The human syndrome of dendritic cell, monocyte, B and NK lymphoid deficiency

Abstract

Congenital or acquired cellular deficiencies in humans have the potential to reveal much about normal hematopoiesis and immune function. We show that a recently described syndrome of monocytopenia, B and NK lymphoid deficiency additionally includes the near absence of dendritic cells. Four subjects showed severe depletion of the peripheral blood HLA-DR(+) lineage(-) compartment, with virtually no CD123(+) or CD11c(+) dendritic cells (DCs) and very few CD14(+) or CD16(+) monocytes. The only remaining HLA-DR(+) lineage(-) cells were circulating CD34(+) progenitor cells. Dermal CD14(+) and CD1a(+) DC were also absent, consistent with their dependence on blood-derived precursors. In contrast, epidermal Langerhans cells and tissue macrophages were largely preserved. Combined loss of peripheral DCs, monocytes, and B and NK lymphocytes was mirrored in the bone marrow by complete absence of multilymphoid progenitors and depletion of granulocyte-macrophage progenitors. Depletion of the HLA-DR(+) peripheral blood compartment was associated with elevated serum fms-like tyrosine kinase ligand and reduced circulating CD4(+)CD25(hi)FoxP3(+) T cells, supporting a role for DC in T reg cell homeostasis.

Figure 1.

Deficiency of peripheral blood DCs, monocytes, and lymphoid cells. Flow cytometry of PBMCs and proportion of lymphoid, monocyte, and DC fractions in the subjects relative to controls. Plots show equivalent numbers of total cells analyzed (75,000 for DCs; 65,000 for lymphocytes). Error bars indicate mean ± SD from a population of normal individuals (n = 11 for lymphoid cells; n = 28 for HLA-DR⁺ lineage⁻ cells). One of at least two independent results is shown for subjects 1–4. (A) CD3⁺ T cells, CD19⁺ B cells, and CD56⁺ NK cells. (B) HLA-DR⁺ lineage⁻ (CD3/19/56) cells further divided into CD14⁺ and CD16⁺ monocyte fractions and double-negative CD14⁻CD16⁻ cells, which comprise CD34⁺ progenitor cells, CD123⁺ PDCs, and CD11c⁺ myeloid DC fractions. (C) Quantitative analysis of monocyte/DC fractions, expressed as percentage of mononuclear cells, and lymphoid cells, expressed as percentage of cells in the lymphoid SSC/FSC gate, compared with healthy controls.

Figure 2.

Dysfunction of the IL-12–IFN-γ axis. Cytokines released into the supernatant when whole blood was stimulated in vitro with LPS, IL-12, or IFN-γ as indicated. A paired “travel control” was run with each subject, and each subject was analyzed at least twice. The graph shows the mean ± SD for subjects (n = 4) compared with controls (n = 4) from one experiment. *, P < 0.05 compared with adjacent control (paired Student’s t test).

Figure 3.

Depletion of tissue DCs, but preservation of LCs and macrophages. (A) Analysis of dermal DC populations by flow cytometry of collagenase-digested dermis. Plots show equivalent number of total cells analyzed (40,000). Gated CD45⁺ HLA-DR⁺ cells normally comprise two fractions separable by autofluorescence (AF) and side scatter (SSC). AF⁻ SSC^low cells (gate 1) contain CD14⁺ DCs, CD1a⁺ DCs, and occasional CD1a^high langerin⁺ migrating LCs (gate 3); AF⁺ SSC^high cells are macrophages (gate 2). CD1a^high cells express langerin (subject 1, inset). Subjects 1 and 3 were analyzed twice, independently; subjects 2 and 4 were analyzed once. (B) Dermal DCs, LCs, and macrophage counts relative to normal controls (n = 22; mean ± SD). (C) Whole-mount immunofluorescence staining for CD1a of epidermal sheets showing representative fields of each subject. (D) Total LC counts averaged from entire low-power image relative to controls (n = 12; mean ± SD). (E) Proportion of Ki-67⁺ LCs in whole-mount epidermal sheets relative to controls (n = 3).

Figure 4.

Depletion of specific compartments of CD34⁺ BM cells. Flow cytometric analysis of CD34⁺ progenitor compartments in BM according to a recent protocol (Doulatov et al., 2010). Control (performed 7 times); subjects 1, 3, and 4, as indicated (performed twice). Total BM mononuclear cells are shown. The minimum number of CD34⁺ cells analyzed was 4,500. Numbers show the percentage of CD34⁺ cells in each gate. Gate 1 contains CD45⁺ cells; gate 2 contains CD34⁺ cells; gate 3 contains committed CD38⁺ fraction; gate 4 contains a primitive CD38⁻ fraction. All definitions according to Doulatov et al., 2010. HSC, hematopoietic stem cell; MPP, multipotent progenitor; B/NK, committed B/NK precursor; CMP, common myeloid progenitor; MEP megakaryocyte/erythroid progenitor. In this analysis, it was not possible to separate Flt-3⁺ CMP from Flt-3⁻ MEP because Flt3 was not expressed by the subjects’ CD34⁺ cells; thus, CMP and MEP were combined.

Figure 5.

Elevation of Flt3L with T reg cell deficiency. (A) Serum Flt3L, M-CSF, and SCF concentrations measured by ELISA. Each subject was analyzed once. (B) Expression of Flt-3/CD135 and SCF-R/CD117 on BM CD34⁺ cells of subjects 1 and 3 compared with healthy controls and isotype (filled histogram). Each subject was analyzed twice. (C) Representative example of T reg cell analysis from subject versus normal control. Number indicates absolute count ×10⁶ /ml. (D) Absolute number of CD4⁺CD25^hiFoxP3⁺ T reg cells compared with HLA-DR⁺ lineage⁻ (CD3, 19, 20, 56) cells in subjects 1–4, as indicated, and controls (n = 21). Each subject was analyzed once. (E) Proportion of CD56⁺ and CD25⁺ CD3⁺ T cells in subjects 1–4 as indicated relative to controls.