LTβR deficiency causes lymph node aplasia and impaired B cell differentiation

Abstract

Secondary lymphoid organs (SLOs) provide the confined microenvironment required for stromal cells to interact with immune cells to initiate adaptive immune responses resulting in B cell differentiation. Here, we studied three patients from two families with functional hyposplenism, absence of tonsils, and complete lymph node aplasia, leading to recurrent bacterial and viral infections. We identified biallelic loss-of-function mutations in LTBR, encoding the lymphotoxin beta receptor (LTβR), primarily expressed on stromal cells. Patients with LTβR deficiency had hypogammaglobulinemia, diminished memory B cells, regulatory and follicular T helper cells, and dysregulated expression of several tumor necrosis factor family members. B cell differentiation in an ex vivo coculture system was intact, implying that the observed B cell defects were not intrinsic in nature and instead resulted from LTβR-dependent stromal cell interaction signaling critical for SLO formation. Collectively, we define a human inborn error of immunity caused primarily by a stromal defect affecting the development and function of SLOs.

Figure 1. Identification of LTβR-deficient patients

(A) Pedigrees of the two unrelated families included in this study. Black solid symbols indicate affected individuals. Genotypes are indicated below the symbols. Squares indicate male and circles female family members. Slashed symbols indicate that the individual has died. Roman numerals indicate generations, and Arabic numbers indicate individuals within a generation. (B) Lymphoscintigraphy images depicting the lower lymphatic system in a healthy control (HC) alongside P1 and P2 (posterior view). P1 and P2 display normal lymphatic duct development but lack inguinal and iliac lymph nodes (blue dashed rectangle). Blue arrows indicate the main lymphatic nodes. The red arrow indicates the injection sites. (C) Howell-Jolly bodies (blue arrows) in erythrocytes in a blood smear from P2. (D) Geometric Mean Fluorescence Intensity (gMFI) of CD47 in lymphocytes. Data representative of N=2; HC (n=7) and patients (n=3). Statistical analysis performed on one of these experiments using Unpaired t-test with Welch’s correction. (E) Innumerous verrucae planae (flat warts) on the neck of P1. (F) Serum values for CXCL13 for P1-P2 and controls (n=4) from two separate Luminex multiplex assays (complete results displayed in Fig. S4). (G) LTβR expression by flow cytometry analysis in fibroblasts from a HC, P1-P3, as well as in P1-derived fibroblasts where the mutation was reverted to wild type by CRISPR-Cas9 editing (CRISPR-KI). (H) Representative immunoblot displaying the expression of p52 before and after stimulation with the lymphotoxin (LT) ligand in a HC, P1 and P1-derived CRISPR-KI fibroblasts. Quantification shown as fold change of p52/HSP90 relative to untreated HC1. Heat shock protein 90 (HSP90) serves as loading control. Unpaired t-test was used for statistical analysis in panel D and F, and G.

Figure 2. In-depth characterization of the immune cell compartment from LT βR-deficient patients

(A) Gating strategy to identify subpopulations of CD19⁺ B cells using flow cytometry and results from patients and HCs (n=9). Subpopulations were classified as Plasmablasts (PB), Pre-/ and Germinal-center (Pre-GC, and GC) Naïve or Memory/DN (double negative). Data shown here is from one experiment, representative of N=5; HC (n=7) and patients (n=3). (B) Representative plots from two HCs and P1-P3, highlighting the reduction of both class-switched (IgD^-CD27⁺) and unswitched (IgD⁺CD27⁺) CD19⁺ B cells in the patients. (C) Distribution of naïve (CD45RA⁺CCR7⁺), central memory (TCM CD45RA^-CCR7⁺), terminally differentiated effector memory T cells re-expressing CD45RA (TEMRA CD45RA⁺CCR7^-) and effector memory (TEM CD45RA^-CCR7^-) subpopulations within CD4⁺ and CD8⁺ T-cell fractions. The data shown here is from one experiment, representative of N=4; HC (n=7) and patients (n=3). (D) Frequency of CD25⁺FOXP3⁺ T-regulatory (Treg) cells. The data shown here is from one experiment, representative of N=2; HC (n=7) and patients (n=3). Statistical analysis was performed on one of these experiments. (E) Frequency of CD45RA^-CCR7^-CXCR5⁺ T-follicular helper (TfH) cells. The data shown here is from one experiment, representative of N=5; HC (n=7) and patients (n=3). Statistical analysis was performed on one of these experiments. (F) Distribution of T-helper (Th) subsets, characterized by cytokine expression on CD3⁺CD4⁺CD25^-CD45RA^- T cells following 5-hour stimulation with phorbol myristate acetate (PMA) and ionomycin. Results show a shift in the patients from IL-4 producing Th2 and IL-17 producing Th17 subsets towards the IFN-γ producing Th1 cells (HCs n=10). The experiment was performed twice with cells from P1 and P2. Statistical analysis was done using analysis of variance (ANOVA) followed by Bonferroni correction in (A) and Unpaired t-test with Welch’s correction in (D-F).

Figure 3. Sequencing analysis reveals LTB downregulation and reduced TCR clonality

(A) Low-dimensional projection (uniform manifold approximation and projection (UMAP) plot) of the combined scRNAseq data set from P1 and P2 and four HCs. In the left panel, colors correspond to the cell type identified by label transfer from a reference data set of healthy PBMCs. The right panel displays the same projection, with colors indicating the distribution of patient cells (blue) with the clusters compared to controls (grey). (B) Distribution of CD8 T-cell compartments in the scRNAseq data. (C) Heatmap showing pseudobulk expression data for genes (shown as columns) that are differentially expressed between P1, P2 and HCs within the CD8+ T-cell population (samples and cell types shown as rows). Numbers on the left indicate cell count per group. Dot plots indicate the degree of significance, and in which subtypes they were observed. (D) TCR clonality results obtained from bulk DNA TCRB sequencing in the 1000 most abundant clones with a productive TCRB VDJ rearrangement and Shannon’s evenness indices below each plot.

Figure 4. Alterations in the LTβR/TNF network and the immunomodulatory effect of DcR3

(A) Heatmap depicting LEGENDplex™ multiplex assay for serum samples from P1 and P2 compared to HC (n=2). Results were individually normalized, with the lowest value set to 0 and the highest value set to 100. All other values were scaled proportionally between these two extremes. Numbers shown are absolute values in pg/mL. (B) ELISA serum analysis for lymphotoxin beta (LTB). Data from one experiment with HCs (n=7) and from P2 (n=2) and P3 (n=1). (C-E) ELISA results for LIGHT/TNFSF14 (C), FasL (D) and DcR3 (E) for serum samples from HCs (n=6) and samples pooled from P1 and P2, from two blood draws and one from P3. In (E) commercially available intravenous immunoglobulin (IVIG) was also tested. Samples from P1 and P2 were taken from two timepoints. (F) ELISA results of DcR3 fold change in treated fibroblasts normalized to untreated cells for HC (n=2), patients (n=3), and cells from P2 where TNFR1 was knocked out. Each datapoint represents the average of N=2. (G) Activation-induced cell death (AICD) following restimulation of feeder-expanded T cells with soluble anti-CD3 (sCD3). Graph representing the percentage of apoptotic cells with or without DcR3 treatment. Dots represent average of individual healthy donors (n=3) over two independent experiments. (H) Effect of DcR3 treatment on the killing capacity of expanded T-cells, based on % of surviving co-cultured p815 target cells treated with 0.1 sCD3 compared to the results in the same condition being treated with 0 sCD3. Data representative of two independent experiments, including two of the HCs repeated as a biological replicate. Statistical analysis performed on one of these experiments. Analysis in (B) was performed using Unpaired t-test with Welch’s correction. Analysis in (C-E) and (G) was performed using Unpaired t-test. Analysis in (F) and (H) was performed using ANOVA followed by Dunnett’s post-hoc test for multiple comparisons.

Figure 5. Functional ex vivo B-cell activation in LTβR deficient cells

(A) The rate of somatic hypermutation (SHM) in the Ig heavy-chain variable regions (IGHV) of the top 200 B-cell clones with productive IGH rearrangements. Each dot represents a unique B-cell clone with the number of mutated nucleotides (nt) plus one on the y axis (logarithmic scale). The percentage of clones with no SHM is shown below each sample in a circular plot. Asterisks represent p-values < 0.001 of Dunnett’s test comparison between each individual patient samples and the aggregate of HC samples. (B) Upregulation of the activation marker CD25 and Activation-induced cytidine deaminase (AID) in CD19⁺ B cells from HCs (n=4) following 7 days of co-culture in various combinations of PBMCs, SCs, DCs and MMR vaccine as indicated in the panel. (C) Differentiation of CD19⁺ B cells from controls (n=4) and patients into CD27⁺ memory or CD38⁺IgD^- GC-like B cells following 12 days of co-culture. Measurements are compared to Day 0. Indicated conditions include the addition of stromal cells (SC), monocyte-derived dendritic cells (DC) or additional treatment with a measles, mumps, and rubella (MMR) vaccine. All conditions were stimulated with B-cell activating factor (BAFF) every other day. Readout was performed using flow cytometry.