Cómo elevar la eficiencia y rentabilidad
V. Determinantes de la eficiencia
Based on our previous fi nding that in vitro exposure of human PB B cells to RTX altered the B-T-cell interaction (16), we analyzed the intracellular cytokine production by T cells after stimulation with LN B cells. LN B cells from RTX-treated or untreated patients were added to allogeneic CFSE-labeled CD3+ T cells. After
7 days of culture, we measured the intracellular cytokine production of IL-2, IL-4, IL-17, and IFNγ by proliferating (CFSElow) T cells. Interestingly, the percentage of
CFSElow CD4+ T cells that produced IL-17 was lower upon stimulation with LN B
cells from RTX-treated patients as compared to LN B cells from untreated patients (2.6±0.6% versus 4.6±1.4%; P=0.003; Figure 4). µg /m l IgG1 0 200 IgG2 0 4 200 * IgG3 0 15 200 IgG4 0 1 200 IgM 0 30 200 ** IgA 0 200 IgE 0 0.1 200 Tx RTX- Tx RTX+
A
B
0 10 0.069 0.076 RTX - + αCD40 IL-21 medium - + A ID m RN A (f o ld c h a n g e )RTX aff ects the Ig-isotype production and enhances the mRNA expression of activation-induced deaminase (AID).
(A) Production of IgM, IgA, IgE, IgG1, IgG2, IgG3 and IgG4 in the supernatant by lymph node (LN) B cells of renal transplant recipients after ex vivo stimulation with αCD40 mAb and IL-21 for 8 days. Shown are the results for RTX-treated (Tx RTX+; n=4; grey bars) and untreated patients (Tx RTX-; n=6; white bars); *P<0.05, **P<0.01. (B) . Real-time quantitative PCR of the AID mRNA expression in human splenic CD19+ B cells after 4 days of culture
in the presence or absence of RTX with or without addition of 1 μg/ml αCD40 mAb and 50 ng/ml IL-21. Results were normalized using the human HPRT1 endogenous control and the expression in unstimulated control condition was set at 1.0. Shown are the relative fold changes in B cells obtained from 3 diff erent donors.
A
IL-2 IL-4 CD4 + CD8 + IL-17 IFN JB
IL-2 0 75 0 75 IL-4 0 30 0 30 IL-17 0 10**
0 10 IFNJ 0 80 0 80 % positive p ro lif e ra te d CD4 + T c e lls 14 2.5 31 13 2.4 43 17 1.7 37 18 3.2 35 12 1.0 5.2 70 1.8 0.5 18 0.7 1.8 76 2.7 0.5 Tx RTX+ Tx RTX- Tx RTX- Tx RTX+ P ro lif e ra te d % positive p ro lif e ra te d CD8 + T c e lls - + Tx RTX - + - + - + - + Tx RTX - + - + - +Intracellular cytokine production by T cells upon stimulation with lymph node (LN) B cells from renal transplant recipients
(A) The intracellular cytokine production of IL-2, IL-4, IL-17 and IFNγ within the proliferated CD4+ (upper panel) and CD8+ (lower panel) T-cell population was analyzed on day 7 of culture. (B) Shown are the percentages of the cytokines produced by the proliferated CD4+ and CD8+ T cells after stimulation with CD19+ LN B cells of RTX-treated (Tx RTX+; grey box- plots) and untreated (Tx RTX-; white box-plots) from 8 to 12 diff erent donors; Signifi cant diff erences are indicated by asterisks: **P<0.01.
5
Discussion
In this study, we showed that at four weeks after administration of a single dose of RTX there is a nearly complete B-cell depletion in PB, but not in LNs of renal transplant recipients. Exposure of human B cells to RTX resulted in a lower percentage of naive (IgD+CD27-) and a higher percentage of switched memory
(IgD-CD27+) B cells. Concomitantly, there was a change in the production of Ig-
subclasses after ex vivo stimulation with αCD40 mAb and IL-21. Finally, CD4+ T cells
showed lower IL-17 production upon stimulation with LN B cells from RTX-treated patients as compared to LN B cells from untreated patients in an in vitro stimulation assay.
The observed persistence of B cells in lymphoid tissues after RTX treatment, has been reported by others before (8, 13, 15, 22-26). However, in contrast to our fi nding of unaff ected numbers of LN B cells, these studies described at least some degree of B-cell reduction in synovial tissue, spleen or lymph nodes. Renal transplant recipients treated with RTX for antibody-mediated rejection, showed a complete depletion of circulating B cells with a 50% reduction of B cells in tertiary lymphoid organs (24). RTX therapy of patients with autoimmune thrombocytopenia resulted in complete B-cell depletion in PB and in a reduction in SPL B cells (13). In patients with rheumatoid arthritis, the number of synovial and bone marrow B cells were decreased after RTX treatment, but there was no complete depletion (8, 22, 23). Genberg et al. (15) reported an average of 50% reduction of the percentage of CD19+ B cells in LNs after treatment with RTX as induction therapy
in renal transplant patients. The variation in B-cell depletion might be explained by diff erences in RTX treatment regimen, choice of immunosuppressive agents, and heterogeneity of the patient populations. The majority of the patients were treated with at least two doses of 375 mg/m2, or 1000 mg for RA patients, with diff erent
time intervals before obtaining secondary lymphoid tissue (8, 22-24), while our patients only received a single dose of 375 mg/m2 four weeks before collecting the
lymph nodes. Notably, there is a trend to use less intensive dosing regimens of RTX for auto-immune diseases, and like in our study, a single dose of RTX is currently applied in several conditions (27, 28).
Why there is such a wide discrepancy between depletion of B cells in PB and LN after RTX treatment is an intriguing question. A possible explanation could be an inability of RTX to reach the B cells in the LNs. However, we clearly demonstrate that LN B cells that remain after RTX treatment were CD19+CD20-, probably due
to modulation of CD20 by the binding of RTX (29-32), which indicates that the LN B cells had been exposed to RTX. Exhaustion of the complement system is another possible explanation, which could not be further addressed in the current study (33). Finally, a high concentration of the B-cell activation factor (BAFF) in LNs, might favor the survival of B cells (24).
Although treatment with RTX had minimal eff ect on the proportion of LN B cells, it induced a striking population shift from a naive to a switched memory
phenotype, which can be the result of two non mutually exclusive processes. First, RTX might selectively deplete and/or inhibit naive but not memory B cells leading to a relative increase of memory B cells. Second, binding of RTX to naive B cells might induce diff erentiation into memory B cells. Using an in vitro nondepleting B-cell stimulation model, we have previously shown that RTX inhibited the proliferation of CD27 naive, but not of CD27+ memory B cells (16). In agreement
with these fi ndings, it has been reported that RTX administered in vivo as part of a desensitization protocol in kidney transplantation decreased the number of splenic naive B cells, but had no eff ect on the number of CD27+ memory B cells
(14). In general, as compared to naive cells, memory B cells are characterized by a high expression of activation and prosurvival molecules, which allows them to respond quickly during an immune response and to persist for long time (34). These properties may also result in a relative resistance of memory B cells to depletion by RTX. In summary, these observations suggest that selective inhibition of naive, but not of memory B cells can indeed contribute to the observed shift from a naive to a switched memory B-cell phenotype. Next to its eff ect on the phenotype of B cells, RTX also aff ected the Ig-isotype production with a decrease in IgM production. Combined with a trend towards increased AID mRNA expression, this suggests that the population shift might be accompanied by class-switch recombination (CSR). Taken together, the LN B-cell population shift from a naive to a switched memory phenotype after treatment with RTX might be due to both a selective depletion of naive B cells and a direct eff ect on signaling cascades resulting in CSR and transition from a naive to a switched memory phenotype.
Treatment with RTX can be eff ective in chronic infl ammatory diseases, such as rheumatoid arthritis (8, 35) and multiple sclerosis (9). The improvement of these conditions by RTX has been associated with a reduced Th17 response (35). In the current study, we found that LN B cells obtained from RTX treated patients resulted in a weaker Th17 response when used as stimulators in an allogeneic mixed lymphocyte reaction. Likewise, it has been observed that RTX reduces the IL-17 production by PBMCs after stimulation with Candida albicans in vitro (35). Moreover, the Th17-cell frequency has been shown to correlate with the frequency of both switched memory B cells and serum BAFF levels (36). These fi ndings suggest a close relationship between Th17-cell homeostasis and B-cell maturation which can be aff ected by RTX. In a previous study, we observed that B cells that were treated with RTX in vitro, were able to induce a Th2-like shift in proliferating T cells. However, it should be noted that in the present study we investigated lymph node derived B cells, whereas in the previous study peripheral blood B cells were used. The B-cell subset distribution between the two is clearly diff erent, which might explain a diff erent T-cell response regardless of any further treatment (e.g. RTX), as
5
was observed by comparing CD27- and CD27+ B cells to stimulate allogeneic CD4+
Tcells (37).
We acknowledge that the changes in the B-cell repertoire that we observed at four weeks after RTX treatment, could theoretically also have been caused by steroids that were given at the time of RTX administration, or by the treatment with immunosuppressive drugs (tacrolimus, mycophenolate mofetil, and prednisone from 2 weeks before transplantation), or IVIG (1 day before transplantation). However, none of these drugs directly targets B cells. It has been shown that IVIG does not aff ect B-cell responses in vitro (38), and treatment with IVIG as part of desensitization protocols had no eff ect on B cells in the spleen (14). In SLE patients treated with mycophenolate mofetil, the number and phenotype of B-cells were similar to that in controls without immunosuppressive therapy (39). Tacrolimus had minimal eff ect on B-cell proliferation and survival after stimulation in vitro (38). Finally, treatment of healthy volunteers with a single dose of prednisolone (30 mg) resulted in a decrease of the absolute counts of total lymphocytes, B cells, T cells and NK cells, but the counts returned to baseline levels within 13 to 26 hours after administration (40). Moreover, the only diff erences between RTX-treated and untreated patients were those found in the B-cell compartment. The fact that LN B cells from treated patients were negative for CD20, strongly indicates that RTX was involved in the observed changes in B-cell phenotype and function.
In summary, we have demonstrated that a single dose of RTX depletes B cells in PB, but not in LNs at four weeks after administration. Exposure of human B cells to RTX results in a relative increase of B cells with a switched memory phenotype.
Consequently, the eff ect of rituximab on the immune response will not only be determined by the extent of B-cell depletion, but also by the functional properties of the remaining B cells.
Acknowledgments
This study was supported by research funding from the Dutch Kidney Foundation (nr C09-2301). The authors thank L. Boon (Bioceros, Utrecht) for kindly providing the αCD40 mAb. Technical support was kindly provided by F.W. Preijers and H. Tijssen (Radboud University Medical Centre). We also thank M.C. Warlé (Radboud University Medical Center), M.M. Idu and E.B. Remmerswaal (Academic Medical Centre, Amsterdam) for harvesting the lymph nodes.
References
1. Jordan SC, Kahwaji J, Toyoda M, Vo A. B-cell immunotherapeutics: emerging roles in solid organ transplantation. Curr Opin Organ Transplant. 2011;16(4):416- 24.
2. Iwata Y, Matsushita T, Horikawa M, et al. Characterization of a rare IL-10- competent B-cell subset in man that parallels mouse regulatory B10 cells. Blood. 2010;117(2):530-41.
3. Blair PA, Norena LY, Flores-Borja F, et al. CD19(+)CD24(hi)CD38(hi) B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic Lupus Erythematosus patients. Immunity. 2010;32(1):129-40.
4. Newell KA, Asare A, Kirk AD, et al. Identifi cation of a B cell signature
associated with renal transplant
tolerance in humans. JClinInvest.
2010;120(6):1836-47.
5. Chow KU, Sommerlad WD, Boehrer S, et al. Anti-CD20 antibody (IDEC-C2B8, rituximab) enhances effi cacy of cytotoxic drugs on neoplastic lymphocytes in vitro: role of cytokines, complement,
and caspases. Haematologica.
2002;87(1):33-43.
6. Glennie MJ, French RR, Cragg MS,
Taylor RP. Mechanisms of killing by anti- CD20 monoclonal antibodies. Molecular Immunology. 2007;44(16):3823-37. 7. Dorner T, Isenberg D, Jayne D, Wiendl
H, Zillikens D, Burmester G. Current status on B-cell depletion therapy in autoimmune diseases other than rheumatoid arthritis. AutoimmunRev. 2009;9(2):82-9.
8. Thurlings RM, Vos K, Wijbrandts CA, Zwinderman AH, Gerlag DM, Tak PP.
Synovial tissue response to rituximab: mechanism of action and identifi cation of biomarkers of response. Annals of the Rheumatic Diseases. 2008;67(7):917-25. 9. Bar-Or A, Fawaz L, Fan B, et al. Abnormal
B-cell cytokine responses a trigger of T-cell-mediated disease in MS? Annals of Neurology. 2010;67(4):452-61.
10. Fuchinoue S, Ishii Y, Sawada T, et al. The 5-year outcome of ABO- incompatible kidney transplantation with rituximab induction. Transplantation. 2011;91(8):853-7.
11. Vo AA, Peng A, Toyoda M, et al. Use of intravenous immune globulin and rituximab for desensitization of highly
HLA-sensitized patients awaiting
kidney transplantation. Transplantation. 2010;89(9):1095-102.
12. Faguer S, Kamar N, Guilbeaud-Frugier C, et al. Rituximab therapy for acute humoral rejection after kidney transplantation. Transplantation. 2007;83(9):1277-80. 13. Audia S, Samson M, Guy J, et
al. Immunological eff ects of
rituximab on the human spleen in
immune thrombocytopenia. Blood.
2011;118(16):4394-400.
14. Ramos EJ, Pollinger HS, Stegall MD, Gloor JM, Dogan A, Grande JP. The eff ect of desensitization protocols on human splenic B-cell populations in vivo. AmJTransplant. 2007;7(2):402-7. 15. Genberg H, Hansson A, Wernerson A,
Wennberg L, Tyden G. Pharmacodynamics of rituximab in kidney allotransplantation. AmJTransplant. 2006;6(10):2418-28. 16. Kamburova EG, Koenen HJ, Boon L,
Hilbrands LB, Joosten I. In vitro eff ects of rituximab on the proliferation, activation
5
and diff erentiation of human B cells. Am J Transplant. 2012;12(2):341-50.
17. Tyden G, Kumlien G, Genberg H,
Sandberg J, Lundgren T, Fehrman I. ABO incompatible kidney transplantations without splenectomy, using antigen-
specifi c immunoadsorption and
rituximab. AmJTransplant. 2005;5(1):145- 8.
18. Koenen HJ, Smeets RL, Vink PM, van Rijssen E, Boots AM, Joosten I. Human CD25highFoxp3pos regulatory T cells diff erentiate into IL-17-producing cells. Blood. 2008;112(6):2340-52.
19. Cragg MS, Bayne MB, Tutt AL, et al. A new anti-idiotype antibody capable of binding rituximab on the surface of lymphoma cells. Blood. 2004;104(8):2540-2. 20. Avery DT, Bryant VL, Ma CS, de Waal
MR, Tangye SG. IL-21-induced isotype switching to IgG and IgA by human naive B cells is diff erentially regulated by IL-4. JImmunol. 2008;181(3):1767-79.
21. Honjo T, Kinoshita K, Muramatsu M. Molecular mechanism of class switch recombination: linkage with somatic hypermutation. Annu Rev Immunol. 2002;20:165-96.
22. Nakou M, Sidiropoulos P, Bertsias G, et al. Rituximab therapy reduces activated B cells in both the peripheral blood and bone marrow of patients with rheumatoid arthritis: depletion of memory B cells
correlates with clinical response.
Arthritis ResTher. 2009;11(4):R131. 23. Vos K, Thurlings RM, Wijbrandts CA, van
SD, Gerlag DM, Tak PP. Early eff ects of rituximab on the synovial cell infi ltrate in patients with rheumatoid arthritis. Arthritis Rheum. 2007;56(3):772-8. 24. Thaunat O, Patey N, Gautreau C, et
al. B cell survival in intragraft tertiary
lymphoid organs after rituximab therapy. Transplantation. 2008;85(11):1648-53. 25. Barbera-Guillem E, Nelson MB, Barr B,
et al. B lymphocyte pathology in human colorectal cancer. Experimental and clinical therapeutic eff ects of partial B cell depletion. Cancer Immunol Immunother. 2000;48(10):541-9. 26. Maloney DG, Liles TM, Czerwinski DK, et
al. Phase I clinical trial using escalating single-dose infusion of chimeric anti- CD20 monoclonal antibody (IDEC- C2B8) in patients with recurrent B-cell lymphoma. Blood. 1994;84(8):2457-66. 27. Sugiura H, Takei T, Itabashi M, et al. Eff ect
of single-dose rituximab on primary glomerular diseases. Nephron Clin Pract. 2011;117(2):c98-105.
28. Wermke M, von Bonin M, Gehrisch S, Siegert G, Ehninger G, Platzbecker U. Successful eradication of acquired
factor-VIII-inhibitor using single
low-dose rituximab. Haematologica.
2010;95(3):521-2.
29. Jilani I, O’Brien S, Manshuri T, et
al. Transient down-modulation of
CD20 by rituximab in patients with chronic lymphocytic leukemia. Blood. 2003;102(10):3514-20.
30. Cragg MS, Bayne MC, Illidge TM, Valerius T, Johnson PW, Glennie MJ. Apparent modulation of CD20 by rituximab:
an alternative explanation. Blood.
2004;103(10):3989-90; author reply 90- 1.
31. Beum PV, Peek EM, Lindorfer MA, et al. Loss of CD20 and bound CD20 antibody from opsonized B cells occurs more rapidly because of trogocytosis mediated by Fc receptor-expressing eff ector cells than direct internalization by the B cells. J Immunol. 2011;187(6):3438-47.
32. Beers SA, French RR, Chan HT, et al. Antigenic modulation limits the effi cacy of anti-CD20 antibodies: implications
for antibody selection. Blood.
2010;115(25):5191-201.
33. Kennedy AD, Beum PV, Solga MD, et al. Rituximab infusion promotes rapid complement depletion and acute CD20 loss in chronic lymphocytic leukemia. JImmunol. 2004;172(5):3280-8. 34. Good KL, Avery DT, Tangye SG. Resting
human memory B cells are intrinsically programmed for enhanced survival and responsiveness to diverse stimuli compared to naive B cells. J Immunol. 2009;182(2):890-901.
35. van de Veerdonk FL, Lauwerys B, Marijnissen RJ, et al. The anti-CD20
antibody rituximab reduces the
Th17 cell response. Arthritis Rheum. 2011;63(6):1507-16.
36. Barbosa RR, Silva SP, Silva SL, et al. Primary B-cell defi ciencies reveal a link between
human IL-17-producing CD4 T-cell
homeostasis and B-cell diff erentiation. PLoS One. 2011;6(8):e22848.
37. Carpenter EL, Mick R, Rech AJ, et al. Collapse of the CD27+ B-cell compartment associated with systemic plasmacytosis in patients with advanced melanoma and other cancers. Clinical Cancer Research. 2009;15(13):4277-87. 38. Heidt S, Roelen DL, Eijsink C, Eikmans
M, Claas FH, Mulder A. Intravenous immunoglobulin preparations have no direct eff ect on B cell proliferation and immunoglobulin production. Clin Exp Immunol. 2009;158(1):99-105.
39. Eickenberg S, Mickholz E, Jung E, Nofer JR, Pavenstadt HJ, Jacobi AM. Mycophenolic acid counteracts B cell proliferation and plasmablast
formation in patients with systemic lupus erythematosus. Arthritis Res Ther. 2012;14(3):R110.
40. Fukuda R, Ichikawa Y, Takaya M, Ogawa Y, Masumoto A. Circadian variations and
prednisolone-induced alterations of
circulating lymphocyte subsets in man. Intern Med. 1994;33(12):733-8.
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Tx RTX+ anti-RTX Tx RTX- CD45+ lymphocytes CD20 isotype anti-RTX CD19 - RTXLymph nodes Spleen
+ RTX
Anti-RTX antibody MB2A4 for the detection of RTX on the surface of cells.
Lymph node (LN) cells from RTX-treated (Tx RTX+) and untreated patients (Tx RTX-) were