JVII Journal

Introduction

Generation of high titer neutralizing antibodies is a desired end-point in the vaccine development. Antibodies binding to target antigens result in the formation of Immune Complexes (ICs), which trigger effector Functions in Fc Receptors (FcRs) bearing cells in particular lymphocytes. These receptors are well studied in innate cells and B lymphocytes, however their role in CD4⁺ T cells has starting to emerge. Antibodies produced against pathogenic antigens during the vaccination facilitate FcR ligation on immune cells, resulting in the massive production of inflammatory cytokines. Antibody Dependent Enhancement (ADE) is a concomitant phenomenon observed in the Ribonucleic Acid (RNA) viral vaccines i.e. HIV-1, RSV, Ebola, Dengue, SARS and MERS. In HIV-1 infection, both neutralizing and nonneutralizing antibodies cause ADE in macrophages. Cells such as monocytes, macrophages and dendritic cells that express FcRs has been implicated in the ADE, but the participation of CD4⁺ T cells has not been examined. The majority of vaccine efforts for corona virus disease 19 (COVID-19) have targeted Spike protein. Antibodies against the Spike protein contribute to the ADE. A neutralizing antibody targeted to Receptor Binding Domain (RBD) of Spike protein from Middle East Respiratory Syndrome (MERS), facilitated the conformational changes and utilized FcR (CD32a) for viral entry [1]. A key role for monocytes and macrophages is suggested in the inflammatory responses observed in COVID-19 [2]. Even though, CD4⁺ T cell lymphopenia is observed both in HIV-1 and COVID-19 infection, thus far, ADE has not been examined in either CD4 or CD8 T cells. A contributing factor to this disengagement is the current accepted paradigm that the CD4⁺ T cells do not express FcRs [3,4]. This notion is incorrect and is not experimentally validated. Several earlier studies have supported the presence of FcRs on CD4⁺ T cells and have hypothesized their role in modulating CD4⁺ T cell responses [5]. In 2011, we reported phosphorylation of T Cell Receptor (TCR) proteins in the human CD4⁺ T cells and Jurkat cell line, in response to in vitro treatment with ICs [6]. Since then we and several other groups have confirmed the presence of FcRs and signaling mediated by these receptors in providing a costimulatory signal that result in the activation and differentiation of human naïve CD4⁺ T cells [6-8]. The costimulation from FcR signaling results in the production of proinflammatory cytokines by human CD4⁺ T cells, that could possibly trigger cytokine storm [9,10]. Now several other groups have shown the presence of various FcR proteins on the human CD4⁺ T cells i.e. FcγRIIa, FcγR1, and FcµR. Upon ligand binding, the FcR signaling in these cells produce IFN-γ [7-12]. We thus commend that it is important to examine the contribution of FcR signaling in the adaptive immune responses manifested by T cells. In the human airways, epithelial cells are protected by humoral proteins such as collectins, defensins and antibodies. Toll-Like Receptor (TLR) signaling in lung epithelium result in the production of anti-microbial peptides for protection. Thus, it is important to consider the outcome from nucleic acid-based vaccines currently being developed for COVID-19. How these new vaccines will enhance the nucleic-acid sensing in the endosomes and on the cell surface remains unknown. It is thus essential to examine the time of retention of these RNA and DNA vectors in the human system both in tissue and circulation. In this review, I will summarize and evaluate the accumulating evidence for the role of FcRs, Complement Receptors (CRs) and nucleic-acid sensing tolllike receptors (NA-TLR signaling in enhancing antibody mediated viral entry into CD4⁺ T cells and the consequence that follows.

Fc receptors on CD4⁺ T cells: Participation in ADE development?

Recognizing and fully understanding the expression pattern of various receptors on cell types and their participation in the ADE during RNA viral infection is critical for vaccine safety. Both FcR and CRs contribute to the process of ADE. These receptor proteins are critical to the viral entry, and they also contribute to the cell death and viral spread in the cell types such as monocytes, macrophages and dendritic cells. A role for CD32a in ADE was recently confirmed in corona virus infection [1]. FcRs are expressed by the activated CD4⁺ T cells, and hence their role in ADE require validation. It is argued that ADE is an event of innate cells such as macrophages and monocytes. Enhanced viral entry via ADE will likely contribute to the lymphopenia, observed for CD4⁺ and CD8⁺ T cells during viral infection. In HIV-1 infected patients, loss of CD4⁺ T cells correlates with increase in the number of IC bound CD4⁺ T cells, which suggest that the FcR signaling is an important contributor to the observed cell death [13]. In HIV-1 patients, CD4⁺ T cell subset is the largest subpopulation that binds to labeled ICs, when compared to CD19⁺ B cell and CD8⁺ T cell subset (unpublished observation, data available on request). The viral particles of SARS-CoV-2 RNA are observed in CD4⁺ T cells. The pro inflammatory responses that are being observed in children infected with COVID-19 could be a result of FcRs engagement by virus-antibody complexes. One possible explanation for inflammation observed in the children is the production of proinflammatory cytokines by activated CD4⁺ T cells [14]. Thus, it is not that far-fetched to suggest that the Fc mediated signaling in CD4⁺ T cells contributes to the proinflammatory cytokine production, which then enhance the viral pathology [8-10]. IFN-γ is a well-recognized proinflammatory cytokine that is produced upon CD16a signaling in CD4⁺ T cells and both moderate and high secretors are observed [9]. Now both, CD32 and CD64 are shown to produce IFN-γ [8,11].

ICs activate classical complement pathway and the byproducts of this pathway are now implicated in COVID-19 infection [15]. Complement receptor proteins are expressed by CD4⁺ T cells, and upon activation from ligand binding, these cells produce inflammatory cytokines. These cytokines do also contribute to the cellular differentiation [16]. A role for complement receptors, in particular CR2 has been suggested in enhancing the HIV-1 infection [17]. The classical complement pathway is triggered by the binding of ICs to C1q and this ultimately leads to the formation of C5b-9 complex on the cell surface. C5b-9 is a pleotropic signaling molecular protein complex, which triggers the inflammation, apoptosis, and tissue necrosis [18]. Sublytic amounts of C5b-9, the terminal complex of the late complement pathway triggers cellular apoptosis. Apoptosis has been suggested as a contributor to the lymphopenia observed in COVID-19 infection. The deposition of higher amounts of C5b-9 on cells leads to the cellular lysis. We confirmed these events in vitro by assembling C5b-9 using purified human late complement proteins on the membranes of purified human CD4⁺ T cells and in Jurkat cells [6]. Sublytic amount of C5b-9 deposition on the cells includes protein synthesis, DNA synthesis, arachidonic acid metabolism, and activation of polymorphonuclear leukocytes [19]. C5b-9 enhances the production of endothelial intercellular adhesion molecule-1 (ICAM-1), E selectin, interleukin 8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1). Other notable events that are trigged by C5b-9 are the activation of cell cycle related genes p53, p21, growth arrest DNA damage-45 (GADD45), checkpoint kinase-1 (CHK-1), and CHK-2, mitogen activated protein kinase (MAPK) p38, janus kinase (JAK) 1, signal transducer and activator (STAT) 3, and STAT 4 in endothelial cells [18,20-23]. IC treatment along with sublytic C5b-9 of human CD4⁺ T cells enhances RNA transcripts from nuclear factor kappa B (NF-kB), Burton’s tyrosine kinase (Btk) and activator protein 1 (AP-1) resulting in the production of pro-inflammatory cytokines i.e. IL-1a, IL-1b, IL10, and IL-12a [10]. These events also occur in cardiac myocytes and muscle cells in response to C5b-9 deposition [24,25]. Both sublytic and lytic amount of C5b-9 deposition does occur on CD4⁺ T cells, which leads to the inflammatory responses and cell death leading to the viral spread. Both classical complement activation and direct activation of complement by viral particles will enhance the assembly of C5b-9. Thus, it is safe to suggest that complement activation participates in enhancing viral pathology in T cells.

Antibody Dependent Cellular Cytotoxicity (ADCC) is exhibited by Natural Killer (NK) cells. Emerging studies have now shown a role for CD16a in driving ADCC in CD4⁺ T cell population. A chimeric construct including the high-affinity CD16 (FCGR3A) V158 variant, CD8a hinge, and transmembrane domains, along with signaling domains from CD3z and 4-1BB TNF Receptor Superfamily Member 9 (TNFRSF9), forming a chimeric receptor termed CD16V-BB-z triggered cell death upon the engagement of antibodies [26]. Forced expression of γ-chain of FcR, induced the expression of CD16a in CD4⁺ T cells and this resulted in enhanced ADCC, establishing a role for this pathway in cell death [26-28]. In our studies that examined the RNA transcript levels, we observed a strong overexpression of granzyme A upon FcRs engagement by ICs, compared to CD28 (Chauhan, unpublished observation, NCBI accession no. GSE127664). Thus, it can be safely stated that both FcR signaling and complement activation contribute to the viral spread and lymphocytic cell death causing lymphopenia. Lesson could be drawn from RV-144 vaccine trial for HIV-1 infection, where a role for FcRs was observed. Decreased risk of infection in this trial was attributed to Fc-mediated functions. Still a role of FcR expressing CD4⁺ T cells in viral pathologies has yet to be examined. FcR bearing CD4⁺ T cells population would be critical in understanding COVID-19 pathology.

Toll-like Receptor’s on CD4⁺ T cells: Contribution to ADE

Pattern recognition receptors (PRRs) in particular NA-TLRs are critical players in the viral infections. In humans, TLR3 binds to dsRNA, and ssRNA is recognized by TLR8. The nucleic-acid sensing is extensively studied in innate cell responses. TLR9 signaling in Plasmacytoid Dendritic Cells (pDCs) upon the ligation of CD32 by its ligand ICs composed of dsDNA, drives the Interferon Signature Genes (ISG) expression. Nucleic-Acid Containing Immune Complexes (NA-ICs) engage FcRs on the cell surface, which are then internalized to endosomes or lysosomes, where the nucleic-acid is recognized by NA-TLRs. Such events trigger signaling, leading to the secretion of proinflammatory cytokines. In a landmark study in 2006, it was shown that when TLR9 is forced to relocate to the cell surface, TLR9 recognized altered nucleic acids and CpG Deoxynucleotide (CpG ODN) on the cell surface [29]. Cellular trafficking events are noted during TLR 9 movement. TLR9 is tracked through endoplasmic reticulum and lysosomes, post ligand binding [30]. Acidification of lysosomes and endosomes by drugs such as chloroquine and bafilomycin AI abrogates TLR9 signaling [31]. The natural ligands that forces the NA-TLRs to the cell surface has never been identified. In a 2017 paper, we showed that indeed the CD16a ligation with ICs isolated from human plasma, induces the overexpression of NA-TLRs and forced their relocation to the cell surface in human CD4⁺ T cells [32]. The three NA-TLRs in CD4⁺ T cells that are activated by plate-bound ICs localized with CD16a on the cell surface and this triggered secretion of several key proinflammatory cytokines [32,33]. In COVID-19 infection, TLR3 and TLR8 that are present on the cell surface will facilitate direct binding of virus particles, which will lead to the cellular activation, enhanced levels of proinflammatory cytokines production and this will also contribute to the ADE. The nucleic acid-based vaccines could also contribute to enhanced nucleic-acid based signaling events from the endosomes. The mRNA introduced into the cells upon vaccination exists for at least two days, which is sufficient time for NA-TLR signaling. FcR signaling when compared to CD28 showed 20-fold increase in TLR3 and TLR8 RNA transcripts [10,32]. A similar increase was observed at protein level (Figure 1) [10,32]. A statistically significant upregulation of Myeloid Differentiation Factor 88 (MyD88) and Nuclear Factor Kappa B (NF-kB) RNA transcripts upon FcγR signaling were also noted [10,32]. In human CD4⁺ T cells, drug hydroxychloroquine disrupted the colocalization of all the three NA-TLRs examined with ICs bound to FcRs on the cell surface [32]. In these cells, ICs by triggering FcR signaling induced the overexpression of MyD-88, a signaling protein required for TLR8 signaling and this protein migrated towards the cell surface [32].

Recycling of TLRs between subcellular compartment and to the cell membrane is governed by various accessory proteins. Trafficking of TLRs3, TLR7, and TLR9 from endoplasmic reticulum to endosomes is essential for TLR signaling [34]. A glycoprotein unc-93 homologue B1 (UNC93B1) is the only recognized protein that interacts and contributes to the NA-TLRs trafficking from endoplasmic reticulum to endolysosomes. Syk kinase contributes to the microorganism mediated intracellular internalization of TLR4. Thus, it is not far-fetched to suggest that Syk activation contribute to the movement of NA-TLRs in the CD4⁺ T cells [10,32].

The viral nucleic acid in the cytoplasm is recognized by Retinoic-Acid Inducible Gene I (RIG-I) Melanoma Differentiation Associated Gene 5 (MDA5) and nucleotidyltransferase cyclic GMP-AMP Synthase (cGAS). Signaling complex formed by these proteins, recruit adaptors including TIR-domain-containing adaptor proteins, Mitochondrial Antiviral-Signalling Protein (MAVS), TIR-Domain-Containing Adapter-Inducing Interferon-Β (TRIF) and stimulator of interferon genes Protein (STING) triggers activation of downstream adaptor molecule MyD88, which leads to the activation of the transcription factor NF-κB and Interferon Regulatory Factor 3 (IRF3) (Figure 2). These events lead to the production of type I Interferons (IFN-α /β) and pro-inflammatory cytokines. In CD4⁺ T cells, these proteins are upregulated upon FcR signaling [10]. We suggest that the formation of joint signaling complex of key receptors such as FcRs, CRs, and TLRs in activated CD4⁺ T cells, will drive the hyperactivation of cellular responses (Figure 1) [5,6,32,35]. Cytoconjugation among antigen presenting cells, B cells and T cells will trigger formation of receptor clusters, which will facilitate cross-talk. Joint signaling from these receptors will produce robust proinflammatory cytokine responses [10,32]. Formation of these cellular clusters, which will also bind to FcR expressing platelets could lead to clogging of arteries and hypertension. A successful disruption of these receptor clusters should be targeted for therapeutic intervention.

In activated CD4⁺ T cells, TLR3 and TLR8 are relocated to the cell surface, where they will recognize the viral RNA from HIV-1 and COVID-19 viruses. Studying these signaling events from the cell surface will open the possibilities to target disruption of signaling complexes for developing therapies and enhancing the efficacy of vaccines. FcR signaling will drive epigenetic changes and microRNA expression in CD4 population [36] and unpublished observation (Chauhan, et. al, NCBI accession no. GSE127664). The effector cell responses generated upon the formation of ICs and their subsequent Fc/FcR interactions, provided the limited benefits observed in RV144 HIV-1 trial and broadly neutralizing antibodies 3BNC117, PG16 clinical trial [37,38]. The existing literature thus suggest a coordinated role for NA-TLRs and FcRs in triggering CD4⁺ T cell responses in RNA-viral infections.

Fc R signaling in CD4⁺ T cells: regulate pro-inflammatory cytokines

In viral infections including SARS-CoV-2, increase in cytokine levels correlate with disease pathology Innate cells i.e. monocytes, macrophages, and dendritic cells are considered as primary contributors to the cytokine storm. IL-1, IL-6, IL-8, IFN-γ, IL1-a, and IL1-b are observed at enhanced levels in infectious pathology. Interferon responses are critical for the resolution of the viral infection; however, these responses also contribute to the pathology [39]. It has been previously suggested that Th2 subset contributes to the lung pathology in SARS-Cov- 2 infection. IFN-γ producer Th1 cells are critical for viral pathology and these cells have been identified in SARS-CoV-2 infection. The emerging data now implicates both the Th1 as well as Th17 cell types exists in COVID-19 pathology [40,41].

Although, not a prominent response, early results from Moderna’s mRNA-1273 vaccine show the development of Th1 and Tfh responses and lack of the Th2 response [42]. We and others have reported production of IFN-α, IFN-β and IFNγ upon FcR signaling in CD4⁺ T cells [8,10,11]. Engagement of FcγgRIIIa by ICs triggers a strong production of IFN-γ, compared to CD28 cosignal [9]. Engagement of FcγRI, FcγRIIa, FcγRIIIa and FcµR by their ligands on CD4⁺ T cells, triggers the release of proinflammatory cytokines such as IL-1a, IL-1b, IL-10, IL-12a, IFN-γ, IL-17A, IL17F, IL-21, IL-22 and IL-23 [7,8,10,11]. IFN gene signature (ISG) upregulation was observed in the human CD4⁺ T cells upon FcR signaling, and some patients show predominant type 1 response [10, 32] Peripheral blood monocytes obtained from COVID-19 patients produced IFN-γ, TNFα, IL-2, IL-5, IL-13, IL-10, IL-9, IL-17A, IL-17F and IL-22 after stimulation with Spike protein peptide MP_S. [41].

Interestingly, peripheral follicular helper CD4⁺ T cells (pTfh) cells which are CXCR5⁺ ICOS⁺ PD1⁺ are induced upon vaccination against influenza virus. Such pTfh cells are also observed before symptomatic recovery of COVID-19 patients and upon vaccination. These cells were observed prominently in convalescing patients on day 20^th [43]. These findings are relevant in the context of our findings, where we have shown that the FcR ligation by ICs in vitro triggers the development of such pTfh cells, and these cells do exists in SLE subjects [44,45]. In COVID-19, we expect the increase in the levels of both IgG and IgM containing ICs, and such ICs will drive the generation of these pTfh cells. A prominent role for proinflammatory cytokine storm in COVID-19 pathology has been suggested. These cytokines have also been suggested to be critical to the inflammation in children’s that are exposed to SARS-CoV-2 virus and present symptoms similar to Kawasaki disease. In summary, we insinuate a prominent and critical role for adaptive immune cells in modulating responses in viral pathology associated with RNA viruses. Increased expression of FcR is observed as early as 48 h post activation of CD4⁺ T cells, which is severally enhanced by day 14^th [32]. We suggest a need for further examination of the FcR expressing CD4⁺ T cells in viral pathology such as COVID-19. It will be of significant interest to examine the predictive value of these cells as a potential biomarker for identifying the subjects that show enhanced disease severity during viral infection. It will be useful to assess if FcR⁺ CD4⁺ T cells can serve as a biomarker for testing the efficacy of various vaccines, currently in the development.

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