Follicular CD8 T cells (fCD8, follicular cytotoxic, defined as CD8+CXCR5+) are a specialized population that has recently been shown to be expanded in chronic viral infections and some forms of cancer.
Comparison of fCD8 isolated from secondary lymphoid organs (SLO) of 6 healthy donors and 3 patients with common variable immunodeficiency (CVID).
We found that fCD8 have higher expression of exhaustion markers PD-1, TIGIT, 2B4 and Eomesodermin, high CD57, Granzyme B and lower expression of CD127 and TCF1 compared to conventional CXCR5- CD8s, suggesting terminal differentiation. In CVID, fCD8 were expanded, less differentiated and proliferated more compared to fCD8 of healthy individuals. Healthy donor, but not CVID fCD8 were able to support IgG production by B cells in vitro, however were less potent than follicular helper CD4s.
RNAseq of CVID fCD8s revealed upregulation of several genes compared to healthy donor fCD8s, including B3GAT1 (inducing CD57 expression), HAVCR2 (Tim3), IL10, CTLA4 and FAIM2. Indeed, CVID fCD8 had higher production of IL-10 in vitro and lower sensitivity to Fas-induced apoptosis.
Furthermore, using multidimensional reduction methods we identified two separate fCD8 populations, CD4like and CD8like. CD4like fCD8s shared phenotypic characteristics with TFH CD4 T cells, expressed CD4, low to medium levels of CD8, high CXCR5 and PD1, and were significantly expanded in CVID patients.
We show for the first time the expansion of follicular CD8 T cells in CVID patients with lymphadenopathy, the exhausted/differentiated phenotype of fCD8s in healthy and CVID SLOs and that two separate fCD8 populations co-exist in human germinal centers.
Common Variable Immunodeficiency (CVID) is characterized by impaired antibody production and poor terminal differentiation of the B cell compartment. We first reported the occurrence of epigenetic alterations in CVID by high-throughput methylation analysis in CVID-discordant monozygotic twins. Data from a recent whole DNA methylome analysis throughout different stages of normal B cell differentiation allowed us to design a new experimental approach.
We selected CpG sites for analysis undergo significant demethylation from naïve to memory B cells in controls. DNA methylation was analyzed by bisulfite pyrosequencing of specific CpG sites in sorted naïve and memory B cell subsets from CVID patients and controls. In the same samples we analyzed the replication history by the k-deleting recombination excision circle (KREC) assay. And the somatic hypermutation using the Igk-restriction enzyme hot spot mutation assay.
We observed impaired demethylation in two thirds of the selected CpGs in CVID memory B cells, in genes that govern B cell-specific processes or participate in B cell signalling. The degree of demethylation impairment associated with the extent of the memory B cell reduction. The impaired demethylation in such functionally relevant genes as AICDA in switched memory B cells correlated with a lower proliferative rate.
Our new results reinforce the hypothesis of altered demethylation during B cell differentiation as a contributing pathogenic mechanism to the impairment of B cell function and maturation in CVID. In particular, deregulated epigenetic control of AICDA could play a role in the defective establishment of a post-germinal center B cell compartment in CVID.
Patients with Common Variable Immunodeficiency Disorders (CVID) present with antibody deficiency and increased risk of malignancy, both of which involve DNA damage and repair (DDR) pathways. The aim of this study was to interrogate DDR in CVID patients.
A custom panel of 285 genes involved in DDR designed using Agilent’s HaloPlexHS Target Enrichment System was analysed in 39 CVID patients. Potentially pathogenic variants were identified by filtering based on quality, functional consequences and allele frequency <1% in public repositories. PBMCs from healthy controls (n=11) and patients (n=34) were exposed to γ-irradiation and viability, γH2AX, phosphorylated (p)ATM and 53BP1 measured by flow cytometry after 1 and 24 hours (h).
From a total of 89,003 variants, we identified 226 rare variants, including 64 novel variants, with a median of 6 variants per individual (range 1-14). EP400, TP53BP1 and POLR2B were the most variable genes. CVID CD4+T cells had higher endogenous levels of γH2AX than controls at 1 and 24h (1h p=0.006; 24h p=0.03). Following γ-irradiation, T cells had a higher γH2AX than controls at 1h (CD4+p<=0.001; CD8+ p=0.002) and 24h (CD4+p<=0.001; CD8+p=0.001), while B cells had higher γH2AX at 24h (p=0.03). A subset of patients’ B cells had lower pATM than controls at 24h (p=0.04). These pATMlo B cells had increased γH2AX, decreased 53BP1 and increased apoptosis (r=-0.91, p<=0.001).
We identified rare variants in genes involved in DNA damage and repair in CVID patients. Our functional analyses have demonstrated evidence of dysregulated DDR and apoptosis in a subgroup of CVID patients.
CD4 T-cells include effector/conventional (Tconv) and suppressive/regulatory (Treg) lineages with thymus origin that egress into naïve (n) compartment with broad-reactivity, and are continuously challenged to differentiate into memory (m) T-cells upon contact with cognate antigens. Naïve compartment homeostasis is, therefore, critical to ensure T-cell diversity, depending on homeostatic proliferation to counteract differentiation/death rates and thymic replenishment decline. Intriguingly, adults thymectomised early in life feature nTreg preservation despite remarkable nTconv contraction. Here, we aim to identify molecular pathways regulating human naïve Treg and Tconv homeostasis.
We used CD25/CD127 to sort Tregs/Tconvs from CD4 single-positive thymocytes (3 thymuses discarded during pediatric corrective cardiac surgery), and from circulating naïve and memory CD4 T-cells (3 healthy young adults). Transcriptomes were generated by RNA-seq and differential expressions quantified at gene level (DEG) for each lineage along sequential transitions between compartments, thymus-to-naïve (TN) and naïve-to-memory (NM).
We find a large consistency between Tregs and Tconvs for expression changes upon thymus egress (TN: 3663 of 4351) and during memory differentiation (NM: 446 of 970), in agreement with a common developmental programme. However, there is also significant DEG specific to each lineage in TN and NM transitions (respectively: Tregs, 406/374; Tconvs, 276/141). Importantly, some genes are specifically upregulated in nTregs upon thymic egress and repressed after mTreg differentiation. Amongst these is FOXP1-AS1, a quiescence regulator, suggesting a Treg-specific mechanism of promoting cell growth in naïve compartment by silencing pathways that inhibit proliferation.
Exploring distinct pathways of naïve Treg and Tconv homeostasis may identify novel targets to modulate immune reconstitution.
Major histocompability class I deficiency is a rare disease with remarkable clinical and biological heterogeneity. This deficiency consists of a group of autosomal recessive diseases caused by mutations in TAP1, TAP2, tapasin and β2 microglobulin, which are important for intracellular loading of antigens into MHC Class I molecules and stabilizing the complex. The clinical spectrum varies from complete absence of symptoms to life-threatening disease. We aimed to present the clinical, laboratory, genetic and follow-up results of 8 patients who were followed up with MHC Class I deficiency.
Patients' data were evaluated.retrospectively
8 patients from 4 families (F/M:4/4); median age of 16,5years (range 10-30y), median age of onset of symptoms 3,5years (range 1-17y), median follow up 12,5years (2-15y). We observed bronchiectasia in 7, skin lesions in 4, uveitis in and retinitis in 1 of patients. MHC Class I expresion was measured by flow cytometry very low. The mutation analysis performed and validated by next generation sequencing (NGS) and Sanger’s methods. We detected TAP1 mutation in six patients, TAP2 mutation in two patients. While one of two sibling was almost asymptomatic the other died due to sepsis caused by severe skin lesions. Overall survival is 87.5%.
MHC Class 1 deficiency should be consider in patients with skin lesions, sinopulmonary infections and bronchiectasis. The measure of HLA ABC expression by routine lymphocyte subgroup analysis allows the diagnosis. Clinical manifestation changes from patient to patient. The knowledge about bone marrow transplantation in MHC Class I deficiency is insufficient
B cells play a central role in adaptive immune processes, mainly through the production of antibodies. Children are born without having had much contact with foreign antigens but through continuous exposure, the human immune system builds a repository of cells bearing diverse antigen-specific adaptive immune receptors that enable an effective secondary immune response. The maturation of the B-cell system through continuous antigen exposure with age is poorly studied.
We investigated the naïve and antigen-experienced B-cell receptor (BCR) repertoire in 46 healthy individuals aged 6m to 50y. Heavy chain BCR transcripts were amplified and sequenced with data analysis assessing repertoire characteristics and the self-reactive and structural nature of BCR transcripts.
The final dataset consisted of ~7M unique sequences. Most dynamics were observed in the first 10y of life characterized by alterations in immunoglobulin gene usage, increase in frequencies of mutated transcripts through positive selection, increased usage of downstream constant region genes and a decrease in the frequency of transcripts with self-reactive properties indicating that somatic hypermutation has driven specificity of these sequences away from self. Structural analysis revealed that the frequency of antibodies different from germline in shape increased with age.
This study demonstrates an extensive maturation of the B-cell system in the first 10 years of life in line with accumulating environmental antigen exposure. Further antibody repertoire alterations continue to be made thereafter, although at a lower rate. This study also provides a reference data set of BCR repertoires and stresses the importance of using well-selected, age-appropriate controls in future studies.
Polymorphism within the T cell receptor beta (TCRB) variable (TRBV) gene is implicated in PID, autoimmune disease and immune-related adverse events (IRAEs) during immunotherapy. Efforts to evaluate TRBV polymorphism by WGS were hampered by the repetitive nature of TCRB locus. We present a novel long-amplicon TCRB repertoire sequencing approach to enable haplotype analysis of the TRB locus from peripheral blood.
Baseline total RNA from peripheral blood leukocytes from 81 Caucasians treated with checkpoint inhibitor for cancer was used for amplification and sequencing of TCRB chains via the Oncomine TCRB-LR assay (spanning complementarity-determining regions 1, 2 and 3) and the Gene Studio S5. VDJ rearrangements were annotated by comparison to the gold-standard IMGT database, then mined to construct TRBV allele profiles for each individual. Principal component analysis (PCA) of variable gene allele profiles and k-means clustering identified TRBV allele haplotypes.
PCA and k-means clustering of TRBV allele profiles revealed presence of 6 major sets of coincident variable gene alleles, which we term haplotype groups. Allele features varied markedly across haplotype groups, with haplotype group 2 members, comprising approximately one third of the cohort, having limited TRBV allelic diversity and few uncommon alleles compared to members of other groups.
We demonstrate a cost-efficient and rapid method for routine analysis of germline-encoded polymorphism within the TRB locus to enable high-resolution studies of genetic variation and its potential link to IRAEs. This technology could potentially be used to analyze the TRBV to aid in studying thymic output defects, and indicate autoimmunity in PID patients.