CHANGES IN THE SUBPOPULATION COMPOSITION AND IMMUNOPHENOTYPIC CHARACTERISTICS OF NORMAL LYMPHOCYTES IN THE BLOOD OF PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA IN THE DYNAMICS OF IMMUNOCHEMOTHERAPY

Immunological changes in subpopulations of normal lymphocytes in the blood of patients with chronic lymphocytic leukemia (CLL) may play a negative role in the progression of the disease. The aim - to study the effect of immunochemotherapy on the subpopulation composition and immunophenotypic characteristics of normal lymphocytes in the blood of patients with CLL. The study included 37 men, 25 women with CLL in stages B, C according to Binet, median age 64 [50; 71] years old, who received 6 cycles of immunochemotherapy: RB (Rituximab+Bendamustine) or FCR (Rituximab+Fludarabine+ Cyclophosphamide). The subpopulation composition of polyclonal B-, T- (T helper cells, T regulatory cells, T cytotoxic cells), NK-lymphocytes, as well as the expression of PD1, PD-L1, LAG3 were studied in the blood of patients before and after treatment. The studies were performed using the method of flow cytometry (Navios 10/3, Beckman Coulter, USA). The statistical analysis is performed in Statistica 13.0. The study showed that all patients with CLL had significant quantitative and functional changes in the subpopulation composition of B-, T-, and NK-lymphocytes before and after therapy. The proportion of normal polyclonal B cells among all lymphocytes before treatment is reduced and is characterized by PD1 expression. The therapy leads to a partial restoration of the population of normal B-lymphocytes, which do not show expression of PD1, PD-L1, LAG3. Before and after treatment, CD4+ and CD8+ T cells show the highest expression of PD1, which indicates a violation of their antitumor functions. After treatment, the T-lymphocyte microenvironment tends to recover due to an increase in their number. However, the preservation of PD1 expression on T-cells apparently prevents the complete restoration of their functional activity, which, along with an increase in the content of T-regulatory cells and the preservation of PD1 and LAG3 expression on NK-lymphocytes, helps to suppress the antitumor immune response and reduce the possibility of long-term remission. In this regard, monitoring of immunological parameters during therapy is useful for detecting the degree of dysregulation of the immune response and, thus, for timely provision along with antitumor therapy and corrective immunotherapy.

1. Klyuchagina Yu.I., Sokolova Z.A., Baryshnikova M.A. The role of the PD1 receptor and its ligands PD-L1 and PDL-2 in tumor immunotherapy. Oncopediatrics, 2017, Vol. 4, no. 1, pp. 49-55 (In Russ.).

2. Oleinik E.K., Shibaev M.I., Ignatiev K.S., Oleinik V.M., Zhulai G.A. Tumor microenvironment: the formation of the immune profile. Medical Immunology (Russia), 2020, Vol. 22, no.2, p.p. 207-220. (In Russ.).

3. Khaidukov S.V., Zurochka A.V., Totolyan A.A., Chereshnev V.A. Basic and small populations of human peripheral blood lymphocytes and their normative values (by multicolored cytometric analysis), Medical immunology, 2014, Vol.11, pp. 227-238 (In Russ.).

4. Ali A., Mahla S.B., Reza V., Hossein A., Bahareh K., Mohammad H., Fatemeh S., Mostafa A.B., Leili R. MicroRNAs: Potential prognostic and theranostic biomarkers in chronic lymphocytic leukemia. EJHaem., 2024, Vol. 5, no. 1, p.p. 191-205.

5. Andrews L.P., Marciscano A.E., Drake C.G., Vignali D.A. LAG3 (CD223) as a cancer immunotherapy target. Immunological reviews, 2017, Vol. 276, no. 1, p.p. 80-96.

6. Augé H., Notarantonio A.B., Morizot R., Quinquenel A., Fornecker L.M., Hergalant S., Feugier P., Broséus J. Microenvironment Remodeling and Subsequent Clinical Implications in Diffuse Large B-Cell Histologic Variant of Richter Syndrome. Front Immunol., 2020, Vol. 11, p. 594841.

7. Bandini S., Ulivi P., Rossi T. Extracellular Vesicles, Circulating Tumor Cells, and Immune Checkpoint Inhibitors: Hints and Promises, 2024, Vol. 13, no.4, p. 337.

8. Buechele C., Baessler T., Wirths S., Schmohl J.U., Schmiedel B.J., Salih H.R. Glucocorticoid-induced TNFR-related protein (GITR) ligand modulates cytokine release and NK cell reactivity in chronic lymphocytic leukemia (CLL). Leukemia, 2012, Vol. 26, no. 5, p.p. 991–1000.

9. Catakovic K., Klieser E., Neureiter D., Geisberger R. T cell exhaustion: from pathophysiological basics to tumor immunotherapy. Cell Commun. Signal., 2017, Vol. 15, no. 1, p. 1.

10. Chihara N., Madi A., Kondo T., Zhang H., Acharya N., Singer M., Nyman J., Marjanovic N.D., Kowalczyk M.S., Wang C., Kurtulus S., Law T., Etminan Y., Nevin J., Buckley C.D., Burkett P.R., Buenrostro J.D., Rozenblatt-Rosen O., Anderson A.C., Regev A., Kuchroo V.K. Induction and transcriptional regulation of the co-inhibitory gene module in T cells. Nature, 2018, Vol. 558, no. 7710, p.p. 454-459.

11. Criado I., Rodríguez-Caballero A., Gutiérrez M.L., Pedreira C.E., Alcoceba M., Nieto W., Teodosio C., Bárcena P., Romero A., Fernández-Navarro P., González M., Almeida J., Orfao A. Primary Health Care Group of Salamanca for the Study of MBL. Low-count monoclonal B-cell lymphocytosis persists after seven years of follow up and is associated with a poorer outcome. Haematologica, 2018, Vol. 103, no. 7, p.p. 1198-1208.

12. de Weerdt I., Hofland T., de Boer R., Dobber J.A., Dubois J., van Nieuwenhuize D., Mobasher M., de Boer F., Hoogendoorn M., Velders G.A., van der Klift M., Remmerswaal E.B.M., Bemelman F.J., Niemann C.U., Kersting S., Levin M.D., Eldering E., Tonino S.H., Kater A.P. Distinct immune composition in lymph node and peripheral blood of CLL patients is reshaped during venetoclax treatment. Blood Adv., 2019, Vol. 3, no. 17, p.p. 2642-2652.

13. Elston L., Fegan C., Hills R., Hashimdeen S.S., Walsby E., Henley P., Pepper C., Man S. Increased frequency of CD4+ PD-1+ HLA-DR+ T cells is associated with disease progression in CLL. Br J Haematol., 2020, Vol. 188, no. 6, p.p. 872–880.

14. Fisher J.G., Doyle A.D.P., Graham L.V., Sonar S., Sale B., Henderson I., Del Rio L., Johnson P.W.M., Landesman Y., Cragg M.S., Forconi F., Walker C.J., Khakoo S.I., Blunt M.D. XPO1 inhibition sensitises CLL cells to NK cell mediated cytotoxicity and overcomes HLA-E expression. Leukemia, 2023, Vol. 37, no. 10, p.p. 2036-2049.

15. Forconi F., Moss P. Perturbation of the normal immune system in patients with CLL. Blood, 2015, Vol. 126, no. 5, p.p. 573–581.

16. Görgün G., Holderried T.A., Zahrieh D., Neuberg D., Gribben J.G. Chronic lymphocytic leukemia cells induce changes in gene expression of CD4 and CD8 T cells. J. Clin. Invest., 2005, Vol. 115, no. 7, p.p. 1797-1805.

17. Graydon C.G., Mohideen S., Fowke K.R. LAG3's Enigmatic Mechanism of Action. Frontiers in immunology, 2021, Vol. 11, p. 615317.

18. Griggio V., Perutelli F., Salvetti C., Boccellato E., Boccadoro M., Vitale C., Coscia M. Immune Dysfunctions and Immune-Based Therapeutic Interventions in Chronic Lymphocytic Leukemia. Front Immunol., 2020, Vol. 11, p. 594556.

19. Grioni M., Brevi A., Cattaneo E., Rovida A., Bordini J., Bertilaccio M.T.S., Ponzoni M., Casorati G., Dellabona P., Ghia P., Bellone M., Calcinotto A. CD4+ T cells sustain aggressive chronic lymphocytic leukemia in Eμ-TCL1 mice through a CD40L-independent mechanism. Blood Adv., 2021, Vol. 5, no. 14, p.p. 2817–2828.

20. Guo Y., Ji X., Liu J., Fan D., Zhou Q., Chen C., Wang W., Wang G., Wang H., Yuan W., Ji Z., Sun Z. Effects of exosomes on pre-metastatic niche formation in tumors, Mol. Cancer, 2019, Vol. 18, no. 1, p. 39.

21. Hadadi L., Hafezi M., Amirzargar A.A., Sharifian R.A., Abediankenari S., Asgarian-Omran H. Dysregulated Expression of Tim-3 and NKp30 Receptors on NK Cells of Patients with Chronic Lymphocytic Leukemia. Oncol. Res. Treat., 2019, Vol. 42, no. 4, p.p. 197-203.

22. Kretz-Rommel A., Qin F., Dakappagari N., Ravey E.P., McWhirter J., Oltean D., Frederickson S., Maruyama T., Wild M.A., Nolan M.J., Wu D., Springhorn J., Bowdish K.S. CD200 expression on tumor cells suppresses antitumor immunity: new approaches to cancer immunotherapy. J. Immunol., 2007, Vol. 178, no. 9, p.p. 5595-5605.

23. Lanasa M.C., Allgood S.D., Bond K.M., Gockerman J.P., Levesque M.C., Weinberg JB. Oligoclonal TRBV gene usage among CD8(+) T cells in monoclonal B lymphocytosis and CLL. Br J Haematol., 2009, Vol. 145, no. 4, p.p. 535-537.

24. Laumont C. M., Brad H. N. B cells in the tumor microenvironment: Multi-faceted organizers, regulators, and effectors of anti-tumor immunity. Cancer cell, 2023, Vol. 41, no. 3, p.p. 466-489.

25. Marin-Acevedo J.A., Dholaria B., Soyano A.E., Knutson K.L., Chumsri S., Lou Y. Next generation of immune checkpoint therapy in cancer: new developments and challenges. J. Hematol. Oncol., 2018, Vol. 11, no.1, p. 39.

26. Maruhashi T., Okazaki I.M., Sugiura D., Takahashi S., Maeda T.K., Shimizu K., Okazaki T. LAG-3 inhibits the activation of CD4+ T cells that recognize stable pMHCII through its conformation-dependent recognition of pMHCII. Nat. Immunol., 2018, Vol. 19, no.12, p.p. 1415-1426.

27. Nunes C., Wong R., Mason M., Fegan C., Man S., Pepper C. Expansion of a CD8(+)PD-1(+) replicative senescence phenotype in early stage CLL patients is associated with inverted CD4:CD8 ratios and disease progression. Clin. Cancer Res., 2012, Vol. 18, no. 3, p.p. 678-687.

28. Pallasch C.P., Ulbrich S., Brinker R., Hallek M., Uger R.A., Wendtner C.M. Disruption of T cell suppression in chronic lymphocytic leukemia by CD200 blockade. Leuk Res., 2009, Vol. 33, no. 3, p.p. 460-464.

29. Palma M., Gentilcore G., Heimersson K., Mozaffari F., Näsman-Glaser B., Young E., Rosenquist R., Hansson L., Österborg A., Mellstedt H. T cells in chronic lymphocytic leukemia display dysregulated expression of immune checkpoints and activation markers. Haematologica, 2017, Vol. 102, no. 3, p.p. 562-572.

30. Pang N., Alimu X., Chen R., Muhashi M., Ma J., Chen G., Zhao F., Wang L., Qu J., Ding J. Activated Galectin-9/Tim3 promotes Treg and suppresses Th1 effector function in chronic lymphocytic leukemia. FASEB J., 2021, Vol. 35, no. 7, Art. e21556.

31. Perutelli F., Jones R., Griggio V., Vitale C., Coscia M. Immunotherapeutic Strategies in Chronic Lymphocytic Leukemia: Advances and Challenges. Front Oncol., 2022, Vol. 12, p. 837531.

32. Purroy N., Wu C.J. Coevolution of Leukemia and Host Immune Cells in Chronic Lymphocytic Leukemia. Cold Spring Harbor perspectives in medicine, 2017, Vol. 7, no. 4, p.p. 1-19.

33. Ramsay A.G., Gribben J.G. Vaccine therapy and chronic lymphocytic leukaemia. Best practice and research. Clinical haematology, 2008, Vol. 21, no. 3, p.p. 421-436.

34. Roessner P.M., Llaó Cid L., Lupar E., Roider T., Bordas M., Schifflers C., Arseni L., Gaupel A.C., Kilpert F., Krötschel M., Arnold S.J., Sellner L., Colomer D., Stilgenbauer S., Dietrich S., Lichter P., Izcue A., Seiffert M. EOMES and IL-10 regulate antitumor activity of T regulatory type 1 CD4+ T cells in chronic lymphocytic leukemia. Leukemia, 2021, Vol. 35, no. 8, p.p. 2311-2324.

35. Roessner P.M., Seiffert M. T-cells in chronic lymphocytic leukemia: Guardians or drivers of disease? Leukemia, 2020, Vol. 34, no. 8, p.p. 2012-2024.

36. Sandova V., Pavlasova G.M., Seda V., Cerna K.A., Sharma S., Palusova V., Brychtova Y., Pospisilova S., Fernandes S.M., Panovska A., Doubek M., Davids M.S., Brown J.R., Mayer J., Mraz M. IL4-STAT6 signaling induces CD20 in chronic lymphocytic leukemia and this axis is repressed by PI3Kδ inhibitor idelalisib. Haematologica, 2021, Vol. 106, no. 11, p.p. 2995-2999.

37. Sordo-Bahamonde C., Lorenzo-Herrero S., Gonzalez-Rodriguez A.P., R Payer Á., González-García E., López-Soto A., Gonzalez S. BTLA/HVEM Axis Induces NK Cell Immunosuppression and Poor Outcome in Chronic Lymphocytic Leukemia. Cancers (Basel), 2021, Vol. 13, no. 8, p. 1766.

38. Sordo-Bahamonde C., Lorenzo-Herrero S., González-Rodríguez A.P., Payer Á.R., González-García E., López-Soto A., Gonzalez S. LAG-3 Blockade with Relatlimab (BMS-986016) Restores Anti-Leukemic Responses in Chronic Lymphocytic Leukemia. Cancers (Basel), 2021, Vol. 13, no. 9, p. 2112.

39. Thieu V.T., Nguyen E.T., McCarthy B.P., Bruns H.A., Kapur R., Chang C.H., Kaplan M.H. IL-4-stimulated NF-kappaB activity is required for Stat6 DNA binding. J. Leukoc. Biol., 2007, Vol. 82, no. 2, p.p. 370-379.

40. Van Attekum M.H., Eldering E., Kater A.P. Chronic lymphocytic leukemia cells are active participants in microenvironmental cross-talk. Haematologica, 2017, Vol. 102, no. 9, p.p. 1469-1476.

41. Wherry E.J., Kurachi M. Molecular and cellular insights into T cell exhaustion. Nature reviews. Immunology, 2015, Vol. 15, no. 8, p.p. 486-499.

42. Yousefi M., Movassaghpour A.A., Shamsasenjan K., Ghalamfarsa G., Sadreddini S., Jadidi-Niaragh F., Hojjat-Farsangi M. The skewed balance between Tregs and Th17 in chronic lymphocytic leukemia. Future Oncol., 2015, Vol. 11, no.10, p.p. 1567-1582.

43. Zheng Z., Liu J., Ma J., Kang R., Liu Z., Yu J. Advances in new targets for immunotherapy of small cell lung cancer. Thorac. Cancer, 2024, Vol. 15, no. 1, p.p. 3-14.