Inherited CD19 deficiency impairs signaling in plasma cells

doi:10.21203/rs.3.rs-1498757/v1

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Inherited CD19 deficiency impairs signaling in plasma cells

https://doi.org/10.21203/rs.3.rs-1498757/v1

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published 29 May, 2023

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Background: The human CD19 antigen is expressed throughout B cell ontogeny with the exception of neoplastic plasma cells and a subset of normal plasma cells. CD19 plays a role in propagating signals from the B cell receptor and other receptors such as CXCR4 in mature B cells. Studies of CD19-deficient patients have confirmed its function during the initial stages of B cell activation, however its role in the later stages of B cell differentiation is unclear.

Objective: Using B cells from a newly identified CD19-deficient individual, we investigated the role of CD19 in the generation and function of plasma cells using an in vitro differentiation model.

Methods: Flow cytometry and long-read nanopore sequencing using locus-specific long-range amplification products were used to screen a patient with suspected primary immunodeficiency. Purified B cells from the patient and healthy controls were activated with CD40L, IL-21, IL-2 and anti-Ig, then transferred to different cytokine conditions to induce plasma cell differentiation. Subsequently, the cells were stimulated with CXCL12 to induce signalling through CXCR4. Phosphorylation of key downstream proteins including ERK and AKT was assessed by Western blotting.

Results: Long-read nanopore sequencing identified the homozygous pathogenic mutation c.622del (p.Ser208Profs*19) which was corroborated by the lack of CD19 cell surface staining. CD19-deficient B cells generate phenotypically normal plasma cells with normal levels of CXCR4, however they have an altered response to CXCL12 resulting in a loss of ERK signalling. Additionally, CD19 ligation on normal plasma cells results in AKT phosphorylation.

Conclusion: CD19 is not required for generation of antibody secreting cells but may alter responses of these populations to CXCL12 and direct CD19 ligands, potentially affecting localisation, proliferation, or survival.

Antigen dependent B cell activation and B cell development and is largely regulated by signals received through the B cell antigen receptor (BCR) and cell surface molecules such as CD19 and CD21^1-3. Together these make up the BCR complex. BCR activation results in a cascade of molecular events including the activation of the spleen tyrosine kinase SYK, phosphorylation of tyrosine residues in the cytoplasmic tail of CD19 and the activation of phosphatidylinositol-3- kinase (PI3K)/AKT and ERK pathways ⁴. Several lines of evidence highlight the importance of CD19 in BCR signalling. Firstly, disruption of the actin cytoskeleton alone has been shown to activate BCR signalling in a CD19-dependent manner ^{5, 6}. Secondly, stimulation of CD19 lowers the threshold needed for activation of B cells by several orders of magnitude ^{2, 7}. Finally, the CD19/CD21 complex has been shown to prolong BCR signalling by stabilizing the BCR in plasma membrane lipid rafts and blocking internalization of the BCR ⁸. Moreover, the BCR complex is thought to integrate signals from multiple other receptors including toll-like receptors, CD40, BAFFR and chemokine receptors, all of which depend on CD19 for signal propagation ^9-13.

Murine and human models indicate that loss of CD19 results in an overall decrease in the humoral response and an increased susceptibility to bacterial infection ^14-16. To date 10 patients with CD19 deficiency causing an absence of CD19 on the cell surface have been reported^17-21. All patients had a low IgG level and recurrent infections of the respiratory tract. Other features included recurrent bacterial conjunctivitis, meningitis, and gastroenteritis ¹⁶.

The affected individuals all had a reduced proportion of class switched memory B cells suggesting a defect in early memory B cell formation. In keeping with this, experimental data show that CD19 deficiency leads to impaired somatic hypermutation and that the memory B cell compartment is more affected than the plasma cell compartment ²². However, several lines of evidence indicate that CD19 expression may effect plasma cell development and function. For example, a small proportion of long-lived bone marrow plasma cells downregulate the expression of CD19. These cells show an increased frequency of V gene somatic mutation, whereas there is an absence of CD19- plasma cells in the bone marrow of infants ^{23, 24}. The plasma cell compartment in CD19 deficiency patients is yet to be fully described. Here we describe a patient with genetically proven CD19 deficiency and investigated the role of CD19 in the generation and function of plasma cells using an in vitro differentiation model.

Patient cohort

The patient, family members and healthy donor volunteers provided written informed consent to participate in this study. Ethical approval was granted by the Leeds East Research Ethics Committee (18/YH/0070 and 07/Q1206/47).

Flow cytometry

Cells were stained in FACs buffer (PBS, 0.5% BSA, 0.05% sodium azide) with anti-CD20-V450 (Thermofisher), anti-CD19-PE, anti-CD38-PECγ7, anti-CD138-APC, 7-AAD (BD Parmingen) and anti-CXCR4-PE (R&D Systems). Cell number was assessed using CountBright™ beads (Thermofisher). Samples were run on a Beckman Coulter Cytoflex S flow cytometer and analysed using FlowJo version 10 (BD Biosciences) and Prism 8 (GraphPad).

Nanopore sequencing

Following written consent, DNA was isolated from peripheral blood lymphocytes of the proband and her relatives, using a Chemagic 360 automated extractor (Perkin Elmer, Waltham, MA, USA). A 7,476-bp long-range PCR amplicon was optimised to amplify the CD19 coding sequence using primers dAGTGTTGTGAGTCTGGAGGG (forward) and dCTGGAAGTGTCACTGGCATG (reverse). Amplification products were resolved on a 1% tris-borate EDTA agarose gel then purified using a QIAquick column, eluting in 30 µL of buffer EB (Qiagen GmbH, Hilden, Germany). Nanopore sequencing was undertaken on the gel-purified amplification products. An end-repair and nickase treatment reaction was first performed then sequencing adapters were ligated to the double-stranded DNA. A 24-hr Flongle sequencing run was initiated using MinKNOW software v.3.6.5 (ONT).

Offline basecalling converted raw data from fast5 to FASTQ format using Guppy v.3.6.0 (http://nanoporetech.com). Adapter sequences were trimmed from the resulting reads using Porechop v.0.2.3 (https://github.com/rrwick/Porechop) before NanoFilt v.2.2.0 was used to remove low quality reads (Q ≤ 6) (https://github.com/wdecoster/nanofilt) ²⁵. Sequence reads were then aligned to an indexed human reference genome (build hg19) using minimap2 v.2.16 (https://github.com/lh3/minimap2) ²⁶ before being sorted by alignment coordinate and calling was performed at the CD19 locus using NanoPolish v.0.13.2 (https://nanopolish.readthedocs.io). Each variant was annotated using Alamut Batch standalone (v.1.11; database version 2020.03.18) (Interactive Biosoftware, Rouen France) before its clinical significance was assessed according to Association for Clinical Genomic Science best practice guidelines ²⁷. Aligned sequence reads were manually inspected using the Integrative Genomics Viewer v.2.4.10 (http://software.broadinstitute.org/software/igv/) ²⁸. Summary read metrics were generated using NanoStat v.1.1.2 (https://github.com/wdecoster/nanostat)²⁵.

To confirm and genotype the putative pathogenic CD19 variant, a 436 bp amplicon was designed and optimised before being Sanger sequenced using an ABI3730, following manufacturer’s protocols (Applied Biosystems, Paisley, UK). Sequence chromatograms were visualised using 4Peaks v.1.8 (http://www.nucleobytes.com).

B cell purification and in vitro differentiation

Peripheral blood was obtained from healthy donors and the affected patient after informed consent. Mononuclear cells were obtained following lymphoprep density centrifugation. B-cell selection was carried out with a Miltenyi memory B-cell isolation kit, negatively selecting for untouched total B cells. B-cells were cultured as previously described ²⁹ in 24-well plates at 1x10⁵ cells/ml in IMDM + 10% FBS with MEM amino acid solution (1:50) and Lipid Mixture 1, chemically defined (1:200) (all Thermo Fisher Scientific) and the addition of 20 U/ml human IL-2 (Roche), 50 ng/ml human IL-21 (Peprotech) and 10 µg/ml F (ab′) 2 goat anti-human IgM/IgG (Jackson Immunoresearch) on previously irradiated CD40L-expressing fibroblasts. After 3 days, B-cells were removed from the CD40L-cells by gentle pipette mixing. B-cells were reseeded at in media containing 20 U/ml human IL-2 and 50 ng/ml human IL-21. On day 6, cells were seeded in media containing human IL-6 (10 ng/ml), human IL-21 (50 ng/ml), 100 U/ml multimeric APRIL (Adipogen) and 100nM gamma secretase inhibitor (GSI) inhibitor (L-685458; Sigma) and re-fed at day 10.

ELISAs

ELISAs were performed with Human IgM ELISA Quantitation Set (E80-100) or Human IgM ELISA Quantitation Set (E80-104) (Bethyl Laboratories Inc) according to the manufacturer’s instructions. ELISA absorbance values were analysed at 450 nm and Ig concentrations calculated from standard curves.

B cell stimulations and protein analysis

Anti-CD19 beads were prepared by pre-coupling biotinylated αCD19 (clone HD37, Absolute Antibody) to anti-biotin MACSiBead particles (Miltenyi) as per manufacturer’s instructions for the T cell activation/expansion kit. Prior to use the antibody-bead mix was resuspended in the same media as the B cell cultures. At day 6 and day 13 cells were washed, counted, and re-suspended at 1-2x10⁶ in 0.5% serum IMDM containing IL-6/IL-21/APRIL. Cells were incubated for 12–20 hours at 37°C. Following this, cells were placed in a 37°C water bath. An unstimulated sample of cells was taken prior to the addition of either 1ng/ml CXCL12 (Peprotech) or αCD19 stimulating beads. Samples were then taken after 1, 5 and 10 minutes post stimulation.

Protein lysates were separated on an SDS-polyacrylamide gel and transferred to nitrocellulose membrane. The membranes were probed with antibodies to phospho-AKT (T308), phospho-AKT (S473), pan AKT, phospho-ERK or pan ERK (all Cell Signaling) and then incubated with an HRP-coupled secondary antibody (Jackson Immunoresearch). HRP was then detected with ECL substrate (Thermofisher). The signal was visualised using a Bio-Rad ChemiDoc™ MP Imaging System, which uses ImageLab™ software for protein quantification.

Case description

A 30-year-old female born to healthy related parents presented with 3 episodes of radiologically confirmed pneumonia and one episode of Streptococcus pneumoniae septicaemia. Additionally, she had an isolated episode of Campylobacter gastroenteritis and a long-standing history of recurrent bacterial ear and sinopulmonary infections.

Immunological investigations showed a low polyclonal immunoglobulin G (3.5 g/l, reference range 6–16), with a normal level of IgA and IgM. She made a poor response to pneumococcal polysaccharide vaccination, in keeping with her clinical picture. Initial lymphocyte profiling showed normal numbers of T and NK cells and an apparent lack of B cells as assessed by CD19 expression (Fig. 1, A). Further flow cytometric evaluation confirmed the presence of a polyclonal CD20 + CD81 + CD19- B cell population and a reduction in memory B cell populations, in contrast to unaffected family members (Fig. 1, B and C).

A novel biallelic variant in CD19 identified by long-read nanopore sequencing

The molecular basis for the complete lack of B cell CD19 was presumed to be autosomal recessive CD19-deficiency and targeted mutation analysis of the CD19 gene was requested.

Long-read nanopore sequencing was performed on locus-specific long-range amplification products. Two possibly relevant DNA sequence variants were initially identified, and their clinical significance was interpreted according to criteria recommended by the Association for Clinical Genomic Science [Ellard et al., (2020)]. The first of these was the homozygous missense variant c.520C > G (NM_001770.5) (p.Leu174Val) which was reported at high frequency in the gnomAD control cohort (rs2904880: 0.2761) and was interpreted to be a benign sequence variant ³⁰. A second homozygous variant, c.622del (NM_001770.5) (p.Ser208Profs*19), was identified in CD19 exon 4 and classified as pathogenic (Fig. 2). The variant results in an out-of-frame translation that is predicted to result in nonsense mediated decay, and deficient gene expression. The variant had not been previously reported on ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), or the LOVD3 CD19 gene-specific variant database (https://databases.lovd.nl/shared/variants/CD19). Consistent with a rare recessive inheritance pattern, there were no observations of this variant in the gnomAD v.2.1 dataset (consisting of 125,748 exome sequences) or in-house control databases.

The zygosity and segregation of the c.622del variant was confirmed by Sanger sequencing; the proband’s mother and sister were determined to be heterozygous carriers (Supplementary Fig. 1). A DNA sample from the patient’s father was not available.

Ex vivo generation of mature plasma cells

Previous studies have documented hypogammaglobulinemia, reduced memory B cell numbers and impaired responses to vaccination as common features of CD19-deficient patients; however, there is substantial variance in the severity of symptoms and ex vivo CD19-deficient B cell function^{17–21, 31, 32}. The capacity of B cells from the newly identified CD19-deficent patient to generate antibody secreting cells in response to a T-dependent like stimulation was evaluated (Fig. 3A). When the CD19-deficient B cells were cultured under these optimised conditions, plasma cells were generated normally as assessed by loss of CD20 and gain of CD38/CD138 (Fig. 3B,C). The CD19-deficient cells exhibited a typical pattern of initial expansion with maximal cell numbers obtained at the plasmablast stage, followed by contraction as mature plasma cells emerge (Fig. 3D). Although the levels of IgM and IgG produced in the CD19-ve cultures were relatively low at day 6, the levels became comparable to those of healthy controls at the day 13 time point (Fig. 3E).

Signalling events in antibody-secreting cells

CD19 is part of a complex that functions as a co-stimulatory element to amplify antigenic signaling via the BCR. In particular, CD19 plays a prominent role in activation of the PI3K pathway after BCR ligation. However, there is evidence that a variety of other cell surface receptors utilize CD19 to propagate PI3K signals, including the chemokine receptor CXCR4 which regulates leukocyte trafficking and survival ¹³. During the in vitro differentiation, expression of CXCR4 is maximally expressed at day 6 but is also maintained on plasma cells on both healthy controls and CD19-ve cells (Fig. 4A). To determine whether CXCR4 signal propagation was affected by the loss of CD19, cells were exposed to the ligand CXCL12 and the phosphorylation of signaling intermediates was assessed (Fig. 4C, D). In healthy controls, both day 6 plasmablasts and day 13 plasma cells showed a rapid induction of AKT and ERK phosphorylation. The response to CXCL12 in CD19-ve plasmablasts was generally preserved, but CD19-ve plasma cells exhibited striking defects, particularly in the lack of ERK phosphorylation.

The loss of signalling in plasma cells prompted us to determine whether ligation of CD19 on antibody secreting cells would result in alterations in signal transduction events as has been documented for B cells ^33–37. Day 6 plasmablasts generated from healthy control B cells were refractory to crosslinking of CD19 following exposure to anti-CD19 coupled beads (Fig. 5A, B). In contrast, phosphorylation of pAKT S473 was detected in stimulated day 13 plasma cells. There was no evidence of changes in either pAKT T308 or phosphorylated ERK (not shown).

This report describes the use of third-generation methods in a patient that presented with clinical characteristics consistent with CD19-deficiency. We used nanopore sequencing to perform rapid low-cost whole-gene analysis and identified a homozygous frameshift mutation, c.622del (p.Ser208Profs*19), establishing a precise molecular diagnosis. The data processing pipeline we established was integrated with our standard variant annotation tools, enabling identified variants to be rapidly interpreted. Targeted genetic assays may therefore be more appropriate for patients presenting with well-defined clinical symptoms, or in whom prior investigations have revealed pathognomonic features, indicative of the underlying molecular lesion. Moreover, this experience highlights how nanopore sequencing could also be deployed as an orthogonal technology for the verification of sequence variants initially identified by short-read sequencing.

To date, as few as 10 CD19-deficient cases have been reported in the medical literature ¹⁶. In agreement with the other cases, the patient described herein presented with hypogammaglobulinaemia and a history of recurrent infections. Previous investigations have attributed antibody deficiency to reduced activation following antigenic stimulation, focussing on the role of CD19 in the regulation of BCR signalling thresholds. B cells from Cd19^−/− mice also respond poorly to mitogenic signals but can still differentiate and class switch when additional signals are provided ³⁸. Similarly, we found that CD19-deficient human B cells are capable of generating fully mature plasma cells that secrete IgG using in vitro conditions optimised to promote maturation and viability.

During the transition to becoming plasma cells, the responding B cells use conserved signalling pathways to integrate external information, which has the potential to impact on the differentiation process. This may be due to collaboration between surface receptors and modulation of signal strength and timing.

CXCR4 is a chemokine receptor present on a multitude of cell types, often mediating cell movement to specific niches crucial for further development and prolonged survival. Indeed, B cells utilise the CXCR4/CXCL12 axis to migrate between the light zones and dark zones of germinal centers ³⁹ and subsequently antibody secreting cells use CXCR4/CXCL12 to migrate towards survival niches within the bone marrow to support long-term immunity ^40–43. CXCR4 signalling can also promote survival, adhesion or proliferation and its expression and downstream signalling capabilities can be modulated by co-receptors present on the cell surface ^{44, 45}.

In B cells there is evidence that CXCR4 works in concert with CD19 ¹³ and the IgD-BCR ¹⁰. However, unlike B cells that express surface IgD, antibody secreting cells are fully capable of transmitting CXCR4 signals without the requirement for surface immunoglobulin. As CD19 acts as a central hub for relaying the downstream signal, we postulated that plasmablasts and plasma cells may continue to utilise CD19 in this capacity. In keeping with this idea, antibody secreting cells from the CD19-deficient patient displayed reduced activation of both PI3K and MAPK pathways. This was particularly evident in mature plasma cells.

As B cells transition to the plasma cell stage, the cells undergo extensive reprogramming to reach a non-dividing, antibody secreting phenotype and with this, the function of CXCR4 and CD19 adapt accordingly. We have shown that AKT and ERK signalling downstream of CXCR4 is generally enhanced as the cells terminally differentiate and also more dependent on the presence of CD19. Given that plasmablasts and plasma cells exhibit distinct differences in cell cycle status, longevity and location in vivo, it is reasonable to suggest that they are potentially wired differently for downstream signalling events.

CD19 down-regulation is consistently observed in a fraction of normal plasma cells and may relate to longevity ^{23, 24, 46}. However, very few differences in gene expression have been detected between CD19 + and CD19- fractions and long-term protective immunity has been documented from both types of cells ^{47, 48}. Our data suggests that CD19 on plasma cells does indeed have the potential to function as a signalling element in this terminally differentiated effector population. In plasma cells, a shift in the amplitude and timing of CXCR4 and CD19 signals could lead to acute or transient changes in gene expression profiles, metabolic activity and functional behaviour.

In summary, identification of a CD19-deficient patient enabled the exploration of CD19 function in late stages of B cell differentiation, providing evidence to support a role for CD19 as a key response regulator throughout B cell ontogeny. Thus, the hypogammaglobulinemia observed in CD19-deficient individuals is likely to reflect not only changes in early activation events but also the impact of diminished signalling competence to a range of ligands required to sustain humoral immunity.

Funding

This research was supported by the National Institute for Health Research (NIHR) Leeds Biomedical Research Centre. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care.

Competing interests

The authors declare no conflicts of interest.

Ethics approval

Ethical approval was granted by the Leeds East Research Ethics Committee (18/YH/0070 and 07/Q1206/47). All participants provided informed written consent
Consent to Participate

Yes
Consent to Publish

Yes
Data and/or Code availability

The data that support the findings of this study are available on request from the corresponding author (SS)
Authors’ contribution statements.

SS and GMD designed the study. CMW and LAC performed the sequencing and analysis of DNA samples. AM, and GA recruited patients, collected DNA samples and provided clinical data. KW, FN and CC performed experiments. CC and AK performed bone marrow/aspirate analysis. AM, GMD and SS wrote the first draft of the manuscript. All authors read and approved the manuscript.

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Supplementary Figure 1. Sanger sequencing chromatogram

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Inherited CD19 deficiency impairs signaling in plasma cells

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Journal Publication

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Abstract

Figures

Introduction

Methods

Patient cohort

Flow cytometry

Nanopore sequencing

B cell purification and in vitro differentiation

ELISAs

B cell stimulations and protein analysis

Results

Case description

A novel biallelic variant in CD19 identified by long-read nanopore sequencing

Ex vivo generation of mature plasma cells

Signalling events in antibody-secreting cells

Discussion

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1