Loss-of-function of the protein kinase C δ (PKCδ) causes a B-cell lymphoproliferative syndrome in humans

Abstract

Defective lymphocyte apoptosis results in chronic lymphadenopathy and/or splenomegaly associated with autoimmune phenomena. The prototype for human apoptosis disorders is the autoimmune lymphoproliferative syndrome (ALPS), which is caused by mutations in the FAS apoptotic pathway. Recently, patients with an ALPS-like disease called RAS-associated autoimmune leukoproliferative disorder, in which somatic mutations in NRAS or KRAS are found, also were described. Despite this progress, many patients with ALPS-like disease remain undefined genetically. We identified a homozygous, loss-of-function mutation in PRKCD (PKCδ) in a patient who presented with chronic lymphadenopathy, splenomegaly, autoantibodies, elevated immunoglobulins and natural killer dysfunction associated with chronic, low-grade Epstein-Barr virus infection. This mutation markedly decreased protein expression and resulted in ex vivo B-cell hyperproliferation, a phenotype similar to that of the PKCδ knockout mouse. Lymph nodes showed intense follicular hyperplasia, also mirroring the mouse model. Immunophenotyping of circulating lymphocytes demonstrated expansion of CD5+CD20+ B cells. Knockdown of PKCδ in normal mononuclear cells recapitulated the B-cell hyperproliferative phenotype in vitro. Reconstitution of PKCδ in patient-derived EBV-transformed B-cell lines partially restored phorbol-12-myristate-13-acetate-induced cell death. In summary, homozygous PRKCD mutation results in B-cell hyperproliferation and defective apoptosis with consequent lymphocyte accumulation and autoantibody production in humans, and disrupts natural killer cell function.

Figure 1

Clinical, immunological, histopathological, and characterization of patient. (A) Pedigree of patient. Parents do not have any known consanguinity. All 3 unaffected children and both parents are heterozygous for the same point mutation in PRKCD, whereas the patient represents the only affected offspring who is homozygous for this change. (B) Coronal computed tomography scan images illustrate massive hepatosplenomegaly and mediastinal, axillary, inguinal lymphadenopathy before treatment (left) and after treatment (right).

Figure 2

Histopathology of the patient lymph node as compared with a reactive node from an otherwise-healthy node. (A) H&E stain reactive follicle with polarized germinal center and mantle; (D) IHC for MIB-1: the proliferating lymphoid cells within the germinal centers are positive with polarization (dark zone); (G) IHC for BCL-6 (the master regulator of the GC formation): the entire reactive germinal center is positive for BCL-6. (J) IHC stain for PKCδ showing diffusely positive cytoplasmic stain. All the remaining photos are from the lymph node biopsy from our patient: (B) and (C) H&E stains of follicles with varying degree of ill-defined germinal center lacking mantles. (E) and (F) IHC for MIB-1: variable degree of proliferation in different germinal centers is seen with lack of polarization. (H) and (I) IHC for BCL-6: a relative small number of positive cells are present within each germinal center in comparison with the reactive follicle. (K) Absence of PKCδ stain in patient’s lymph node, compared with (J).

Figure 3

Identification of PKCδ mutation and protein expression. (A) Sequencing of PRKCD with genomic DNA from blood from the index patient, parents, and 3 siblings. (B) Immunoblot analysis of PKCδ and of the control protein β-actin in the cells from the patient and normal volunteer. Purified B cells were activated with F(ab′)₂ specific for IgM (10 μg/mL), IL-4 (50 ng/mL), and CD40 ligand (10 μg/mL) for 5 days, and then cell lysates were subjected to western blot (WB) analysis. The protein lysates from PBMCs and EBV-transformed B cells were used without stimulation. (C) PRKCD gene expression levels in the PBMCs and EBV-transformed B cells are expressed as a relative expression of normal cells. Data are represented as means ± SE of n = 3 separate experiments.

Figure 4

Cell proliferation and cytokine generation. (A) Enriched B cells were stained with CFSE (1 μM) and stimulated with F(ab′)₂ specific for anti-IgM (10 μg/mL), IL-4 (50 ng/mL), and CD40 ligand (10 μg/mL) for 5 days. Percent cells having undergone at least one cellular division, (numbers above the lines), assessed by CFSE dilution. Gray solid peak is an unstimulated cell. (B) EBV-transformed B cells from 3 normal and 2 different batches of patient were counted for 7 days. (C) PBMCs were stained with CFSE (1 μM) and stimulated Dynabeads T-cell activator CD3/CD28 for 3 days. PHA, phytohemagglutinin. (D) and (E) Enriched B cells were stimulated as indicated. After 48 hours, cell-free supernatants were harvested and cytokines measured as described in the Cytokine measurement section in the Materials and methods. Data from panels A and C are representative of 3 independent experiments. Data from panels D and E were represented as means ± SE of n = 3 separate experiments.

Figure 5

Knockdown of PKCδ-induced B-cell proliferation. (A) B cells or PBMCs were transfected with siRNA-universal negative control or siRNA-PRKCD (300 nM) with Amaxa electrophoration according to the Materials and Methods. After 3 days, cell lysates were prepared and subjected to the western blots (WB). (B) B cells or PBMCs were transfected with siRNA-universal negative control or siRNA-PRKCD (300 nM). The next day cells were stained with CFSE (1 μM) and stimulated with F(ab′)2 specific for anti-IgM (10 μg/mL), IL-4 (50 ng/mL), and CD40 ligand (10 μg/mL) for 5 days (for B cells) or Dynabeads T cell activator CD3/CD28 for 3 days (for PBMCs). Percent cells having undergone at least one cellular division, (numbers above the lines), assessed by CFSE dilution. Gray solid peak is unstimulated cells. Data are representative of 3 independent experiments.

Figure 6

Patients’ B cells were resistant to PMA-induced cell death. (A) and (C) EBV-transformed B cells or PBMCs were cultured with PMA (100 ng/mL) or APO-1-3/protein A (1 μg/mK each). After 24 hours (for EBV B cells) or 48 hours (for PBMCs), cells were stained with DiOC6 (40 nM) for 15 minutes and live cells were counted by flow cytometry by constant time acquisition. (B) EBV-transformed B cells were stimulated with or without PMA or APO-1-3 for 6 hours, stained with propidium iodide (PI; 50 ng/mL) for 15 minutes, and analyzed by flow cytometry. (D) EBV-transformed B cells were stimulated with or without PMA for 3 hours, and cells were stained with FITC-activated caspase-3 (BD Pharmingen) according to the manufacturer’s instructions. (E) and (F) The patient’s EBV-transformed were transduced with pLVX-IRES-ZsGreen control vector or pLVX-IRES-ZsGreen-RRKCD using lentivirus system. Overexpression of PKCδ was analyzed by western blots (WB; E). Cells were stimulated with PMA (100 ng/mL) for 24 hours, live cells were counted by flow cytometry by constant time acquisition (F). Analysis was performed by gating on live ZsGreen expressed cells. Data are represented as means ± SE of 3 separate experiments. *P < .05 by Student t test for comparison with untreated cells.