammatory monocyte population concomitant with viral clearance by adaptive immune cells. Genetic ablation of C1q led to reduced CD8+ T-cell function. Production of C1q by a myeloid lineage was necessary to enhance CD8+ T-cell function. Activated and dividing CD8+ T cells expressed a C1q receptor, gC1qR. Perturbation of gC1qR signaling led to altered CD8+ T-cell IFN-γ production, metabolic capacity, and cell proliferation. Autopsy specimens from fatal respiratory viral infections in children exhibited diffuse production of C1q by an interstitial population. Humans with severe coronavirus disease (COVID-19) infection also exhibited upregulation of gC1qR on activated and rapidly dividing CD8+ T cells. Collectively, these studies implicate C1q production from monocytes as a critical regulator of CD8+ T-cell function following respiratory viral infection.

Keywords

human metapneumovirus
complement
antiviral immunity
COVID-19
National Institute of Allergy and Infectious Diseases 10.13039/100000060 AI085062 National Heart, Lung, and Blood Institute 10.13039/100000060 1F30HL159915 National Institute of General Medical Sciences 10.13039/100000060 T32GM008208 Eunice Kennedy Shriver National Institute of Child Health and Human Development 10.13039/100000060 K12HD000850 Henry L. Hillman Foundation 10.13039/100000060
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pmcClinical Relevance

The current manuscript shows a novel role for a complement protein (C1q) in regulating and potentiating antiviral CD8 function in preclinical mouse models of human metapneumovirus. The study also has human data from children who succumbed to respiratory viral infection showing increased C1q+ cells and transcriptional evidence of the same signaling pathway in COVID-19 patients.

Lower respiratory tract infections are a leading infectious cause of morbidity and mortality globally, with viral pathogens representing key causative agents (1, 2). This was undoubtedly highlighted by the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, which accounted for more than 6 million deaths globally between 2020 and 2022. In 2022, viruses such as respiratory syncytial virus, human metapneumovirus (HMPV), and influenza reemerged in the setting of waning public health mitigation measures (3, 4). Given the constant threat of new and old respiratory viral pathogens, it is critical to better understand the fundamental pathways involved in lung protection and immunopathology.

CD8+ antiviral T cells and the humoral compartment are key contributors to acute viral clearance and memory formation (5, 6). Prior animal models have demonstrated the formation of protective antigen-specific CD8+ T cells in response to viral pathogens, which are subsequently inhibited by a number of mechanisms to limit immunopathology (7–12). These mechanisms include viral inhibition of T-cell receptor signaling on CD8+ T cells (7) and PD-1–mediated signaling, which causes CD8+ T cell functional impairment (9, 10). Additionally, the presence of CD4+ CD25+ Foxp3+ regulatory T cells recruits CD8+ T cells to the lung during the acute phase of infection (12) while infiltrating effector CD4+ and CD8+ T cells produce IL-10 and proinflammatory cytokines in the periphery to help ameliorate and reduce lung damage (11). When regulatory T cells are depleted from the lung or IL-10 production is blocked, CD8+ T cell influx into the lung is significantly impaired and there is delayed viral clearance, resulting in severe lung damage (11, 12).

Immunopathology following respiratory viral pathogens has also been attributed to inflammatory monocyte (iMono) recruitment (13–15). As more studies have explored the pathogenesis of SARS-CoV-2, potential pathologic contributions of CD8+ T cells and monocytes have been described (16–21). However, the mechanisms and signaling mediators governing monocyte/CD8+ T cell interactions are not fully defined.

In this study, we identify a novel role of C1q-expressing iMonos during respiratory viral infection. In a murine model of HMPV, C1q was required for the optimal function of CD8+ T-cell effectors, which express a complement receptor, gC1qR. C1q and gC1qR have been demonstrated to play a role in systemic antiviral immunity, although their respective roles in acute respiratory viral infection are unclear (22, 23). In this study, we also show that in vitro blockade of gC1qR during HMPV infection led to reduced metabolic capacity, diminished effector function, and reduced proliferation. In human single-cell transcriptomic samples, the C1q:gC1qR axis was expressed on recruited macrophages and CD8+ T cells, respectively, following severe respiratory viral infection.

Some of the results of these studies have been previously reported in the form of a preprint (bioRxiv, 6 Jun 2023, www.biorxiv.org/content/10.1101/2023.06.04.543430v1).

Methods

Mice and Virus Stocks

C57BL/6 (strain 664), 6(Cg)- C1qatm1 d(EUCOMM)Wtsi/TennJ (strain 31675, referred to as C1qa−/−), B6.129S4-C3tm1Crr/J (strain 29661, referred to as C3−/−), Rag1−/− (strain 2216), and B6.SJL-Ptprca Pepcb/BoyJ (strain 2014, referred to as CD45.1) mice were purchased from the Jackson Laboratory. At 6–8 weeks of age, mice were anesthetized with 3% isoflurane and infected with 2.0 × 106 plaque-forming units of HMPV strain TN/94-49 (genotype A2) in 100 μl of sterile PBS solution. HMPV was grown in LLC-MK2 cells and purified as previously described (24). Mice were treated with a mock-infected LLC-MK2 lysate as a negative control. In select experiments, mice were infected with 1.0 × 105 plaque-forming units of clinical isolate C2-202 (genotype B1) (frozen and thawed a maximum of three times) (25, 26). See data supplement for a description of the plaque assay that was used to determine the viral titer (24, 27). Clinical scoring was performed, with 1 point added for each of the following variables: hunched posture, ruffled fur, rapid breathing, and decreased activity. See data supplement for descriptions of the bone marrow (BM) transplantation and adoptive transfer models. All animals were handled according to protocols approved by the University of Pittsburgh Institutional Animal Care and Use Committee.

Human Lung Tissue Staining

Lung samples from children who died of respiratory viral infections (autopsy cases) were selected on retrospective review of specimens from the department of pathology at the Children’s Hospital of Pittsburgh (University of Pittsburgh Medical Center). Normal lung tissues resected as perilesional lung in patients with pleural blebs were used as normal controls. Use of cadaveric tissue was approved by the University of Pittsburgh Committee for Oversight of Research and Clinical Training Involving Decedents.

Single-Cell RNA Sequencing

See data supplement for descriptions of the generation and processing of single-cell suspensions of lung tissue. Single-cell RNA sequencing analysis was performed using Seurat 4.0 with R (version 4.1.1; R Foundation for Statistical Computing). Quality control (QC) metrics included nFeature_RNA more than 200, nFeature_RNA less than 5,000, and percent.mt less than 20, followed by normalization. Samples were then demultiplexed by hashtag-oligos (HTOs), and doublets identified by HTOs were removed before downstream analysis. QC metrics were assessed for each HTO after demultiplexing (see Figure E1 in the data supplement), and the analysis was continued with QC cutoffs of nFeature-RNA>200, nFeature-RNA>5000 and percent.mt>20). Poor-quality droplets were excluded from subsequent analysis if a deficient number of genes or a high percentage of mitochondrial genes was detected. For human single-cell RNA sequencing analysis, publicly available dataset GSE145926 was mined (19).

Flow Cytometry/ImageStream

Following generation of a single-cell suspension from lung as above, cells were then plated for ex vivo peptide stimulation or tetramer staining in parallel as described previously, including a full gating strategy (9, 28, 29). See data supplement for a full description of the methods.

IFN-γ ELISpot

Following generation of a single-cell suspension from lung, 50,000 cells were plated per well in triplicate on an IFN-γ ELISpot plate (no. EL485; R&D Systems), with 10 μM of irrelevant (LCMV gp66-77 peptide) or MHC class I–restricted immunodominant HMPV epitope (N11). Antibody blockade was performed with 10 μg/ml anti-gC1qR (clone 74.5.2; no. sc-23884-L; Santa Cruz) or anti–PD-L1 (clone 10F.9G2; no. BE0101; BioXcell). Recombinant murine C1q (10 μg/ml) was used for treatment (M099; ComplementTech). Cell stimulation cocktail (PMA/I) was used for nonspecific T-cell activation. The plate was incubated for 48 hours at 37°C and developed per the manufacturer’s instructions. Supernatants were collected and analyzed via Luminex.

gC1qR Blockade in Vivo

On Days 5 and 6 after infection, 50 μg of anti-gC1qR antibody was administered to anesthetized mice via the oropharyngeal route. An equal volume of PBS solution was used as control. CD8 endpoints were assessed as above.

Metabolic Profiling

A single-lung cell suspension was generated as above, followed by further enrichment of CD8+ T cells by magnetic bead separation (no. 130-104-075; Miltenyi Biotec) per the manufacturer’s instructions. CD8+ T cells were then counted using a BD Accuri cytometer and plated at 200,000 cells per well with 10 μg/ml anti-gC1qR. Metabolic status was assessed using an Agilent Seahorse XF Cell Mito Stress Test Kit (no. 103010-100) per the manufacturer’s instructions on an Agilent Seahorse XFe96 analyzer.

Immunofluorescence Imaging

Staining was performed per a previously described protocol. See data supplement for full details (30).

C1q Staining on Autopsy Specimens

Formalin-fixed, paraffin-embedded tissues were stained on a Ventana BenchMark Ultra automated staining platform. Slides were pretreated with ultraCC1 (Roche Tissue Diagnostics) and stained using a polyclonal FITC-labeled anti-C1q primary antibody (no. 05267994001; Roche Tissue Diagnostics); sections were counterstained with DAPI. Slides were viewed using an Olympus BX63 microscope equipped with appropriate FITC and DAPI filter sets.

Quantification and Statistical Analysis

All data are displayed as mean ± SEM. Analyses with two groups were analyzed using unpaired Student’s t tests, whereas analyses with three or more groups were analyzed using a one-way ANOVA with Tukey’s multiple comparisons. Analyses with two time points (e.g., Day 1 and Day 7) were analyzed using a two-way ANOVA with Holm-Šidák multiple comparisons. Significance was defined at P < 0.05 for all analyses. All statistical analyses for mouse experiments were performed using GraphPad Prism (v. 9.3.0). For single-cell RNA sequencing analysis, differential gene expression was determined by assessing log2FC with an adjusted P value of <0.05 using a nonparametric Wilcoxon rank sum test with Bonferroni correction for multiple comparisons. Permutation tests were used to assess differences in cell abundance; results of these tests are shown in the figures in the data supplement. Global differential gene assessment was performed via pseudobulk analysis between mock- versus HMPV-infected animals. All single-cell analysis was performed using the Seurat pipeline in R studio.

Results

C1q Is Produced by an Inflammatory Macrophage Population following HMPV in Mice

Our group demonstrated previously that murine HMPV infection with the TN/94-49 strain leads to minimal clinical disease but robust antigen-specific CD8+ T-cell responses by Day 7 after infection (9, 25, 31). This time point also represents a period of rapid viral clearance due to adaptive immunity with functional, albeit PD-1–mediated impaired, CD8+ T-cell response (9, 10, 31). Single-cell RNA sequencing using the 10x Genomics platform was performed on lung tissue from mock-infected versus HMPV-infected mice at Day 7 to assess factors contributing to the antiviral CD8+ T-cell response. An average of 5,730 cells were captured/sequenced per sample, with an average of 24,964 reads per cell. Using clustering analysis, nine unique cellular populations were identified: T cells, B cells, an iMono, endothelial cells, monocytes, fibroblasts, macrophages, natural killer cells, and neutrophils (Figure 1A). These clusters were manually annotated based on expression of canonical markers (Figure 1B). Notably, the iMono population shared expression of several genes associated with monocytes, but were differentiated by expression of C1qa, C1qb, and C1qc (Figures 1A and 1B). These clusters were then visualized via Uniform Manifold Approximation and Projection nonlinear dimensional reduction, with cells from every cluster observed in mock- and HMPV-infected animals (Figure 1C). However, HMPV-infected mice had a statistically significant expansion of the T-cell and iMono compartments compared with mock-infected animals (Figures 1D and E2A).

Figure 1. Identification of a C1q signature on Day 7 after human metapneumovirus (HMPV) infection. (A) Heat map demonstrating clustering of nine different cell populations after single-cell RNA sequencing. (B) Dot plot with expression of marker genes used for manual annotation for individual clusters. (C) Uniform Manifold Approximation and Projection (UMAP) representation of cell populations in mock-infected versus HMPV-infected animals demonstrating increased inflammatory monocyte (iMono) and T-cell populations after infection. (D) Percent abundance of cell populations by cluster in mock- and HMPV-infected animals. (E) Feature plot of C1qa, C1qb, and C1qc shown as UMAPs. (F) CellChat analysis showing the number of inferred interactions (top) and interaction strength (bottom) in mock- and HMPV-infected animals. (G) Pathway analysis of information flow using CellChat. Single-cell RNA sequencing was performed as a single replicate with two mice in each group. Endo = endothelial cells; Fibro = fibroblasts; Mac = macrophages; Mono = monocytes; Neut = neutrophils; NK = natural killer (cells).

For the T-cell compartment, this expansion was partially composed of CD8+ cells that expressed granzyme B (Gzmb), PD-1 (Pdcd1), and IFN-γ (Ifng) (see Figures E2B and E2C), consistent with prior reports (9, 32). Because PD-1 is prominently upregulated in this model, we next sought to characterize signaling pathways and downstream targets in an unbiased manner using NicheNet (33). This predictive package identified 18 prioritized ligands, including PD-L1 (i.e., Cd274), the ligand for PD-1 (see Figure E2D). Further corroborating prior studies, PD-L1 was predicted to regulate several target genes, including Ifng (see Figure E2D). These data demonstrate that the single-cell RNA sequencing methodology and subsequent unbiased pathway analysis adequately captured biologic processes consistent with our prior work, validating use of this dataset for discovery purposes.

As described above, the iMono population was noted to have marked expression of C1qa, C1qb, and C1qc (Figure 1E) that was not observed in other populations. Using differential expression analysis, C1qa, C1qb, and C1qc were significantly upregulated in iMonos compared with monocytes and macrophages. As an alternative statistical approach, global differential gene expression was also calculated between HMPV-infected animals and mock-infected animals. In this analysis, C1qa, C1qb, and C1qc were significantly upregulated in HMPV-infected lungs (P < 1 × 10−50).

Given the interesting expansion of C1q+ iMonos and T cells, we next sought to analyze cell-to-cell communication with an emphasis on these two populations. To model this, we used CellChat, a package that quantitatively infers connections and signaling pathways between single-cell RNA sequencing cell clusters (34). HMPV-infected animals had increased numbers of interactions as well as increased interaction strength (Figure 1F). CellChat pathway analysis also revealed a strong complement signature in HMPV-infected animals, although this database does not include ligand:receptor interactions related to C1q (Figure 1G). In the HMPV-infected lung, iMonos were the greatest contributors of outgoing signals by number, followed closely by macrophages and fibroblasts (Figures E3A and E3B). As for receiving signals, T cells had the most incoming signals from iMonos. iMonos had several methods of communicating with T cells when analyzed at a ligand:receptor level, including through chemotactic mechanisms (see Figures E3C and E3D). Collectively, these single-cell RNA sequencing data demonstrate that HMPV-infected mice have evidence of a C1q-expressing iMono population and expansion of CD8+ effector T cells, with predicted cell-to-cell communication occurring between the two cell types.

To validate these single-cell RNA sequencing findings, we next assessed C1q production at the protein level. C1q was upregulated in BAL fluid over the course of HMPV infection, peaking at Day 7 after infection (Figure 2A). Additionally, using validated surface markers for various monocyte/macrophage populations, we next identified the iMono population via flow cytometry (Figures 2B and E4A and E4B). The number of iMonos was significantly increased in HMPV-infected mice at Day 7 after infection (Figures 2B and E4C). Similarly, there was a significant increase in C1q-producing iMonos at Day 7 after HMPV infection (Figures 2B and E4D). C1q production was not seen in other myeloid cells, such as neutrophils (see Figure E4E). Additionally, if staining for C1q was performed before permeabilization, the signal in the iMono population was absent, suggesting intracellular production rather than detection of bound C1q (see Figure E4F). To further visualize the production of C1q, we used ImageStream technology and flow cytometry staining to capture high-resolution images of individual cells from the single-cell suspension in mock- or HMPV-infected mice 7 days after exposure. iMonos from HMPV-infected mice had a robust C1q intracellular signal (Figure 2C, top). C1q staining was not observed in CD3+CD8+ T cells, however (Figure 2C, bottom). Minimal C1q production was noted in mock-infected animals in both populations (Figure 2C). Further assessing colocalization, immunofluorescent staining on fixed lung specimens from Day 7 after infection demonstrated C1q and CD68 overlap in the B6 HMPV specimen, but not B6 mock-infected or C1q-deficient mice (Figures 2D and E5). Collectively, these data suggest that HMPV-infected animals have a robust C1q-producing iMono population present late in HMPV infection, coincident with adaptive immunity.

Figure 2. Validation of C1q production by iMonos in HMPV infection. (A) C1q protein quantity in BAL specimens from mock- or HMPV-infected animals at various time points (*P < 0.05 and **P < 0.01 by two-way ANOVA with multiple comparisons; n = 1–3 for mock groups; n = 4–12 for infected groups). (B) Enumeration by flow cytometry of iMonos (left) and C1q+ iMonos (right) isolated from the lung at Day 7 after infection (**P < 0.01 by two-way ANOVA with multiple comparisons; n = 3 per group at Day 1 [n = 1 replicate]; n = 5 at Day 7 [n = 2 replicates]). (C) ImageStream analysis of C1q production in iMonos (top) versus CD8 T cells (bottom) at Day 7 after infection (n = 3 per group, with 10,000 events captured from each animal). (D) Immunofluorescent staining of C1q, CD68, and DAPI showing colocalization of C1q with CD68 in the B6 HMPV-infected group alone (white arrowheads) at Day 7 after infection (n = 3–5 per group). Scale bars, 10 μm.

C1q Is Required for Optimal CD8 Effector Function

We next assessed CD8+ T-cell responses in the absence of C1q using 6(Cg)C1qatm1d(EUCOMM)Wtsi/TennJ (referred to as C1qa−/−) mice following infection with HMPV (TN/94-49) as described above. The lack of C1q induction was confirmed in C1qa−/− mice via ELISA (Figure E6A). There was no difference in weight loss or viral titer between C1qa−/− and B6 mice (see Figures E6B and E6C). At Day 7 after infection, there was also no difference in the frequency or absolute cell numbers of epitope-specific CD8+ T cells using an MHC-I tetramer bearing one of the immunodominant H2-Kb HMPV epitopes, N11-18 (Figure 3A). Similarly, inhibitory receptors (i.e., PD-1, TIM-3, LAG-3, and 2B4), activation states (CD44, CD62L), and transcription factors (i.e., T-bet, GATA3, Foxp3, EOMES, TOX, and TCF1) on virus-specific CD8+ T cells were unchanged between the two groups (see Figure E6D–E6F). B6 and C1qa−/− mice displayed a similar extent of immunopathology following TN/94-49 infection (see Figures E6G and E6H). However, CD8+ T cells from C1qa−/− mice exhibited a striking decrease in function, producing significantly less granzyme B, IL-2, and IFN-γ (Figure 3B). Polyfunctionality was assessed by analyzing CD8+ T cells producing two or more functional cytokines via Boolean gating (Figure 3C). Combinatorial analysis assessing CD107a, IFN-γ, granzyme B, IL-2, and perforin production revealed that C1qa−/− CD8+ T cells were less polyfunctional compared with CD8+ T cells from B6 mice, further demonstrating altered CD8+ functionality in the absence of C1q (Figure 3C).

Figure 3. Absence of C1q leads to less functional CD8+ T cells. Mice were infected with 2.8 × 106 plaque-forming units of HMPV and lung cells isolated at Day 7 after infection. (A) N11 tetramer–positive CD3+CD8+ leukocytes in the lung are similar between B6 and C1qa−/− mice in cell percentages and absolute cell numbers. (B) CD8+ T cells in C1qa−/− mice had impaired production of effector cytokines, granzyme B, IL-2, and IFN-γ following ex vivo peptide stimulation. (C) Combinatorial analysis of functional markers revealed that CD8+ T cells from C1q−/− mice had a significantly reduced portion of cells with 3 or 4 markers. Bar graphs (left) show raw data, and pie charts (right) reflect a summary of raw data. (D) Increased weight loss was seen in C1qa−/− mice infected with C-202 (*P < 0.05 by two-way ANOVA with multiple comparisons). (E) Increased clinical score in C1qa−/− mice infected with C-202 calculated by the quantification of hunched posture, fur grooming, respiratory rate, and activity (*P < 0.05 by two-way ANOVA with multiple comparisons). (F) Experimental schematic for four-way transplant. (G) iMono cell number isolated from the lungs at Day 7 after infection in the transplant model. (H) The recipients that received B6 bone marrow (BM) had significantly more C1q-expressing iMonos compared with recipients that received C1qa−/− BM. (I) Recipient mice that received C1qa−/− BM had CD8+ T cells that produced less granzyme B compared with mice that received B6 BM (*P < 0.05, **P < 0.01, and ***P < 0.005 by two-way ANOVA with multiple comparisons). Data in A–C represent two experimental replicates (n = 3 mice per group per replicate). Data in D–G represent one experimental replicate (n = 3–5 mice per group). PFU = plaque-forming units.

To determine the effects of C1q signaling to CD8+ T cells during severe HMPV infection, we used a clinical isolate of HMPV, C-202, that is known to cause severe disease in young adult mice (25). C1qa−/− mice were able to clear the virus, but lost significantly more weight (Figure 3D) and had higher clinical scores compared with B6 mice (Figure 3E). C1qa−/− mice also had enhanced histopathology following C-202 infection compared with B6 mice (Figure E7A and E7B). These data indicate that the absence of C1q signaling has detrimental effects on CD8+ T-cell function and lung pathology during severe respiratory disease.

To determine if this CD8+ T-cell phenotype required the complement pathway downstream of C1q, we used C3−/− mice, which lack C3, a common protein used by all three complement pathways (Figure E8A). There was no difference in weight loss (see Figure E8B). In contrast to WT B6 mice, C3−/− mice had a detectable titer (see Figure E8C) at Day 7 after infection. However, there were no differences in epitope-specific CD8+ T-cell frequency in C3−/− mice (see Figure E8D). There was also no difference in inhibitory receptor (see Figure E8E), transcription factor expression (Figure E8E), or polyfunctionality of CD8+ T cells (see Figures E8F and E8G) between C3−/− and B6 mice, suggesting that C3-mediated complement activation is not involved in the CD8 dysfunction.

To assess if myeloid-derived C1q was required for enhanced CD8+ T-cell function, we used a four-way reciprocal BM chimera approach. First, C1qa−/− or B6 BM was transplanted into lethally irradiated CD45.1 recipients or the reciprocal whereby CD45.1 BM was transplanted into lethally irradiated C1qa−/− and B6 recipients (Figure 3F). For clarity, the experimental groups are referred to as BM→host (i.e., C1qa−/− BM into CD45.1 recipient is represented as C1qa−/−BM→CD45.1H). There was no difference in viral titer between transplant groups. There were equal numbers of iMonos in the C1qa−/−BM→CD45.1H and B6BM→CD45.1H groups (Figure 3G), but the C1qa−/−BM→CD45.1H group had negligible C1q+ inflammatory macrophages compared with the B6BM→CD45.1H group (Figure 3H). This was also reflected in the C1q protein level in whole-lung homogenate, with C1qa−/−BM→CD45.1H mice having minimal C1q detection (Figure E9A). Compared with B6BM→CD45.1H, CD8+ T cells from the C1qa−/−BM→CD45.1H group tended to produce less granzyme B (Figure 3I), whereas there was no difference in CD8+ production of IL-2 or IFN-γ in either group (see Figure E9B).

To demonstrate that C1qa−/− CD8+ T cells were not intrinsically defective, a competitive adoptive transfer experiment of congenically labeled CD45.1 B6 versus CD45.2 C1qa−/− CD8+ T cells into the same Rag1−/− recipient reconstituted with CD45.1 CD4+ T and B lymphocytes was performed (see Figure E9C). No difference was observed in granzyme B or IFN-γ production in B6 versus C1qa−/− CD8+ T cells in this model (see Figure E9D).

Blockade of gC1qR Leads to Reduced CD8 Effector Function

Because mice lacking myeloid-derived C1q showed a trend toward reduced CD8+ T-cell function following HMPV infection, we next assessed if C1q was directly acting on CD8+ T cells. Recombinant C1q bound strongly to purified lung CD8+ T cells from Day 7 after HMPV infection in C1qa−/− animals (Figure 4A). To assess possible receptors for C1q on CD8+ T cells, we further analyzed the T-cell compartment from the single-cell RNA sequencing dataset, identifying naive, effector, dividing, and nonspecific subsets (Figure 4B). One extracellular receptor for C1q, gC1qR (i.e., C1qbp), was expressed on a subset of dividing, cytotoxic CD8+ T cells with increased expression of Mik67 and on Gzmb CD8+ T cells (Figures 4C and E10A) (35). The dividing and nondividing CD8 effector populations were significantly expanded in HMPV-infected animals by permutation testing (Figures 4D and E10B). Additional C1q receptors were also assessed, with calreticulin (Calr) representing the only other notable receptor expressed within T cells (see Figure E10A). Calreticulin, however, is primarily surface-bound on apoptotic cells and plays a critical role in complement-mediated clearance of cell debris (36, 37). Because these analyses suggested gC1qR as a surface-bound C1q receptor on CD8+ T cells, we subsequently used a blocking antibody on ex vivo–stimulated lung cells from mice infected with HMPV. Production of IFN-γ was abrogated in cells treated with N11 HMPV peptide (a dominant MHC class I peptide) and α-gC1qR antibody (Figure 4E). As described previously, IFN-γ production was enhanced following blockade of PD-L1 (Figure 4E) (10). However, αgC1qR treatment reduced IFN-γ production even in the presence of PD-L1 blockade, suggesting two distinct pathways of IFN-γ regulation in CD8+ T cells (Figure 4E). IL-2, IFN-γ, and downstream IFN response gene CXCL10 and CXC9 protein levels were also decreased when cells were treated with αgC1qR and combination treatment; again, αgC1qR blockade reduced production of these cytokines despite the presence of PD-L1 blockade (Figure E11). Moreover, cells treated with recombinant C1q also exhibited an upregulation of IFN-γ production, which was again blunted by αgC1qR blockade (Figure 4E). C1q has previously been shown to regulate CD8+ T-cell metabolism and function in a chronic viral infection model (22). To that end, purified CD8+ T cells isolated from the lungs at Day 7 after HMPV infection showed a diminished spare respiratory capacity when treated with αgC1qR antibody (Figure 4F). Furthermore, administration of anti-gC1qR in vivo significantly reduced granzyme B production during HMPV infection, with a trend toward reduced IFN-γ as well (Figure 4G). Treatment of naive splenocytes with anti-gC1qR in the setting of PMA/I stimulation did not lead to reduction in IFN-γ, but rather was associated with increased IFN-γ (Figure E12). A lack of response to anti-gC1qR in this context suggests the importance of the concomitant inflammatory milieu, site of disease, and/or requirement of an antigen-specific T-cell receptor signaling event for disruption of optimal CD8+ function. Collectively, these data demonstrate a critical role for the gC1qR receptor in maintaining optimal CD8+ T-cell effector function and metabolic capacity that functions independently of PD-L1 signaling in the setting of respiratory viral infection.

Figure 4. gC1qR blockade leads to reduced CD8+ T cell function. (A) Recombinant C1q bound to cultured murine CD8+ T cells. Representative flow plots (left) and quantification of C1q-FITC+ CD8+ T cells (right). Data of one replicate are shown (n = 3 per group). (B) UMAP visualization of T cells by single-cell RNA sequencing split by infection status from Day 7 after infection. (C) Violin plots showing expression of C1qbp (i.e., gC1qR), Cd8a, Sell, Cd4, Gzmb, and Mki67. (D) Abundance plots of T-cell subsets. (E) ELISpot of IFN-γ in murine lung lymphocytes undergoing ex vivo class I peptide stimulation on Day 7 after infection with or without αgC1qR treatment. Additionally, PDL1 blockade increased IFN-γ production, and combination treatment resulted in decreased IFN-γ production (top). The bottom panel shows IFN-γ ELISpot with the addition of recombinant C1q with or without αgC1qR. Two replicates (n = 3 per group per replicate) were performed in triplicate. (F) Purified CD8+ T cells on Day 7 after infection had diminished spare respiratory capacity when treated with αgC1qR (*P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001 by unpaired t test or two-way ANOVA with multiple comparisons). (G) Administration of αgC1qR oropharyngeally in vivo on Days 5 and 6 after infection reduces CD8 T-cell effector function (n = 2 replicates; n = 2–3 per group per replicate; *P < 0.05 by Student’s t test). FCCP = carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; Gzmb = granzyme B; OCR = oxygen consumption rate; SRC = spare respiratory capacity.

C1q in Human Respiratory Viral Infection

The myeloid populations capable of producing C1q represent one of myriad differences between murine and human immunology. Human alveolar macrophages produce C1q at steady state, whereas C1q production in mice is often limited to interstitial macrophages and recruited alveolar macrophages after an inflammatory stimulus (38–40). However, we tested whether respiratory viral infection altered C1q production in the lung by using immunofluorescent staining on archived pediatric lung specimens. In a child who had a partial lung resection as a result of pleural blebs, C1q was visualized in the alveolar spaces (Figure 5A). In a case of fatal HMPV infection, the location of C1q+ cells markedly shifted to the interstitial and perivascular spaces (Figure 5B). Likewise, staining from a fatal case of mixed rhinovirus/parainfluenza virus pneumonia showed numerous interstitial and perivascular inflammatory cells producing C1q (Figure 5C).

Figure 5. C1q-producing cells are present in the lungs of children with fatal respiratory viral infection. Immunofluorescent staining of human lung tissue (A, resection from child with pleural blebs; B, autopsy specimen from a child who died of HMPV; C, autopsy specimen from a child who died of rhinovirus/parainfluenza infection). C1q (green) is present in alveolar spaces in a healthy child, with redistribution to interstitial spaces in infectious conditions. Scale bars, 1 mm. PIV = parainfluenza virus.

We next queried publicly available single-cell RNA sequencing data from bronchoalveolar immune cells in humans with severe coronavirus disease (COVID-19) infection to assess for evidence of C1q:gC1qR signaling (19). The macrophage populations identified previously were isolated for analysis and compared based on infection status (Figure 6A). Healthy controls had two clear macrophage clusters in the BAL fluid, both of which had several markers of alveolar macrophages and had high expression levels of C1QA, C1QB, and C1QC (Figures 6A and 6B). The macrophages in patients with COVID-19 were transcriptionally disparate compared with those in healthy controls, as COVID-19 macrophages clustered in six other discrete populations (Figure 6A). Importantly, several of these clusters produced C1QA, C1QB, and C1QC (Figure 6B). Moreover, these macrophage populations had transcriptional evidence of markers associated with inflammatory macrophages or monocytes, interstitial macrophages, and recruited macrophages (Figure 6B). Collectively, these data support the notion that respiratory viral infection leads to recruitment of a monocyte/macrophage population that produces C1q.

Figure 6. Transcriptional evidence C1q:gC1qR axis activation in severe coronavirus disease (COVID-19) cases. (A) UMAP of macrophage/monocyte populations identified in BAL fluid from healthy controls, moderate COVID-19 cases, and severe COVID-19 cases demonstrating eight clusters. (B) Stacked violin plots showing expression levels of C1QA, C1QB, C1QC, and various transcriptional markers of macrophage states from clusters identified from healthy controls (left) or patients with COVID-19 (right). (C) UMAPs of the subset of T cells from the overall dataset separated by disease state. (D) Abundance plots of various subsets in moderate versus severe COVID-19 disease. (E) Coexpression of MKI67 (marker of rapid division) and C1QBP is largely confined to the rapidly dividing CD8 cluster. HC = healthy control; S/C = severe COVID-19 cases.

Evaluation of the T-cell compartment in this data set demonstrated an increase in dividing CD8+ and CD4+ T cell populations in moderate and severe COVID-19 disease (Figures 6C and 6D and E13A). Notably, gC1qR (gene C1QBP) expression could be noted on all T-cell populations, but was most abundantly expressed on the rapidly dividing CD8+ T-cell population (see Figure E13B). Using differential gene expression analysis, C1QBP expression was significantly upregulated in dividing CD8+ T cells in severe COVID-19 disease compared with moderate disease (see Figure E13C). Similar to the animal model of HMPV, C1QBP and MKI67 expression largely colocalized to the rapidly dividing CD8 cluster (Figure 6E). Collectively, these data demonstrate transcriptional evidence of C1q expression from a recruited macrophage population and gC1qR expression on rapidly dividing CD8+ T cells, recapitulating several features found in the murine HMPV model.

Discussion

Taken together, our data identify an integral role of C1q signaling in respiratory viral infection. Three key findings from this study are as follows: 1) there is a novel iMono population that produces C1q concomitant with adaptive immune-mediated clearance of virus, 2) C1q directly impacts CD8+ T-cell function in our murine model, and 3) the C1q/gC1qR axis presents a possible mechanism as to how monocyte-derived C1q regulates CD8+ T-cell function in mice and humans across multiple respiratory viral pathogens.

C1q is canonically thought of as the initiator of the classical complement pathway in the innate immune response (41). However, C1q has also been implicated in homeostasis and inflammatory conditions, including clearance of apoptotic debris and recognition of damage-/pathogen-associated molecular patterns with resultant phagocytosis of extracellular pathogens (42). Previous studies in cancer and autoimmunity models in mice identified iMonos and macrophages as key producers of C1q, which facilitates a unique role of C1q independent of the complement cascade (22, 43–45).

Here, we demonstrate a novel role of a C1q-expressing myeloid population that promotes CD8+ T-cell function following respiratory viral infection in our murine model. During HMPV infection, the absence of C1q resulted in diminished effector CD8+ T-cell polyfunctionality and severe clinical disease when infected with a virulent strain of HMPV. In contrast to our data, in chronic LCMV infection in C1qa−/− mice, CD8+ T cells differentiated into short-lived effector cells with diminished metabolic capacity but more potent effector activity (22). These data suggest that the duration of infection/exposure to antigen may play a role in this regulatory pathway on CD8+ T cells, as pathogen-specific antibody and/or immune complex formation may alter the effects of C1q on CD8+ T cells.

There are differences between murine and human C1q production in the lung. In the human lung, alveolar macrophages produce C1q at baseline, whereas mice have negligible C1q production in the absence of infection (38–40). On staining of human lung tissue, C1q could be distinctly seen in the alveolar spaces of a lung section from an uninfected child; this distribution changed dramatically to an interstitial population in the setting of lethal respiratory viral infection, consistent with our animal model. Additionally, single-cell sequencing of the myeloid compartment in humans infected with COVID-19 demonstrated C1q as a differentially expressed gene in alveolar macrophages, although some expression was observed in monocyte-derived macrophages (46). Similarly, C1q expression was present in various recruited/interstitial macrophage populations in the setting of COVID-19 infection in the reanalysis of the COVID-19 BAL samples described above. Pathway analyses performed on the same COVID-19 BAL samples reanalyzed for this study found upregulation of complement in several myeloid cell types (19). Another study of the single-cell landscape on lung tissue from COVID-19 infection in humans also found evidence of C1QBP upregulation as part of the assessment of procoagulation (47). These data, coupled with the immunofluorescent images shown in our study, may suggest that the cell type and location of C1q production shifts during acute respiratory viral infection in humans.

The next question we addressed is how the communication between C1q and CD8+ T cells might be occurring. C1q has previously been shown to bind to human CD8+ T cells, with increased binding following in vitro stimulation (48). Similarly, our data show that, following HMPV infection, murine CD8+ T cells readily bound C1q. C1q can bind to apoptotic cells to promote phagocytosis and the Fc receptor of IgG antibodies to initiate the classical complement pathway (49). However, the most notable receptor for C1q in the context of the immune response, gC1qR (i.e., C1q binding protein, C1qBP), recognizes the globular head of the C1q polypeptide and has been shown to play a critical role in mitochondrial fitness (35, 50, 51). In addition to C1q, gC1qR has numerous ligands, including high-molecular-weight kininogen (HK), factor XII (Hageman factor), fibrinogen, thrombin, calreticulin, and multimeric vitronectin (52, 53). However, we show a unique role of gC1qR whereby rapidly dividing CD8+ T cells during HMPV infection upregulated gC1qR expression in our murine model and in patients with severe COVID-19. These findings are consistent with a prior study that demonstrated that gC1qR is abundantly expressed on human CD8+ T cells in the setting of chronic hepatitis C infection (23). Subsequent in vitro blockade of gC1qR led to reduced effector function and cell proliferation in our model. Blocking gC1qR signaling in CD8+ T cells from the murine lung following HMPV infection also led to reduced metabolic capacity, similar to a prior study assessing the metabolomics of C1qa−/− CD8+ T cells in the LCMV chronic model (22). Collectively, these data identify gC1qR as at least one of the critical C1q receptors required for optimal CD8+ T-cell function in multiple respiratory viral models.

There are limitations to the present study, as well as possible future directions. First, C1qa−/− mice cleared HMPV comparably to wild-type mice despite the diminished CD8+ T-cell effector function. However, in our C-202 model and in prior LCMV models, C1qa−/− mice have increased pathology (22). These findings may suggest effective, but pathologic, alternative mechanisms of clearance in the absence of C1q, which would warrant further study of other antiviral cell types such as natural killer cells. Second, certain control groups or experiments were performed as a single replicate with a relatively low sample size. As an example, restoration of CD8+ T-cell functionality after using a B6 hematopoietic stem cell transplant approach for a single four-way transplant was noted with granzyme B production, but not other effector functions. These data support C1q’s role in CD8+ T-cell responses, but may be limited by the negative effects of whole-body radiation in the absence of C1q (54). The interplay between C1q, CD8+ T-cell function, and pathogen-specific antibody could also be explored mechanistically by passive convalescent serum transfer in C1qa−/− mice and/or rechallenge experiments. The latter would also be fascinating in the context of CD8+ T-cell memory formation in the absence of C1q. Further evaluation of the C1q/gC1qR axis in our mouse model and human samples are warranted. gC1qR loss of function is poorly tolerated in T cells, with an in vitro knockdown approach in mice resulting in diminished proliferative capacity, increased mitochondrial membrane permeability, and, ultimately, increased apoptotic cell death (51). This makes it technically challenging to completely knock out the gC1qR receptor on CD8+ T cells, as this will have detrimental effects to the survival of CD8+ T cells, but also underscores the importance of this receptor in CD8+ T-cell fitness. In a chimeric antigen receptor T-cell model, heterozygosity of gC1qR led to reduced CD8+ T-cell effector function and increased tumor burden (51), which supports our hypothesis that gC1qR signaling on CD8+ T cells is crucial to their function. One future study could assess serum C1q levels in acute disease or ex vivo manipulation of gC1qR from cells isolated from infected patients, which could provide insights into the potential diagnostic and/or therapeutic potential of this pathway.

Collectively, the present study identifies a novel C1q+ monocyte population and elucidates a potential mechanism of how C1q signaling via gC1qR on CD8+ T cells can regulate optimal CD8+ cytotoxic antiviral effector function during respiratory viral infections.

Acknowledgment

The authors thank the University of Pittsburgh Unified Flow Core for help with flow cytometry and the NIH Tetramer Core Facility (contract no. 75N93020D00005) for providing HMPV tetramers.

Supported by National Institute of Allergy and Infectious Diseases grant AI085062 (J.V.W.), National Heart, Lung, and Blood Institute grant 1F30HL159915 (O.B.P.), National Institute of General Medical Sciences grant T32GM008208 (O.B.P. and J.S.), Eunice Kennedy Shriver National Institute of Child Health and Human Development grant K12HD000850 (T.E.), NIH grant R01HL137709 (K.C.), the Henry L. Hillman Foundation (J.V.W.), and the Marjory K. Harmer endowment for research in Pediatric Pathology (M.R.-M.).

Author Contributions: T.E. and O.B.P. designed, executed, interpreted, and drafted the work equally. D.L., L.F., H.M.Z., and K.C. designed, executed, and interpreted single-cell RNA sequencing work. J.S. and A.E. contributed significant data. L.S., C.M.S., M.R.-M., and T.D.O. contributed significant expertise and data related to pathology. M.J.R. and C.B. contributed significant expertise and data. J.V.W. made critical contributions in conception, design, and analysis of the data and drafting/revising of the manuscript. All authors reviewed the manuscript and approved of the final submission.

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Originally Published in Press as DOI: 10.1165/rcmb.2024-0004OC on May 2, 2024

Author disclosures are available with the text of this article at www.atsjournals.org.
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