論文の公開元へB cell depletion with anti-CD20 mAb exacerbates anti-donor CD4+ T cell responses in highly sensitized transplant recipients
概要
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B cell depletion with anti‑CD20
mAb exacerbates anti‑donor CD4+
T cell responses in highly sensitized
transplant recipients
Asuka Tanaka, Kentaro Ide*, Yuka Tanaka, Masahiro Ohira, Hiroyuki Tahara &
Hideki Ohdan*
Pretransplant desensitization with rituximab has been applied to preformed donor-specific antihuman leukocyte antigen antibody (DSA)-positive recipients for elimination of preformed DSA. We
investigated the impact of pretransplant desensitization with rituximab on anti-donor T cell responses
in DSA-positive transplant recipients. To monitor the patients’ immune status, mixed lymphocyte
reaction (MLR) assays were performed before and after desensitization with rituximab. Two weeks
after rituximab administration, the stimulation index (SI) of anti-donor CD4+ T cells was significantly
higher in the DSA-positive recipients than in the DSA-negative recipients. To investigate the
mechanisms of anti-donor hyper responses of CD4+ T cells after B cell depletion, highly sensitized mice
models were injected with anti-CD20 mAb to eliminate B cells. Consistent with clinical observations,
the SI values of anti-donor CD4+ T cells were significantly increased after anti-CD20 mAb injection
in the sensitized mice models. Adding B cells isolated from untreated sensitized mice to MLR
significantly inhibited the enhancement of anti-donor CD4+ T cell response. The depletion of the CD5+
B cell subset, which exclusively included IL-10-positive cells, from the additive B cells abrogated such
inhibitory effects. These findings demonstrate that IL-10+ CD5+ B cells suppress the excessive response
of anti-donor CD4+ T cells responses in sensitized recipients.
It has been reported that preformed DSAs are associated with detrimental effects in transplant r ecipients1. In
kidney transplantation, preformed DSAs, regardless of whether they are HLA-class I or II, can trigger hyper
acute, accelerated acute, and early acute antibody-mediated rejection (ABMR)2, 3. Acute and chronic ABMR are
major factors of renal allograft dysfunction and loss4. In contrast to other types of organ transplantation, liver
transplant (LT) recipients are considered resistant to ABMR caused by D
SAs5, 6. However, recent studies suggest
that liver allografts have a relative resistance to ABMR, but specific situations can override the liver’s natural
resistance and defense m
echanisms7, 8. In detail, preformed class I and/or II DSAs, detected by single-antigen
bead analyses, with a mean fluorescence intensity (MFI) ≥ 5000, are independently correlated with a greater risk
of death9. Furthermore, preformed class I DSA (MFI ≥ 5000) also disproportionately affects patients transplanted
with a high calculated model for end-stage liver disease (MELD) score and those who received lower quality
organs [donor risk index (DRI) > 1.5]10.
To eliminate the preformed DSAs, several desensitization protocols comprising plasmapheresis, splenectomy,
intravenous immunoglobulins (IVIG), and/or anti-B cell immunosuppressant treatment in the recipients have
been reported for successful transplant11–13. Among these, B cell depletion with the prophylactic use of rituximab
has also been applied in preformed DSA-positive r ecipients14, 15. However, depletion of B cells may influence T
cell allo-responses because B cells are effective antigen-presenting cells that can activate allo-specific T c ells16.
The cytokine release syndrome induced by rituximab may also enhance T cell activation17. Reportedly, rituximab
can modulate the immunoresponse by secretion of IL-10 and macrophage inflammatory protein-1β18. However,
few studies have clearly investigated whether immune modification after administration of rituximab promotes
or inhibits T cell allo-response, even though T cells are also sensitized with alloantigens in DSA-positive sensitized patients.
Department of Gastroenterological and Transplant Surgery, Graduate School of Biomedical and Health Sciences,
Hiroshima University, 1‑2‑3 Kasumi Minami‑ku, Hiroshima 734‑8551, Japan. *email: ideken@hiroshima-u.ac.jp;
hohdan@hiroshima-u.ac.jp
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Recipient age, years, median (range)
ABO-incompatible (n = 45)
DSA-positive (n = 17)
53.0 (20–71)
61.0 (24–70)
Gender
P value
0.50
< 0.01
Male
31
3
Female
14
14
Kidney
32
11
Liver
13
6
Donor age, years, median (range)
55.0 (23–78)
58.0 (28–74)
Organ
0.63
Gender
0.43
< 0.01
Male
17
14
Female
28
3
Child
9
4
Parent
8
3
Sibling
2
1
Spouse
26
9
Relationship
0.98
HLA allele MM
A (0: 1: 2)
10: 19: 16
3: 9: 5
0.75
B (0: 1: 2)
3: 22: 20
0: 8: 9
0.51
DR (0: 1: 2)
7: 21: 17
0: 12: 5
0.12
Table 1. Characteristics of ABO-incompatible and DSA-positive patients. A difference was considered
significant if the p-value was <0.05.
We desensitized DSA-positive patients with rituximab and plasmapheresis, which was adopted from a protocol for ABO-blood type incompatible (ABO-I) transplant r ecipients19. We have recently shown that pretransplant
desensitization with rituximab has a minimal effect on the alloreactive T cell responses by comparing ABO-I
and ABO-compatible g roups20. However, the previous study excluded DSA-positive recipients who might have
preformed donor-reactive T cells. Hence, the objective of this study was to elucidate the impact of pretransplant
desensitization with rituximab on the subsequent response of T cells to donor-antigens in DSA-positive transplant recipients. Moreover, a highly sensitized murine model was applied to investigate the mechanisms of the
significant impacts of B cell depletion by injecting anti-CD20 monoclonal antibodies (mAbs) on anti-donor T
cell responses.
Results
T cell immune responses before and after rituximab administration in desensitized
patients. The baseline characteristics of the desensitized patients are listed in Table 1.
Of these, 17 patients were DSA-positive (kidney, n = 11; liver, n = 6) and the remaining 45 were DSA-negative
ABO-I patients (kidney, n = 32; liver, n = 13). Of the DSA-positive group, 9 patients were CDC cross match
(XM)-positive (kidney, n = 5; liver, n = 4), and the remaining eight were CDC XM-negative flow cytometry
crossmatch (FCXM)-positive patients (kidney, n = 6; liver, n = 2). In the DSA-positive group, there were more
female recipients than males, and male donors than females. Similar to our previous report20, the proportion of
peripheral blood I gM+ CD19+ B cells in all the recipients decreased below 0.1% at 2 weeks after administration of
rituximab (data not shown). To evaluate the T cell immune status of recipients, we performed mixed lymphocyte
reaction (MLR) assays using a carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeling technique before
rituximab administration and 2 weeks after rituximab administration (before transplantation). There were no
significant differences in the SI values for the C
D4+ T cell responses between the two groups before rituximab
administration (Fig. 1A,C).
Of note, after rituximab administration, the SI values for the CD4+ T cell responses to donor stimulation
were significantly higher in the DSA-positive group than those in the ABO-I group (p < 0.05, Fig. 1A,C). There
were no significant differences in the SI values for the CD8+ T cell responses to donor between the two groups
(Fig. 1B,D). After transplantation, kidney transplant recipients did not suffer from T cell-mediated rejection
(TCMR), probably because they received either rabbit anti-thymocyte globulin (ATG) or basiliximab as induction therapy. In contrast, two out of five DSA-positive liver transplant recipients suffered from severe TCMR
requiring rabbit ATG treatment.
Kinetics of anti‑allo Ab titers of a highly sensitized murine model. To investigate the mechanisms
of anti-donor hyper responses of CD4+ T cells after B cell depletion, naïve Balb/c mice were transplanted with
C57BL/6 skin grafts to prepare a highly sensitized model, and the sensitized mice were injected with anti-CD20
mAb to eliminate B cells. To confirm allo-sensitization by allogenic skin transplantation, the production of anti-
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Figure 1. T cell immune responses before and after rituximab administration in desensitized patients. Sixtytwo patients received pretransplant desensitization with rituximab. Of these, 17 patients were DSA-positive
(kidney, n = 11; liver, n = 6) and the remaining 45 were DSA-negative ABO-I patients (kidney, n = 32; liver,
n = 13). To evaluate the T cell immune status of recipients, CFSE-MLR assays were performed before rituximab
administration and 2 weeks after rituximab administration (before transplantation). The stimulation index (SI)
values of each CD4+ T cells (A, C) and CD8+ T cells (B, D) before and after rituximab are shown. White box,
ABO-I group; gray box, DSA-positive group. Data are shown as median, 25th and 75th percentiles, and range.
The Wilcoxon-Mann–Whitney test was used to evaluate differences between before and after desensitization.
C57BL/6 Abs in the sera of recipient Balb/c mice was serially evaluated. The levels of anti-C57BL/6 IgG titers
gradually increased and reached a peak at nine weeks after the first skin transplantation (Figs. 2 and S1).
In the anti-CD20 mAb group (n = 5), unlike clinical desensitization therapy, plasma exchange was not performed in mouse experiments, and thus no dramatic decrease in anti-C57BL/6 IgG titers was observed. Instead,
titers gradually decreased after injection of anti-CD20 mAb at week 6 after the first skin transplantation. The
differences in anti-C57BL/6 IgG titers between the groups reached statistical significance at week 12 (p < 0.05). ...
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