The splenic T cell receptor repertoire during an immune response against a complex antigen: Expanding private clones accumulate in the high and low copy number region.

Large cellular antigens comprise a variety of different epitopes leading to a T cell response of extreme diversity. Therefore, tracking such a response by next generation sequencing of the T cell receptor (TCR) in order to identify common TCR properties among the expanding T cells represents an enormous challenge. In the present study we adapted a set of established indices to elucidate alterations in the TCR repertoire regarding sequence similarities between TCRs including VJ segment usage and diversity of nucleotide coding of a single TCR. We combined the usage of these indices with a new systematic splitting strategy regarding the copy number of the extracted clones to divide the repertoire into multiple fractions for separate analysis. We implemented this new analytic approach using the splenic TCR repertoire following immunization with sheep red blood cells (SRBC) in mice. As expected, early after immunization presumably antigen-specific clones accumulated in high copy number fractions, but at later time points similar accumulation of specific clones occurred within the repertoire fractions of lowest copy number. For both repertoire regions immunized animals could reliably be distinguished from control in a classification approach, demonstrating the robustness of the two effects at the individual level. The direction in which the indices shifted after immunization revealed that for both the early and the late effect alterations in repertoire parameters were caused by antigen-specific private clones displacing non-specific public clones. Taken together, tracking antigen-specific clones by their displacement of average TCR repertoire characteristics in standardized repertoire fractions ensures that our analytical approach is fairly independent from the antigen in question and thus allows the in-depth characterization of a variety of immune responses.

Sleep restriction prior to antigen exposure does not alter the T cell receptor repertoire but impairs germinal center formation during a T cell-dependent B cell response in murine spleen.

It is well known that sleep promotes immune functions. In line with this, a variety of studies in animal models and humans have shown that sleep restriction following an antigen challenge dampens the immune response on several levels which leads to e.g. worsening of disease outcome and reduction of vaccination efficiency, respectively. However, the inverse scenario with sleep restriction preceding an antigen challenge is only investigated in a few animal models where it has been shown to reduce antigen uptake and presentation as well as pathogen clearance and survival rates. Here, we use injection of sheep red blood cells to investigate the yet unknown effect on a T cell-dependent B cell response in a well-established mouse model. We found that 6 â€‹h of sleep restriction prior to the antigen challenge does not impact the T cell reaction including the T cell receptor repertoire but dampens the development of germinal centers which correlates with reduced antigen-specific antibody titer indicating an impaired B cell response. These changes concerned a functionally more relevant level than those found in the same experimental model with the inverse scenario when sleep restriction followed the antigen challenge. Taken together, our findings showed that the outcome of the T cell-dependent B cell response is indeed impacted by sleep restriction prior to the antigen challenge which highlights the clinical significance of this scenario and the need for further investigations in humans, for example concerning the effect of sleep restriction preceding a vaccination.

Regulatory T cells suppress Th17 cell Ca2+ signaling in the spinal cord during murine autoimmune neuroinflammation.

T lymphocyte motility and interaction dynamics with other immune cells are vital determinants of immune responses. Regulatory T (Treg) cells prevent autoimmune disorders by suppressing excessive lymphocyte activity, but how interstitial motility patterns of Treg cells limit neuroinflammation is not well understood. We used two-photon microscopy to elucidate the spatial organization, motility characteristics, and interactions of endogenous Treg and Th17 cells together with antigen-presenting cells (APCs) within the spinal cord leptomeninges in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Th17 cells arrive before the onset of clinical symptoms, distribute uniformly during the peak, and decline in numbers during later stages of EAE. In contrast, Treg cells arrive after Th17 cells and persist during the chronic phase. Th17 cells meander widely, interact with APCs, and exhibit cytosolic Ca2+ transients and elevated basal Ca2+ levels before the arrival of Treg cells. In contrast, Treg cells adopt a confined, repetitive-scanning motility while contacting APCs. These locally confined but highly motile Treg cells limit Th17 cells from accessing APCs and suppress Th17 cell Ca2+ signaling by a mechanism that is upstream of store-operated Ca2+ entry. Finally, Treg cell depletion increases APC numbers in the spinal cord and exaggerates ongoing neuroinflammation. Our results point to fundamental differences in motility characteristics between Th17 and Treg cells in the inflamed spinal cord and reveal three potential cellular mechanisms by which Treg cells regulate Th17 cell effector functions: reduction of APC density, limiting access of Th17 cells to APCs, and suppression of Th17 Ca2+ signaling.

Effects of sleep on the splenic milieu in mice and the T cell receptor repertoire recruited into a T cell dependent B cell response.

Sleep is known to improve immune function ranging from cell distribution in the naïve state to elevated antibody titers after an immune challenge. The underlying mechanisms still remain unclear, partially because most studies have focused on the analysis of blood only. Hence, we investigated the effects of sleep within the spleen in female C57BL/6J mice with normal sleep compared to short-term sleep-deprived animals both in the naïve state and after an antigen challenge. Lack of sleep decreased the expression of genes associated with immune cell recruitment into and antigen presentation within the spleen both in the naïve state and during a T cell dependent B cell response directed against sheep red blood cells (SRBC). However, neither T cell proliferation nor formation of SRBC-specific antibodies was affected. In addition, the T cell receptor repertoire recruited into the immune response within seven days was not influenced by sleep deprivation. Thus, sleep modulated the molecular milieu within the spleen whereas we could not detect corresponding changes in the primary immune response against SRBC. Further studies will show whether sleep influences the secondary immune response against SRBC or the development of the B cell receptor repertoire, and how this can be compared to other antigens.

Deep Sequencing Reveals Transient Segregation of T Cell Repertoires in Splenic T Cell Zones during an Immune Response.

Immunological differences between hosts, such as diverse TCR repertoires, are widely credited for reducing the risk of pathogen spread and adaptation in a population. Within-host immunological diversity might likewise be important for robust pathogen control, but to what extent naive TCR repertoires differ across different locations in the same host is unclear. T cell zones (TCZs) in secondary lymphoid organs provide secluded microenvironmental niches. By harboring distinct TCRs, such niches could enhance within-host immunological diversity. In contrast, rapid T cell migration is expected to dilute such diversity. In this study, we combined tissue microdissection and deep sequencing of the TCR ß-chain to examine the extent to which TCR repertoires differ between TCZs in murine spleens. In the absence of Ag, we found little evidence for differences between TCZs of the same spleen. Yet, 3 d after immunization with sheep RBCs, we observed a >10-fold rise in the number of clones that appeared to localize to individual zones. Remarkably, these differences largely disappeared at 4 d after immunization, when hallmarks of an ongoing immune response were still observed. These data suggest that in the absence of Ag, any repertoire differences observed between TCZs of the same host can largely be attributed to random clone distribution. Upon Ag challenge, TCR repertoires in TCZs first segregate and then homogenize within days. Such "transient mosaic" dynamics could be an important barrier for pathogen adaptation and spread during an immune response.