Any extracts used in the following article are for non-commercial research and educational purposes only and may be subject to copyright from their respective owners.
Handwritten. No made-up citations or woke propaganda.
In Part I I reviewed 3 case reports of IgG4-RA and myocarditis; Part II reviewed 7 case reports of other diseases associated with IgG4 class switching after COVID-19 vaccination; and Part III started to review the mechanisms behind IgG4-RA, with a deep dive into macrophage priming via the fragment chain receptors(FcRs)
Part IV investigated FcR priming in greater detail, the mechanisms behind how IgG4 induces immunosuppression, and the relevance of an oncomiR called miR-222.
Part V will discuss long-lived vs. short-lived plasma cells and how this relates to IgG4-RD; a possible explanation for the Th2 shift associated with Pfizer’s BNT162b2; and a further discussion about T cell involvement with IgG4-RD. This was touched upon in Part IV, as it may explain why some relapses occur.
Discussion
Long-lived vs short-lived plasma cells (LLPCs vs SLPCs)
A hat tip to James Lyons-Weiler for posting a walkthrough of a study published 27th September 2024 by Nguyen et al.:“SARS-CoV-2-specific plasma cells are not durably established in the bone marrow long-lived compartment after mRNA vaccination”1.
The synopsis is that the mRNA vaccine developers knew from the outset that durable, long-term immunity was never going to be the outcome of vaccination, as only short-lived plasma cells were developed, rather than long-lived, durable PCs in the bone marrow.
“… While serum IgG titers specific for influenza and tetanus correlated with IgG LLPCs, serum IgG levels for SARS-CoV-2, which waned within 3–6 months after vaccination, were associated with IgG non-LLPCs. In all, our studies suggest that rapid waning of SARS-CoV-2-specific serum antibodies could be accounted for by the absence of BM LLPCs after these mRNA vaccines.”
From: “SARS-CoV-2-specific plasma cells are not durably established in the bone marrow long-lived compartment after mRNA vaccination” (2024)
Plasma cells develop from B lymphocytes (B cells). They are a type of white blood cell that produce antibodies (immunoglobulin) and play a significant role in the adaptive immune system as they are the main cells responsible for humoral (body fluid) immunity.
On reading James’s account, my first hypothetical question was “How does this relate to IgG4-RD? Are IgG4 autoAbs derived from long-lived PCs in the bone marrow, or short-lived peripheral plasmablasts?”
We got a clue to the answer in Part IV, from the study into follicular dendritic cells (FDCs), and how immune complexes are repeatedly presented to B-cells, sustaining IgG4 class switched Abs indefinitely.
Put another way, these geniuses have created the worst of all worlds: short-lived anti-viral immunity that quickly wanes, but IgG4 class switching that persists indefinitely. It recalls the challenges faced when trying to create a successful HIV vaccine:
Any extracts used in the following article are for non-commercial research and educational purposes only and may be subject to copyright from their respective owners.
A review from 2021 by Zografou et al. helped to confirm the hypothesis: “Short- and Long-Lived Autoantibody-Secreting Cells in Autoimmune Neurological Disorders”2.
Key takes:
Abstract
As B cells differentiate into antibody-secreting cells (ASCs), short-lived plasmablasts (SLPBs) are produced by a primary extrafollicular response, followed by the generation of memory B cells and long-lived plasma cells (LLPCs) in germinal centers (GCs).
Generation of IgG4 antibodies is T helper type 2 (Th2) and IL-4, -13, and -10-driven and can occur parallel to IgE, in response to chronic stimulation by allergens and helminths.
Although IgG4 antibodies are non-crosslinking and have limited ability to mobilize complement and cellular cytotoxicity, when self-tolerance is lost, they can disrupt ligand-receptor binding and cause a wide range of autoimmune disorders including neurological autoimmunity.
In myasthenia gravis with predominantly IgG4 autoantibodies against muscle-specific kinase (MuSK), it has been observed that one-time CD20+ B cell depletion with rituximab commonly leads to long-term remission and a marked reduction in autoantibody titer, pointing to a short-lived nature of autoantibody-secreting cells.
This is also observed in other predominantly IgG4 autoantibody-mediated neurological disorders, such as chronic inflammatory demyelinating polyneuropathy and autoimmune encephalitis with autoantibodies against the Ranvier paranode and juxtaparanode, respectively, and extends beyond neurological autoimmunity as well.
Rituximab mechanisms were discussed in Part IV, as far as is known: “… Rituximab is a monoclonal antibody (MAB) that attaches itself to CD20 proteins on B cells. It causes B cell death via multiple mechanisms”.
IgG4 autoAbs may quickly wane when you are treated with rituximab, but relapses may be just as quick, as we have seen in some of the case reports:
Although IgG1 autoantibody-mediated neurological disorders can also respond well to rituximab induction therapy in combination with an autoantibody titer drop, remission tends to be less long-lasting and cases where titers are refractory tend to occur more often than in IgG4 autoimmunity.
The long-lived autoimmune plasma cells referred to are more likely to originate from IgG1 autoAbs rather than IgG4:
Moreover, presence of GC-like structures in the thymus of myasthenic patients with predominantly IgG1 autoantibodies against the acetylcholine receptor and in ovarian teratomas of autoimmune encephalitis patients with predominantly IgG1 autoantibodies against the N‐methyl‐d‐aspartate receptor (NMDAR) confers increased the ability to generate LLPCs.
From the discussion:
IgG4 B cells seem to be equally ineffective in producing LLPCs.Compared to IgG1 B cells, human IgG4 B cells express less CXCR4, a chemokine important for bone marrow chemotaxis, and low numbers of IgG4 cells are observed in human secondary lymphoid organs (84).
Data from IgG4 related disease (IgG4-RD) also support the notion that the generation of LLPCs is diminished in IgG4 responses as levels of circulating IgG4 SLPBs correlate with total IgG4 levels.
IgE is commonly known as the allergy antibody. It plays a central role in allergy sensitisation and atopic disorders such as allergic rhinitis, asthma, and atopic dermatitis. LLPC involvement is, however, indicated:
In addition, rituximab treatment affects a significant drop in IgG4 (and IgE) levels (85), but the drop in IgG1 titers is not as pronounced (86). It should be noted though that as the response to rituximab treatment can be partial, some LLPCs most likely do exist.
Treg involvement is suspected:
GC: lymph node germinal centre.
Regulatory T cell (Treg) involvement may be different in IgG4 and IgG1 responses. Apart from extrafollicular Tregs that could control GC initiation, follicular regulatory T cells (Tfr) may balance Tfh cells and participate in determining the fate of B cells.
Tfr cells may either directly suppress GC B cells through CTLA-4-mediated inhibition of CD80/CD86 co-stimulatory signaling or indirectly do so through IL-10 secretion acting on Tfh cells (87).
If you take allergen-specific regulatory T cells from a healthy individual, put them in a petri dish and expose them to anti-inflammatory interleukin-10 or Transforming Growth Factor-β then you cause them to suppress IgE and increase IgG4 production, i.e. you can induce class switching and allergy tolerance via these cytokines and their effects on Tregs:
Importantly, allergen-specific Tregs from healthy individuals can suppress IgE and induce IgG4 production ex vivo via IL-10 and TGF-β (88, 89). During human helminth infection (which causes IgE and IgG4 elevation), Tregs that produce IL-10 and inhibit effector T cells can be found in the peripheral blood and may play a role in limiting inflammation (90), while in murine models of helminth infection, Tregs expand, produce IL-10, and can limit the Th1 more than the Th2 response (91).
To complete the circle, it’s not surprising to find more Tregs in IgG4-RD patients:
Similarly, Tregs are expanded in the peripheral blood of IgG4-RD patients (along with IgG4 and Th2 cells) (92, 93) and infiltrate target organs (along with IgG4 cells) (94).
MG: myasthenia gravis, a chronic autoimmune disease that causes muscle weakness in the body. Dysfunctional Tregs are suspected:
Conversely, in MG mediated by predominantly IgG1 autoantibodies against AChR, patients have been shown to harbor dysfunctional Tregs (95), and further, induction of Tregs via GM-CSF effectively suppressed experimental autoimmune MG (96).
PV: pemphigus vulgaris, a chronic autoimmune disease that causes blisters in the mouth and on the skin.
In pemphigus vulgaris (PV) mediated by predominantly IgG4 autoantibodies against desmoglein, contradicting data that Tregs have both not been able to suppress (97) and have suppressed autoimmune responses (98) are reported. These results underline the need for further investigations into the role of Tregs in autoantibody-mediated autoimmunity.
HLA class antigen presentation
Human leukocyte antigens are protein markers that help our immune systems to see which cells belong in our body (self) vs those that are foreign:
From “HLA class I and II antigen-presentation pathways. The HLA class I pathway (above) is responsible for degrading endogenous antigens into peptides via the multi-catalytic proteasome complex. Peptides are then transported into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). Here peptides bind based on their relative affinity to the allotypes present. HLA-I peptide complex is then transported through the Golgi apparatus to the cell surface where it is scrutinised by CD8+ T cells. The HLA class II pathway (below) involves the degradation of exogenous antigens in the endosome compartment. In the ER immature HLA-II proteins are stabilised by the invariant chain and released into the HLA class II compartment. Here, the enzyme HLA-DM removes the class II invariant chain peptide (CLIP) from the binding pocket allowing for antigenic peptide binding. Mature HLA-II molecules bound to their peptide cargo are then transported to the cell surface for CD4+ T cell recognition.” Source: https://www.researchgate.net/publication/371272257_SAPrIm_a_semi-automated_protocol_for_mid-throughput_immunopeptidomics/figures?lo=1
A further interesting aspect of autoantibody-mediated neurological diseases is HLA restriction seen in patients as compared to healthy controls, likely meaning that specific antigenic peptides are better presented to T cells by specific HLA alleles (217, 218). This is relevant to B cell function, as B cells can pick up antigens with the B cell receptor (BCR) and process them and effectively present them via MHC II to T cells (219).
Interesting to see reference to IgG4 and the lectin pathway here. I hypothesised that IgG4 CSR results from anti-inflammatory cytokines induced by repeated exposure to the galectin-3-like fold on Spike S1 NTD, although in this case, it is more the tail wagging the dog:
The presence of complement deposition points to the putative role of other, coexisting, non-IgG4 antibodies as complement activating factors, or alternatively, points to the ability of IgG4 antibodies to mobilize complement despite classical views, possibly via altered IgG4 glycosylation and the lectin pathway (295–299), or IgG4 aggregation (79).
Galectins are a type of lectin, - the most widely expressed type in an organism. They are glycans, play a part in cell surface adhesion, cancer advancement and metastasis3.
The relationship between galectin, glycosylation, IgG4-RD, autoimmune disorders, and cancer keeps presenting itself:
From a study of patients with ulcerative colitis (UC) and metabolic syndrome (MetS):
… Clinically and histologically milder disease with higher serum level of immunosuppressive cytokine interleukin-10 (IL-10) and fecal content of Galectin-3 (Gal-3) was observed in subjects with UC and MetS, compared to subjects suffering from UC only. This was accompanied with predomination of IL-10 over pro-inflammatory cytokines tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interleukin-17 (IL-17) in the sera as well as Gal-3 over TNF-α and IL-17 in feces of UC patients with MetS. Further, the patients with both conditions (UC and MetS) had higher percentage of IL-10 producing and Gal-3 expressing innate and acquired immune cells in lamina propria.
… Gal-3 is produced mainly by monocytes/macrophages and in UC is expressed on CD68+ colon-infiltrating macrophages[12,13].
… The presence of MetS may attenuate colon inflammation, possibly by deviating local inflammatory response toward enhanced participation of immunosuppressive cells and molecules. The increase in systemic IL-10 and local Gal-3 production as well as expression on Tregs and immunocompetent cells accumulating in affected colon tissue implicate on IL-10 and Gal-3 dependent immunomodulation. The precise mechanism of Gal-3 effect in MetS and UC comorbidity is still to be clarified.
From: “Metabolic syndrome attenuates ulcerative colitis: Correlation with interleukin-10 and galectin-3 expression“ (2019)
IgG4 is associated with more severe cases of UC. In another study, a third of patients had IgG4-positive plasma cells:
3. Distribution of IgG4-positive cells in the colonic mucosa of UC patients
Abundant infiltration of IgG4-positive plasma cells was detected in 10 UC patients (IgG4-present patients, 31%) (Fig. 1). Number of IgG4-positive plasma cells was not significantly difference in biopsy colonic locations. Abundant infiltration of IgG4-positive plasma cells was observed in no patients with irritable colon syndrome or ischemic colitis. Numbers of IgG4-positive plasma cells/HPF in specimens of colonic mucosa from UC patients (9.3±5.7; range, 0 to 20) were greater than those from patients with irritable colon syndrome (2.3±1.8; range, 0 to 5; p<0.01), ischemic colitis (1.5±3.1, range, 0 to 9; p<0.01) and AIP (5.5±6.7, range, 0 to 20; p<0.05) (Fig. 2).
Fig. 1. Abundant infiltration of immunoglobulin G4 (IgG4)-positive plasma cells in the colonic mucosa of a patient with ulcerative colitis (IgG4 immunostaining, ×400).
Fig. 2. Numbers of immunoglobulin G4-positive plasma cells/high power field in specimens of colonic mucosa from ulcerative colitis (UC) patients were greater than those from patients with irritable colon syndrome (p<0.01), ischemic colitis (p<0.01), or autoimmune pancreatitis (AIP) (p<0.05). ♦, outlier; IBS, irritable bowel syndrome.
IgG4-present and IgG4-absent patients showed no significant differences in age, sex, extent of colitis, clinical course, or endoscopic severity. A significant difference was identified in terms of disease activity, with 72% of IgG4-absent UC patients categorized with mild disease activity, and 70% of IgG4-present patients categorized with moderate to severe disease activity (p<0.05) (Table 1). More severe inflammation according to histological grading was associated with greater IgG4-positive plasma cell infiltration in colonic mucosa (p<0.01) (Table 2). Administration of azathioprine was needed in three IgG4-present patients and three IgG4-absent patients, but one acute-onset UC patient presented with pancolitis showing abundant infiltration of IgG4-positive plasma cells (18/HPF) needing total colectomy due to refractory colitis.
From: “Ulcerative Colitis and Immunoglobulin G4“ (2014)
Returning to our study of SLPCs, some long-lived plasma cells can be generated from IgG4-RDs, and short-lived PCs from IgG1:
Moreover, in disorders with IgG1 autoantibodies, the generation of antigen-specific LLPCs seems to occur to a greater extent as compared to IgG4 disorders. It should be noted, however, that a significant degree of variability exists and that both antigen-specific LLPCs can be generated in some patients with IgG4 autoantibody-mediated disorders, and SLPBs—perhaps more frequently—can be significant producers of autoantibodies in some patients with IgG1 disorders.
Back to the case studies. Diagnosis is challenging, and effective long-term treatment is harder still:
Variability in relation to the nature of autoantibody-producing cells could also occur at different times in the same patient. This variability underscores the need for personalized medical approaches.
Exceptions aside, a generally reduced ability to establish LLPCs in IgG4 responses is strongly supported by immunological observations on the longevity of ACSs from the field of allergy (both in humans and animal models) and IgG4-related disease.
This study points to IgG4 autoimmunity as a disorder resulting from extrafollicular B cell responses, rather than from bone marrow-originating plasma cells:
It could be the case that IgG4 autoantibody-mediated autoimmunity constitutes a mainly extrafollicular response, but follicular hyperplasia (in the absence of pathogenic antigen-specificity) has been observed in IgG4-RD (316).
The tendency to generate predominantly IgG1 or IgG4 autoimmune responses may stem from HLA and/or non-HLA genetic differences (317, 318), but incomplete GWAS data (due to disease rarity) would have to be complemented by functional studies to better support such an argument.
On the other hand, many aspects of IgG1 and IgG4 autoimmunity are similar, such as B cell tolerance defects resulting in autoreactive naïve B cells (234, 319–321) and T cell-assisted autoantigen affinity maturation, as evidenced by the presence of somatic mutations in most ASCs.
mRNA COVID vaccines do not generate long-term T cell immunity, but they may mediate long-term T cell involvement with autoimmune disorders:
In further support of the role of T cell help, autoreactive T cells have been observed in both IgG1 and IgG4 autoantibody-mediated disorders (264, 322, 323).
Differences between IgG4 and IgG1 autoimmune responses are not clinically trivial and can inform therapeutic decisions, especially since IgG4 autoantibody pathology responds impressively well to rituximab induction.
More specifically, in IgG4 autoantibody-mediated disorders, prompt induction with rituximab 375 mg/m2 once a week for 4 to 6 weeks can result in a long-lasting favorable response and is highly recommended.
The same induction can be applied in IgG1 disorders. It is important to obtain a pre-rituximab baseline and a post-B cell depletion autoantibody titer in all patients at regular intervals.
If it’s not working, give the patient more!
In the case of a persistence of high titers of either IgG1 or, less frequently, IgG4 autoantibodies, which indicates the presence of LLPCs, repeated rituximab (or other CD20-depleting drug) dosing can be applied to enforce a deeper depletion of lymph node B cells and prevent the formation of new LLPCs, while existing ones slowly wane.
An alternative strategy in such refractory cases is the administration of inebilizumab (or other CD19 depleting drug), which would neutralize CD19+ CD20- ASCs that lie more towards the LLPC end of the ASC spectrum, or the application of daratumumab, an anti-CD38 agent more broadly targeting LLPCs.
IgG4 associated cytokine shifts from Th1 to Th2
Our final hat tip goes to Dr Bine for her review of animal-testing data by J&J, AZ & Pfizer. In revealing what they tested for, they exposed that Pharma did know of the risks of mediating cytokine shifts from pro-inflammatory (antiviral) to anti-inflammatory (tolerance), and that their products were, fingers crossed & pinky swear, not inducing this in animal models.
Translated:
Possible explanation for Th2 shift in BNT162B2Possible explanation for Th2 shift in BNT162B2
AstraZeneca has a possible explanation to offer.
Sometimes explanations only become apparent when you read the problem over and over again in different contexts.
This is what happened to me with the Th2 shift in the immune response, which can be observed in many BNT162B2 treated patients.
The problem was known to the competition, both Johnson&Johnson and AnstraZeneca, and they made it a point to avoid the problem.
The data I am referring to here was published by Scoops Gazette. So far, however, hardly anyone has taken a closer look at this data, even though it finally includes animal testing by JJ, AZ and Novavax.
A problem known to JJ and AZ was a potential Th2 shift in the immune response, which had to be prevented at all costs.
In the normal or ideal case, the Th1 and Th2 responses are in BALANCE!
Th1 is the stronger, more pathogenic reaction, Th2, the more protective reaction of the immune system.
… A possible explanation why a Th2 shift still occurs in BNT162B2 is thus provided by AstraZeneca:
ERD may result from immunization with an antigen that is not processed in the cytoplasm.
Where is the spike protein produced in BNT162B2?
In the endoplasmic reticulum and NOT in the plasma. After the Th2 shift of the treated subjects, the spike protein seems to be channeled directly into the membrane and out of the cell by the Golgi. And this is what the manufacturer itself writes in its documents:
These experiments defined a pathway taken by secreted proteins, the secretory pathway: rough ER → Golgi → secretory vesicles → cell exterior. Further studies extended these results and demonstrated that this pathway is not restricted to proteins destined for secretion from the cell. Plasma membrane and lysosomal proteins also travel from the rough ER to the Golgi and then to their final destinations. Still other proteins travel through the initial steps of the secretory pathway but are then retained and function within either the ER or the Golgi apparatus.
The entrance of proteins into the ER thus represents a major branch point for the traffic of proteins within eukaryotic cells. Proteins destined for secretion or incorporation into the ER, Golgi apparatus, lysosomes, or plasma membrane are initially targeted to the ER. In mammalian cells, most proteins are transferred into the ER while they are being translated on membrane-bound ribosomes (Figure 9.3). In contrast, proteins destined to remain in the cytosol or to be incorporated into the nucleus, mitochondria, chloroplasts, or peroxisomes are synthesized on free ribosomes and released into the cytosol when their translation is complete.
Figure 9.3 Overview of protein sorting. In mammalian cells, the initial sorting of proteins to the ER takes place while translation is in progress. Proteins synthesized on free ribosomes either remain in the cytosol or are transported to the nucleus, mitochondria, chloroplasts, or peroxisomes. In contrast, proteins synthesized on membrane-bound ribosomes are translocated into the ER while their translation is in progress. They may be either retained within the ER or transported to the Golgi apparatus and, from there, to lysosomes, the plasma membrane, or the cell exterior via secretory vesicles.
The answer appears to be that there is indeed a link between ER processing of proteins and IgG4-RD, according to part of a study from 2023 by Lu et al.: “Single-cell transcriptome analysis and protein profiling reveal broad immune system activation in IgG4-related disease“4
It is also a great study into how supposedly tolerant/anti-inflammatory immune responses can become destructive, as we see in IgG4-related aortitis.
Key takes:
PBMC: Peripheral blood mononuclear cell, - any peripheral blood cell having a round nucleus.
We performed single-cell RNA-seq and surface proteome analyses on 61,379 PBMCs from 9 treatment-naive IgG4-RD patients and 7 age- and sex-matched healthy controls. Integrative analyses were performed for altered gene expression in IgG4-RD, and flow cytometry and immunofluorescence were used for validation.
We observed expansion of plasmablasts with enhanced protein processing and activation, which correlated with the number of involved organs in IgG4-RD.
γδT cells: Gamma delta T cells. These are found in the highest abundance in the gut mucosa. Their role isn’t fully understood, but they are thought to bridge the gap between the rapidly responding innate immune system and the slower but long-lasting and antigen-specific B and T cell responses. They act as a first line of defence.
Increased proportions of CD4+ cytotoxic T lymphocytes (CTLs), CD8+ CTLs-GNLY (granulysin), and γδT cells with enhanced chemotaxis and cytotoxicity but with suppressed inhibitory receptors characterize IgG4-RD.
CTL: Cytotoxic T lymphocyte.
Prominent infiltration of lymphocytes with distinct compositions were found in different organs of IgG4-RD patients. Transcription factors (TFs), including PRDM1/XBP1 and RUNX3, were upregulated in IgG4-RD, promoting the differentiation of plasmablasts and CTLs, respectively.
Monocytes in IgG4-RD have stronger expression of genes related to cell adhesion and chemotaxis, which may give rise to profibrotic macrophages in lesions. The gene activation pattern in peripheral immune cells indicated activation of multiple interaction pathways between cell types, in part through chemokines or growth factors and their receptors. Specific upregulation of TFs and expansion of plasmablasts and CTLs may be involved in the pathogenesis of IgG4-RD, and each of these populations are candidate targets for therapeutic interventions in this disease.
Moreover, the predominant infiltration of M2 macrophages in multiple organs was thought to play an important role in the fibrosis noted in IgG4-RD lesions (12, 13).
Other immune cells such as basophils, mast cells, plasmacytoid dendritic cells (pDCs), and some T cell subsets are also involved (5).
Despite the increased knowledge, factors regulating these immune cells and the exact pathophysiology underlying this fibroinflammatory condition remain enigmatic. Advances in high-resolution single-cell RNA sequencing (scRNA-seq) have provided an avenue to identify disease-related cell subsets and explore transcriptional features at a cellular resolution in blood and tissue samples.
They found that, compared to healthy controls, IgG4-RD patients had a trend towards less memory B cells and more dividing plasmablasts:
Given the rise of IgG4+ plasma cells found in IgG4-RD lesions and their critical role in the pathogenesis, a more precise and detailed understanding of the features of B cells in circulation is essential.
We identified 5 transcriptionally distinct B cell subsets, including naive B (MS4A1+IGHD+TCL1A+), memory B (MS4A1+CD27+), intermediate memory B (IGHD+CD27+), plasmablast (MZB1+), and dividing plasmablasts (MZB1+MKI67+) (Figure 2A and Supplemental Figure 2, A–C). Notably, the proportion of the memory B subset decreased significantly in patients with IgG4-RD compared with HCs, while plasmablasts and dividing plasmablasts showed an increasing trend (Figure 2, B and C, and Supplemental Figure 2D), suggesting a possible transition from memory B to antibody-secreting plasmablasts in IgG4-RD.
More plasmablasts = more IgG4 Abs.
Plasmablast ratios were more useful to diagnose IgG4-RD than blood (serum) IgG4 Ab counts. This is the first time I have seen this.
Should we be asking more for dividing plasmablast counts(MZB1+MKI67+), rather than IgG4?
Concentrations of IgG4 were positively correlated with proportions of plasmablast and dividing plasmablasts (Figure 2D). Proportions of plasmablasts and dividing plasmablasts werepositively correlated with organs involved, whereas concentrations of IgG4 or ratios of IgG4/IgG and IgG4/IgM were not (Figure 2D and Supplemental Figure 2E).
This finding is consistent with the prior finding of poor diagnostic utility of serum IgG4 or IgG4/IgG in IgG4-RD (15), and indicates that the roles of plasmablasts/dividing plasmablasts in the pathogenesis of the disease are not fully reflected in systemic immunoglobin (Ig) levels.
Extract from “Figure 2. The transcriptional features of B cells in IgG4-RD. (A) UMAP visualization of B cell subsets shows 5 clusters that were identified and color coded. (B and C) Comparison of the proportions of B cell subtypes, separated by condition and donor. HC n = 7, IgG4-RD n = 9. HC, healthy control; P, patient. Results are shown as mean ± SD. Exact 2-sided P values are given. Significance was determined by Student’s t test (naive B, intermediate memory B) or Mann-Whitney test (memory B, plasmablast, and dividing plasmablast).” Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10544205
Another correlation with engineered mRNA SARS-CoV-2 transfection:
Gene Ontology (GO) analysis showed that genes involved in protein processing in endoplasmic reticulum (ER), ER stress response, and protein folding were significantly upregulated, suggesting enhanced protein synthesis during related processes in IgG4-RD (Figure 2, E and F).
Extract from “Figure 2. (E) Volcano plot showing the differentially expressed genes between B cells from IgG4-RD and HCs. (F) GO analysis using Metascape for genes that were upregulated in B cells from IgG4-RD versus HCs.” Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10544205
Not only does ER processing correlate with IgG4-RD, it appears to be essential to support high rates of protein expression required for plasmablast division and other disease mechanisms. Tumour-like traits?
This observation was validated by gene set enrichment analysis (GSEA) (Figure 2G). Interestingly, oxidative phosphorylation was highly enriched in IgG4-RD (Figure 2G), suggesting a high consumption of energy to support protein synthesis. Moreover, both GO analysis and GSEA showed enrichment of genes involved in antigen processing and presentation via MHC class I (cross presentation), indicating the role of B cells in activation of CD8+ T cells in IgG4-RD (Figure 2G).
I cited this in our paper - IgG4-RD is closely related to incidence rates of some cancer types. We need to keep a close watch on this in clinical settings, as the immune system shifts are predicting increased numbers being diagnosed:
SIRs: standardized incidence ratios.
The overall SIR estimates suggested an increased risk of overall cancer in IgG4-RD patients (SIR 2.57 95% CI 1.72-3.84) compared with the general population. The specific SIRs for pancreas and lymphoma were higher than those of the general population in IgG4-RD patients (SIR 4.07 95% CI 1.04-15.92, SIR 69.17 95% CI 3.91-1223.04, respectively). No significant associations were revealed in respiratory and gastric cancer (SIR 2.14 95% CI 0.97-4.75, SIR 0.95 95% CI 0.24-3.95, respectively).
From: “The risk of malignancy in patients with IgG4-related disease: a systematic review and meta-analysis“ (2022)
Genes encoding for IgG4, IgG1 and IgE were highly upregulated, but only genes for IgG4 and IgE in the plasmablasts, not IgG1:
Notably, Ig-encoding genes, including IGHG4, IGHG1, and IGHE were most highly upregulated in IgG4-RD (Figure 2E). Single-cell validation showed that IGHG4 and IGHE, but not IGHG1, were significantly upregulated in plasmablasts/dividing plasmablasts in IgG4-RD patients compared with HCs (Supplemental Figure 2F), which is in line with elevated serum IgG4 and IgE in these patients (4).
If you see a gene or protein prefixed “HS” then it probably is a heat shock protein. HSPs are a family of proteins produced when a cell is exposed to stressful conditions. By now, you won’t be surprised that HSPs also help cancer. They do this by protecting the tumour cell from apoptosis due to surgery or chemotherapy.
Cancer and autoimmunity mechanisms of almost every kind keep being discussed in the literature that are also invoked by the engineered mRNA technologies being used:
Furthermore, higher expression of ER stress–related genes such as HSP90B1, HSPA5, and UBE2J1 were found in IgG4-RD patients (Figure 2E and Supplemental Figure 2F).
I will discuss HSPs further in a future Substack, as a literature search revealed another series of Pfizer-induced autoimmune pathways.
Returning to our discussion on plasmablasts, although these aren’t long-lived, they keep being generated and the source B-cells are made more persistent by “survival cytokines”:
B cell activation pathways were also enriched, and representative genes, including XBP1, PRDM1, and BLK were highly expressed in B cells from IgG4-RD (Supplemental Figure 2F). B cell survival cytokines, including B cell–activating factor (BAFF, encoded by TNFSF13B) and a proliferation-inducing ligand (APRIL, encoded by TNFSF13), are survival factors for B cells that also control B cell maturation (16).
The endoplasmic reticulum (ER) functions as a quality-control organelle for protein homeostasis, or “proteostasis”. The protein quality control systems involve ER-associated degradation, protein chaperons, and autophagy. ER stress is activated when proteostasis is broken with an accumulation of misfolded and unfolded proteins in the ER. ER stress activates an adaptive unfolded protein response to restore proteostasis by initiating protein kinase R-like ER kinase, activating transcription factor 6, and inositol requiring enzyme 1.
ER stress is multifaceted, and acts on aspects at the epigenetic level, including transcription and protein processing. Accumulated data indicates its key role in protein homeostasis and other diverse functions involved in various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration, retinitis pigmentosa, achromatopsia, cataracts, ocular tumors, ocular surface diseases, and myopia.
From: “Endoplasmic reticulum stress: molecular mechanism and therapeutic targets“ (2023)
IgG4-RD would be less symptomatic if the plasmablasts & B cell lymphocytes stayed in the bone marrow rather than migrating to the periphery and affected organs. In other words, chemotaxis (the directed migration of a cell in response to a chemical stimulus, such as a growth factor) to the sites of lesions (or arteries) is a feature of the disease:
To understand the source of plasmablasts/dividing plasmablasts and regulatory mechanisms involved in expansion of these cells in IgG4-RD, trajectory analysis and TF analysis were applied.
There was a transition from naive B, intermediate memory B, and memory B cells to IgG4-secreting plasmablasts and dividing plasmablasts (Figure 2H).
Extract from “Figure 2. (H) Trajectory of differentiation from naive B cells to plasmablasts predicted by monocle and heatmap show upregulated or downregulated genes in the differentiation process.” Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10544205
During this process, markers of plasma cells, including CD38, MZB1, and Ig-coding genes were upregulated (Figure 2H). CD74, CXCR4, and CXCR5 were decreased along stages of B cell maturation, while MIF, COPA, and SLC7A1 were upregulated gradually, indicating that distinct chemokines were involved in chemotaxis of B cells at different stages (Figure 2H).
CXCR4/CXCL12 promote the retention of immune cells in the bone marrow, while CCR2 and CX3CR1 direct their movement from circulation to the tissue (20). Interestingly, plasmablasts/dividing plasmablasts downregulated CXCR4 but upregulated CCR2 and CX3CR1 (Supplemental Figure 2J), which promotes their trafficking to inflamed tissues in a coordinated, step-wise fashion.
Extract from: “Supplementary Figure 2. J, Plasmablasts and dividing plasmablasts have upregulated expression of CX3CR1, CCR2, and IL32, but absence of CXCR4 expression. Significance was determined by student’s T test or Mann–Whitney test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.“ Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10544205/bin/jciinsight-8-167602-s116.pdf
These are probably the key genes responsible for the transition from a B cell to a plasma cell:
We found that IGHG4, XBP1, and PRDM1 were coexpressed by plasmablasts/dividing plasmablasts (Figure 2J), and the expression levels of XBP1 and PRDM1 were positively correlated with IGHG4, proportion of plasmablasts, and marginally with the number of organs involved (Figure 2K). Therefore, XBP1 and PRDM1 may play critical roles in regulating B cell development toward plasmablasts, and could be candidate biomarkers for IgG4-RD.
They are quite prolific:
Plasma cells release antibodies in response to antigens. Once a B cell becomes a mature plasma cell, it can release up to 2,000 antibodies per second. Plasma cells are also called plasmacytes or effector cells. They have a shorter lifespan than memory cells.
T cells are the dominant cell type in the lymphoplasmacytic infiltrate in IgG4-RD.
Proportions of CD4+ CTLs, CD8+ CTL-GNLY, and γδT cells increased, while proportions of CD4+ naive T, CD4+ effector memory T cells, CD8+ naive T, and MAIT cells decreased in patients with IgG4-RD compared with HCs (Figure 3C and Supplemental Figure 3C).
Consistent with this result, higher proportions of CD4+ CTLs, CD8+ CTL-GNLY, and γδT cells were detected in PB of IgG4-RD by flow cytometry in a larger cohort (Supplemental Figure 3, D and E).
10/10 for the title of this paper:
Granzyme A (Gr A) is a highly selective tryptic protease that triggers a rapid form of cell death that exhibits all of the morphological features associated with apoptosis: membrane blebbing, chromatin condensation and nuclear fragmentation [47]. However, Gr A does not activate caspase family members, caspase inhibition fails to prevent Gr A-induced death and Bcl-2 overexpression does not inhibit Gr A cytolysis [47,48]. In addition Gr A does not cleave many key caspase targets including lamin B and PARP [47].
During cell death Gr A rapidly accumulates in the nucleus, where it appears to target the nuclear envelope protein lamin, the chromatin structural protein Histone H1 and DNA [49–52]. Indeed, DNA damage is a defining characteristic of the caspase-independent Gr A cell death pathway, marked by single-stranded nicks resulting in large DNA fragments [52].
From: “A more serine way to die: Defining the characteristics of serine protease-mediated cell death cascades“
Granzyme B mediates allergic inflammation. Granzymes A and B are both increased in cases of asthma that may prove fatal:
In addition, the CD4+ CTLs and γδT cells had upregulated granzyme A expression, while CD8+ CTL-GNLY did not (Supplemental Figure 3E). The expansion of cytotoxic T subsets and reduction of naive T subsets in PB of IgG4-RD indicate that T cells may be activated and differentiated into cell-killing subsets.
Different T cells associated with IgG4-RD travel to different sites:
In tissues, distinct components of infiltrated T cells were found in different organs (Figure 3, D and E). CD4+ and CD8+ T were predominant T cells in lesional sites, whereas the inflamed tissues only had a small number of γδT cells.
In addition, a higher frequency of CD8+ T cells than CD4+ T cells was found in the salivary glands and pancreas, while it was opposite in lymph nodes and lacrimal glands. The constituent ratio of CD4+ CTL and CD8+ CTL-GNLY in tissues was similar.
Another topic for a future Substack (as research shows that engineered mRNA depletes it), programmed cell death protein 1 (PD-1) is an inhibitory checkpoint receptor. When bound to its ligand PD-L1 on cytotoxic T lymphocytes (CTLs) and regulatory T cells (Tregs) it suppresses immune responses in two ways, which can allow cancers to progress:
It promotes apoptosis of CTLs.
It reduces apoptosis in Tregs.
In this study, transcriptome analysis found that one of the reasons for the prevalence of IgG4-associated Tregs was that protein levels of PD-1 (=PDCD-1) were lower than in the healthy controls:
Consistently, 2 other data sets also showed increased chemotaxis and cytotoxicity signals in both tissues (GSE40568) and blood (GSE66465) from IgG4-RD patients (Supplemental Figure 4C).
Dark green indicates increased pro-inflammatory chemotaxis and cytotoxicity in IgG4-RD:
In contrast, the CTLs together with γδT showed lower expression of inhibitory receptors, including TIGIT, CD244, PECAM1, and PDCD-1 (Figure 3H and Supplemental Figure 4, A and D). The protein level of PD-1 was validated with AbSeq data (Supplemental Figure 4E).
Then we have another trait of IgG4-RD: upregulation of genes linked to fibrosis. Fibrosis is common to both IgG4-RD and the effects of Gal-3 on tissues:
We also found the cytotoxic T subsets have high expression of FGFBP2 and CX3CR1 (Supplemental Figure 4F).
FGFBP2 is an important modulator of fibroblast growth factor (FGF) signaling by chaperoning FGFs through the extracellular matrix to FGF receptors (27).
High prevalence of FGFBP2 variants was also found in IgG4-RD (28).
Meanwhile, these cells express the fractalkine receptor CX3CR1, which has been associated with fibrosis in many organs (29). Therefore, these expanded cytotoxic T subsets are likely to migrate to tissues and promote fibrosis.
On the immune response shift to Th2:
Follicular helper T cells (Tfh) are a special subset of CD4+ T cells that expedite B cell and plasma cell differentiation, resulting in germinal center formation. Among them, the number of type 2 Tfh (Tfh2) cells was found to be specifically increased in IgG4-RD and was correlated with serum IgG4 levels and plasmablast counts (30).
Through our single-cell AbSeq data (Supplemental Figure 4G), IgG4-RD patients had a higher proportion of Tfh2 cells (11.5%) (CD45RA–CD4+PDCD1+CXCR5+CXCR3–CCR6–) than HCs did (8.3%) (P < 0.001), but not a significant elevation of Tfh cells (CD45RA–CD4+PDCD1+CXCR5+) (18.0%) compared with HCs (16.8%) (P > 0.05) (Figure 3J).
However, the levels of IL21 and CXCL13 mRNA in these circulating Tfh cells were very low in this data set (data not shown). Treg cytokines such as IL-10 and TGF-β are proposed to be involved in the class switching of plasma cells and fibrosis in IgG4-RD.
We found that the proportion of Treg cells was comparable between IgG4-RD and HCs; however, the expression of FOXP3 was higher in Treg cells in IgG4-RD (Supplemental Figure 4H), which is in line with published data (31).
Just because you don’t find your cytokines it doesn’t mean they aren’t there. You may be digging in the wrong place.
The RNA expression of IL-10 and TGF-β in these circulating Treg cells was barely detectable (data not shown). We speculated that these cells might expand in lymphoid organs andare finally activated locally in the lesional sites.
A possible link between gut dysbiosis and IgG4-RD?
Keep taking your kefir:
Moreover, GSEA showed that pathways related to the defense response to Gram-negative bacteria and regulation of B cell differentiation were upregulated in T cell genes in IgG4-RD (Supplemental Figure 4I), suggesting that T cells in IgG4-RD might be activated by some pathogens and facilitated B cell differentiation to plasmablasts.
TOR signaling and Toll-like receptor signaling pathways exhibited similar trends (Supplemental Figure 4I). Although no direct evidence has demonstrated that IgG4-RD is caused by infection,gut microbiota disturbance may be a trigger for the immune dysregulation in IgG4-RD (32, 33).
They also investigated immune cell-to-cell interactions in IgG4-RD, when they finally put their mobiles down.
We investigated the interaction network among cell types identified in PBMCs using CellphoneDB (40) and CellChat (41).
MNs: Mononuclear cells.
DCs: Dendritic cells.
CD14+ MNs, CD16+ MNs, and DCs showed the most interactions with other cells, especially among each other (Figure 6A).
Cell-cell interaction based on chemokine (C-C motif) ligands (CCLs) occurred predominantly among CD14+ MNs, CD4+ CTLs, CD8+ CTL-GZMK, CD8+ CTL-GNLY, γδT, MAIT, and NK cells (Figure 6B).
BAFF: B cell–activating factor.
BAFF-based interaction was mainly found between MNs and 5 B cell subsets, while macrophage migration inhibitory factor–based (MIF-based) interaction was observed between dividing plasmablasts and others (Figure 6B).
Furthermore, we found CD4+ CTL interactions with CD14+ MNs, CD16+ MNs, and DCs via CCL5-CCR1/CCR5, while CD4+ CTLs interacted with CD8+ CTL-GZMK, MAIT, and γδT cells via CCL4/CCL5-CCR5.
Moreover, CCL5 in T/NK cells was positively correlated with CCR1 in MNs (Supplemental Figure 5I).
Good to know, but what does all this mean? It means that immune cells in inflammatory lesions signal to other IgG4-RD immune cells and they migrate to the site, making the inflammation and tissue damage worse, and signalling to yet more plasmablasts in a vicious circle:
Altogether, the interaction data revealed that abnormal B and T subsets present in IgG4-RD display a strong interaction with each other via CD74, CCL4, and CCL5, which confer signals contributing to the abnormal inflammatory responses in IgG4-RD.
Previous studies have revealed that cytotoxic T subtypes, especially CD4+ CTLs, play an important role in the pathogenesis of IgG4-RD. These cells are clonally expanded both in the PB and at the lesional sites.
The number of tissue-infiltrating CD4+ CTLs correlates with the extent of organ involvement (9, 11, 45).
In the inflammatory environment, CTLs may induce apoptosis of mesenchymal cells and secrete profibrotic factors, amplifying tissue fibrosis and organ dysfunction (10).
In addition to CD4+ CTLs, we also noticed expansion and activation of other cytotoxic effector T subsets, including CD8+ CTL-GNLY and γδT.
These CTLs have enhanced cytotoxicity and chemotaxis, suggesting that they may have synergistic effects in inducing apoptosis of mesenchymal cells and in promoting fibrosis.
We also detected different distributions of T cell subsets in various tissues; however, the actual reason for the tissue preference remains unclear. It could be due to a combination of distinct chemokine profiles in tissues, special tissue composition, and antigen specificity.
Back to macrophages. The immune system, and especially macrophages, are quite plastic. In this respect, effectively treating IgG4-RD long-term may be like trying to kill a multi-headed hydra.
IgG4-RD patients have a slight increase in the number of CD14+ MNs. As the main source of BAFF in PB, CD14+ MNs help preserving the survival of B cells. While migrating to inflamed tissues, these cells might develop into profibrotic macrophages, as evidenced by enrichment of M2 macrophage markers.
In addition to their contribution to fibrosis through production of profibrotic factors, including CCL18 and IL-10, M2 macrophages initiate Th2 immune responses via IL-33 secretion (12, 13). Inhibition of MN migration or blockage of M2 polarization may influence tissue damage.
A very useful study published in 2023, long after the engineered mRNA products were rushed out under EUA:
In conclusion, we demonstrate that peripheral immune cells were broadly activated in IgG4-RD. Increased numbers of MNs were found in the circulation, which showed stronger chemotaxis signals.
B cells are activated and have a higher transformation rate to Ig-secreting plasmablast/dividing plasmablast in IgG4-RD. TFs such as XBP1 and PRDM1 may be responsible for this transformation.
Cytotoxic T subpopulations, including CD4+ CTLs, CD8+CTL-GNLY, and γδT were expanded in IgG4-RD. These cells have stronger cell-killing ability, and chemotaxis, and could be more profibrotic.
Broad communication exists within the peripheral immune system. Understanding these pathways will not only guide clinicians in the diagnosis of rare autoimmune disorders but also facilitate the development of targeted treatments.
This was a Chinese-funded study, and no conflicts of interest were declared.
The studies reviewed in part V of this series further increase our understanding of IgG4-RD.
We now know more about the relevance of short-lived plasma cells, the impact of HLA presentation, cytokine shifts from Th1 to Th2, on chemotaxis, granzymes, gamma delta T cells, and the importance of communication within the peripheral immune system.
Publishing this Substack has taken longer than planned, as the section on heat shock proteins is being written in parallel. There is too much important discussion to include it all here. I wish to thank my friend Dr. Annelise Bocquet for her assistance and peer review of my work on this.
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Nguyen DC, Hentenaar IT, Morrison-Porter A, et al. SARS-CoV-2-specific plasma cells are not durably established in the bone marrow long-lived compartment after mRNA vaccination.Nat Med. Published online September 27, 2024:1-10. doi:10.1038/s41591-024-03278-y
Gurav MJ, Manasa J, Sanji AS, Megalamani PH, Chachadi VB. Lectin-glycan interactions: a comprehensive cataloguing of cancer-associated glycans for biorecognition and bio-alteration: a review.Glycoconj J. Published online September 2, 2024. doi:10.1007/s10719-024-10161-y
Lu C, Li S, Qing P, et al. Single-cell transcriptome analysis and protein profiling reveal broad immune system activation in IgG4-related disease.JCI Insight. 8(17):e167602. doi:10.1172/jci.insight.167602
Bioweapon
Oh, and thank you for all of this digging and compiling