Relationship Between Expression of TLRs and Disease Activity in CLL.

Toll-like receptors (TLRs) are major agents of innate immunity and initiators of adaptive immune response by acting as costimulatory signals for B cells and inducing maturation, proliferation and antibody production after pathogen recognition. They are also involved in the self-antigen recognition and could play a role in autoimmune phenomena. It is well known that chronic lymphocytic leukemia (CLL) is characterized by an increased incidence of autoimmune phenomena and immunodeficiency, which can greatly influence the disease outcome leading to a variable clinical course.

To evaluate the correlation between the gene expression of TLRs in 41 patients with CLL (mean age±SD 66±18 years, range 42-90, 15 female and 26 male), the clinical course of the disease and the expression of prognostic factors (mutational status of IgVH region, CD38 and ZAP70 expression, and cytogenetic alterations).

The gene expression of TLR4, TLR9, and TLR10 was studied. Total RNA was extracted from B cells of patients and controls (pool of 10 healthy donors), cDNA was synthesized, and real-time PCR was performed. For each reaction 50 ng of cDNA were mixed with 1.25μl of TaqMan primer/probe set and 10.25μl of TaqMan Universal Master Mix. The TLR4, TLR9, and TLR10 expression was normalized according to GAPDH as an internal control gene and it was expressed as percentage of control.

TLR4 gene expression was significantly lower in CLL patients compared to controls (18±3%, mean±SE) while TLR9, and TLR10 gene expression was higher (3457±500% and 2897±440%, respectively). Moreover, considering “progressive” CLL patients (n=20), TLR4 gene expression was significantly lower compared with “indolent” ones (n=21) (10.2±2.2% vs 24.9±4.9%, p=0.01). Patients with unmutated IgVH expression and unfavorable cytogenetic alterations (mostly 11q-) showed even more reduced TLR4 gene expression, but the small numbers did not allow statistical evaluation. TLR4 gene expression was lower in patients with infective diathesis, although not significantly (11.5±2.9% vs 20.0±3.9).The 6 “progressive” CLL patients with autoimmune diseases (hemolytic anemia, Evan's syndrome and phemphigus) showed significantly higher TLR4 gene expression compared with “progressive” patients without autoimmune phenomena (17.7±4.4% vs 6.9±2.1%, p=0.02), possible expression of activated immune system.

The reduced TLR4 gene expression in CLL patients with “progressive” disease, unmutated IgVH status, and unfavourable cytogenetic alterations is consistent with a reduced ability to a proper immune response to Gram-negative bacterial lipopolysaccarides and consequent increased susceptibility to infections.

No relevant conflicts of interest to declare.