Characterization and Monitoring of Antigen-Responsive T Cell Clones Using T Cell Receptor Gene Expression Analysis

High-throughput T-cell receptor repertoire sequencing constitutes a powerful tool to study T cell responses at the clonal level. However, it does not give information on the functional phenotype of the responding clones and lacks a statistical framework for quantitative evaluation. To overcome this, we combined datasets from different experiments, all starting from the same blood samples. We used a novel, sensitive, UMI-based protocol to perform repertoire analysis on experimental replicates. Applying established bioinformatic routines for transcriptomic expression analysis we explored the dynamics of antigen-induced clonal expansion after in vitro stimulation, identified antigen-responsive clones, and confirmed their activation status using the expression of activation markers upon antigen re-challenge. We demonstrate that the addition of IL-4 after antigen stimulation drives the expansion of T cell clones encoding unique receptor sequences. We show that our approach represents a scalable, high-throughput immunological tool, which can be used to identify and characterize antigen-responsive T cells at clonal level.

Human IgA binds a diverse array of commensal bacteria

In humans, several grams of IgA are secreted every day in the intestinal lumen. While only one IgA isotype exists in mice, humans secrete IgA1 and IgA2, whose respective relations with the microbiota remain elusive. We compared the binding patterns of both polyclonal IgA subclasses to commensals and glycan arrays and determined the reactivity profile of native human monoclonal IgA antibodies. While most commensals are dually targeted by IgA1 and IgA2 in the small intestine, IgA1+IgA2+ and IgA1−IgA2+ bacteria coexist in the colon lumen, where Bacteroidetes is preferentially targeted by IgA2. We also observed that galactose-α terminated glycans are almost exclusively recognized by IgA2. Although bearing signs of affinity maturation, gut-derived IgA monoclonal antibodies are cross-reactive in the sense that they bind to multiple bacterial targets. Private anticarbohydrate-binding patterns, observed at clonal level as well, could explain these apparently opposing features of IgA, being at the same time cross-reactive and selective in its interactions with the microbiota.

P5384Postnatal mouse aorta contains yolk sac-derived haemangioblasts with myeloid and endothelial plasticity and vasculogenic capacity

Background Macrophages and endothelial cells share an intimate relationship during neovessel formation in different pathophysiological conditions. Recent studies have determined that in some tissues, both cell types are derived embryonically from yolk sac (YS) progenitor cells and are maintained postnatally without contribution from circulating sources. The mechanism by which this local “self-maintenance” occurs is unknown. Purpose We previously identified that mouse arteries contain macrophage and endothelial progenitor cells in their adventitial Sca-1+CD45+ compartment. Here we investigated at a clonal level for the existence of postnatal adventitial haemangioblasts and studied their developmental origins. Methods and results Single cell digests were prepared from murine aortas to perform colony-forming unit (CFU) assays in methylcellulose. Aortic cells from C57BL/6J mice selectively generated macrophage colonies (CFU-M) which contained progenitor cells that displayed >95% positive for expression of CD45, Sca-1, c-Kit, CX3CR1 and CSF1R, but negative for Lineage markers, as well as mature monocyte/macrophage (CD11b, F4/80) and endothelial (CD144) markers. Secondary replating of CFU-M progenitors from adult aortas revealed their self-renewal capacity, with 1 in 10 cells forming new CFU-M. Lineage mapping using Flt3CrexRosamT/mG mice demonstrated that aortic CFU-M progenitors were FLT3-ve, indicating that they were not derived from definitive bone marrow haematopoiesis. CFU-M prevalence in C57BL/6J aortas was highest in neonatal mice and diminished progressively with increasing age (∼100 per 105 cells at P1, ∼15 at 12w, ∼5 at 52w, P<0.01, n>4/gp), consistent with prenatal seeding. Embryonic profiling determined that CFU-M progenitors first appeared in extra-embryonic yolk sac around E9.5 and in aorta-gonad-mesonephros at E10.5, before the emergence of definitive haematopoietic stem cells. Inducible fate-mapping then confirmed that aortic CFU-M progenitors originated from CX3CR1+ and CSF1R+ cells in E9.5 yolk sac. Both yolk sac and postnatal aortic CFU-M progenitors generated vascular-like networks when cultured in Matrigel in vitro, containing M2-like macrophages (CD11b+F4/80+CD206+) and endothelial cells (CD31+CD144+). They produced similar progeny and rescued adventitial vascular sprouting when seeded around aortic rings whose adventitia had been stripped. Finally, adoptive transfer of CFU-M progenitors into a mouse model of hindlimb ischaemia resulted in 80% augmentation in hindlimb perfusion compared to cell-free control, with de novo transformation of donor cells into macrophages, endothelial cells and perfused neovessels (n=6). Conclusion To the best of our knowledge, this is the first ever definitive proof at a clonal level for the existence of haemangioblasts in postnatal tissue. Adventitial haemangioblasts originate from extra-embryonic YS and are a source of vasculogenesis in the arterial wall, relevant to vasa vasorum formation. Acknowledgement/Funding NHMRC of Australia (GNT1086796, CDF1161506), NHFA (FLF100412, FLF102056) Royal Australasian College of Physicians

Clonal Level Lineage Commitment Pathways of Hematopoietic Stem CellsIn Vivo

ABSTRACTWhile hematopoietic stem cells (HSCs) have been extensively studied at the population level, little is known about the lineage commitment of individual clones. Here, we provide comprehensive maps ofin vivoHSC clonal development in mice under homeostasis and after depletion of the endogenous hematopoietic system. Under homeostasis, all donor-derived HSC clones regenerate blood homogeneously throughout all measured stages and lineages of hematopoiesis. In contrast, after the hematopoietic system has been depleted by irradiation or by an anti-ckit antibody, only a small fraction of donor-derived HSC clones differentiates while dominantly expanding and exhibiting lineage bias. We identified the cellular origins of clonal dominance and lineage bias, and uncovered the lineage commitment pathways that lead HSC clones to differential blood production. This study reveals surprising alterations in HSC regulation by irradiation, and identifies the key hematopoiesis stages that may be manipulated to control blood production and balance.SIGNIFICANCE STATEMENTHematopoietic stem cells (HSCs) sustain daily blood production through a complex step-wise lineage commitment process. In this work, we present the first comprehensive study of HSC lineage commitment at the clonal level and identify new HSC regulatory mechanisms that are undetectable by conventional population level studies. First, we uncover distinct HSC clonal pathways that lead to differential blood production and imbalances. Second, we reveal that HSC regulation under physiological conditions is strikingly different from that after injury. Third, we present a comprehensive map of HSC activities in vivo at the clonal level.

Identifying the Clonal Origins of Gvhd-Causing T Cells

In graft-versus-host disease (GVHD) donor αβ T cells in the allograft recognize host tissues as non-self and cause multi-organ damage through direct and indirect mechanisms. This multi-organ disease is mediated by T cells acquiring diverse phenotypes, including expression of homing receptors, adhesion molecules, cytokines and other effector molecules. How this diversity is generated at a clonal level is not understood. In the one extreme, each type of effector could be derived from a single T cell expressing a unique antigen receptor that may even target a unique antigen. Alternatively, a single T cell targeting a single antigen may be able to differentiate into a variety of effectors (Fig. 1). A second related question is whether GVHD is maintained within affected organs. That is, what is the importance of the continued recruitment of new blood-derived alloreactive effectors in maintaining GVHD. To answer these questions, we needed 1) a system in which GVHD-inducing T cells could be unambiguously tracked, as in polyclonal systems it is difficult if not impossible to know which T cells are disease causing; and 2) a way to distinguish GVHD-inducing T cells at the clonal level. To address the first point, we used a CD4 T cell receptor (TCR) transgenic (Tg) model of GVHD wherein GVHD is induced by the transfer of <1000 BALB/c RAG2-/- TS1 TCR Tg T cells, which recognize the S1 peptide derived from HA, into BALB/c RAG2-/- HA104 Tg recipients that ubiquitously express HA. GVHD is manifest by weight loss, death, and TS1-infiltrative pathology of the skin, liver, small intestine and colon. In order to determine the clonal contributions, we crossed TS1 Tg mice to combinations of CD45.1, CD45.2, Thy1.1, Thy1.2 and GFP backgrounds to create a matrix of up to 9 distinguishable TS1 cells. Using this model, we first characterized the variety of TS1 effector phenotypes being generated across tissues at day 14 and 21 post-transplant and will be presenting data detailing these TS1 phenotypes, including the expression of CCR9, α4β7 integrin, and cutaneous lymphocyte antigen (CLA). To confirm that different matrix cells perform similarly, we assessed the development of total TS1 effector cells in mice that had received an equal number of 5 distinct TS1 cell combinations. We found that within individual mice (n=5), there is comparable distribution of GVHD-causing TS1 effector cells (% of total TS1 cells) from all 5 TS1cell matrices across tissues 14-21 days post-transplant (Table 1). We next tested our ability to detect total effector cells derived from transferring low numbers of TS1 cells (5-500 cells) of a given matrix combination. We found that within individual mice (n=9), the transfer of 5 and 10 TS1 clones of a given matrix results in disparate distribution of TS1 effectors (% of total TS1 cells) across tissues 14-21 days post-transplant. As an example for one mouse at day 14, the fraction of TS1effectors derived from 10 TS1clones of one matrix was 0.8% (spleen), 1.9% (mLN), 1.4% (BM), 0.6% (colon), 4.4% (SI IEL), 1.1% (SI LP), 1.2% (skin) and 1.5% (liver). In that same mouse, the fraction of TS1effectors derived from 5 TS1clones of a different matrix was 0.7% (spleen), 0.9% (mLN), 0.4% (BM), 1.1% (colon), 1.1% (SI IEL), 0.6% (SI LP), 0.6% (skin) and 2.7% (liver). We made similar observations in mice at day 21, where the percentage of TS1 effectors derived from 5 TS1 clones in one mouse was 2.5% (spleen), 2.0% (BM), 2.8% (mLN), 0.2% (SI IEL), 2.3% (SI LP), 1.1% (skin) and 1.6% (liver). When focusing on specific TS1 phenotypes derived from 5 and 10 TS1clones, we also found unequal distributions of these cells across different tissues. For example, the distribution of CD69+CD103+ TS1 cells (% of total TS1 cells) derived from 5 TS1clones was 2.9% (colon), 1.5% (SI IEL) and 1.9% (SI LP) in one mouse at day 14. Similarly, TS1 cells expressing α4β7 integrin also varied in distribution across tissues, with one mouse having 2.2% (spleen), 0.5% (mLN) and 0% (BM) α4β7+ TS1 cells. These data suggest that tissue GVHD effectors are not simply in equilibrium with blood, consistent with some local GVHD maintenance. Furthermore, the presence of 5 TS1 clones in more than one effector type suggests that single cells can differentiate into multiple effectors. We are now directly addressing these possibilities by analyzing progeny derived from many sorted single cells. Disclosures No relevant conflicts of interest to declare.