Essential Thrombocytosis Patients Have At Diagnosis Prevalence Of CD8+ and CD45RO+CCR7- Lymphocytes and High Proportions Of Double Positive IL-17+/CD15+ Cells In The Marrow Cell Population

Thirty-one patients with essential thrombocytosis (ET, F/M: 20/11 age 19-86, median 60 years, platelets: 499-1724x10^3/μl (median: 652x10^3), JAK2 V617F mutation: 26 positive, marrow cellularity: 6.1–184x10^3/μl (median: 31x10^3/μl)) were studied at the time of the diagnostic procedure for the presence in the marrow (i) cells and (ii) some transcripts associated with the immune response. Five techniques were employed: (i) marrow smears analysis, (ii) four color cytometric (BD, San Jose; e-biosciences, San Diego, CA, USA) analysis, (iii) RT-PCR relative quantification of IL-17, ROR gamma t, IFN gamma and CXCL10 mRNA against four housekeeping genes (ABL, HPRT, b-actin, beta 2 microglobulin), (iv) trephine biopsies specimens were in addition to the routine analysis (PAS, HE and reticulin silver stain) stained for CD34, CD15, CD68, CD3, CD4, CD8, IL-17 and (v)immunofluorescence double staining (IL-17/CD15). For transcripts study the ET patients results were compared with those of CML patients (6 patients at diagnosis). Nine healthy volunteers served as a control group for the peripheral blood mononuclear cells IL-17 study. Statistical analysis employed the paired sample Wilcoxon Signed Rank Test and Mann-Whitney U Test, as appropriate.

The following observations were made:

(i) The marrow lymphocyte population (gated according to the physical parameters and CD45 positivity) differed as compared to blood with respect to: lower CD4+/CD8+ ratio (1.30±0.115 vs 2.125±0.202, p<0.001), lower CD45RO+CCR7+/CD45RO+CCR7- ratio (0.243±0.032 vs 0.322±0.036, p=0.007).

(ii) IL-17+ blood mononuclear cells (subjected to 4 hours of Ionomycin, Brefeldin A, and PMA stimulation) werepresent in higher proportions in ET patients in both CD4+ and CD4- lymphocyte subpopulations as compared to healthy volunteers (CD4+IL-17+: 0.412±0.129% vs 0.063±0.022%, p=0.002; CD4-IL-17+: 0.627±0.290% vs 0.034±0.007%, p=0.029).

(iii) IL-17 (4.19E-04±2.44E-04 vs 6.0E-07±3.0 E-07, p=0.027) and ROR gamma t (3.32E-02±1.22E-02 vs 2.60 E-03±3.57E-04, p=0.003) transcripts levels in marrow cells population enriched in mononuclear cells with the use of density gradient separation were significantly higher in ET patients as compared to those found in CML patients (at diagnosis).

(iv) IFN gamma (8.60E-01±6.48E-01 vs 1.95E-02±1.04E-02, p=0.018) and CXCL10 (5.76E-01±4.01E-01 vs 1.59E-02±5.56E-03, p=0.054) transcripts also prevailed in marrow of ET patients as compared to CML group.

(v) Trephine biopsies staining revealed:

• high numbers of CD8+ cells in 22 out of 30 patients (> 40 CD8+ cells/HPF),

• high proportions of cells positive for IL-17 in 25 patients out of 30 (30 to 180 IL-17+ cells/HPF). Double staining for IL-17 and CD15 documented that IL-17+ cells were in the majority also CD15 positive cells.

We conclude:

• in the lymphocyte marrow population of ET patients there is an increase of cytotoxic lymphocytes and those of effector/memory cells phenotype what may suggest the presence of an active immune response at the marrow site in at early stage of the disease prior to the treatment,

• IL-17 engagement in ET associated processes is suggested by an increase of IL-17+ lymphocytes in blood and high proportions of IL-17 producing cells in the marrow the majority of which are CD15 positive. The latter observation suggests that the innate immunity is involved in initiation of the immune system recognition of ET associated factors.

Supported by N N402 43 0039 grant from the Polish Ministry of Science & Higher Education.