Abstract
During unresolved infections, some viruses escape immunological control and establish a persistant reservoir in certain cell types, such as human immunodeficiency virus (HIV), which persists in follicular helper T cells (TFH cells), and Epstein-Barr virus (EBV), which persists in B cells. Here we identified a specialized group of cytotoxic T cells (TC cells) that expressed the chemokine receptor CXCR5, selectively entered B cell follicles and eradicated infected TFH cells and B cells. The differentiation of these cells, which we have called 'follicular cytotoxic T cells' (TFC cells), required the transcription factors Bcl6, E2A and TCF-1 but was inhibited by the transcriptional regulators Blimp1, Id2 and Id3. Blimp1 and E2A directly regulated Cxcr5 expression and, together with Bcl6 and TCF-1, formed a transcriptional circuit that guided TFC cell development. The identification of TFC cells has far-reaching implications for the development of strategies to control infections that target B cells and TFH cells and to treat B cell–derived malignancies.
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Acknowledgements
We acknowledge the facilities, scientific and technical assistance of Flowcore, Monash Micro Imaging, and Monash Bioinformatics Platform (S. Archer and K. Tsyganov) at Monash University, and University of Birmingham's Human Biomaterials Resource Centre (supported through Birmingham Science City - Experimental Medicine Network of Excellence project). We thank L. Ye (Third Military Medical University) for Thy1.1 reporter constructs; C. Vinuesa (Australian National University) and S. Nutt (Walter and Eliza Hall Institute of Medical Research) for mice; R. Gloury and L. Mackiewicz for technical support; C. Dong for the list of Bcl6-bound genes; and H. Xue for the list of TCF-1-bound genes. Supported by the National Health and Medical Research Council of Australia (Y.A.L. and S.R.L.; GNT1085509 to D.Y.; and GNT1085151 to A.K.), Monash University (D.Y.), the amfAR Research Consortium on HIV Eradication (109327-59-RGRL, D.Y., S.R.L. and A.L.L.), The Creative and Novel Ideas in HIV Research Program of The International AIDS Society (D.Y.), Australian Centre for HIV and Hepatitis Virology Research (2015-69 to D.Y.), The Priority Research Program of Shandong Academy of Sciences (D.Y.), Shandong Province Taishan Scholar Program (D.Y.), the Sylvia and Charles Viertel Foundation (A.K.), the Delaney AIDS Research Enterprise to find a cure, Martin Delaney Collaboratories, the National Institute for Allergy and Infectious Diseases of the US National Institutes of Health (U19 AI096109 to S.R.L., T.W.S. and J.D.E.), Bloodwise, UK (15021 to H.M.L. and B.J.M.), the National Cancer Institute of the US National Institutes of Health (HHSN261200800001E), and the Victorian State Government Operational Infrastructure Support and Australian Government NHMRC Independent Research Institute Infrastructure Support scheme. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.
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Y.A.L. planned and performed most experiments with the support from Y.C. and H.S.O.; Y.C. and A.A. performed experiments related to retrovirus-mediated gene overexpression; D.W. and J.X. performed bioinformatics analysis; K.M. performed experiments to generate chimeric mice; C.D, J.G.C., G.J.B., T.W.S. and J.D.E. performed immunofluorescence staining and quantification of HIV samples; M.M., M.B. and A.K. performed and/or analyzed the ChIP-seq experiments; B.J.M., U.P. and H.M.L. performed analysis of EBV samples; Y.W., Z.H., L.S., P.W., and Y.T. performed flow cytometry on HIV samples; D.Z. and A.K. performed experiments on lymphoma; K.A.F., I.C. and S.R.M. performed experiments on influenza infection; S.P., C.C.A., J.G.T. and M.P. provided support for experiments on LCMV and relevant models; H.K.L. provided support for experiment to quantify viral RNA; A.L.D. provided Bcl6fl/fl mice; A.L.L. and S.R.L. contributed to scientific planning; G.T.B. provided support on the MuHV-4 model; D.Y. and A.K. oversaw and designed the study; Y.A.L. and D.Y. analyzed data; and Y.A.L., A.K. and D.Y. wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 CXCR5-expressing TFC cells are generated in immune responses and are localized in B cell follicles.
(a) Staining of anti-CD3 (red), anti-CD8 (green) and anti-B220 (blue) of splenic sections from uninfected mice or mice at day 8 post-infection (p.i.) of LCMV. CD8+ T cells (yellow) indicated by higher magnification of B cell follicles. Data are representative of 2 independent experiments.
(b) Congenically marked CD45.1+ P14 cells were adoptively transferred into CD45.2+ wildtype mice, which were then infected with LCMV (DOCILE). Flow cytometric analysis of CCR7 expression on P14 cells (red box) or endogenous activated CD44+ TC cells (blue box) at day 8 p.i.
(c) CD45.1 CXCR5+/+ or CXCR5−/− P14 cells were adoptively transferred into congenically marked (CD45.2) wildtype mice, which were then infected with LCMV (DOCILE). Flow cytometric analysis of CXCR5 expression on P14 cells at day 8 p.i..Numbers adjacent to each outlined area indicate percentage of population. **P < 0.01 (Mann Whitney's U-test)
(d, e) OT-I cells (CD45.2) were adoptively transferred into congenically marked (CD45.1) wildtype mice, which were then intravenously infected with OVA-expressing influenza virus (d) or immunized subcutaneously at hock with OVA in CFA. Flow cytometric analysis of CXCR5 expression on OT-I cells in in spleens at day 10 p.i (d) or popliteal lymph nodes at day 8 post immunization (e). Numbers adjacent to each outlined area indicate percentage of population.
Supplementary Figure 2 CD4+ T cells are significantly infected by LCMV (DOCILE).
(a) Intracellular staining of LCMV nucleoprotein (NP) in splenic CD4+ T cells from uninfected, day 8 post OVA-CFA immunization, day 8 p.i. with LCMV (WE) or LCMV (Docile) mice. Each symbol represents one mouse, bars represent means. **P < 0.01 (Mann Whitney's U-test)
(b) Mice were infected with LCMV (DOCILE). At day 15 p.i., TH subsets in spleens (gated as in Fig. 4b) were purified and pooled. The LCMV viral RNA was measured by qPCR. Data are representative of two independent experiments. Each symbol represents one experimental replicate, bars represent means. **P < 0.01 (Mann Whitney's U-test)
Supplementary Figure 3 Control of TFH cell infection by TFC cells.
(a) Schematic of the experiment.
(b) Quantification of splenic TFH differentiation.
(c) Quantification of P14 cells in spleens.
(d, e) Viral loads in sera (d) and spleens (e), as measured by plague forming assays. Data are representative of two independent experiments. Each symbol represents one mouse, bars represent means. NS = not significant, P > 0.05 (Mann Whitney's U-test).
Supplementary Figure 4 Population expansion and activation of transferred CD8+ T cells in mice infected with MuHV-4.
(a) Representative FACS plots showing the total CD8 T cells with transferred cells marked by congenic marker CD45.1.
(b) Quantification of transferred cells (left) or activated transferred cells (right) in total CD8+ T cells. Data are representative of two independent experiments. Each symbol represents one mouse, bars represent means. NS, not significant, P > 0.05 (Mann Whitney's U-test).
Supplementary Figure 5 The top 50 genes up- and downregulated in TFC cells and non-TFC cells.
Heatmaps for normalized log2 data of the top 50 lists of upregulated (a) or downregulated (b) genes.
Supplementary Figure 6 The phenotype and function of TFC cells.
CD45.1+ P14 cells were adoptively transferred into congenically marked (CD45.2) wildtype mice, which were then infected with LCMV (DOCILE). The expression of indicated proteins in naïve (grey), CXCR5+ TFC (red), CXCR5- non-TFC (blue), B220+ B (black) cells at day 8 p.i.
(a) The expression of ICOSL on TFC cells.
(b) IL-21-GFP reporter mice were infected with LCMV Docile and the expression of IL-21-GFP in indicated populations were analysed at day 15 p.i..
(c) Ex vivo killing of LCMV GP33-41 peptide-pulsed non-TFH or TFH SMARTA cells, or B cells by CXCR5+ TFC or CXCR5− non-TFC P14 cells purified at day 8 p.i.
Each symbol represents one mouse (A, B and D) or one sample (C), bars represent means. GMFI: geometric mean fluorescence intensity. **P < 0.01 (Mann-Whitney's U-test). Data are representative of two independent experiments.
Supplementary Figure 7 Bcl6 regulates the phenotype and function of TC cells.
(a) Gene set enrichment test of genes differentially expressed in TFC cells as compared to non-TFC cells in differentially expressed genes in TFH cells as compared to non-TFH cells. Red and blue bars designate up and down-regulated genes in TFC cells, respectively. Correlation of up (top) and down (bottom)-regulated genes were shown by rotation gene set test P values and the percentages.
(b-e) P14 cells were transduced with a GFP retroviral empty vector (RV) or the vector expressing Bcl6.
(b, c) Transduced cells were transferred into congenically marked recipient mice which were subsequently infected with LCMV (DOCILE). The expression of indicated proteins on transduced cells was measured by flow cytometry at day 8 p.i.,
(d) Transduced cells were co-cultured with LCMV gp33–41 peptide-pulsed splenocytes and the killing of target cells were measured by flow cytometry.
(e) Transduced cells were purified and the indicated genes were measured by quantitative PCR.
*P < 0.05, **P < 0.01 (Mann Whitney's U-test). Data are representative of two independent experiments.
Supplementary Figure 8 Transcriptional regulation of TFC cell differentiation.
(a-c) Enrichment analysis of the differentially expressed genes in TFC cells. Gene set enrichment test of genes differentially expressed in TFC cells as compared to non-TFC cells in differentially expressed genes in Blimp1-deficient TC cells (a), Id3-deficient TC cells (b) and Id2-deficient TC cells (c) as compared to counterpart wildtype TC cells. Red and blue bars designate up and down-regulated genes in TFC cells, respectively. Correlation of up (top) and down (bottom)-regulated genes were shown by rotation gene set test P values and the percentages.
(d, f-h) Correlation between transcription factor-bound genes and TFC signature genes. Gene set enrichment test of genes bound by Bcl6 (d), E2A (f), Blimp1 (g) or TCF-1 (h) among transcripts differentially expressed in TFC cells versus non-TFC cells. Red and blue bars designate upregulated and downregulated genes in TFC cells, respectively. Significant correlation of binding sites (barcode plots) with differentially expressed genes is shown by P value. Percentages show proportion of genes bound by each transcription factor that were also differentially expressed in TFC vs non-TFC.
(e) Binding of Blimp1 and E2A at the indicated genes. ChIP-seq analysis of Blimp1 and E2A binding was performed with CD8+ effector T cells and total thymocytes, respectively. Binding regions, which were identified by MACS peak calling, are indicated by black rectangles below the horizontal axis. RPM; reads per million.
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Leong, Y., Chen, Y., Ong, H. et al. CXCR5+ follicular cytotoxic T cells control viral infection in B cell follicles. Nat Immunol 17, 1187–1196 (2016). https://doi.org/10.1038/ni.3543
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DOI: https://doi.org/10.1038/ni.3543
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