Primär myelofibros (PMF) är en typ av myeloproliferativ neoplasm (MPN) som leder till en progressiv och irreversibel benmärgsfibros. En somatisk mutation, Jak2V617F, har hittats hos 50 % av patienterna med MPN i hematopoetiska stamceller. Nyligen har man upptäckt grupp 2 av medfödda lymfoida celler (ILC2) som tillhör det medfödda systemet. De är T-cellernas motsvarighet men saknar TCR-receptorn. ILC2 reagerar på IL-33 och producerar Il-13. Under de senaste åren har man upptäckt att dessa två cytokiner är inblandade i PMF. För att undersöka ILC2:s roll i utvecklingen av benmärgsfibros in vivo producerade vi retrovirus som uttrycker Jak2 vildtyp (JAK2_WT) eller Jak2V617F (JAK2_V617F) och transducerade benmärg vildtyp (BM_WT) eller benmärg ILC2KO (BM_ILC2KO). Benmärgen transplanterades till subletalt bestrålade immunbristande möss (NOG). Klinikopatologiska drag som är karakteristiska för sjukdomens första stadier, som förhöjda hemoglobinnivåer, megakaryocythyperplasi och betydande trombocytos, uppstod inte under studieperioden. Ökade vita blodkroppar uppstod dock på grund av avsaknaden av ILC2 i JAK2_V617F-expressiva möss. Flödescytometeranalys visade ursprunget till den markerade leukocytosen som ett resultat av expansionen från lymfocytlinjen, mer specifikt B-celler, men resultaten är inte entydiga eftersom de förhöjda nivåerna av B-celler kan vara en följd av ILC2 knock-out fenotypen som förvärras av närvaron av mutationen. Granulocytnivåerna från de inympade cellerna hölls låga till följd av att stamcellerna i värdens benmärg var inblandade på grund av subletal bestrålning. Vi drar slutsatsen att frånvaron av ILC2 i JAK2_V617F-uttryckta benmärgsprogenitorer har en tendens att förvärra den myeloproliferativa fenotypen i sjukdomens tidiga skeden, vilket tyder på en möjlig skyddande roll för ILC2 vid utvecklingen av MPN. / Primary myelofibrosis (PMF) is one type of myeloproliferative neoplasm (MPN) that leads to a progressive and irreversible bone marrow fibrosis. A somatic mutation, Jak2V617F has been found in 50% of patients with MPN in hematopoietic stem cells. Group 2 innate lymphoid cells (ILC2) belonging to the innate system has been recently discovered. They are the counter part of T cells but lacking the TCR receptor. ILC2 response to IL-33 producing Il-13. In recent years, the involvement of these two cytokines in the PMF has been uncovered. To investigate the role of ILC2 in the progression of bone marrow fibrosis in vivo we produced retrovirus expressing Jak2 wild-type (JAK2_WT) or Jak2V617F (JAK2_V617F) and transduced bone marrow wild type (BM_WT) or bone marrow ILC2KO (BM_ILC2KO). The bone marrow was transplanted into sub-lethally irradiated immunodeficient mice (NOG). Clinicopathologic features characteristic from the first stages of the disease, as elevated hemoglobin levels, megakaryocyte hyperplasia and significant thrombocytosis did not emerge during the study period. However, increased in white blood cells arise from the absence of ILC2 in JAK2_V617F expressing mice. Flow cytometer analysis revealed the origin of the marked leukocytosis as a result of the expansion from the lymphocyte lineage, more specifically B cells, but the results are inconclusive as the elevated levels of B-cells could be a consequence of the ILC2 knock-out phenotype aggravated by the presence of the mutation. Granulocyte levels from engrafted cells were kept low because of the involvement of host bone marrow stem cells due to sublethal irradiation. We conclude that the absence of ILC2 in JAK2_V617F-express bone marrow progenitors has a tendency to aggravate the myeloproliferative phenotype in the early stages of the disease, indicating a possible protective role of ILC2 in the development of MPNs.

Introduction: Atopic dermatitis (AD) is characterized by chronic pruritic relapsing eczematous lesions of the skin. Eosinophilic inflammation in AD is driven by activation of type 2 inflammatory cells including CD4+ T cells and type 2 innate lymphoid cells (ILC2s). We have shown that type 2 cytokines, namely interleukin (IL)-5 and IL-13, stimulate migration and terminal differentiation of eosinophil progenitor cells (EoPs). We propose that these cytokines are important drivers of tissue eosinophilia in AD lesional skin. This study aimed to quantify, by flow cytometry, cells that produce type 2 cytokines in lesional skin compared to peripheral blood from moderate-severe AD patients. Methods: In a cross-sectional study of patients with moderate-to-severe AD (n=16), type 2 inflammatory cells were enumerated in blood and cells extracted from excised skin biopsies. By flow cytometry, live, singlet CD45+cells were identified as ILC2 (lin-CD127+CD294+), EoP (CD34+125+), and CD4+ T cells (Lin+CD3+CD4+). Intracellular expression of type 2 cytokines (IL-5 and IL-13) were evaluated in each cell population. In addition, we developed a protocol to enumerate ILC2s by fluorescence immune-histochemistry in lesional versus non-lesional skin samples and skin biopsies taken 24h post-intradermal challenge with allergen versus diluent. Data are expressed as median (interquartile range [IQR]) unless otherwise stated. Cross compartmental comparisons were made using the Wilcoxon rank-sum test and where applicable, correlational analyses were performed using a Spearman’s rank-correlational test. Results: There was a significantly higher number of total ILC2s in lesional skin compared to blood from AD subjects (556 [99 – 5501] vs 235 [67 – 569] cells/mL, p=0.03). Similarly, IL-5+, IL-13+ ILC2s, were significantly greater in skin compared to blood (6 [1 – 666] vs 1 [1 – 19] cells/mL, p=0.03; 28 [1 – 1357] vs 1 [1 – 7] cells/mL, p=0.01, respectively). We found higher numbers of total and type 2 cytokine positive EoP in lesional skin biopsies from AD patients compared to blood (Total EoP: 815 [285 – 2794] vs 112 [46 – 247] cells/mL, p<0.01; IL-5+EoP: 36 [1 – 129] vs 1 [1 – 23] cells/mL, p=0.07; IL-13+EoP: 92 [10 – 182] vs 1 [1 – 8] cells/mL, p<0.01 and IL-5+IL-13+ILC2: 70 [1 – 158] vs 1 [1 – 12] cells/mL, p=0.02, respectively). In contrast, significantly higher numbers of total and type 2 cytokine positive CD4+ cells were found in blood compared to lesional skin biopsies from AD patients (Total CD4+: 1092 [650 – 1742] vs 58.3 [35.3– 152.4] x 103 cells/mL, p<0.01 and IL-5+IL-13+CD4+ cells: 13.5 x 103 [2.1 x 103 – 42.9 x 103] vs 3.8 x 103 [1.6 x 103 – 4.9 x 103] cells/mL, p=0.02, respectively). For IF staining, there was a significant higher number of ILC2s in lesional compared to non-lesional skin biopsies and biopsies taken 24h post allergen- compared to diluent challenge (1 [0 – 2] vs 0 [0 - 0] cells/mm2, p=0.008, and 2 [1 – 2] vs 0 [0 – 0] cells/mm2, p=0.0002, respectively). Interestingly, in sex analyses we found significantly greater levels of blood ILC2 in females compared to males, but this not was found in the skin. Importantly, we found a significant correlation between lesional skin levels of ILC2 measured by flow cytometry and clinical measures of disease severity/symptoms as reported/calculated from the Patient-Oriented Eczema Measure questionnaire (POEM) score (total ILC2: r=0.55, p=0.04; IL-13+ ILC2s, r=0.61, p=0.02 and IL-5+ IL-13+ ILC2s: r=0.75, p=0.002). Conclusions: Preferential increases in skin-resident ILC2 that produce a type 2 rich environment were found in AD subjects. These levels correlated with patient-oriented measure of disease severity. We propose that this increase may encourage recruitment of mature eosinophils and EoP and possibly drive localized differentiation of EoP into mature eosinophils that may drive the pathology of AD lesions. Furthermore, immunofluorescence staining may be a suitable alternative to flow cytometry for identification of ILC2 in the event of a low cell count. These techniques can be used in future studies that target ILC2 biology to fully understand the role of these cells in driving AD. / Thesis / Master of Science (MSc)