Non-response to rituximab therapy in rheumatoid arthritis is associated with incomplete disruption of the B cell receptor repertoire.

OBJECTIVE: To gain more insight into the dynamics of lymphocyte depletion and develop new predictors of clinical response to rituximab in rheumatoid arthritis (RA). METHODS: RNA-based next-generation sequencing was used to analyse the B cell receptor (BCR) repertoire in peripheral blood and synovial tissue samples collected from 24 seropositive patients with RA treated with rituximab. Clonal expansion, mutation load and clonal overlap were assessed in samples collected before, at week 4 and at week 16 or 24 after treatment and correlated to the patients' clinical response. RESULTS: After 4 weeks of rituximab-induced B cell depletion, the peripheral blood BCR repertoire of treated patients consisted of fewer, more dominant and more mutated BCR clones. No significant changes in the synovial tissue BCR repertoire were detected until week 16 post-treatment, when a reduced clonal overlap with baseline and an increased mutation load were observed. In patients who were non-responders at month 3 (n=5) using the European League Against Rheumatism response criteria, peripheral blood samples taken at week 4 after rituximab treatment showed more dominant clones compared with moderate responders (n=9) (median (IQR): 36 (27-52) vs 18 (16-26); p<0.01) and more clonal overlap with the baseline (median (IQR): 5% (2%-20%) vs 0% (0%-0%); p≤0.01). CONCLUSION: Significant changes in BCR clonality are observed in peripheral blood of patients 4 weeks after rituximab treatment, while changes in synovial tissue were observed at later time points. Incomplete depletion of the dominant baseline peripheral blood BCR repertoire in the first month of treatment might predict clinical non-response at 3 months.

In Rheumatoid Arthritis, Synovitis at Different Inflammatory Sites Is Dominated by Shared but Patient-Specific T Cell Clones.

Genetic and immunological evidence clearly points to a role for T cells in the pathogenesis of rheumatoid arthritis (RA). Selective targeting of such disease-associated T cell clones might be highly effective while having few side effects. However, such selective targeting may only be feasible if the same T cell clones dominate the immune response at different sites of inflammation. We leveraged high-throughput technology to quantitatively assess whether different T cell clones dominate the inflammatory infiltrate at various sites of inflammation in this prototypic autoimmune disease. In 13 RA patients, we performed quantitative next-generation sequencing-based human TCRß repertoire analysis in simultaneously obtained samples from inflamed synovial tissue (ST) from distinct locations within one joint, from multiple joints, and from synovial fluid (SF) and peripheral blood (PB). Identical TCRß clones dominate inflammatory responses in ST samples taken from different locations within a single joint and when sampled in different joints. Although overall ST-SF overlap was comparable to higher ST-ST values, the overlap in dominant TCRß clones in ST-SF comparisons was much lower than ST-ST and comparable to the low ST-PB overlap. In individual RA patients, a limited number of TCRß clones dominate the immune response in the inflamed ST regardless of the location within a joint and which joint undergoes biopsy; in contrast, there is limited overlap of ST with SF or PB TCR repertoires. This limited breadth of the T cell response in ST of the individual RA patient indicates that development of immunotherapies that selectively modulate dominant T cell responses might be feasible.

Computational Model Reveals Limited Correlation between Germinal Center B-Cell Subclone Abundancy and Affinity: Implications for Repertoire Sequencing.

Immunoglobulin repertoire sequencing has successfully been applied to identify expanded antigen-activated B-cell clones that play a role in the pathogenesis of immune disorders. One challenge is the selection of the Ag-specific B cells from the measured repertoire for downstream analyses. A general feature of an immune response is the expansion of specific clones resulting in a set of subclones with common ancestry varying in abundance and in the number of acquired somatic mutations. The expanded subclones are expected to have BCR affinities for the Ag higher than the affinities of the naive B cells in the background population. For these reasons, several groups successfully proceeded or suggested selecting highly abundant subclones from the repertoire to obtain the Ag-specific B cells. Given the nature of affinity maturation one would expect that abundant subclones are of high affinity but since repertoire sequencing only provides information about abundancies, this can only be verified with additional experiments, which are very labor intensive. Moreover, this would also require knowledge of the Ag, which is often not available for clinical samples. Consequently, in general we do not know if the selected highly abundant subclone(s) are also the high(est) affinity subclones. Such knowledge would likely improve the selection of relevant subclones for further characterization and Ag screening. Therefore, to gain insight in the relation between subclone abundancy and affinity, we developed a computational model that simulates affinity maturation in a single GC while tracking individual subclones in terms of abundancy and affinity. We show that the model correctly captures the overall GC dynamics, and that the amount of expansion is qualitatively comparable to expansion observed from B cells isolated from human lymph nodes. Analysis of the fraction of high- and low-affinity subclones among the unexpanded and expanded subclones reveals a limited correlation between abundancy and affinity and shows that the low abundant subclones are of highest affinity. Thus, our model suggests that selecting highly abundant subclones from repertoire sequencing experiments would not always lead to the high(est) affinity B cells. Consequently, additional or alternative selection approaches need to be applied.

Somatic Variation of T-Cell Receptor Genes Strongly Associate with HLA Class Restriction.

Every person carries a vast repertoire of CD4+ T-helper cells and CD8+ cytotoxic T cells for a healthy immune system. Somatic VDJ recombination at genomic loci that encode the T-cell receptor (TCR) is a key step during T-cell development, but how a single T cell commits to become either CD4+ or CD8+ is poorly understood. To evaluate the influence of TCR sequence variation on CD4+/CD8+ lineage commitment, we sequenced rearranged TCRs for both α and ß chains in naïve T cells isolated from healthy donors and investigated gene segment usage and recombination patterns in CD4+ and CD8+ T-cell subsets. Our data demonstrate that most V and J gene segments are strongly biased in the naïve CD4+ and CD8+ subsets with some segments increasing the odds of being CD4+ (or CD8+) up to five-fold. These V and J gene associations are highly reproducible across individuals and independent of classical HLA genotype, explaining ~11% of the observed variance in the CD4+ vs. CD8+ propensity. In addition, we identified a strong independent association of the electrostatic charge of the complementarity determining region 3 (CDR3) in both α and ß chains, where a positively charged CDR3 is associated with CD4+ lineage and a negatively charged CDR3 with CD8+ lineage. Our findings suggest that somatic variation in different parts of the TCR influences T-cell lineage commitment in a predominantly additive fashion. This notion can help delineate how certain structural features of the TCR-peptide-HLA complex influence thymic selection.

Clonal evolution of CD8+ T cell responses against latent viruses: relationship among phenotype, localization, and function.

UNLABELLED: Human cytomegalovirus (hCMV) infection is characterized by a vast expansion of resting effector-type virus-specific T cells in the circulation. In mice, interleukin-7 receptor α (IL-7Rα)-expressing cells contain the precursors for long-lived antigen-experienced CD8(+) T cells, but it is unclear if similar mechanisms operate to maintain these pools in humans. Here, we studied whether IL-7Rα-expressing cells obtained from peripheral blood (PB) or lymph nodes (LNs) sustain the circulating effector-type hCMV-specific pool. Using flow cytometry and functional assays, we found that the IL-7Rα(+) hCMV-specific T cell population comprises cells that have a memory phenotype and lack effector features. We used next-generation sequencing of the T cell receptor to compare the clonal repertoires of IL-7Rα(+) and IL-7Rα(-) subsets. We observed limited overlap of clones between these subsets during acute infection and after 1 year. When we compared the hCMV-specific repertoire between PB and paired LNs, we found many identical clones but also clones that were exclusively found in either compartment. New clones that were found in PB during antigenic recall were only rarely identical to the unique LN clones. Thus, although PB IL-7Rα-expressing and LN hCMV-specific CD8(+) T cells show typical traits of memory-type cells, these populations do not seem to contain the precursors for the novel hCMV-specific CD8(+) T cell pool during latency or upon antigen recall. IL-7Rα(+) PB and LN hCMV-specific memory cells form separate virus-specific compartments, and precursors for these novel PB hCMV-specific CD8(+) effector-type T cells are possibly located in other secondary lymphoid tissues or are being recruited from the naive CD8(+) T cell pool. IMPORTANCE: Insight into the self-renewal properties of long-lived memory CD8(+) T cells and their location is crucial for the development of both passive and active vaccination strategies. Human CMV infection is characterized by a vast expansion of resting effector-type cells. It is, however, not known how this population is maintained. We here investigated two possible compartments for effector-type cell precursors: circulating acute-phase IL-7Rα-expressing hCMV-specific CD8(+) T cells and lymph node (LN)-residing hCMV-specific (central) memory cells. We show that new clones that appear after primary hCMV infection or during hCMV reactivation seldom originate from either compartment. Thus, although identical clones may be maintained by either memory population, the precursors of the novel clones are probably located in other (secondary) lymphoid tissues or are recruited from the naive CD8(+) T cell pool.