Cytotoxicitet och blodsjukdomar

Transkript

1 Cytotoxicitet och blodsjukdomar DAPI Perforin Actin From Stephanie Wood

2 Hemofagocyterande Lymfohistiocytos (HLH) Okontrollerad hyperinflammation på basen av olika ärftliga eller förvärvade störningar i immunsystemet. Familjär Hemofagocyterande Lymfohistiocytos Sekundär Hemofagocyterande Lymfohistiocytos

3 Diagnostiska kriterier 1. Känd familjär/annan genetisk HLH 2. Kliniska och laboratoriemässiga kriterier (5/8 krävs för diagnos) * Feber * Splenomegali * Cytopeni 2 cellinjer * Hypertriglyceridemi (faste-tg > 3 mmol/l) och/eller hypofibrinogenemi (fibrinogen < 1,5 g/l) * Ferritinstegring (>500 µg/l) * scd25 (>2400U/ml) * Minskad eller upphörd NK-cellsaktivitet * Hemofagocytos i histologisk/cytologisk undersökning av benmärg, eller lymfkörtlar

4 Gener vid FHL Fyra kända loci. 9q (FHL1) 10q21 (FHL2) 17q25 (FHL3) 6q24 (FHL4) Tre kända gener. okänd PRF MUNC13-4 STX11

5 Molekylära mekanismer vid FHL/HLH Ménasche et al. Immunological reviews 2005; 203:

6 Lymphocyte biology Mediated through regulated release of secretory lysosomes containing several cytotoxic effector proteins (e.g. perforin, granzymes, Fas ligand) Mechanisms shared by CD8 + effector T cells and natural killer (NK) cells Triggered by T cell receptor, Fc receptors (ADCC) and natural cytotoxicity receptor engagement

7 Diagnostic assays for evaluation of lymphocyte cytotoxic function Target cell lysis Induction of LAMP-1/CD107a plasma membrane Release of 51 Cr from radioactive labeled target cells as a measure of effector cell cytotoxicity (K562 target cells are susceptible to NK cell-mediated cytotoxicity) Flow cytometric assessment of LAMP-1/CD107a surface expression as a measure of effector cell degranulation upon triggering by multiple stimuli

8 Cytolytic activity by FHL4 lymphocytes is restored by IL-2 Resting IL-2 activated Bryceson et al. (2007) Blood

9 Establishment of assay for measurement of degranulation from different lymphocyte subsets by different means of stimulation Bryceson et al. (2007) Blood

10 Familial hemophagocytic lymphohistocytosis (FHL) Unstimulated NK cells 72 hour IL-2 stimulated NK cells Bryceson et al. (2007) Blood Mutations in PRF1, UNC13D, and STX11 have been described in FHL, the two latter are associated with defective cytotoxic lymphocyte degranulation Observations in Huddinge IVA patient are reminiscent of mutations in STX11 (FHL4) FHL4 displays later onset and longer periods of remission relative to FHL2 and FHL3

11 Preliminary observations in EBVinduced HLH compared to FHL

12 Treatment Chemo-immunotherapy HSCT

13

14 Case 1 EBV 17-årig flicka insjuknar i mononukleos. -inlagd med TPN -tonsillektomi p.g.a andningssvårigheter -post op IVA p.g.a blödning och svullnad

15 Hög feber -kyldräkt Koagulationspåverkan -trombocyter -faktorer Septiska inslag -antibiotika -vasopressor -PRISMA-behandling Kraftig hepatosplenomegali Case 1 forts.

16 Case 1 forts. 19/2: EBV-PCR kopior 25/2: EBV-PCR kopior -Mabthera och i.v immunoglobulin

17 Case 1 forts. 29/2: NK-cells analys Lymphocyte distribution: CD3 CD56 7.5%; CD %; CD3 CD %. The cytotoxicity in the patient was pathological (<10 lytic units). Degranulation by patient NK cells was defective. Patient cells gained cytotoxic function after incubation with IL-2, reminiscent of FHL type 4.

18 Case 1 forts. Unstimulated 6 hour IL-2 NK cells No stimulus K562 cells stimulated No stimulus K562 cells NK cells Patient FL4-H: CD56 APC FL4-H: CD56 APC FL4-H: CD56 APC FL4-H: CD56 APC FL1-H: CD107a FITC FL1-H: CD107a FITC FL1-H: CD107a FITC Control FL4-H: CD56 APC 10 2 FL4-H: CD56 APC 10 2 FL4-H: CD56 APC 10 2 FL4-H: CD56 APC FL1-H: CD107a FITC 10 0 CD107a FL1-H: CD107a FITC FL1-H: CD107a FITC FL1-H: CD107a FITC Defective degranulation in unstimulated patient NK cells (K562 stimulation shown) Partial restoration of degranulation by IL-2 stimulation CD107a

19 Case 1 forts. 2/3: påbörjar behandling med Vepesid och steroider Diflukanprofylax Maxipime dag 6 efter påbörjad behandling p.g.a neutropeni. Förbättras kliniskt EBV-PCR neg. 28/3.

20 Case 1 forts. A molecular diagnosis consistant with FHL- negativ. Diagnostic criteria for HLH fulfilled (5 out of 8 criteria below). Fever- positiv Splenomegaly- positiv Cytopenias (affecting 2 of 3 lineages in peripheral blood).- positiv Hypertriglyceridemia and/or hypofibrinogenemia- positiv Hemophagocytosis in bone marrow or spleen or lymph nodes- negativ Low or absent NK-cell activity- positiv Ferritin >500 microgram/l- positiv Soluble CD25 >2400U/ml- oklart

21 Case 2 Flicka v.24 -Vikt: 732g -Apgar: dagar Serratia marcescens sepsis.

22 Case 2 forts. Behandling: -Meropenem -Netilmicin -Vancomycin -Metronidazol

23 A) CRP L / g m Days of age Max CRP: 322mg/L dag 25 Normaliserat dag 74

24 L / l o m o r c i m C) Bilirubin 0L 9/ 1 * B) Platelets Days of age Days of age

25 Case 2 forts. A molecular diagnosis consistant with FHL- negativ. Diagnostic criteria for HLH fulfilled (5 out of 8 criteria below). Fever- negativ Splenomegaly- positiv Cytopenias (affecting 2 of 3 lineages in peripheral blood).- positiv Hypertriglyceridemia and/or hypofibrinogenemia- positiv Hemophagocytosis in bone marrow or spleen or lymph nodes- oklart Low or absent NK-cell activity- positiv Ferritin >500 microgram/l- positiv Soluble CD25 >2400U/ml- positiv

26 TACK!