Expression of TLRs in B-CLL: Relationship with Disease Activity

Abstract 4597

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 established that B-chronic lymphocytic leukemia (B-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.

The aim of this study was to evaluate the gene expression of TLRs in 97 B-CLL patients (median age 74 yrs, range 41–89, 41 female and 57 male) and to relate it with the clinical course of the disease (median follow-up 107 months, range 36–336), in particular with infections and autoimmunity, and the expression of prognostic factors (mutational status of IgVH region, CD38 and ZAP70 expression, and cytogenetic alterations).

The gene expression of TLR4 and TLR9 was evaluated in total RNA of B cell from B-CLL patients and controls (pool of 10 healthy donors) by real-time PCR performed with a model 7300 real-time PCR system (Applied Biosystems). The TLR4 and TLR9 relative quantification expression (RQ) was normalized according to GAPDH (internal control gene) and to control mRNAs.

We found that TLR4 gene expression was decreased (RQ=16.1+/−1.56) and TLR9 increased (RQ=2725+/−165) in B-CLL patients vs controls (RQ=100). TLR4 gene expression was reduced in: a) stage Binet B-C/Rai II-III-IV (n=20) versus low risk cases (Binet A/Rai 0-I) (10.8+/−2.2 vs 17.4+/−1.9, p=0.048); b) patients treated with I line therapy (n=25) and II line therapy (n=14) vs untreated patients (n=58) (13.1+/−2 vs 19.2+/−2.3, p=0.05, and 8.3+/−2.1 vs 19.2+/−2.3, p=0.03, respectively); c) patients untreated but with ITT and treated with stable disease or progressive disease (n=28) vs untreated and treated with DFS>24 months at the time of the investigation (n=69) (12+/−2 vs 17.8+/−2, p=0.048); d) patients with grade 2–4 infections (n=52), according to Common Terminology Criteria for Adverse Events, vs patients with grade 0–1 infections (13.2+/−1.5 vs 19.51+/−2.9, p=0.04); e) patients with autoimmune phenomena (n=12) vs cases without (8.8+/−2.5 vs 17.12+/−1.7, p=0.04). As far as TLR9 is concerned, we found that it was significantly reduced in untreated and treated with II line therapy patients vs patients treated with I line therapy (2549+/−183 vs 3528+/−356, p=0.01, and 1893+/−277 vs 3528+/−356, p=0.01, respectively). Finally, we found that patients with an unmutated IgVH region status (n=15) showed TLR4 gene expression significantly reduced compared with patients with mutated IgVH region status (n=29) (9.57+/−2.1 vs 18.8+/−3.2, p=0.028). (Data are expressed as mean+/−SE for all data).

These results show that in B-CLL patients the reduced expression of TLR4 is consistent with a reduced ability to mount a proper immune response and it is even more pronounced in “active” patients that show high prevalence of infectious episodes and autoimmune phenomena.