HLA-E restricted responses in Tuberculosis Simone A. Joosten Dept. of Infectious Diseases LEIDEN UNIVERSITY MEDICAL CENTER
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, 1114 1123 (2015) doi:10.1038/ni.3298
Unconventional immunity αβ T-cells Insert 3 > Header & footer DI Godfrey, AP Uldrich, J McCluskey, J Rossjohn & DB Moody Nature Immunology 16, 1114 1123 (2015) doi:10.1038/ni.3298 3-Feb-16
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 (~10 2 10 5 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, 1114 1123 (2015) doi:10.1038/ni.3298
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
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): e1000374
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-A2 + + + Pietra, Eur J Imm, 2001 VTAPRTVLL HLA-B15 + + + Romagnani, PNAS, 2002 VMAPRTLVL HLA-A02 + + + VMAPRTLIL HLA-Cw3 & UL40 + + + VMAPRALLL HLA-Cw7 + + + VMAPRTLFL HLA-G1 + + + SQAPLPCVL Epstein Barr Virus + + + VMAPRTLIL, VMHLA-Cw*04011; A*0101 + + + + + + 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, 2002 69 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
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
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
accession HLA-E discrimination diversity binding # sequence derived from number* motif score a score b ranking c (in um) 1 SMADRAENL Rv1286 Q10600 268 >50 2 SMAGRAGQL Rv3282 P96887 268 >50 3 IMANRAQVL Rv3532 Q6MWW4 268 0,4 4 KMNAKAATL Rv2911 Q7D6F2 263 15 5 QMKFYAVAL Rv2119 O33254 260 1,5 6 PMKRTALAL Rv0959 P0A5D7 259 18 7 GMNVTAPAL Rv1617 O06134 257 8 8 VMADRTRHL Rv0418 P96264 255 5 9 WMCDRAVDL Rv2954c Q50461 254 >50 10 LMVVRALFL Rv3101c P96293 254 nt 11 EMVLRADQL Rv0191 O07435 253 >50 12 DMLGRAGGL Rv3015c O53262 253 >50 13 MMKYLAFGL Rv0206c O53657 246 0,4 14 RMAATAQVL Rv2932 Q10978 237 2 15 FLAADALVL Rv1047 P96354 185 >50 16 VEAFRTRPL Rv1047 P96354 189 >50 17 LMGAEADAL Rv1047 P96354 210 >50 18 GLDSRAYRL Rv0146 O86321 212 >50 19 GLDARAYRL Rv0145 P96356 212 >50 20 PMAPLAPLL Rv3871 O69736 226 3 21 EMKTDAATL Rv3874 P0A566 246 >50 22 PMQQLTQPL Rv3873 Q79F92 205 >50 23 PMADIAAAL Rv1253 Q11039 225 >50 24 ALPPRAFEL Rv3428c Q50700 212 0,27 25 AMVNTTTRL Rv2074 Q10682 209 20 26 LMGALAVVL Rv2075c Q10683 211 21 27 VSNLRTGKL Rv1967 O53968 208 >50 28 TSADRAVVL Rv1973 O53974 203 39 29 LMHYRGELL Rv1965 O53966 201 20 30 NMMARGMDL Rv0221 P96403 201 >50 31 LPAERAHEL Rv0222 P96404 202 >50 32 VMMSEIAGL Rv1813c P64889 348 13 33 TMITFRLRL Rv1733c P71991 373 2,4 34 VMTTVLATL Rv1734c P71992 348 1,8 35 GMGMVGTAL Rv1737c P71995 348 >50 36 AMAGSIDLL Rv2006 Q10850 367 34 37 EMLTSRGLL Rv1997 P63687 352 >50 38 QMRACARRL Rv2004c P0A5F9 210 1,5 39 AMEYFRQVL Rv2006 Q10850 348 28 40 SMFAAVQAL Rv2030c O53475 375 5 41 HMAQTLGSL Rv2030c O53475 185 >50 42 SMFAAVQAL Rv2030c O53475 357 2,5 43 EMGRAPLDL Rv2627c O06185 373 >50 44 RLPAKAPLL Rv1484 P0A5Y6 1,347 0,03 45 VMAPLGPIL Rv1405c P64841 NA 0,8 46 QLAPGLQLI Rv1563c Q10768 1,248 >50 47 RLAPGGTTI Rv0068 O53613 1,238 >50 48 RLANLLPLI Rv3189 O53335 1,231 32 49 ILPSDAPVL Rv3823c O07800 NA 0,2 50 VLLPGLPYL Rv3847 P96230 1,208 8 51 REPRRGPRL Rv0142 P96819 1,169 1 >50 52 RLRPELAGL Rv3772 P72039 0,755 3 46 53 RELPGRVLL Rv3094c O05773 0,759 4 >50 54 FLLPRGLAI Rv0056 P66315 0,867 5 >50 55 VMATRRNVL Rv1518 Q50590 0,924 6 16 56 RLGRLLNRI Rv3737 O69704 0,825 7 >50 57 IEPRGAQAL Rv0171 O07415 0,985 8 >50 58 DLPSRLGKI Rv3087 O53304 0,802 9 >50 59 RPANLAFFL Rv1188 O50444 NA 1,3 60 FMTRLGPLL Rv0035 Q7DAJ8 0,829 11 37 61 RLASCRDAL Rv2761c O33303 0,775 12 >50 62 RMPPLGHEL Rv2997 O53244 1,19 13 4 63 VMAPDAVRI Rv0324 O08446 1,014 14 31 64 RSCPRLTIL Rv0894 P64749 0,895 15 >50 65 RLARRARNI Rv1513 P71792 0,773 16 >50 66 RLGLCALAL Rv3370c O50399 NA 14 67 VLPACLGIL Rv0206c O53657 0,878 18 12 68 VLRPGGHFL Rv1523 Q50584 0,767 19 1,8 69 WLPPLLTNL Rv0249c O53671 0,963 20 39 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):e1000782
# peptides recognized peptide donor Peptide induced proliferation 30 p=0.0027 p=0.028 20 10 0 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):e1000782
peptide 44 63 62 125 101 139 100 82 10 9 0 0 10 0 10 0 0 40 0 4 0 0 0 0 0 1 0 5 33 1 1 1 0 0 0 0 2 0 23 23 0 4 0 0 0 2 0 10 0 0 54 0 8 3 0 0 0 0 1 0 5 37 0 0 0 0 0 9 0 4 0 19 38 0 4 0 0 0 3 0 0 0 0 35 0 10 0 0 0 3 0 1 0 54 34 0 3 0 0 0 3 1 13 0 114 46 3 2 4 0 0 8 0 8 0 0 30 0 2 0 0 BCG vaccination resulted in recognition of HLA-E peptides BCG vaccinated infants (Cape Town), stimulation with 10 selected peptides PPD CD8 31 3 24 14 5 37 14 48 22 54 12 55 25 59 25 61 1 62 7 68 14 donor 1 2 3 4 5 6 7 8 9 10 11 12 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):e1000782
% 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/B7 50 20 15 Donor 2, T cell line against p62 K562/E + 62 K562/E + 68 K562 +62 (no HLA E) 10 25 5 0 12:1 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):e1000782
62.500 125.000 250.000 62.500 125.000 250.000 62.500 125.000 250.000 62.500 125.000 250.000 62.500 125.000 250.000 62.500 125.000 250.000 62.500 125.000 250.000 62.500 125.000 250.000 62.500 125.000 250.000 62.500 32.250 15.625 62.500 125.000 250.000 62.500 32.250 15.625 % 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. 30 25 D2 #54 D2 #62 D4 #55 D6 #68 control - BCG 20 15 10 5 0 effector cells Joosten SA, et al. PLoS Pathog. 2010; 6(2):e1000782
- 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 40000 30000 20000 10000 0 D2 #62 D4 #55 D6 #68 LAP inhibits suppression: membrane bound TGF involved Joosten SA, et al. PLoS Pathog. 2010; 6(2):e1000782
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
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. 2500 2000 440 420 py292 py319 1500 1000 500 400 380 360 0 p62 p68 p62 p68 340 Van Meijgaarden KE, et al. PLoS Pathog. 2015, 11(3): e1004671
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. 100 80 60 40 20 0 0 20 40 60 80 100 lysis(%) Suppression(%) Van Meijgaarden KE, et al. PLoS Pathog. 2015, 11(3): e1004671
% perforin HLA-E restricted T-cell clones can inhibit intracellular growth of Mtb CFU % perforin + granulysin + 30000 20000 10000 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 D6-2B4 100 60 % perforin + granzymeb + 80 80 60 40 20 R 2 = 0.77 40 20 R 2 = 0.72 60 40 20 R 2 = 0.80 0 0 20 40 60 80 % specific Mtb growth inhibition 0 0 20 40 60 80 % specific Mtb growth inhibition 0 0 20 40 60 80 % specific Mtb growth inhibition Van Meijgaarden KE, et al. PLoS Pathog. 2015, 11(3): e1004671
HLA-E restricted T-cell clones can inhibit intracellular growth of Mtb 100 80 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 60 40 20 0 0 20 40 60 80 100 0 20 40 60 80 100 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): e1004671
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-13 200-1000 10-500 10-100 <1 1000-2500 500-1000 100-500 1-5 2500-5000 1000-2000 500-1000 5-10 5000-10000 2000-5000 1000-2000 10-20 >10000 5000-10000 2000-3500 >20 C. peptide Th1 Th2 Th1 Th2 Th1 Th2 D2-1B4 D6-1F11 D2-1B9 B. IL-13 IL-5 IL-4 0.2 0.2 2.4 5.3 1.9 2.3 6.9 23 14 6.5 4.9 17 32 11 5.4 9.8 0.2 0.03 0.4 1.2 3.9 0.5 1.9 3.6 1.6 0 0 BCG Th1 Th2 IFNγ Van Meijgaarden KE, et al. PLoS Pathog. 2015, 11(3): e1004671
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 + D6-2F6 17 10 8 20 4 43 B. CD80 CD86 CD25 14 20 26 CD86 13 18 2 22 27 4 12 63 2 C. CD25 D. E. Van Meijgaarden KE, et al. PLoS Pathog. 2015, 11(3): e1004671
B-cells in TB disease are decreased in numbers and respond poorly to polyclonal activation % B cells Total B cells plasma cells im 30 25 20 15 p=0.001 * 10 8 6 4 2.0 30 25 20 15 10 10 8 6 4 2 0 Controls LTBI TB TB treated 1.5 1.0 0.5 0.0 Controls LTBI TB TB treated 10 5 0 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
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: 1069 1081
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: 1069 1081
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 1.0 0.5 0.0 0.44 0.53 0.01 p62 p68 p62 0.38 0.70 0.006 p68 D 0.6 p=0.005 p=0.0005 0.8 p=0.006 0.4 0.2 0.6 0.4 0.2 16 hour peptide stimulation followed by ICS 0 - + - + - + - + TNF IFN-γ IL-4 IL-13 T M 0 - + - + - + - + TNF IFN-γ IL-4 IL-13 T M Van Meijgaarden KE, et al. PLoS Pathog. 2015, 11(3): e1004671
HLA-E TM binding cells can produce Th2 cytokines Teresa Prezzemolo, work in progress
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?
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
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)
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