Paul L. Klarenbeek

Human T-cell memory consists mainly of unexpanded clones

The immune system is able to respond to millions of antigens using adaptive receptors, including the alphabeta-T-cell receptor (TCR). Upon antigen encounter a T-cell may proliferate to produce a clone of TCR-identical cells, which develop a memory phenotype. Previous studies suggested that most memory clones are clearly expanded. In accordance, the beta-chain repertoire of T-cell memory subsets was reported to be 10 times less diverse than those of naive subsets, reflecting stringent selection. However, due to technological limitations detailed information was lacking regarding the size of clonal expansions and the diversity of the TCR-repertoire in naive and memory T-cell populations. Here, using high-throughput sequencing, we show that the memory repertoire in human peripheral blood contains only few expanded clones and consists mainly of low frequency clones. Additionally, the memory repertoire is much more diverse than expected. In two healthy persons we observed that only 2-7% of the CD4 and CD8 memory clones found were clearly expanded. In line with this observation we show that the beta-chains repertoire size of the CD4 memory compartment is only two times smaller, and that of the CD8 memory compartment is only 3-10 times smaller than the naive compartments. Our results show that the T-cell memory compartment has a very different distribution of clones than anticipated. This has important implications for the current dogma of immunological memory, and changes the interpretation of repertoire aberrations in (patho-)physiological situations such as ageing and auto-immunity. It raises new questions on the factors that steer maturation of memory phenotype and determine the size of memory clones.

Immunoglobulin G4+ clones identified by next‐generation sequencing dominate the B cell receptor repertoire in immunoglobulin G4 associated cholangitis

Immunoglobulin G4 (IgG4)‐associated cholangitis (IAC) is a manifestation of the recently discovered idiopathic IgG4‐related disease. The majority of patients have elevated serum IgG4 levels and/or IgG4‐positive B‐cell and plasma cell infiltrates in the affected tissue. We hypothesized that clonally expanded, class‐switched IgG4‐positive B cells and plasma cells could be causal to these poorly understood phenomena. In a prospective cohort of six consecutive IAC patients, six healthy controls, and six disease controls, we used a novel next‐generation sequencing approach to screen the B‐cell receptor (BCR) repertoires, in blood as well as in affected tissue, for IgG4+ clones. A full repertoire analysis of the BCR heavy chain was performed using GS‐FLX/454 and customized bioinformatics algorithms (>10,000 sequences/sample; clones with a frequency ≥0.5% were considered dominant). We found that the most dominant clones within the IgG+ BCRheavy repertoire of the peripheral blood at baseline were IgG4+ only in IAC patients. In all IAC patients, but none of the controls, IgG4+ BCR clones were among the 10 most dominant BCR clones of any immunoglobulin isotype (IgA, IgD, IgM, and IgG) in blood. The BCR repertoires of the duodenal papilla comprised the same dominant IgG4+ clones as the paired peripheral blood samples. In all IAC patients, after 4 and 8 weeks of corticosteroid therapy the contribution of these IgG4+ clones to the IgG+ repertoire as well as to total BCR repertoire was marginalized, mirroring sharp declines in serum IgG4 titers and regression of clinical symptoms. Conclusion: The novel finding of highly abundant IgG4+ BCR clones in blood and tissue of patients with active IAC, which disappear upon corticosteroid treatment, suggests that specific B cell responses are pivotal to the pathogenesis of IAC. (HEPATOLOGY 2013 )

Inflamed target tissue provides a specific niche for highly expanded T-cell clones in early human autoimmune disease

Objective To profile quantitatively the T-cell repertoire in multiple joints and peripheral blood of patients with recent onset (early) or established rheumatoid arthritis (RA) using a novel next-generation sequencing protocol to identify potential autoreactive clones. Methods Synovium of patients with recent onset (early) RA (<6 months) (n=6) or established RA (>18 months) (n=6) was screened for T-cell clones by sequencing over 10 000 T-cell receptors (TCR) per sample. T cells from paired blood samples were analysed for comparison. From two patients synovial T cells were obtained from multiple inflamed joints. The degree of expansion of each individual clone was based on its unique CDR3 sequence frequency within a sample. Clones with a frequency of over 0.5% were considered to be highly expanded clones (HEC). Results In early RA synovium, the T-cell repertoire was dominated by 35 HEC (median, range 2–70) accounting for 56% of the TCR sequenced. The clonal dominance in the synovium was patient specific and significantly greater than in established RA (median of 11 HEC (range 5–24) in established RA synovium accounting for 9.8% of T cells; p<0.01). 34% (range 28–40%) of the most expanded T-cell clones were shared between different joints in the same patients, compared with only 4% (range 0–8%) between synovium and blood (p=0.01). Conclusions In RA, a systemic autoimmune disease, the inflamed synovium forms a niche for specific expanded T-cell clones, especially in early disease. This suggests that, at least in RA, autoreactive T cells should be addressed specifically in the inflamed tissue, preferably in the early phase of the disease.

Enhanced gene transfer to arthritic joints using adeno-associated virus type 5: implications for intra-articular gene therapy

Background: Gene therapy of the joint has great potential as a new therapeutic approach for the treatment of rheumatoid arthritis (RA). The vector chosen is of crucial importance for clinical success. Objective: To investigate the tropism and transduction efficiency in arthritic joints in vivo, and in synovial cells in vitro, using five different serotypes of recombinant adeno-associated virus (rAAV) encoding β-galactosidase or green fluorescent protein genes. Methods: rAAV was injected into the ankle joints of rats with adjuvant arthritis after the onset of disease. Synovial tissue was examined at different time points for β-galactosidase protein and gene expression by in situ staining and polymerase chain reaction (PCR) analysis, respectively. In addition, the ability of rAAV to transduce primary human fibroblast-like synoviocytes from patients with RA was investigated in vitro. Results: Intra-articular injection of the rAAV5 serotype resulted in the highest synovial transduction, followed by much lower expression using rAAV2. Expression of the transgene was already detectable 7 days after injection and lasted for at least 4 weeks. Only background staining was seen for serotypes 1, 3, and 4. Importantly, there was a minimal humoral immune response to rAAV5 compared with rAAV2. Additionally, it was found that both rAAV2 and rAAV5 can efficiently transduce human fibroblast-like synoviocytes obtained from patients with RA. Conclusion: Intra-articular rAAV mediated gene therapy in RA might be improved by using rAAV5 rather than other serotypes.

Deep Sequencing of Antiviral T-Cell Responses to HCMV and EBV in Humans Reveals a Stable Repertoire That Is Maintained for Many Years

CD8+ T-cell responses against latent viruses can cover considerable portions of the CD8+ T-cell compartment for many decades, yet their initiation and maintenance remains poorly characterized in humans. A key question is whether the clonal repertoire that is raised during the initial antiviral response can be maintained over these long periods. To investigate this we combined next-generation sequencing of the T-cell receptor repertoire with tetramer-sorting to identify, quantify and longitudinally follow virus-specific clones within the CD8+ T-cell compartment. Using this approach we studied primary infections of human cytomegalovirus (hCMV) and Epstein Barr virus (EBV) in renal transplant recipients. For both viruses we found that nearly all virus-specific CD8+ T-cell clones that appeared during the early phase of infection were maintained at high frequencies during the 5-year follow-up and hardly any new anti-viral clones appeared. Both in transplant recipients and in healthy carriers the clones specific for these latent viruses were highly dominant within the CD8+ T-cell receptor Vβ repertoire. These findings suggest that the initial antiviral response in humans is maintained in a stable fashion without signs of contraction or changes of the clonal repertoire.

Rheumatoid arthritis synovial tissue harbours dominant B-cell and plasma-cell clones associated with autoreactivity

Objective To identify potential autoreactive B-cell and plasma-cell clones by quantitatively analysing the complete human B-cell receptor (BCR) repertoire in synovium and peripheral blood in early and established rheumatoid arthritis (RA). Methods The BCR repertoire was screened in synovium and blood of six patients with early RA (ERA) (<6 months) and six with established RA (ESRA) (>20 months). In two patients, the repertoires in different joints were compared. Repertoires were analysed by next-generation sequencing from mRNA, generating >10 000 BCR heavy-chain sequence reads per sample. For each clone, the degree of expansion was calculated as the percentage of the total number of reads encoding the specific clonal sequence. Clones with a frequency ≥0.5% were considered dominant. Results Multiple dominant clones were found in inflamed synovium but hardly any in blood. Within an individual patient, the same dominant clones were detected in different joints. The majority of the synovial clones were class-switched; however, the fraction of clones that expressed IgM was higher in ESRA than ERA patients. Dominant synovial clones showed autoreactive features: in ERA in particular the clones were enriched for immunoglobulin heavy chain gene segment V4–34 (IGHV4–34) and showed longer CDR3 lengths. Dominant synovial clones that did not encode IGHV4–34 also had longer CDR3s than peripheral blood. Conclusions In RA, the synovium forms a niche where expanded—potentially autoreactive—B cells and plasma cells reside. The inflamed target tissue, especially in the earliest phase of disease, seems to be the most promising compartment for studying autoreactive cells.

Memory CD4+CCR5+ T cells are abundantly present in the gut of newborn infants to facilitate mother-to-child transmission of HIV-1

Despite potential clinical importance, target cells for mother-to-child transmission of HIV-1 have not yet been identified. Cord blood-derived CD4(+) T cells are largely naive and do not express CCR5, the mandatory coreceptor for transmitted HIV-1 R5 strains in infants. In the present study, we demonstrate that in the human fetal and infant gut mucosa, there is already a large subset of mucosal memory CD4(+)CCR5(+) T cells with predominantly a Th1 and Th17 phenotype. Using next-generation sequencing of the TCRβ chain, clonally expanded T cells as a hallmark for memory development predominated in the gut mucosa (30%), whereas few were found in the lymph nodes (1%) and none in cord blood (0%). The gut mucosal fetal and infant CD4(+) T cells were highly susceptible to HIV-1 without any prestimulation; pol proviral DNA levels were similar to infected phytohemagglutinin-stimulated adult PBMCs. In conclusion, in the present study, we show that extensive adaptive immunity is present before birth and the gut mucosa is the preferential site for memory CD4(+) T cells. These CD4(+)CCR5(+) T cells in the infant mucosa provide a large pool of susceptible cells for ingested HIV-1 at birth and during breastfeeding, indicating a mucosal route of mother-to-child transmission that can be targeted in prevention strategies.

Alterations of the synovial T cell repertoire in anti-citrullinated protein antibody-positive rheumatoid arthritis.

OBJECTIVE The association of HLA-DRB1 alleles with anti-citrullinated protein antibodies (ACPAs) in rheumatoid arthritis (RA) suggests the potential involvement of T lymphocytes in ACPA-seropositive disease. The purpose of this study was to investigate this hypothesis by systematic histologic and molecular analyses of synovial T cells in ACPA+ versus ACPA- RA patients. METHODS Synovial biopsy samples were obtained from 158 RA patients. Inflammation was determined histologically and immunohistochemically. RNA was extracted from peripheral blood mononuclear cells and synovial tissues obtained from 11 ACPA+ RA patients, 7 ACPA- RA patients, and 10 spondylarthritis (SpA) patients (arthritis controls). T lymphocyte clonality was studied by combined quantitative and qualitative T cell receptor CDR3 length distribution (LD) analysis and direct sequencing analysis. RESULTS ACPA+ and ACPA- RA patients were similar at both the clinical and histologic levels. At the molecular level, however, patients with ACPA+ synovitis displayed a marked elevation of qualitative CDR3 LD alterations as compared with those with ACPA- synovitis and with the SpA controls. These differences in CDR3 LD were not observed in the peripheral blood, indicating a selective recruitment and/or local expansion of T cells in the synovial compartment. The CDR3 LD alterations reflected true monoclonal or oligoclonal expansions, as confirmed by direct sequencing of the T cell receptor. The CDR3 LD alterations in RA synovium did not correlate with B cell clonal expansions but were inversely associated with synovial lymphoid neogenesis. CONCLUSION The T cell repertoire is specifically restricted in RA patients with ACPA+ synovitis. Whereas the origin and role of these clonal alterations remain to be determined, our data suggest the preferential involvement of T lymphocytes in ACPA-seropositive RA.

Immunoglobulin G4+ B‐cell receptor clones distinguish immunoglobulin G 4‐related disease from primary sclerosing cholangitis and biliary/pancreatic malignancies

Immunoglobulin G4 (IgG4)‐related disease (IgG4‐RD) of the biliary tree and pancreas is difficult to distinguish from sclerosing cholangitis and biliary/pancreatic malignancies (CA). An accurate noninvasive test for diagnosis and monitoring of disease activity is lacking. We demonstrate that dominant IgG4+ B‐cell receptor (BCR) clones determined by next‐generation sequencing accurately distinguish patients with IgG4‐associated cholangitis/autoimmune pancreatitis (n = 34) from those with primary sclerosing cholangitis (n = 17) and CA (n = 17). A novel, more affordable, and widely applicable quantitative polymerase chain reaction (qPCR) protocol analyzing the IgG4/IgG RNA ratio in blood also achieves excellent diagnostic accuracy (n = 125). Moreover, this qPCR test performed better than serum IgG4 levels in sensitivity (94% vs. 86%) and specificity (99% vs. 73%) and correlates with treatment response (n = 20). Conclusions: IgG4+ BCR clones and IgG4/IgG RNA ratio markedly improve delineation, early diagnosis, and monitoring of IgG4‐RD of the biliary tree and pancreas. (Hepatology 2016;64:501‐507)

Analysis of apoptosis in peripheral blood and synovial tissue very early after initiation of infliximab treatment in rheumatoid arthritis patients

OBJECTIVE Infliximab treatment results in a decrease in synovial cellularity as early as 48 hours after initiation of therapy in patients with rheumatoid arthritis (RA). This study was undertaken to investigate whether infliximab induces apoptosis within the first 24 hours after infusion. METHODS The percentage of apoptotic cells was determined by flow cytometry in blood drawn from 21 patients directly before, 1 hour after, and 24 hours after infliximab infusion. Synovial tissue samples obtained before, 1 hour after (n = 5), or 24 hours after (n = 5) initiation of therapy were subjected to immunohistochemistry to detect active caspase 3 and to TUNEL assay and electron microscopy to detect apoptosis. In addition, plasma levels of nucleosomes (generated during apoptosis) and C4b/c (an indicator of complement activation) were measured. RESULTS There were no signs of apoptosis induction in peripheral blood monocytes or lymphocytes after infliximab treatment. Circulating lymphocyte counts were increased within 1 hour after infusion (P < 0.05). There was no definite evidence of apoptosis induction in the synovium, except in 1 patient 24 hours after the infliximab infusion. Consistent with these results, there was no increase in nucleosome levels nor were there signs of complement activation. CONCLUSION Our findings indicate that the rapid decrease in synovial cellularity observed after initiation of anti-tumor necrosis factor antibody therapy cannot be explained by apoptosis induction at the site of inflammation. It is tempting to speculate that the striking effects on synovial inflammation may be explained by other mechanisms, such as decreased migration toward the synovial compartment and reduced retention in the inflamed synovium.