Altered B cells homeostasis in child-onset immunoglobulin A vasculitis

AbstractBackgroundImmunoglobulin A vasculitis (IgAV), also called Henoch–Schönlein purpura, is a systemic small vessels vasculitis with immunoglobulin A1-dominant immune deposits. B-cells are a heterogeneous population with unique subsets distinguished by their phenotypes and cytokine production. Here, we explored the status of B cell subsets in patients with IgAV.MethodsThirty IgAV patients and fifteen age- and sex-matched healthy individuals were enrolled in this study. Fresh blood samples were collected from both healthy and IgAV patients. Upon the distinct expressions of CD3, CD19, CD20, CD38, CD27 and IgD, peripheral blood mononuclear cells (PBMCs) were initially categorized into plasmablasts and memory B cells. Subsequently, using surface markers including CD138 and IgM, and intracellular markers containing IgM and IgG, plasmablasts and memory B cells were further divided into distinct subgroups. A total of eleven populations were detected using multiple flow cytometry.ResultsCD3-CD19+IgD+CD27-, CD3-CD19+CD20-CD38+, CD3-CD19+CD20-CD38+IgM+, and CD3-CD19+CD20-CD38+CD138+ B cells were larger in patients with IgAV than in the HCs. Only CD3-CD19+IgD-CD27+IgM+ B cell counts were reduced in IgAV. The elevated B cell numbers returned to normal after treatment. Plasma and plasmablast B cell numbers correlated with plasma IgA levels. On the contrary, CD3-CD19+IgD-CD27+IgM+ B cell numbers were negatively proportional to the plasma IgA levels while naïve B cell numbers correlated with plasma and plasmablast B cell counts.ConclusionsWe hypothesized that immunoglobulin production was abnormally elevated in IgAV and could be explained by altered B-cell subset homeostasis.

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Two populations of circulating PD-1hiCD4 T cells with distinct B cell helping capacity are elevated in early rheumatoid arthritis

Rheumatology ◽

10.1093/rheumatology/kez169 ◽

2019 ◽

Vol 58 (9) ◽

pp. 1662-1673 ◽

Cited By ~ 9

Author(s):

Paula Fortea-Gordo ◽

Laura Nuño ◽

Alejandro Villalba ◽

Diana Peiteado ◽

Irene Monjo ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Memory B Cells ◽

Peripheral Blood Mononuclear ◽

Cell Numbers ◽

Tfh Cells ◽

Two Populations

Abstract Objective A novel population of B helper cells, phenotypically CD4+CXCR5−PD-1hi, has been described in the synovial tissues and peripheral blood of seropositive RA patients, and termed ‘peripheral helper T’ (Tph) cells. Contrary to CD4+CXCR5+PD-1hi follicular helper T (Tfh), Tph cells are not located in lymphoid organs but accumulate in inflamed tissues. Our objective was to study the frequency of circulating Tph (cTph) and circulating Tfh cell counterparts (cTfh) in patients with early RA (eRA). Methods Freshly isolated peripheral blood mononuclear cells from 56 DMARD-naïve eRA patients and 56 healthy controls were examined by flow cytometry. Autologous cocultures of naïve or memory B cells were established with isolated peripheral blood Tph or Tfh cells. Results Seropositive (RF+ and/or ACPA+, n = 38) but not seronegative eRA patients (n = 18) demonstrated increased frequencies and absolute numbers of cTph and cTfh cells. cTph but not cTfh cells expressed CCR2. Those eRA patients who experienced a significant clinical improvement at 12 months demonstrated a marked decrease of their cTph cell numbers whereas their cTfh cell numbers remained unchanged. Both isolated Tph and isolated Tfh cells were able to induce maturation of memory B cells, whereas only Tfh cells could differentiate naïve B cells. Conclusion Two populations of PD-1hiCD4 T cells with distinct phenotype and B cell helping capacity are increased in the peripheral blood of seropositive eRA patients. Whereas cTph cells are present only in patients with an active disease, cTfh cells seem to be constitutively elevated.

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OP0186 CHANGES IN CIRCULATING B CELL LEVELS AND IMMUNOPHENOTYPE ARE ASSOCIATED WITH DEVELOPMENT OF ARTHRITIS FOLLOWING TREATMENT WITH CHECKPOINT INHIBITORS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.908 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 112.2-113

Author(s):

M. Gatto ◽

S. Bjursten ◽

C. Jonell ◽

C. Jonsson ◽

S. Mcgrath ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

Adverse Events ◽

B Cell ◽

Mononuclear Cells ◽

Checkpoint Inhibitors ◽

Therapy Monitoring ◽

Cell Subset ◽

Peripheral Blood Mononuclear ◽

Transitional B Cells

Background:Inflammatory arthritis (IA) is frequent among rheumatic side effects induced by checkpoint inhibitor (CPI) therapy for metastatic malignancies1. While T cells are likely to sustain the inflammatory process2, fewer data are available concerning the role of B cells3.Objectives:To investigate the phenotype of circulating B cells in patients who develop CPI-induced IA (CPI-IA) and to compare it with features of B cells in patients not developing immune-related adverse events (irAE) upon CPI treatment.Methods:B cell subsets at baseline (before CPI initiation) and during CPI treatment were analyzed in CPI-IA patients and in patients receiving CPI but who did not develop irAE (non-irAE). Peripheral blood mononuclear cells (PBMC) were analyzed by flow cytometry and B cells were identified as CD19+ and divided into naïve (CD27-IgD+), memory (CD27+IgD+/-), double negative (CD27-IgD-) and transitional (CD10+CD24+CD38+/hi) B cells. Levels of CD21, an activation marker on transitional B cells, were also analyzed. Non-parametric tests were used for analysis of differences between groups.Results:Six CPI-IA and 7 non-irAE patients matched for age, gender and CPI treatment were included, who had received CPI treatment due to metastatic melanoma. Flow cytometry revealed a significant increase of circulating B cells (p=0.002) (Figure 1A) and especially of transitional B cells in CPI-IA patients vs. non-irAE (median %, range: 7.8 (4.5-11.4) vs. 3.2 (1.6-4.3),p=0.007) (Figure 1B), while no remarkable changes were seen across other subsets. Transitional B cell levels significantly decreased from active to quiescent CPI-IA in all patients (p=0.008). In two CPI-IA patients for whom baseline sampling was available, the increase of transitional levels occurred early after CPI treatment and before CPI-IA onset. Levels of expression of CD21 on transitional B cells were increased in CPI-IA vs. non-irAE (p=0.01).Conclusion:Transitional B cells are expanded in CPI-IA patients and seem to increase early after start of CPI therapy. Monitoring this B cell subset might lead to closer follow-up and earlier diagnosis of CPI-IA.References:[1]Ramos-Casals M, Brahmer JR, Callahan MK, et al. Immune-related adverse events of checkpoint inhibitors. Nat Rev Dis Primers 2020;6:38[2]Murray-Brown W, Wilsdon TD, Weedon H, et al. Nivolumab-induced synovitis is characterized by florid T cell infiltration and rapid resolution with synovial biopsy-guided therapy. J Immunother Cancer 2020;8:e000281[3]Das R, Bar N, Ferreira M, et al. Early B cell changes predict autoimmunity following combination immune checkpoint blockade. J Clin Invest. 2018;128:715-2Disclosure of Interests:None declared

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Belimumab alters transitional B-cell subset proportions in patients with stable systemic lupus erythematosus

Lupus ◽

10.1177/0961203319869468 ◽

2019 ◽

Vol 28 (11) ◽

pp. 1337-1343 ◽

Cited By ~ 1

Author(s):

A Benitez ◽

K Torralba ◽

M Ngo ◽

L M Salto ◽

K S Choi ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

B Cells ◽

B Cell ◽

Lupus Erythematosus ◽

Mononuclear Cells ◽

Cell Subset ◽

Systemic Lupus ◽

Peripheral Blood Mononuclear ◽

Healthy Donors ◽

Immature B Cells

Objective We evaluated the effects of the B-cell activating factor (BAFF)-targeting antibody Belimumab on human nonmemory B-cell pools. Human B-cell pools were identified using surface markers adapted from mouse studies that specifically assessed reductions in immature B cells due to BAFF depletion. Patients with systemic lupus erythematosus (SLE) have high levels of both BAFF and immature B cells. Mechanistic mouse studies provide a framework for understanding human responses to therapies that target B cells. Methods Peripheral blood mononuclear cells were isolated from healthy donors and SLE patients on Belimumab or standard-of-care therapy (SCT). Cells were stained for flow cytometry to identify B-cell subsets based on CD21/CD24. Differences in subset proportions were determined by one-way ANOVA and Tukey’s post hoc test. Results Patients treated with Belimumab show alterations in the nonmemory B-cell pool characterized by a decrease in the Transitional 2 (T2) subset ( p = 0.002), and an increase in the proportion of Transitional 1 (T1) cells ( p = 0.005) as compared with healthy donors and SCT patients. The naïve B-cell compartment showed no significant differences between the groups ( p = 0.293). Conclusion Using a translational approach, we show that Belimumab-mediated BAFF depletion reduces the T2 subset in patients, similar to observations in mouse models with BAFF depletion.

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MO251HIGHLY SENSITIVE FLOW CYTOMETRIC DETECTION OF RESIDUAL B-CELLS AFTER RITUXIMAB IN ANTI-NEUTROPHIL CYTOPLASMIC ANTIBODY-ASSOCIATED VASCULITIS PATIENTS*

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab104.009 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Y K O Teng ◽

L Van Dam ◽

Jelle Oskam ◽

S W A Kamerling ◽

E J Arends ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Mononuclear Cells ◽

Plasma Cells ◽

Memory B Cells ◽

Cell Depletion ◽

B Cell Depletion ◽

Peripheral Blood Mononuclear

Abstract Background and Aims B-cell depletion with rituximab (RTX) is an effective treatment for anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) patients. Nevertheless, relapses are frequent after RTX, often preceded by B-cell repopulation suggesting that residual autoreactive B-cells persist despite therapy. Therefore, this study aimed to identify minimal residual autoimmunity (MRA) in the B-cell compartment of AAV patients treated with RTX. Method EuroFlow-based highly-sensitive flow cytometry (HSFC) was employed to study B-cell and plasma cell (PC) subsets in-depth in AAV patients before and after RTX treatment. Additionally, peripheral blood mononuclear cells (PBMCs) of these RTX-treated AAV patients were cultured and in vitro stimulated with CpG, IL-2, and IL-21 to induce antibody-secreting cells (ASC). (ANCA)-IgG was measured in these supernatants by ELISA. Results By employing EuroFlow-based HSFC, we detected circulating CD19+ B-cells at all timepoints after RTX treatment, in contrast to conventional low-sensitive flow cytometry. Pre-germinal center (Pre-GC) B-cells, memory B-cells and CD20+CD138− plasmablasts (PBs) were rapidly and strongly reduced, while CD20−CD138− PrePC and CD20-CD138+ mature (m)PCs were reduced slower and remained detectable. Both memory B-cells and CD20− PCs remained detectable after RTX. Serum ANCA-IgG decreased significantly upon RTX. Changes in ANCA levels strongly correlated with changes in naive, switched CD27+ and CD27− (double-negative) memory B-cells, but not with plasma cells. Lastly, we demonstrated in vitro ANCA production by AAV PBMCs, 24 and 48 weeks after RTX treatment reflecting MRA in the memory compartment of AAV patients. Conclusion We demonstrated that RTX induced strong reductions in circulating B-cells, but never resulted in complete B-cell depletion. Despite strongly reduced B-cell numbers after RTX, ANCA-specific memory B-cells were still detectable in AAV patients. Thus, MRA is identifiable in AAV and can provide a potential novel approach in personalizing RTX treatment in AAV patients.

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Highly Sensitive Flow Cytometric Detection of Residual B-Cells After Rituximab in Anti-Neutrophil Cytoplasmic Antibodies-Associated Vasculitis Patients

Frontiers in Immunology ◽

10.3389/fimmu.2020.566732 ◽

2020 ◽

Vol 11 ◽

Author(s):

Laura S. van Dam ◽

Jelle M. Oskam ◽

Sylvia W. A. Kamerling ◽

Eline J. Arends ◽

O. W. Bredewold ◽

...

Keyword(s):

Flow Cytometry ◽

B Cells ◽

B Cell ◽

Mononuclear Cells ◽

Memory B Cells ◽

Cell Depletion ◽

B Cell Depletion ◽

Peripheral Blood Mononuclear ◽

Highly Sensitive

BackgroundB-cell depletion with rituximab (RTX) is an effective treatment for anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) patients. Nevertheless, relapses are frequent after RTX, often preceded by B-cell repopulation suggesting that residual autoreactive B-cells persist despite therapy. Therefore, this study aimed to identify minimal residual autoimmunity (MRA) in the B-cell compartment of AAV patients treated with RTX.MethodsEuroFlow-based highly-sensitive flow cytometry (HSFC) was employed to study B-cell and plasma cell (PC) subsets in-depth in AAV patients before and after RTX treatment. Additionally, peripheral blood mononuclear cells (PBMCs) of these RTX-treated AAV patients were cultured and in vitro stimulated with CpG, IL-2, and IL-21 to induce antibody-secreting cells (ASC). (ANCA)-IgG was measured in these supernatants by ELISA.ResultsBy employing EuroFlow-based HSFC, we detected circulating CD19+ B-cells at all timepoints after RTX treatment, in contrast to conventional low-sensitive flow cytometry. Pre-germinal center (Pre-GC) B-cells, memory B-cells and CD20+CD138− plasmablasts (PBs) were rapidly and strongly reduced, while CD20−CD138− PrePC and CD20-CD138+ mature (m)PCs were reduced slower and remained detectable. Both memory B-cells and CD20− PCs remained detectable after RTX. Serum ANCA-IgG decreased significantly upon RTX. Changes in ANCA levels strongly correlated with changes in naive, switched CD27+ and CD27− (double-negative) memory B-cells, but not with plasma cells. Lastly, we demonstrated in vitro ANCA production by AAV PBMCs, 24 and 48 weeks after RTX treatment reflecting MRA in the memory compartment of AAV patients.ConclusionWe demonstrated that RTX induced strong reductions in circulating B-cells, but never resulted in complete B-cell depletion. Despite strongly reduced B-cell numbers after RTX, ANCA-specific memory B-cells were still detectable in AAV patients. Thus, MRA is identifiable in AAV and can provide a potential novel approach in personalizing RTX treatment in AAV patients.

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POS0412 CD21-/LOW B-CELLS ARE INCREASED IN PATIENTS WITH ANKYLOSING SPONDYLITIS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.2957 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 435.2-436

Author(s):

R. Wilbrink ◽

A. Spoorenberg ◽

S. Arends ◽

F. G. M. Kroese ◽

G. M. Verstappen

Keyword(s):

Ankylosing Spondylitis ◽

B Cells ◽

Autoimmune Disease ◽

B Cell ◽

Lupus Erythematosus ◽

Mononuclear Cells ◽

Cell Subset ◽

Memory B Cells ◽

Clinical Parameters ◽

Color Flow

Background:Ankylosing spondylitis (AS) is a chronic immune mediated inflammatory rheumatic disease, in which primarily the sacroiliac joints and the spine are affected. Extra-skeletal manifestations (ESM) include uveitis, psoriasis, inflammatory bowel disease and peripheral arthritis. In studies into the pathogenesis of AS, B-cells have received little attention most likely due to the lack of auto-antibodies1. A B-cell subset that has been particularly associated with autoreactivity is characterized by low expression of CD21. These CD21-/low B-cells are increased in systemic autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus and primary Sjögren syndrome (pSS)2. At least part of the CD21-/low B-cells are considered to represent anergic, autoreactive B-cells, that fail to become activated through conventional B-cell receptor and CD40 signaling2.Objectives:To phenotypically study the peripheral B-cell compartment in in the blood of AS patients compared to pSS patients, a typical B-cell-associated autoimmune disease, and healthy controls (HC). Special emphasis was given to the CD21-/low compartment.Methods:The proportions and phenotype of peripheral B-cells were assessed in cryopreserved peripheral blood mononuclear cells of 45 AS patients (62% male, mean age 49.2±13.2 years, mean ASDAS 2.5±1.0), 20 age-matched patients with pSS (20% male, mean age 50.6±12.0, median (IQR) ESSDAI 3±6.25) and 30 age- and sex-matched HCs, using 15-color flow-cytometry analysis. Differences between groups were tested using the Independent Samples t-test or Mann-Whitney U test depending on the distribution of variables. Associations between CD21-/low B-cells and clinical parameters were explored using the Pearson or Spearman correlation coefficient.Results:The percentage of total B-cells in AS patients did not differ from pSS patients and HCs. In AS patients, percentages of CD27+ memory B-cells and CD27-IgD+ naïve B-cells were also similar to HCs, whereas CD27+IgD- memory B-cells were significantly reduced in pSS patients, as expected. The proportions of CD27-CD38lowCD21-/low B-cells among total B-cells were significantly increased in both AS (median 6.4%, p<0.0001) and pSS patients (median 7.8%, p<0.0001) compared to HCs (median 4.9%). Interestingly, only in AS patients, expression of chemokine receptors CXCR3 and CXCR5 was significantly elevated on CD27-CD38lowCD21-/low B-cells compared to HCs (p<0.001 and p<0.01, respectively). In comparison to HCs the expression of the immune markers T-bet and CD11c by CD27-CD38lowCD21-/low B-cells was significantly lower in AS patients (p<0.01 and p<0.01, respectively). The distribution of IgM and IgD expression within the CD27-CD38lowCD21-/low B-cell population was similar between all three study groups. Regarding the association between CD27-CD38lowCD21-/low B-cells and clinical parameters in AS patients, we observed a positive correlation with age (r=0.347, p=0.02) and erythrocyte sedimentation rate (ρ=0.386, p=0.01). Furthermore, AS patients with ESM showed increased proportions of CD27-CD38lowCD21-/low B-cells compared to patients without ESM (p<0.05).Conclusion:In this cross-sectional study, we observed an increased proportion of circulating CD27-CD38lowCD21-/low B-cells in AS patients, similar as in patients with pSS, a typical B-cell-mediated autoimmune disease. The elevated expression of CXCR3 on CD27-CD38lowCD21-/low B-cells in AS patients is suggestive for active involvement in the inflammatory response. These findings are indicative of B-cell involvement in the pathogenesis of AS, against current dogma.References:[1]Ranganathan V et al. Nat Rev Rheumatol. 2017;13(6):359-367.[2]Thorarinsdottir K et al. Scand J Immunol. 2015;82(3):254-261.Disclosure of Interests:Rick Wilbrink: None declared, Anneke Spoorenberg: None declared, Suzanne Arends: None declared, Frans G.M. Kroese Speakers bureau: BMS, Roche, Janssen-Cilag, Consultant of: BMS, Grant/research support from: BMS, Gwenny M. Verstappen: None declared

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Aberrant Frequency of IL-10-Producing B Cells and Its Association with Treg/Th17 in Adult Primary Immune Thrombocytopenia Patients

BioMed Research International ◽

10.1155/2014/571302 ◽

2014 ◽

Vol 2014 ◽

pp. 1-10 ◽

Cited By ~ 10

Author(s):

Fanli Hua ◽

Lili Ji ◽

Yanxia Zhan ◽

Feng Li ◽

Shanhua Zou ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Immune Thrombocytopenia ◽

Mononuclear Cells ◽

Cell Subset ◽

Brefeldin A ◽

Interleukin 10 ◽

T Cell Subsets ◽

Peripheral Blood Mononuclear ◽

B10 Cells

Background. Regulatory B cells (Breg) are a distinct B cell subset with immunoregulatory properties. Pivotal to Breg function is interleukin-10. This study was to investigate the role of IL-10-producing B cell (B10) and its association with Treg and Th17 subsets in immune thrombocytopenia (ITP) patients.Methods. Peripheral blood mononuclear cells from ITP patients and controls were stimulated with PMA, ionomycin, and Brefeldin A. The frequencies of CD19+IL-10+B cells, CD3+CD4+IL-17+Th17 cells, and CD4+CD25hiFoxp3+Treg cells were analyzed by flow cytometry. The mRNA expression of Foxp3 and RORγt was detected by real-time quantitative PCR.Results. The number of B10 cells was elevated in ITP patients. After first-line therapies, it remained at high level in patients who achieved complete or partial response but decreased in those who acquired no response. There was a positive correlation between B10 cells and Tregs in ITP both before and after therapies. The ratio of Treg/Th17 decreased in ITP, and it strongly correlated with B10 cells.Conclusions. The frequency of B10 cells is elevated in ITP and it correlates with both the Tregs counts and the Treg/Th17 ratio. B10 cells to regulate functional T cell subsets might be impaired in patients with ITP.

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Elevated STAT1 expression but not phosphorylation in lupus B cells correlates with disease activity and increased plasmablast susceptibility

Rheumatology ◽

10.1093/rheumatology/keaa187 ◽

2020 ◽

Vol 59 (11) ◽

pp. 3435-3442 ◽

Cited By ~ 2

Author(s):

Arman Aue ◽

Franziska Szelinski ◽

Sarah Y Weißenberg ◽

Annika Wiedemann ◽

Thomas Rose ◽

...

Keyword(s):

B Cells ◽

Disease Activity ◽

B Cell ◽

Mononuclear Cells ◽

Janus Kinase ◽

T Cell Subsets ◽

Type I ◽

Type I Ifn ◽

Peripheral Blood Mononuclear ◽

B Cell Subsets

Abstract Objectives SLE is characterized by two pathogenic key signatures, type I IFN and B-cell abnormalities. How these signatures are interrelated is not known. Type I-II IFN trigger activation of Janus kinase (JAK) – signal transducer and activator of transcription (STAT). JAK-STAT inhibition is an attractive therapeutic possibility for SLE. We assess STAT1 and STAT3 expression and phosphorylation at baseline and after IFN type I and II stimulation in B-cell subpopulations of SLE patients compared with other autoimmune diseases and healthy controls (HD) and related it to disease activity. Methods Expression of STAT1, pSTAT1, STAT3 and pSTAT3 in B and T cells of 21 HD, 10 rheumatoid arthritis (RA), seven primary Sjögren’s (pSS) and 22 SLE patients was analysed by flow cytometry. STAT1 and STAT3 expression and phosphorylation in PBMCs (peripheral blood mononuclear cells) of SLE patients and HD after IFNα and IFNγ incubation were further investigated. Results SLE patients showed substantially higher STAT1 but not pSTAT1 in B- and T-cell subsets. Increased STAT1 expression in B-cell subsets correlated significantly with SLEDAI and Siglec-1 on monocytes, a type I IFN marker. STAT1 activation in plasmablasts was IFNα dependent while monocytes exhibited dependence on IFNγ. Conclusion Enhanced expression of STAT1 by B-cell candidates as a key node of two immunopathogenic signatures (type I IFN and B-cells) related to important immunopathogenic pathways and lupus activity. We show that STAT1 is activated upon IFNα exposure in SLE plasmablasts. Thus, Jak inhibitors, targeting JAK-STAT pathways, hold a promise to block STAT1 expression and control plasmablast induction in SLE.

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Evidence for progenitors of chronic lymphocytic leukemia B cells that undergo intraclonal differentiation and diversification

Blood ◽

10.1182/blood.v87.4.1586.bloodjournal8741586 ◽

1996 ◽

Vol 87 (4) ◽

pp. 1586-1594 ◽

Cited By ~ 27

Author(s):

M Dono ◽

S Hashimoto ◽

F Fais ◽

V Trejo ◽

SL Allen ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Dna Sequences ◽

Mononuclear Cells ◽

Lymphocytic Leukemia ◽

Specific Primers ◽

Region Gene ◽

Peripheral Blood Mononuclear ◽

Ancestral Gene

Peripheral blood mononuclear cells from five patients with IgG+ B-type chronic lymphocytic leukemia (B-CLL) were analyzed for the presence of clone-specific Ig H chain variable region gene mRNA transcripts linked to C mu and/or C alpha. This was assessed by (1) comparing the lengths of portions of the VHDJH of the IgG+ CLL clones with those of the mu and alpha isotype-expressing B cells, (2) performing clone-specific endonuclease digestion studies, and (3) determining the DNA sequences of the mu and alpha isotype-expressing cDNA. Thus, when B-cell mRNA from these five patients were reverse transcribed with C gamma-specific primers and then amplified by polymerase chain reaction, dominant cDNA were found with lengths corresponding to those of the IgG+ CLL B cell. In addition, in four cases, cDNA of lengths identical to those of the CLL B cell were detected when mRNA was reverse transcribed and amplified using c mu- and/or C alpha-specific primers, strongly suggesting clonal relatedness. These CLL-related mu- and alpha- expressing cDNA were present in greater amounts that unrelated (non- CLL) mu- and alpha-expressing cDNA from normal B cells that used genes of the same VH family. When the sequences of these CLL-related C mu- and C alpha-expressing cDNA were compared with those of the IgG+ CLL clones, it was clear that they were derived from the same ancestral gene as the IgG-expressing CLL B cell, thus documenting their common origin. Finally, nucleotide point mutations were observed in the mu- and alpha-expressing cDNA of certain patients, indicating divergence with the CLL. These data suggest that IgM+ B cells, which are precursors of the leukemic B cells, exist in increased numbers in the blood of most patients with IgG+ B-CELL and that these cells may differentiate, accumulate V genes mutations, and undergo isotype switching in vivo. In addition, the data are consistent with a sequential-hit model for the evolution of CLL.

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Long Term Humoral Immune Reconstitution Kinetics After Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) In Children

Blood ◽

10.1182/blood.v122.21.4627.4627 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4627-4627

Author(s):

Amro Elshoury ◽

Neena Kapoor ◽

Ami J Shah ◽

Bhakti Mehta ◽

Kris M. Mahadeo ◽

...

Keyword(s):

B Cells ◽

Serum Level ◽

B Cell ◽

Immune Reconstitution ◽

Serum Levels ◽

Multivariate Regression Analysis ◽

Memory B Cells ◽

Post Transplantation ◽

Humoral Immune ◽

Cell Counts

Background HSCT recipients have increased incidence of acquiring infections, particularly by encapsulated bacteria such as Streptococcal pneumoniae and Haemophilus influenzae. Delayed immune reconstitution has a pivotal role in these complications. Although T-cell immune reconstitution has been well studied, long-term B-cell reconstitution remains less characterized. Patients and Methods We studied 73 patients, who received allogeneic HSCT at Childrens Hospital Los Angeles. Patients were in complete remission of their underlying disorder and with median follow up 4.15 years [yrs] (range 6 month -18yrs, mean 5 yrs) post-HSCT. All subjects were< 18 years of age. B (naive [IgD+CD27-CD19+], memory [IgD+CD27+CD19+] and switched memory [IgD-CD27+CD19]); and T (CD3+, CD3+CD4+, CD3+CD8+, CD4+CD25+CD127dim (T Regulatory) [Tregs], RA+CD4+) cell subtypes, quantitative Immunoglobulins levels and antibodies to both polyribosyle polyphospate (PRP) and tetanus toxoid (TT) antigens were assessed longitudinally after HSCT. Results Naive B Cells were the first B cell subtype to return to normal value at 6 month post-HSCT, while memory B cells were persistently low up to two years post-HSCT. PRP levels continued to be low up to 10 years post -HSCT in unrelated donor HSCT recipients. TT antibodies level was normal at 6 month post-HSCT following immunization with TT in patients not receiving IVIG therapy. Switched memory B cell counts correlated positively with RA+CD4+ counts at 6 month post-HSCT (r=0.459, P=0.021) and with CD3+CD+4 counts at 6 months (r=0.530, P=0.006) and 2-years post-HSCT (r=0.398, P=0.016). However, switched memory B cells did not correlate with Tregs at any time post-HSCT. Switched memory B cells correlated positively with serum level of IgG at 1 (r=0.443, P=0.039), and 2 years post transplantation (r=0.617, P=0.001) and with serum level of IgA at 2 years post-HSCT(r=0.567. P=0.004). Memory B-cells counts correlated positively with serum levels of IgM at 1 year post-HSCT (r=0.478, P=0.021) and with serum levels of both IgG and IgA (r=0.431 P=0.035, and r=0.416, P=0.043, respectively) at 2 years post-HSCT. However, memory B-cell counts did not correlate with RA+CD4+, CD3+CD4+, CD3+CD8+ or Tregs cell counts. The use of Total body irradiation (TBI) was associated with lower switched memory B cells at 2 years (P=0.01) post-HSCT. TBI recipients also have lower PRP levels at 6-month post-HSCT compared to patients who did not receive TBI. Age of the recipient at time transplantation is another independent factor affecting all the B cell subsets recovery after transplantation; increase in age at transplantation is associated with lower B cell recovery. Decreased memory B cells post-HSCT was observed in patients with history of acute graft versus host disease (GVHD) (P=0.034) and chronic GVHD (P=0.01), even after resolution of clinical manifestations of active GVHD at 6 month and 2 years follow up, respectively. Compared to cord blood graft recipients, Bone marrow graft recipients have increased switched memory B-cells at 6 month and higher (P=0.0001) PRP antibodies titer at 3 years post-HSCT, respectively. Patients who did not receive ATG or Alemtuzumab have increased recovery of naive B-cells (P=0.024) at 2 years post-transplantation. Patients received ATG have higher both naive B cells in univariate analysis and PRP levels (in multivariate analysis) than those received Alemtuzumab at 6 months post-HSCT. Multivariate regression analysis showed that patients received Alemtuzumab have higher TT antibodies titer at 6 month post -HSCT compared to those received ATG. Conclusion We have found that memory and switched memory B-cells and serum PRP levels are deficient post-HSCT in children. Switched memory B cells correlated positively with serum level of IgG and IgA and memory B-cells correlated positively with serum levels of IgM, IgG and IgA. This confirms that HSCT recipients have impaired humoral immune reconstitution, hindering both B-cells development and generation of immunoglobulins. We also studied the different factors affecting humoral immune reconstitution using backwards multivariate regression analysis model and found that the use of TBI, age of the recipient at transplantation, GVHD status and source of stem cells can affect the kinetics of humoral immune reconstitution after HSCT in children. Disclosures: No relevant conflicts of interest to declare.

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