Overview of methodologies for T-cell receptor repertoire analysis

Abstract Background The T-cell receptor (TCR), located on the surface of T cells, is responsible for the recognition of the antigen-major histocompatibility complex, leading to the initiation of an inflammatory response. Analysing the TCR repertoire may help to gain a better understanding of the immune system features and of the aetiology and progression of diseases, in particular those with unknown antigenic triggers. The extreme diversity of the TCR repertoire represents a major analytical challenge; this has led to the development of specialized methods which aim to characterize the TCR repertoire in-depth. Currently, next generation sequencing based technologies are most widely employed for the high-throughput analysis of the immune cell repertoire. Results Here, we report on the latest methodological advancements in the field by describing and comparing the available tools; from the choice of the starting material and library preparation method, to the sequencing technologies and data analysis. Finally, we provide a practical example and our own experience by reporting some exemplary results from a small internal benchmark study, where current approaches from the literature and the market are employed and compared. Conclusions Several valid methods for clonotype identification and TCR repertoire analysis exist, however, a gold standard method for the field has not yet been identified. Depending on the purpose of the scientific study, some approaches may be more suitable than others. Finally, due to possible method specific biases, scientists must be careful when comparing results obtained using different methods

Gut and Liver B Cells of Common Clonal Origin in Primary Sclerosing Cholangitis-Inflammatory Bowel Disease

B cells express an antigen‐specific B‐cell receptor (BCR) and may contribute to liver inflammation by recognizing shared antigens in the gut and liver. Herein, we used high‐throughput BCR sequencing of the immunoglobulin heavy chain, specifically the complementarity‐determining region 3 (CDR3), to characterize the B‐cell repertoire of freshly‐frozen paired gut and liver tissue samples from patients with primary sclerosing cholangitis (PSC) and concurrent inflammatory bowel disease (IBD) (PSC‐IBD, n = 10) and paired formalin‐fixed paraffin‐embedded (FFPE) tumor‐adjacent normal colon and liver tissue from patients with colorectal liver metastases (controls, n = 10). We observed significantly greater numbers of B cells (P < 0.01) and unique B‐cell clonotypes (P < 0.05) in gut samples compared to liver samples of patients with PSC‐IBD, whereas BCR sequences in FFPE normal gut and liver samples were nearly absent (14 ± 5 clonotypes; mean ± SD; n = 20). In PSC‐IBD, an average of 8.3% (range, 1.6%‐18.0%) of B‐cell clonotypes were found to overlap paired gut and liver samples following the exclusion of memory clonotypes reported in the blood of healthy controls. Overlapping gut and liver clonotypes showed stronger evidence of antigen‐driven activation compared to non‐overlapping clonotypes, including shorter CDR3 lengths and higher counts of somatic hypermutation (P < 0.0001). Conclusion: A proportion of gut and liver B cells originate from a common clonal origin (i.e., likely to recognize the same antigen) in patients with PSC which suggests B‐cell antigens are shared across the gut–liver axis. (Hepatology Communications 2018; 00:000‐000

T-cell receptors and human leukocyte antigens in primary sclerosing cholangitis

The measurements of total number concentration and number size distribution of aerosols in size ranges of 16-700 nm and 0.5-20 &#956;m diameters made from 14&#176;N to 56&#176;S in the Indian ocean during January 23 to March 31, 2004, are reported. The average values of total number (mass) concentration of micrometer aerosols (0.5-20 &#956;m) are 7.2 &#177; 3 cm-3 (8.89 &#956;g m-3) from 14&#176;N to the Inter-Tropical Convergence Zone (ITCZ), 4.6 &#177; 2 cm-3 (4.9 &#956;g m-3) in the ITCZ, 3.0 &#177; 1.4 cm-3 (5.78 &#956;g m-3) in the trade wind region from 8&#176;-30&#176;S, 3.8 &#177; 2.6 cm-3 (7.9 &#956;g m-3) from 30&#176;-40&#176;S, and 5.8 &#177; 3.5 cm-3 (9.65 &#956;g m-3) in the roaring forties from 40&#176;-56&#176;S. Latitudinal distribution of such aerosols shows that their number concentration is minimum (0.5 cm-3) at 11&#176;S and increases on either side of this location. Size distributions of micrometer aerosols in all latitudinal belts show a maxima in coarse mode at 0.5-1.5 &#956;m diameter. The correlation coefficient in the aerosol concentration-wind speed relations of these aerosols is observed to differ in different latitudinal belts and has maximum value in the belt of strongest winds. Also, diurnal variations of the average aerosol concentration and wind speed show some similarity in belts of strong winds. Total number concentration of submicrometer (16-700 nm) particles is also minimum in the southern trade wind region and their size distribution is bimodal with maxima in Aitken mode (~ 50 nm) and accumulation mode (~ 130 nm). South of the ITCZ, concentrations of both, total number and Aitken mode particles normally increase with latitude. The increase in Aitken mode particles at high latitudes is large enough for the Aitken mode maxima to superimpose the accumulation mode maxima. Observations suggest that addition of Aitken particles is so fast that process of coagulation is not able to reach equilibrium to develop a distinct accumulation maxima. Observations show that pristine air of the trade wind region in the Southern Hemisphere can be advected to mid-latitudes along the ridge developed between anticyclonic and cyclonic systems

Single-cell m6 A mapping in vivo using picoMeRIP–seq

Current N6-methyladenosine (m6A) mapping methods need large amounts of RNA or are limited to cultured cells. Through optimized sample recovery and signal-to-noise ratio, we developed picogram-scale m6A RNA immunoprecipitation and sequencing (picoMeRIP–seq) for studying m6A in vivo in single cells and scarce cell types using standard laboratory equipment. We benchmark m6A mapping on titrations of poly(A) RNA and embryonic stem cells and in single zebrafish zygotes, mouse oocytes and embryos