3.2 Results
3.2.4 Obesity-associated T cell exhaustion persists after WL
Adaptive T-lymphocytes are important regulators and drivers of inflammation in AT. We explored AT T cells by scRNA-seq (Fig. 3.9a) and identified specific subsets of α/β- T Cells by expression of conventional T cell markers (Cd3ε, Cd4, Cd8b1), phenotype
Figure 3.7: Adipose tissue immune cell populations observed by CITE-seq.a Unbi- ased clustering of 33,322 single cells labeled broadly by cell type category and colored by high-resolution cell type identities via Uniform Manifold Approximation and Projection (UMAP). Populations include lipid associated macrophages (LAMs), conventional den- dritic cells (cDCs), plasmacytoid DCs (pDCs), monocyte-derived DCs (moDCs), T helper (Th) cells, T regulatory cells (Tregs), γ/δ (gd) T cells, natural killer (NK) cells, and type 2 innate-like lymphoid cells (ILC2s). Selected markers of specific cell subsets based on b gene expression andc surface protein. d Phylogenetic tree of high-resolution cell type identities. Figure 2a was generated using Biorender.com
markers (Foxp3,Cxcr3, Ccr7, Sell) [259, 260], and markers of cell cycling (Stmn1, Pclaf) [148, 261] (Fig. 3.9b). A population of γ/δ-T cells was identified by expression of the common T cell δ chain (Trdc) and both NK and NKT were identified by expression of
Figure 3.8: CITE-seq recapitulates obesity-associated immune cell changes in adi- pose tissue. a Differential abundance by log fold change (FC) of metacells (index cells representing a neighborhood (Nhood) of cells connected by proximity in Uniform Man- ifold Approximation and Projection; UMAP) comparing cells from obese mice to lean mice. b Differential abundance of annotated cell types comparing cells from obese mice to lean mice.cPermutation testing of high-resolution clusters to calculate the proportional difference comparing cells from obese mice to lean mice by false discovery rate (FDR).
Populations include lipid associated macrophages (LAMs), conventional dendritic cells (cDCs), plasmacytoid DCs (pDCs), monocyte-derived DCs (moDCs), T helper (Th) cells, T regulatory cells (Tregs), γ/δ (gd) T cells, natural killer (NK) cells, and type 2 innate- like lymphoid cells (ILCs). d, eCounts per hundred cells sequenced for lean-associated immune cell subsets and obesity-associated immune cell subsets in lean and obese mice (mean±SEM; n=4 mice; two-tailed t-test with indicated p values).
killer lectin receptorsKlrb1bandKlrb1cwith and without expression ofCd3ε, respectively [262]. Th1 CD4+ and all three CD8+ T cells subsets were not elevated in WC but CD8+ effector memory T cells were increased in AT from WL mice (Fig. 3.9c). Tregs expressing Foxp3trended towards being reduced in proportion by obesity and remain low following WL and WC. Using differential expression, we observed that obesity not only affected Treg number but also reduced the expression ofIl1rl1, which codes for the IL-33 receptor, ST2 (Fig. 3.9d).
Because CD8+ T cells have the capacity to clonally expand in response to antigen, we were interested in exploring if cycling T cells (marked by expression ofPclaf, Stmn1, and Mki67) [148] are precursors for AT memory T cell populations. Therefore, we uti- lized RNA velocity, which estimates cell state progression by comparing the frequency of spliced and unspliced mRNA sequences between cells. RNA velocity indicates cycling CD8+T cells are likely upstream of other effector and memory populations in AT, but also that bidirectional transition between effector memory and circulating memory can occur (Fig. 3.10a). Differential expression across diet groups also identified that cycling and ef- fector memory (TEM) CD8+T cells express the activation/exhaustion signature genePdcd1 (coding for PD-1). To further explore whether AT T cells contain an exhausted signature following obesity, we generated an exhaustion module containing multiple established gene expression features of T cell exhaustion: Pdcd1, Tox, Entpd1, Tigit, and Lag3[263, 264].
We observed that T cells from AT of obesity, WL, and WC were all enriched for our ex- haustion module and that CD8+ TEM were most associated with an exhausted phenotype (Fig. 3.10b). This was further confirmed by a T cell exhaustion module based on pro- tein expression of PD-1 (CD279) and TIGIT from our CITE-sequencing antibodies (Fig.
3.10c). T cell exhaustion is frequently studied in models of viral infection and in the tumor microenvironment, so we utilized ProjecTILs V1.0.0, a published scRNA-seq reference at- las [265], to further confirm our exhaustion profile. We observed that cells captured in our experiments projected accurately onto an LCMV chronic infection atlas (Fig. 3.10d). Con-
cordant with our exhaustion module, more CD8+T cells aligned with the LCMV exhausted precursor cells in obese, WL, and WC groups than in the lean control group.
Figure 3.9: Obesity-induced changes in adipose tissue T cells are not reversed by weight loss. a Uniform Manifold Approximation and Projection (UMAP) of T cell sub- clusters by diet group for lean (grey), obese (blue), weight loss (WL; green), and weight cycled (WC; orange) mice. b Expression of markers enriched in T cell subclusters. c Counts per hundred cells sequenced forα/β T cell subclusters (mean±SEM; n=4 mice).
Box indicates interquartile range (25th-75th percentile) with 50th percentile indicated by solid line and mean indicated by large circle. Range of whiskers indicates largest and smallest values within 1.5 times the interquartile range and values outside of the range are indicated by small circles. d Expression of the Treg markersFoxp3and Il1rl1 within the Treg subcluster across diet groups. Pairwise two-tailed t-tests with Bonferroni correction for multiple comparisons were used to compare groups for cell counts; significant p values shown.
Figure 3.10: Adipose tissue T cells are retained and express markers of exhaustion in mice that have gained, lost, and regained weight. aEmbedding of RNA velocity dis- played on the uniform maniphold approximation and projection (UMAP) for CD8+T cells.
b CD8+ T cells colored by an exhaustion module containing the mRNA features Pdcd1, Tox, Tigit, Lag3, and Entpd1. cCD8+ T cells colored by an exhaustion module contain- ing the CITE-seq features PD-1 (CD279) and TIGIT. dCD8+ T cells plotted onto a viral infection CD8+ T cell reference atlas using ProjecTILs. ProjectTILs populations CD8+ include terminally-exhausted (Tex), CD8+ precursor-exhausted (Tpex), short-lived effector cells (SLEC), effector interim (Eff Interm), and memory precursors (Memory Prec).
3.2.5 Monocytes are abundant in AT, but do not have an altered transcriptional pro-