HLA-E restricted responses in Tuberculosis

Transcription

1 HLA-E restricted responses in Tuberculosis Simone A. Joosten Dept. of Infectious Diseases LEIDEN UNIVERSITY MEDICAL CENTER

2 Unconventional immunity Conventional immunity: T cells reactive to complexes of peptide and major histocompatibility complex (MHC) proteins Unconventional immunity: other types of T cells that do not fit this paradigm These include CD1-restricted T cells, MR1-restricted mucosal associated invariant T cells (MAIT cells), MHC class Ib reactive T cells, and γδ T cells. these T cells are considered 'unconventional', in part because they can recognize lipids, small-molecule metabolites and specially modified peptides. generally show simplified patterns of T cell antigen receptor (TCR) expression, rapid effector responses and 'public' antigen specificities. DI Godfrey, AP Uldrich, J McCluskey, J Rossjohn & DB Moody Nature Immunology 16, (2015) doi: /ni.3298

3 Unconventional immunity αβ T-cells Insert 3 > Header & footer DI Godfrey, AP Uldrich, J McCluskey, J Rossjohn & DB Moody Nature Immunology 16, (2015) doi: /ni Feb-16

4 Characteristics of immune responses Precursor frequency Time to effector function in vivo MHC-restricted T cells Low (~1 10 per million) Days to weeks Unconventional T cells Higher (~ per million) Hours to days Memory response Yes (highly durable) Controversial Antigen-presenting molecule polymorphism Very high Inter-individual TCR diversity Very high Population response to antigen Private (individuals respond differently) Low (functionally monomorphic) Varies for each subset (Species-wide TCR patterns known) Public (most individuals in a species typically respond similarly) DI Godfrey, AP Uldrich, J McCluskey, J Rossjohn & DB Moody Nature Immunology 16, (2015) doi: /ni.3298

5 HLA-E 3 alleles, 2 functional proteins, single amino acid difference: HLA-E*0101: position 107= arginine > E R HLA-E*0103: position 107= glycine > E G E R and E G frequencies in population are about equal No significant differences in crystal structure E G has slightly increased cell surface expression E G has stronger peptide affinity E G has increased thermal stability Functional significance? 107 Strong et al, The Journal of Biological Chemistry, 2003, 278, 5082 Grimsley et al, Human Immunology, 1997, 52, 33 Hoare, Nat Immunol, 2006, 7(3), 256

6 HLA-E in tuberculosis Only 2 variants described in humans: single peptides can be presented by most (all) individuals potentially interesting vaccine target HLA-E is not down-regulated by HIV-nef, potentially interesting to use HLA-E peptides for post-exposure vaccination against Mtb in HIV + setting Cohen et al, Immunity, 1999, 10, 661 HLA-E is enriched within Mtb phagosome compared to HLA-A2, HLA-E may preferentially present phagosomal antigens Grotzke, Lewinsohn et al Plos Pathogens, 2009, 5(4): e

7 CD3 CD8 TCRαβ CD25 LAG3 CD39 CTLA4 CD45RA CD45RO CCR7 CD27 CD28 CD56 CD57 CD62L CD94 Perforin granzyme A granzyme B granulysin CD107a TRAIL TCR Vβ16 TCR Vβ22 IFNγ TNFα IL-2 IL-4 IL-5 IL-10 IL-13 IL-17 TGF-β T-bet GATA3 RORC FOXP3 EOMES specific lysis pathogen killing T-cell suppression TM binding HLA-E restricted T-cells in literature antigen origin T-cell phenotype cytokine profile transcription factors T-cell function reference VMAPRTLVL HLA-A Pietra, Eur J Imm, 2001 VTAPRTVLL HLA-B Romagnani, PNAS, 2002 VMAPRTLVL HLA-A VMAPRTLIL HLA-Cw3 & UL VMAPRALLL HLA-Cw VMAPRTLFL HLA-G SQAPLPCVL Epstein Barr Virus VMAPRTLIL, VMHLA-Cw*04011; A* Garcia, Eur J Imm, 2002 SQAPLPCVL Epstein Barr Virus QMRPVSRVL HSP60p Jiang, J Clin Invest, 2010 SLELGDSAL TCR Vβ1 + + Li, J Immunol, 2001 LLLGPGSGL TCR Vβ2 + + SQAPLPCVL Epstein Barr Virus + + Jørgensen, Plos One, 2012 VMAPRTLLL Cytomegalovirus lo hi - - +/ ? + Allard, Plos One, 2012 VMAPRTLIL, VMCytomegalovirus Hoare, Nat Imm, 2006 VMAPRTLIL Cytomegalovirus Pietra, PNAS, 2003 VMAPRTLIL Cytomegalovirus Romagnani, Hum Imm, 2004 VMAPRTLIL Cytomegalovirus Mazzarino, Eur J Imm, 2005? Mycobacterium tuberculosis + + Lewinsohn, J Exp Med, 1998? Mycobacterium tuberculosis + + Heinzel, J Exp Med, peptides Mycobacterium tuberculosis / /- * Joosten, Plos Path, 2010 RMPPLGHEL Mycobacterium tuberculosis / VLRPGGHFL Mycobacterium tuberculosis / /- + +/- +/- + +/ / VMTTVLATL Mycobacterium tuberculosis /- - +/ / Caccamo, Eur J Imm, 2015 RLPAKAPLL Mycobacterium tuberculosis /- - +/ / VMATRRNVL Mycobacterium tuberculosis /- - +/ / RMPPLGHEL Mycobacterium tuberculosis /- - +/ / VLRPGGHFL Mycobacterium tuberculosis /- - +/ / /- + +/- +/- + +/ / van Meijgaarden, Plos Path, 2015 Joosten, in progress

8 HLA-E is potentially interesting novel target for vaccine development, however, phenotype, quality and functional properties of Mtb specific HLA-E restricted T-cells needs to be determined first

9 Research strategy Identification of Mtb sequences that can be presented by HLA-E Binding to HLA-E Recognition by Mtb infected donors 69 epitopes Generated T-cell lines specific for Mtb epitopes presented in HLA-E T-cell phenotype T-cell function 6 epitopes Generated T-cell clones specific for Mtb epitopes presented in HLA-E Assess functional diversity Investigate TCR composition 2 epitopes Generation of HLA-E TM Enumerate frequency and phenotype in clinical cohorts 6 epitopes

10 accession HLA-E discrimination diversity binding # sequence derived from number* motif score a score b ranking c (in um) 1 SMADRAENL Rv1286 Q >50 2 SMAGRAGQL Rv3282 P >50 3 IMANRAQVL Rv3532 Q6MWW ,4 4 KMNAKAATL Rv2911 Q7D6F QMKFYAVAL Rv2119 O ,5 6 PMKRTALAL Rv0959 P0A5D GMNVTAPAL Rv1617 O VMADRTRHL Rv0418 P WMCDRAVDL Rv2954c Q >50 10 LMVVRALFL Rv3101c P nt 11 EMVLRADQL Rv0191 O >50 12 DMLGRAGGL Rv3015c O >50 13 MMKYLAFGL Rv0206c O ,4 14 RMAATAQVL Rv2932 Q FLAADALVL Rv1047 P >50 16 VEAFRTRPL Rv1047 P >50 17 LMGAEADAL Rv1047 P >50 18 GLDSRAYRL Rv0146 O >50 19 GLDARAYRL Rv0145 P >50 20 PMAPLAPLL Rv3871 O EMKTDAATL Rv3874 P0A >50 22 PMQQLTQPL Rv3873 Q79F >50 23 PMADIAAAL Rv1253 Q >50 24 ALPPRAFEL Rv3428c Q ,27 25 AMVNTTTRL Rv2074 Q LMGALAVVL Rv2075c Q VSNLRTGKL Rv1967 O >50 28 TSADRAVVL Rv1973 O LMHYRGELL Rv1965 O NMMARGMDL Rv0221 P >50 31 LPAERAHEL Rv0222 P >50 32 VMMSEIAGL Rv1813c P TMITFRLRL Rv1733c P ,4 34 VMTTVLATL Rv1734c P ,8 35 GMGMVGTAL Rv1737c P >50 36 AMAGSIDLL Rv2006 Q EMLTSRGLL Rv1997 P >50 38 QMRACARRL Rv2004c P0A5F ,5 39 AMEYFRQVL Rv2006 Q SMFAAVQAL Rv2030c O HMAQTLGSL Rv2030c O >50 42 SMFAAVQAL Rv2030c O ,5 43 EMGRAPLDL Rv2627c O >50 44 RLPAKAPLL Rv1484 P0A5Y6 1,347 0,03 45 VMAPLGPIL Rv1405c P64841 NA 0,8 46 QLAPGLQLI Rv1563c Q ,248 >50 47 RLAPGGTTI Rv0068 O ,238 >50 48 RLANLLPLI Rv3189 O , ILPSDAPVL Rv3823c O07800 NA 0,2 50 VLLPGLPYL Rv3847 P , REPRRGPRL Rv0142 P ,169 1 >50 52 RLRPELAGL Rv3772 P , RELPGRVLL Rv3094c O ,759 4 >50 54 FLLPRGLAI Rv0056 P ,867 5 >50 55 VMATRRNVL Rv1518 Q , RLGRLLNRI Rv3737 O ,825 7 >50 57 IEPRGAQAL Rv0171 O ,985 8 >50 58 DLPSRLGKI Rv3087 O ,802 9 >50 59 RPANLAFFL Rv1188 O50444 NA 1,3 60 FMTRLGPLL Rv0035 Q7DAJ8 0, RLASCRDAL Rv2761c O , >50 62 RMPPLGHEL Rv2997 O , VMAPDAVRI Rv0324 O , RSCPRLTIL Rv0894 P , >50 65 RLARRARNI Rv1513 P , >50 66 RLGLCALAL Rv3370c O50399 NA VLPACLGIL Rv0206c O , VLRPGGHFL Rv1523 Q , ,8 69 WLPPLLTNL Rv0249c O , Mtb derived peptides binding HLA-E In Silico peptide prediction Binding assays: HPLC based competition assay to measure binding to recombinant human HLA-E (HLA-E G ). About half of peptides bind in this assay * UniProtKB/ Swissprot accession number A Identified through "motif" score, i.i. weighting positions differently. B Top five from a discriminant analysis: binder vs non-binder C Top Twenty-odd from diversity analysis of peptide sequences with high discriminant scores NA: not available Joosten SA, et al. PLoS Pathog. 2010; 6(2):e

11 # peptides recognized peptide donor Peptide induced proliferation 30 p= p= PPD responder non-responder UCB proliferation <10% proliferation >10% T cell lines generated Peptides are predominantly recognized by sensitized donors Joosten SA, et al. PLoS Pathog. 2010; 6(2):e

12 peptide BCG vaccination resulted in recognition of HLA-E peptides BCG vaccinated infants (Cape Town), stimulation with 10 selected peptides PPD CD donor proliferation < 5% proliferation 5-10% proliferation >10% BCG vaccination does induce T cells that recognize peptides with an HLA-E binding motif Joosten SA, et al. PLoS Pathog. 2010; 6(2):e

13 % specific lysis % specific lysis HLA-E and peptide are needed for cytotoxicity T cell lines generated by stimulation with peptide, CD8 + sort (MACS) and repeat stimulation with peptide pulsed feeders Cytox assay: peptide or control loaded K562 T cells, chromium labelled, co-culture with T cell lines for 5 hours, lysis measured by chromium release B Donor 4, T cell line against p55 75 K562/E + 55 K562/E + 68 K562/E/B Donor 2, T cell line against p62 K562/E + 62 K562/E + 68 K (no HLA E) :1 25:1 50:1 100:1 E:T 0 6:1 12:1 25:1 50:1 E:T Only target cell lysis when correct peptide is presented in the context of HLA-E Joosten SA, et al. PLoS Pathog. 2010; 6(2):e

14 % specific lysis BCG infected monocytes are lysed by HLA-E specific T cells Cytox assay: monocytes plated at day -7 (complete HLA class I mismatch to all donors), infection with live BCG at day -1 (MOI 5), labelling with 51 Cr, co-culture with T cell lines for 5 hours, lysis measured by chromium release D2 #54 D2 #62 D4 #55 D6 #68 control - BCG effector cells Joosten SA, et al. PLoS Pathog. 2010; 6(2):e

15 - 0.6x10e4 1.25x10e4 2.5x10e4 5x10e4-0.6x10e4 1.25x10e4 2.5x10e4 5x10e4-0.6x10e4 1.25x10e4 2.5x10e4 5x10e4 cpm T cell lines are suppressive CD8 + T cell lines, co-culture with Th1 clone (Rp15): measure proliferation D2 #62 D4 #55 D6 #68 LAP inhibits suppression: membrane bound TGF involved Joosten SA, et al. PLoS Pathog. 2010; 6(2):e

16 T-cells lines show dual activity, Lyse infected target cells and suppress T-cell proliferation > are these the same cells? Generate T-cell clones by limited dilution cloning from 2 different donors that recognize different Mtb peptides presented by HLA-E CD8 + T-cells isolated using MACS from PBMC Stimulation with peptide and allogeneic feeders in presence of IL7, IL15 and IL2 Limiting dilution to 0.3 c/well Selection of proliferating T-cell clones Characterization of T-cell clones including specificity testing > 10 clones for peptide 62 and 6 clones for peptide 68

17 Peptide specific HLA-E restricted CD8+ T-cell clones are activated through TCR ligation by specific peptide/hla-e MFI CD137 MFI zap70 ΔMFI zap70 T-cell clones were stimulated for 16 hours with K562 cells either expressing or lacking HLA-E, which had been pre-loaded with the specific or control peptide. T-cell clones were stimulated for 5 minutes with peptide pulsed HLA-E expressing Meljuso cells and stained for 2 different phosphorylation sites of ZAP70 (Y292 in black, Y319 in grey) as indicator of TCR activation. A. D2-1B4 D6-1G6 C. D2-2B3 D6-1F11 - p62 p68 - p62 p68 - p62 p68 - p62 p68 K562/- K562/E K562/- K562/E B. D py292 py p62 p68 p62 p Van Meijgaarden KE, et al. PLoS Pathog. 2015, 11(3): e

18 HLA-E restricted T-cell clones possess either suppressive or cytolytic activity A. D2-4G5 D2-3H11 D2-2B3 B. D2-1C9 D2-4A1 D6-1F11 C. nt nt nt nt T-cell clones added in different ratios to an unrelated reporter Th1 cell clone (Rp15 1-1; Mtb hsp65 p3-13 specific, HLA-DR3 restricted) T-cell clones were titrated onto 51Cr labelled adherent HLA-A2 negative monocytes that were infected with live BCG, 5 hours lysis(%) Suppression(%) Van Meijgaarden KE, et al. PLoS Pathog. 2015, 11(3): e

19 % perforin HLA-E restricted T-cell clones can inhibit intracellular growth of Mtb CFU % perforin + granulysin HLA-A2 negative macrophages were infected with live H37Rv and HLA-E restricted CD8+ T cell clones were added (5:1 ratio) for 24 hours. Subsequently cells were lysed and plated to determine the number of colony forming units 0 no T cells D6-2B1 D2-4A1 D6-2B % perforin + granzymeb R 2 = R 2 = R 2 = % specific Mtb growth inhibition % specific Mtb growth inhibition % specific Mtb growth inhibition Van Meijgaarden KE, et al. PLoS Pathog. 2015, 11(3): e

20 HLA-E restricted T-cell clones can inhibit intracellular growth of Mtb D2-1 B9 D2-1 D1 D2-4 A 1 D2-3 H 1 1 D2-2 F 6 D2-2 B3 D2-2 A 9 D2-4 G 5 D2-1 C9 D2-1 B4 D6-2 B4 D6-2 B1 D6-1 H3 D6-1 G 6 D6-1 F 1 1 D6-1 A 3 nt nt nt nt suppression (%) lysis(%) specificmtbgrowth inhibition (%) nt nt nt nt nt Clones that can lyse target cells presenting peptide in HLA-E can generally also inhibit intracellular Mtb survival Van Meijgaarden KE, et al. PLoS Pathog. 2015, 11(3): e

21 Mtb specific HLA-E restricted T-cell clones produce Th2 cytokines A. MLPA Luminex (pg/ml) ICS (%) (norm. peak area) IFNγ/IL-4/IL-5 TNF/IL-2 IL-10/IL < > >20 C. peptide Th1 Th2 Th1 Th2 Th1 Th2 D2-1B4 D6-1F11 D2-1B9 B. IL-13 IL-5 IL BCG Th1 Th2 IFNγ Van Meijgaarden KE, et al. PLoS Pathog. 2015, 11(3): e

22 IL-6 (pg/ml) MFI CD86 MFI CD80 HLA-E restricted Mtb specific T-cell clones utilize IL-4 to provide B-cell help A. B cells + D2-1B4 + D6-2F B. CD80 CD86 CD CD C. CD25 D. E. Van Meijgaarden KE, et al. PLoS Pathog. 2015, 11(3): e

23 B-cells in TB disease are decreased in numbers and respond poorly to polyclonal activation % B cells Total B cells plasma cells im p=0.001 * Controls LTBI TB TB treated Controls LTBI TB TB treated Controls LTBI and TB patients have an increased proportion of atypical B-cells Also cytokine and immunoglobulin production are impaired in LTBI and TB patients Joosten SA, et al. submitted

24 Patients with active pulmonary TB have highest frequency of HLA-E TM+ cells 11 LTBI subjects (black columns) 24 TB patients with active (grey columns) 24 cured (white columns) TB disease. Caccamo N, et al. Eur J Immunol. 2015, 45:

25 TB patients recognize HLA-E TM strongest before therapy, in contrast HLA-A2 TM are increasingly recognized during treatment Caccamo N, et al. Eur J Immunol. 2015, 45:

26 TB patients recognize HLA-E TM containing Mtb peptides & produce Th2 cytokines in response to peptide stimulation % cytokine + CD8 + T cells % cytokine + CD8 + T cells TM p62 TM p68 A B 1.5 C IL-4 IL-13 IFN-γ % CD8 + TM + T cells p62 p68 p p68 D 0.6 p=0.005 p= p= hour peptide stimulation followed by ICS TNF IFN-γ IL-4 IL-13 T M TNF IFN-γ IL-4 IL-13 T M Van Meijgaarden KE, et al. PLoS Pathog. 2015, 11(3): e

27 HLA-E TM binding cells can produce Th2 cytokines Teresa Prezzemolo, work in progress

28 Predictive Biomarkers Cohort of intravenous drug-user with longitudinal follow up PBMCs ~ 6 months 1 year prior to diagnosis (n=14) dcrt-mlpa for gene expression analysis Patients that have signature of IL13 and AIRE abundantly express type I interferon related genes BM can predict TB disease development months before diagnosis Role for Th2 immunity?

29 summary Mtb peptides can be bound and presented by human HLA-E BCG vaccination may already prime HLA-E responses Mtb peptides presented in HLA-E are recognized by human CD8 + T-cells through a TCR dependent mechanism CD8 + T-cells that recognize HLA-E + peptide can have multiple functions, they can suppress T-cell proliferation, exert cytotoxicity towards peptide loaded cells and/or inhibit the intracellular growth of Mtb Peptide activation of CD8 + T-cells results in a type 2 cytokine profile (IL-4, IL-5, IL- 13) HLA-E restricted Mtb specific CD8 + T-cells can provide B-cell help through IL-4 Pulmonary TB patients recognize HLA-E Tm loaded with Mtb peptides and produce Th2 cytokines in response to peptide stimulation HLA-E Tm recognition is strongest before initiation of treatment and decreases over treatment, an opposite pattern as observed for HLA-A2 Tm

30 discussion Mtb specific HLA-E restricted T-cells are unconventional, but have some properties similar to conventional T-cells Inhibition of intracellular Mtb outgrowth is interesting property, mechanism of Mtb control is yet unresolved (work in progress) TCRs seem highly diverse, no conservation observed (work in progress)

31 Leiden University Medical Center, Leiden, The Netherlands Krista E van Meijgaarden Simone A Joosten Tom HM Ottenhoff University of Palermo, Palermo, Italy Teresa Prezzemolo Nadia Caccamo Francesco Dieli