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Correlation between CD4+CD25+FOXP3+ Regulatory T Cells (T-reg) and ZAP-70 Expression in Patients with Chronic Lymphocytic Leukemia (CLL). Background

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  1. Correlation between CD4+CD25+FOXP3+ Regulatory T Cells (T-reg) and ZAP-70 Expression in Patients with Chronic Lymphocytic Leukemia (CLL)

  2. Background • Naturally occurring T-regulatory (T-reg) cells are a distinct subset of CD4+/CD25high/FOXP3+ T lymphocytes that play a central role in self tolerance. • T-regs prevent autoimmune and inflammatory diseases and are thought to hinder anti-tumor and anti-leukemia immune response (AML-Wang X et al, Eur J Haematol 2005, MM- Beyer M et al Blood 2006, MDSKordasti et al Blood 2007). • A quantitative increase of peripheral T-reg cells was noted in CLL patients which correlated with a more advanced clinical stage (Beyer et al Blood 2005, Motta M, et al Leukemia 2005).

  3. Expression of ZAP-70, the tyrosine kinase that mediates TCR signaling and is structurally homologous to Syk, plays a central role in BCR signaling in CLL. • ZAP-70 expression in B-CLL cells, is the hallmark of B cell activation and is a negative prognostic factor in B-CLL . • We previously reported that high ZAP-70 expression in B-CLL cells correlated with high ZAP-70 expression in T cells, implying prominent T-cell activation. • This positive correlation between ZAP-70 expression in B-CLL cells and T-cells in CLL implies cross-talk, involving T-cell/B -CLL cell interactions (Herishanu et al, Leukemia 2005).

  4. As this lymphocyte trans-activation may affect the clinical outcome in these patients, • our aim was • To determine whether there is a correlation between T-regs and ZAP-70 status in CLL patients.

  5. Methods • Blood samples were collected from 32 newly diagnosed untreated CLL patients and healthy individuals. • The diagnosis of CLL was based on standard morphologic and immunophenotypic criteria. CD4, CD25, the transcription factor FOXP3. • Viability of the lymphocyte populations were measured by flow cytometry with 7AAD and cycling with KI-67. • Quantitative analysis of the intracellular levels of ZAP-70 was performed by flow cytometry using MESF (Kay et al, Cytometry B Clin Cytom, 2006).

  6. Higher expression of ZAP-70 in both T and B Cells of CLL patients 33

  7. Positive linear correlation of ZAP-70 expression in B cells and T cells of CLL patients

  8. A A A A * * * * 12 12 12 12 25 25 25 25 . . . . * * * * . . . . 10 10 10 10 20 20 20 20 8 8 8 8 15 15 15 15 CD4+CD25+FOXP3+(%) CD4+CD25+FOXP3+(%) CD4+CD25+FOXP3+(%) CD4+CD25+FOXP3+(%) 6 6 6 6 CD4+FOXP3+ cells (%) CD4+FOXP3+ cells (%) CD4+FOXP3+ cells (%) CD4+FOXP3+ cells (%) 10 10 10 10 4 4 4 4 2 2 2 2 5 5 5 5 0 0 0 0 0 0 0 0 CLL CLL CLL CLL NPB NPB NPB NPB CLL CLL CLL CLL NPB NPB NPB NPB B B B B 600 600 600 600 250 250 250 250 . . . . 500 500 500 500 200 200 200 200 400 400 400 400 150 150 150 150 Number of Number of Number of Number of CD4+CD25+FOXP3+/ul CD4+CD25+FOXP3+/ul CD4+CD25+FOXP3+/ul CD4+CD25+FOXP3+/ul CD4+FOXP3+/ul CD4+FOXP3+/ul CD4+FOXP3+/ul CD4+FOXP3+/ul Number of Number of Number of Number of 300 300 300 300 100 100 100 100 200 200 200 200 50 50 50 50 100 100 100 100 0 0 0 0 0 0 0 0 CLL CLL CLL CLL NPB NPB NPB NPB CLL CLL CLL CLL NPB NPB NPB NPB The relative proportion of T-regs is upregulated in CLL patients The percentage of CD4+FOXP3+ and CD4+CD25+FOXP3+ T-regulatory lymphocytes is higher in CLL patients compared to T cells from normal controls (p<0.001). Figure 2: CD4+FOXP3+ and CD4+CD25+FOXP3+ populations in T cells Blood from CLL Patients (n=35) and normal subjects (NPB) (n=12) were measured by flow cytometry. Figure 2a demonstrates that the relative proportion CD4+FOXP3+ and CD4+CD25+FOXP3+ T-regulatory lymphocytes is higher in CLL patients compared to T cells from normal controls (p<0.001). The actual number of T-regulatory cells is also increased as seen in figure 2B. However, due to the wide variability of T cell counts in the patients, statistical significance of the differences in T-reg numbers was not reached Figure 2: CD4+FOXP3+ and CD4+CD25+FOXP3+ populations in T cells Blood from CLL Patients (n=35) and normal subjects (NPB) (n=12) were measured by flow cytometry. Figure 2a demonstrates that the relative proportion CD4+FOXP3+ and CD4+CD25+FOXP3+ T-regulatory lymphocytes is higher in CLL patients compared to T cells from normal controls (p<0.001). The actual number of T-regulatory cells is also increased as seen in figure 2B. However, due to the wide variability of T cell counts in the patients, statistical significance of the differences in T-reg numbers was not reached Figure 2: CD4+FOXP3+ and CD4+CD25+FOXP3+ populations in T cells Blood from CLL Patients (n=35) and normal subjects (NPB) (n=12) were measured by flow cytometry. Figure 2a demonstrates that the relative proportion CD4+FOXP3+ and CD4+CD25+FOXP3+ T-regulatory lymphocytes is higher in CLL patients compared to T cells from normal controls (p<0.001). The actual number of T-regulatory cells is also increased as seen in figure 2B. However, due to the wide variability of T cell counts in the patients, statistical significance of the differences in T-reg numbers was not reached Figure 2: CD4+FOXP3+ and CD4+CD25+FOXP3+ populations in T cells Blood from CLL Patients (n=35) and normal subjects (NPB) (n=12) were measured by flow cytometry. Figure 2a demonstrates that the relative proportion CD4+FOXP3+ and CD4+CD25+FOXP3+ T-regulatory lymphocytes is higher in CLL patients compared to T cells from normal controls (p<0.001). The actual number of T-regulatory cells is also increased as seen in figure 2B. However, due to the wide variability of T cell counts in the patients, statistical significance of the differences in T-reg numbers was not reached

  9. 600 250 . 500 200 400 150 Number of 300 Number of CD4+FOXP3+/ul CD4+CD25+FOXP3+/ul 100 200 50 100 0 0 CLL NPB CLL NPB The number of circulating T-regs is higher in CLL patients The actual number of T-regulatory cells is also increased . However, due to the wide variability of T cell counts in the patients, statistical significance of the differences in T-reg numbers was not reached.

  10. Figure 3: Comparison of CD4+FOXP3+ and CD4+CD25+FOXP3+ populations in ZAP-70 positive vs zap-70 negative patients CD4+FOXP3+ and CD4+CD25+FOXP3+ T-reg lymphocytes are upregulated in peripheral blood of ZAP-70 positive CLL patients compared to ZAP-70 negative patients. A A A A A A * * * * * * 12 12 12 12 12 12 25 25 25 25 25 25 . . . . . . * * * * * * . . . . . . 10 10 10 10 10 10 20 20 20 20 20 20 8 8 8 8 8 8 15 15 15 15 15 15 CD4+CD25+FOXP3+(%) CD4+CD25+FOXP3+(%) CD4+CD25+FOXP3+(%) CD4+CD25+FOXP3+(%) CD4+CD25+FOXP3+(%) CD4+CD25+FOXP3+(%) 6 6 6 6 6 6 CD4+FOXP3+ cells (%) CD4+FOXP3+ cells (%) CD4+FOXP3+ cells (%) CD4+FOXP3+ cells (%) CD4+FOXP3+ cells (%) CD4+FOXP3+ cells (%) 10 10 10 10 10 10 4 4 4 4 4 4 2 2 2 2 2 2 5 5 5 5 5 5 0 0 0 0 0 0 0 0 0 0 0 0 CLL CLL CLL CLL CLL CLL NPB NPB NPB NPB NPB NPB CLL CLL CLL CLL CLL CLL NPB NPB NPB NPB NPB NPB B B B B B B 600 600 600 600 600 600 250 250 250 250 250 250 . . . . . . 500 500 500 500 500 500 200 200 200 200 200 200 400 400 400 400 400 400 150 150 150 150 150 150 Number of Number of Number of Number of Number of Number of CD4+CD25+FOXP3+/ul CD4+CD25+FOXP3+/ul CD4+CD25+FOXP3+/ul CD4+CD25+FOXP3+/ul CD4+CD25+FOXP3+/ul CD4+CD25+FOXP3+/ul CD4+FOXP3+/ul CD4+FOXP3+/ul CD4+FOXP3+/ul CD4+FOXP3+/ul CD4+FOXP3+/ul CD4+FOXP3+/ul Number of Number of Number of Number of Number of Number of 300 300 300 300 300 300 100 100 100 100 100 100 200 200 200 200 200 200 50 50 50 50 50 50 100 100 100 100 100 100 0 0 0 0 0 0 0 0 0 0 0 0 Figure 2: CD4+FOXP3+ and CD4+CD25+FOXP3+ populations in T cells Blood from CLL Patients (n=35) and normal subjects (NPB) (n=12) were measured by flow cytometry. Figure 2a demonstrates that the relative proportion CD4+FOXP3+ and CD4+CD25+FOXP3+ T-regulatory lymphocytes is higher in CLL patients compared to T cells from normal controls (p<0.001). The actual number of T-regulatory cells is also increased as seen in figure 2B. However, due to the wide variability of T cell counts in the patients, statistical significance of the differences in T-reg numbers was not reached CLL CLL CLL CLL CLL CLL NPB NPB NPB NPB NPB NPB CLL CLL CLL CLL CLL CLL NPB NPB NPB NPB NPB NPB T-regs are upregulated in B-CLL ZAP-70 positive patients T-regs are upregulated in B-CLL ZAP-70 positive patients T-regs are upregulated in B-CLL ZAP-70 positive patients T-regs are upregulated in B-CLL ZAP-70 positive patients T-regs are upregulated in B-CLL ZAP-70 positive patients T-regs are upregulated in B-CLL ZAP-70 positive patients

  11. cycling Non-cycling No difference found in the proportion of cycling circulating T regs in ZAP-70 positive and negative patients

  12. No difference found in the viability of circulating CD4+FoxP3+ cells in ZAP-70 positive and negative patients

  13. No significant difference found in the cycling of circulating CD4+FoxP3+ cells in ZAP-70 positive and negative patients

  14. More cycling CD4+ T cells in ZAP-70+ patients

  15. Conclusions • Higher expression of ZAP-70 in both T and B cells of CLL patients indicates activation of all the lymphocytes. • The positive correlation found between ZAP-70 expression in B-CLL cells and T-cells in CLL may imply dual activation, involving T-cell/B -CLL cell interaction, suggesting that this crosstalk may affect the clinical outcome in these patients. • The proportion of circulating CD4+CD25+FOXP3+ T-reg cells is increased in CLL patients compared to normal

  16. We report for the first time that this is more prevalent in the patients with elevated ZAP-70 levels, showing a correlation between ZAP-70 expression and the proportion of T-regulatory cells. • Our findings also may suggest that the increase proportion of T-regs in CLL and particularly in ZAP-70+ CLL patients may contribute to a reduced anti-tumor response.

  17. THANKS Sigi Kay Yair Herishanu Ori Rogovski Rosa Ruchlemer Osnat Bairey Boris Tartakovsky Shoshy Baron Tal Ohayon Ella Napartstek Aaron Polliack

  18. CD127 • While the co-expression of CD4 and CD25 has become widely used as an indicator of T regs, this method of identification may also include cells without suppressive activity. • It has clearly been shown that CD4+CD25+ cells that have down-regulated the expression of CD127 are significantly enriched for the regulatory T population, as defined by expression of the T reg-specific transcription factor Foxp3 and the suppressive activity of these cells, in vitro • CD127 (IL-7Rα) complexes with CD132, also known as the common γ chain (γc), to form the multi-functional IL-7 receptor (IL-7R). CD127 is a type I glycoprotein with a molecular weight of 75-80 kDa and is expressed by immature B cells through the early pre-B stage, by thymocytes during several stages of their development, and on most mature T cells, with transient down-regulation upon activation. • Binding of IL-7 results in signal tansduction which occurs through several tyrosine kinase pathways including the Jak/STAT pathway. • IL-7 is indispensible for the development of lymphocytes, and the control of homeostatic proliferation of T-cells in the periphery. In addition, IL-7R signaling is known to be involved in the regulation of T cell receptor (TCR) locus rearrangment in γδ T cells • CD127 expression is down-regulated on CD4+CD25+ regulatory T cells .

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