Alloreactive CD8 T cell tolerance requires recipient B cells, dendritic cells, and MHC class II.
ABSTRACT Allogeneic bone marrow chimerism induces robust systemic tolerance to donor alloantigens. Achievement of chimerism requires avoidance of marrow rejection by pre-existing CD4 and CD8 T cells, either of which can reject fully MHC-mismatched marrow. Both barriers are overcome with a minimal regimen involving anti-CD154 and low dose (3 Gy) total body irradiation, allowing achievement of mixed chimerism and tolerance in mice. CD4 cells are required to prevent marrow rejection by CD8 cells via a novel pathway, wherein recipient CD4 cells interacting with recipient class II MHC tolerize directly alloreactive CD8 cells. We demonstrate a critical role for recipient MHC class II, B cells, and dendritic cells in a pathway culminating in deletional tolerance of peripheral alloreactive CD8 cells.
- SourceAvailable from: sciencedirect.comImmunity 05/2001; 14(4):417-24. · 19.80 Impact Factor
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ABSTRACT: Both the direct and indirect antigen presentation pathways are important mechanisms for T cell-mediated allograft rejection. Studies using knockout mice and monoclonal antibodies have demonstrated that CD4+ T cells are both necessary and sufficient for the rejection of allogeneic tissues, including skin, heart, and islet. Furthermore, combined blockade of the CD28/B7 and CD154/CD40 costimulatory pathways induces tolerance in multiple CD4+ T-cell dependent allograft models. In this study, we addressed the T-cell requirement for costimulation in direct antigen presentation. We demonstrated that class II-specific alloreactive T-cell receptor transgenic T cells were sufficient to mediate allograft rejection independent of costimulatory blockade. Analysis of the costimulatory capacity of different antigen presenting cell (APC) populations demonstrated that APCs resident within the donor skin, Langerhans cells, are potent stimulators not requiring CD28- or CD154-dependent costimulation for direct major histocompatibility complex (MHC) antigen presentation. These results complement previous work examining the role of costimulation on CD8+ T cells, supporting a model in which the effectiveness of costimulatory blockade in the setting of transplantation may be selective for the indirect pathway of MHC alloantigen presentation.American Journal of Transplantation 08/2002; 2(6):510-9. · 6.19 Impact Factor
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ABSTRACT: Single H2Kb, H2Db and double H2KbDb homozygous knockout (KO) mice were generated and their peripheral CD8+ T cell repertoires compared to that of C57BL/6 (B6) mice. Limited (10-20%, H2Db), substantial (30-50%, H2Kb) and profound (90%, H2KbDb) reduction of peripheral CD8+ T cells was observed in KO mice, without Vbeta diversity alteration. Classical class Ia molecules therefore ensure most but not all of the peripheral CD8+ T cell repertoire education. As expected, H2Kb but also H2Db KO mice developed choriomeningitis following intracranial infection by lymphocytic choriomeningitis virus with the same kinetics, lethality and CD8+ cell implication as wild-type B6 mice. By contrast, H2KbDb (class Ia-Ib+) KO mice survived. Choriomeningitis of H2Db KO mice was linked to the development of a subdominant (in normal B6 mice) H2Kb-restricted cytotoxic T lymphocyte response. Mice expressing a restricted set of histocompatibility class I molecules should represent useful tools to evaluate the immunological potentials of individual MHC class I molecules.European Journal of Immunology 05/1999; 29(4):1243-52. · 4.97 Impact Factor
Alloreactive CD8 T Cell Tolerance Requires Recipient B Cells,
Dendritic Cells, and MHC Class II1
Thomas Fehr,2,3Fabienne Haspot,2Joshua Mollov, Meredith Chittenden, Timothy Hogan,
and Megan Sykes4
Allogeneic bone marrow chimerism induces robust systemic tolerance to donor alloantigens. Achievement of chimerism
requires avoidance of marrow rejection by pre-existing CD4 and CD8 T cells, either of which can reject fully MHC-
mismatched marrow. Both barriers are overcome with a minimal regimen involving anti-CD154 and low dose (3 Gy) total
body irradiation, allowing achievement of mixed chimerism and tolerance in mice. CD4 cells are required to prevent marrow
rejection by CD8 cells via a novel pathway, wherein recipient CD4 cells interacting with recipient class II MHC tolerize
directly alloreactive CD8 cells. We demonstrate a critical role for recipient MHC class II, B cells, and dendritic cells in a
pathway culminating in deletional tolerance of peripheral alloreactive CD8 cells.
merism achieves robust tolerance, permitting acceptance of or-
gan and tissue allografts without long-term immunosuppression
(1–3). If used to induce tolerance, chimerism must be achieved
with minimal host conditioning without toxicity or graft-vs-host
disease. Immune barriers posed by both CD4 and CD8 T cells,
each of which can reject fully MHC-mismatched marrow grafts,
must be overcome (4). These barriers are overcome using anti-
CD154 mAb and low dose (3 Gy) total body irradiation (TBI)5
with fully MHC-mismatched allogeneic bone marrow trans-
plantation (BMT), which achieves durable mixed chimerism in
mice (5). Engrafted donor hematopoietic stem cells continually
supply donor APCs to the thymus, permitting life-long central
Tolerization of pre-existing peripheral donor-reactive CD4 and
CD8 T cells in this model is characterized by rapid deletion (7, 8).
The Journal of Immunology, 2008, 181:
pecific immune tolerance obviates the need for chronic
immunosuppressive therapy with its attendant toxicities
and prevents chronic rejection of allografts. Mixed chi-
Whereas we could not find any evidence for a role for regulatory
cells in CD4 tolerance induction (7, 9), CD8 T cell tolerance is
clearly and critically dependent on the presence of a CD4 T cell
population, which however is CD25 negative, IL-2-independent
and therefore distinct from “natural T regulatory cells” (Tregs).
Although it might be speculated that the critical CD4 cells are an
induced type of Tregs, their requirement is most evident in the first
few days post transplant, and CD4 cells are no longer required by
2 wk post-BMT. This is also the time by which peripheral donor-
reactive CD8 cells have been specifically deleted, obviating any
further need for regulation (8). These results implicate CD4 cells
in the pathway leading to rapid deletional tolerance of pre-existing
donor-reactive CD8 T cells. We have hypothesized that this path-
way involves conditioning of donor-derived APCs by CD4 cells,
making APCs tolerogenic for alloreactive CD8 cells encountering
donor Ag on these APCs. However, no absolute requirement for
any specific donor class II?APC population has been demonstra-
ble (T. Fehr, S. Wang, F. Haspot, P. Blaha, T. Hogan, M. Chit-
tenden, T. Wekerle, and M. Sykes, manuscript submitted). We
therefore investigated the possibility that interactions of recip-
ient CD4 cells with recipient APCs might be involved in the
pathway leading to recipient CD8 T cell tolerance. The present
studies provide evidence for such interactions and suggest a
novel pathway whereby tolerance of directly alloreactive CD8
cells requires interactions between recipient CD4 cells and re-
cipient class II?APCs. Recipient class II MHC, recipient B
cells, and recipient dendritic cells (DCs) are involved in the
tolerization of peripheral alloreactive CD8 cells.
Materials and Methods
Studies were performed under an institutionally approved protocol in ac-
cordance with the National Institutes of Health guide. Female C57BL/6
(B6: H-2b, CD45.2), CD45.1 congenic B6, B10.A (H-2a), B10.RIII (H-2r),
CIITA-deficient (H-2b), B cell-deficient ?MT(H-2b) and CD11c promoter-
driven diphtheria toxin receptor (DTR)-transgenic (H-2b) mice were pur-
chased from Frederick Cancer Research Center or from The Jackson
Laboratory. MHC class II-deficient (I-A??/?, H-2b), ?2m/I-A? double-
deficient (?2m?/?/I-A??/?, H-2b) and KbDbdouble-deficient mice were
purchased from Taconic Farms. CIITA/KbDbtriple-deficient mice were
bred in our facility. All mice were housed in a specific pathogen-free mi-
Transplantation Biology Research Center, Massachusetts General Hospital/Harvard
Medical School, Boston, MA 02129
Received for publication February 19, 2008. Accepted for publication April 29, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grants R01 HL49915 and
P01 HL18646. T.F. was supported by the Swiss Foundation for Medical and Biolog-
ical Grants/Novartis Switzerland and the Walter and Gertrud Siegenthaler Foundation
(University of Zurich). F.H. was supported by the Fondation pour la Recherche Medi-
cale, France and the American Society of Transplantation/American Society of Blood
and Marrow Transplantation.
2T.F. and F.H. contributed equally and are listed alphabetically.
3Current address: Clinic of Nephrology, University Hospital/Zurich Medical School,
4Address correspondence and reprint requests to Dr. Megan Sykes, Transplantation
Biology Research Center, Massachusetts General Hospital, MGH East, Building 149-
5102, 13th Street, Boston, MA 02129. E-mail address: megan.sykes@tbrc.
5Abbreviations used in this paper: TBI, total body irradiation; BM, bone marrow;
BMT, BM transplantation; Treg, regulatory T cell; DC, dendritic cell; DT, diphtheria
toxin; DTR, DT receptor; FCM, flow cytometric; WT, wild type; CML, cell-mediated
lympholysis; WBC, white blood cell.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
Conditioning and BMT
Eight- to twelve-week-old mice received 3 Gy TBI from a137Cesium ir-
radiator on day –1. Anti-mouse CD154 mAb (MR1; 2 mg; produced at the
National Cell Culture Center) was administered i.p. on day 0, before i.v.
injection of 20 ? 106fully MHC-mismatched B10.A BM cells. For the
experiments shown in Figs. 3, 5, and 6, donor BM was depleted of T cells
using magnetic beads coated with anti-CD4 and anti-CD8 Abs and the
SuperMACS system (Miltenyi Biotec) with CS columns.
BM reconstitution experiments
Ten- to twelve-week-old B6 mice received 10.25 Gy TBI from a137Cesium
irradiator followed 6–8 h later by reconstitution with 10 ? 106T cell-
depleted syngeneic BM cells from WT B6, I-A??/?, ?2m?/?/I-A??/?,
CIITA?/?, CIITA/KbDbtriple-deficient or DTR-transgenic mice. For re-
constitution with syngeneic MHC class I-deficient marrow, recipient mice
were additionally depleted of NK cells by injection of mAb PK136 (0.15
mg i.p.) on day ?1 before TBI and BMT.
B cell infusions
Splenic B cells from 8 to 12-wk-old WT or MHC class II-deficient B6 mice
were purified using a non-touch B cell isolation kit and SuperMACS
(Miltenyi Biotec). Twenty to 23 million highly purified B cells (purity
?97%) were i.v. injected into ?MTrecipient mice on the day of BMT.
In vivo T cell depletion
To deplete CD8 or CD4 T cells in vivo, anti-CD8 (2.43, 1.44 mg/mouse)
or anti-CD4 mAb (GK1.5, 1.76 mg/mouse) produced at NCCC was in-
jected i.p. on day ?1 before BMT.
In vivo DC depletion
To deplete CD11c?DCs from reconstituted DTR-transgenic mice, 4 ng/g
body weight DT was injected i.p. starting on day ?1 before allo-BMT and
then three times/week for 17 days. Efficiency of DC depletion was 90–95%
in mice reconstituted with DTR-transgenic marrow, as shown by flow cy-
tometric (FCM) analysis of splenocytes from a non-chimeric mouse from
each group on day 18 (see Fig. 7A).
Flow cytometric analysis of multilineage chimerism
among white blood cells (WBCs)
Multilineage chimerism was assessed by three-color FCM analysis using
FITC-conjugated anti-DdmAb 34-2-12 for H-2aor anti-DbmAb KH95 for
H-2b(BD Pharmingen) with PE- or allophycocyanin-conjugated anti-CD4,
-CD8, -B220 (BD Pharmingen), and -Mac1 (Caltag) mAbs. Negative con-
trol mAbs included HOPC1-FITC (prepared in our laboratory) and rat anti-
mouse IgG2a-PE or -allophycocyanin (BD Pharmingen). Propidium iodide
staining was used to exclude dead cells. A lineage was defined as chimeric
when ?5% cells were donor MHC class I positive.
Bone marrow reconstitution after lethal TBI
Lethally irradiated congenic B6 CD45.1 mice were reconstituted with wild-
type (WT), transgenic or knockout B6 CD45.2 BM. Peripheral WBCs were
stained 7 wk later for three-color FCM with anti-CD45.2 mAb 104-FITC,
anti-CD45.1 mAb A20-biotin (BD Pharmingen), and PE- or PerCP-Cy
5.5-conjugated anti-CD4, -CD8, -B220, or -Mac1 mAbs. Propidium iodide
staining was used to exclude dead cells. MHC class I or class II expression
was assessed on B220?or Mac1?cells using FITC-KH95 anti-Dband
25-9-17 anti-I-A?, respectively.
Cell-mediated lympholysis (CML) assay
CML assay was performed as previously described (10). Where indicated,
human recombinant IL-2, 5 IU/ml, was added. After 5 days, cytolytic ac-
percent specific lysis was calculated as described (10).
51Cr-labelled concanavalin-A-stimulated splenocytes and
Full thickness tail skin (0.5–1.0 cm2) from B10.A (donor-specific) or
B10.RIII (third-party) mice was grafted to the lateral thorax and considered
rejected when ?10% of the graft remained viable.
CD4 recognition of recipient class II MHC is required for CD8
T cell tolerance induction
For tolerization of pre-existing peripheral CD8 T cells by fully
MHC-mismatched BMT with anti-CD154, CD4 T cells are re-
quired (8). We investigated the possibility that CD4 T cell inter-
actions with recipient class II MHC might play a role in this path-
way. MHC class II-deficient I-A??/?mice lack CD4 T cells and
therefore could serve this purpose (11). Instead, we generated syn-
geneic chimeras by transplanting T cell-depleted WT or I-A??/?
B6 BM (both CD45.2) to lethally irradiated CD45.1 congenic WT
B6 recipients (Fig. 1A). CD4 T cells developed normally due to
MHC class II expression on thymic epithelium, but MHC class
II?host APCs were absent (Fig. 1, B and C). Eight weeks later,
these reconstituted mice received fully MHC-mismatched
B10.A BMT with 3 Gy TBI/anti-CD154 (Fig. 2, A and B). All
B63B6 reconstituted mice developed stable multilineage chi-
merism. In contrast, I-A?
failed to achieve multilineage mixed chimerism unless recipient
CD8 cells were depleted (Fig. 2, C–E). Similar results were
obtained when B6 mice were initially reconstituted with bone
marrow from CIITA-deficient mice (CIITA?/?(12)), which
also lack class II MHC expression. Therefore, recognition of
recipient class II MHC by recipient CD4 T cells is critical to
prevent marrow rejection by CD8 T cells and allow their toler-
ization by donor marrow and anti-CD154.
?/?3B6 reconstituted recipients
host hematopoietic cells, but with a normal peripheral CD4 compartment.
A, Congenic CD45.1 B6 mice were lethally irradiated and reconstituted
with MHC class II-deficient CD45.2 BM. Seven to 8 wk later, PBL re-
constitution was tested by FCM and syngeneic chimeras then received
allogeneic B10.A BMT with 3 Gy TBI and anti-CD154. B, CD45.1
expression in WBC lineages (CD4 and B cells in the lymphocyte gate,
Mac1?cells in the granulocyte gate), representing residual recipient-
derived cells. Representative (B63B6) and (I-A??/?3B6) chimeras
are shown (top and bottom, respectively). A radioresistant population of
CD4 and CD8 T cells (10–30%) persisted, whereas B cells, granulo-
cytes, and monocytes (data not shown) were ?99% donor-derived. C,
Histograms of MHC class II (I-A?) expression on peripheral B cells for
the same two animals shown in A.
Generation of mice lacking MHC class II expression on
166ALLOSPECIFIC CD8 TOLERANCE REQUIRES HOST MHC II?APCs
CD4 cell interactions with recipient class II are required to
tolerize directly alloreactive CD8 cells
Because direct CD8 cell-mediated rejection is likely to eliminate
allogeneic cells, we next addressed whether cross-talk from CD4 T
cells interacting with recipient APCs may be needed to tolerize
directly alloreactive CD8 cells. We generated mice lacking both
class I and class II MHC by crossing KbDbdouble-deficient mice
(KbDb?/?(13)) to CIITA?/?(12) mice and used BM from F2
mice that completely lacked class I and class II MHC (i.e., CIITA/
KbDbtriple-deficient) to reconstitute lethally irradiated WT B6
mice. In CIITA?/?KbDb?/?3B6 mice, normal peripheral CD4
and CD8 T cell reconstitution occurred due to T cell selection on
MHC?thymic epithelium, but neither MHC class I nor class II
was detectable on peripheral WBCs (Fig. 3A). After 8 wk, these
mice received 3 Gy TBI, anti-CD154 and T cell-depleted fully
MHC-mismatched B10.A BMT. Eighty percent of control
B63B6 reconstituted recipients developed multilineage mixed
CIITA?/?3B6 mice achieved allogeneic chimerism unless
they were depleted of CD8 cells (Fig. 3, B and C). Similar
results were obtained using mice reconstituted with I-A?/?2m
MHC class I and class II-deficient BM (data not shown). These
data demonstrate, surprisingly, that recipient CD4 T cell rec-
ognition of host MHC class II on a recipient professional APC
population is needed to tolerize directly alloreactive CD8 T
B10.A BMT. A, The incidence of chimerism in the B cell lineage at various time points. Chimerism was defined as ?5% donor cells among B220?B cells.
One representative experiment out of three is shown (7–8 animals/group/experiment). B, Chimerism levels (?SEM) over time for B cells and CD4 T cells
in PBL. C, Mice treated as in A also received depleting anti-CD8 mAb on day ?1. CD8 T cells were ?0.5% of PBL by 2 wk post-BMT. The incidence
of B cell chimerism is shown at indicated times. One representative experiment of two is shown (six to seven animals/group/experiment). D, Chimerism
levels (?SEM) over time are shown for blood B cells and CD4 T cells for groups in C. E, Representative FACS plots for measuring chimerism are shown
6 wk post-BMT for a chimeric WT mouse (upper row) and a non-chimeric mouse lacking MHC class II on recipient APCs (lower row) from the experiment
shown in A and B. Donor cells are marked with FITC-labeled anti-DdmAb 34-2-12. The panels showing CD4 and CD8 T cells and B220?B cells are gated
on lymphocytes; the panels showing CD11b?cells are gated on granulocytes. Percentages indicate the relative frequencies in the total lymphocyte or
granulocyte population, respectively. Chimerism was then calculated as the percentage of donor MHC-positive cells vs total cells in a certain lineage (e.g.,
for CD4 cells: [14.8/(14.8 ? 11.5)] ? 100 ? 56.3%).
Requirement for CD4 cell interaction with host class II for CD8 tolerance. BM-reconstituted mice prepared as in Fig. 1 received allogeneic
167The Journal of Immunology
Early donor-specific CD8 unresponsiveness is overcome by
addition of IL-2 only when CD4 T cells are depleted
To analyze the mechanism of early CD8 unresponsiveness more
precisely, we performed cell-mediated lympholysis assays in
mice receiving 3Gy TBI, anti-CD154, and allo-BMT, with or
without in vivo CD4 depletion. Splenocytes harvested on day 4
post-BMT were stimulated in vitro with donor B10.A or third
party B10.RIII cells, with or without exogenous IL-2. Although
deletion of donor-reactive CD8 cells is not yet near completion
by day 4 (47), mice receiving BMT without CD4 depletion
showed donor-specific unresponsiveness with measurable third-
party responses (Fig. 4A). Adding IL-2 to the culture did not
overcome this donor unresponsiveness (Fig. 4B). When recip-
ient CD4 T cells were depleted in vivo before allo-BMT, donor-
specific responses were detected in four of four mice, but only
when exogenous IL-2 was also added to the cultures (Fig. 4, C
and D). Thus, donor-specific CTL tolerance was apparent by 4
days post-BMT, and the most robust tolerance depended on the
presence of recipient CD4 cells.
Requirement for recipient B cells for CD8 T cell tolerance
We next sought to identify the MHC class II?recipient APC pop-
ulation(s) needed for CD8 tolerance induction. To examine the role
of recipient B cells, we compared B cell-deficient ?MTand WT B6
mice as recipients of T cell-depleted MHC-mismatched B10.A
BMT. Whereas all but one WT mouse developed durable multi-
lineage chimerism, none of the ?MTrecipients was chimeric (Fig.
5, A and B). However, when CD8 T cells were depleted, both WT
and ?MTmice achieved robust mixed chimerism (Fig. 5, C and D).
In a repeat experiment, CD8-depleted WT and ?MTchimeras ac-
cepted donor-type skin grafts, whereas third-party grafts were
promptly rejected (data not shown). These data demonstrate that
recipient B cells are required in the pathway leading to tolerization
of pre-existing donor-reactive CD8 cells.
To confirm the ability of recipient B cells to promote alloreac-
tive CD8 cell tolerance and to exclude the possibility that the ab-
sence of Abs in ?MTmice might be sufficient to preclude donor
engraftment, we evaluated the effect of providing WT B cells to
?MTrecipients of allogeneic marrow with our regimen. Adoptive
transfer of 20–23 ? 106B cells from WT B6 mice at the time of
allogeneic BMT allowed the achievement of durable mixed chi-
merism (Fig. 6, A and B) and donor-specific skin graft tolerance
(Fig. 6C) in ?MTrecipients. Infused B cells were identified in the
adoptive recipients as host-type B cells and represented 5 ? 2.9%
(SD) of lymphocytes at 1.5 wk and 2.9 ? 0.5% of lymphocytes at
4 wk after BMT. Similar outcomes were achieved in a second
experiment (data not shown). These results confirm the ability of
recipient B cells to promote donor engraftment and make it un-
likely that natural Abs play a critical role in the tolerance pathway,
because such Abs were not present at the time of transplant in ?MT
mice receiving adoptively transferred B cells.
We next examined the requirement for class II expression on the
adoptively transferred B cells to promote CD8 tolerance. As shown
in Fig. 6D, adoptive transfer of MHC class II-deficient B cells
promoted the achievement of mixed chimerism as effectively as
WT B cells in ?MTrecipients. Thus, recipient B cells promote
CD8 T cell tolerance independently of their ability to present Ag
on MHC II to recipient CD4 cells.
Requirement for recipient DCs for CD8 T cell tolerance
We then examined the role of recipient DCs in tolerance induction
by using DTR-transgenic mice (14), which express the primate
DTR as a transgene under the murine CD11c promoter. Injection
with DT selectively depletes DCs from these mice for 48–72 h.
Because repeated DT injections induce neurotoxicity in these
transgenic mice, we generated CD45 congenic BM chimeras ex-
pressing DTR only on hematopoietic cells. These mice tolerated
repetitive DT injections well, allowing 90–95% DC depletion
II-restricted CD4 cells for directly alloreactive CD8 T cell
tolerance induction. Lethally irradiated B6 mice were re-
constituted with either WT B6 (B63B6 chimeras), MHC
class II-deficient (CIITA?/?3B6 chimeras) or MHC
class I and II-deficient (CIITA?/?KbDb?/?3B6 chime-
ras) BM. Eight weeks later, they received allogeneic
B10.A BMT with 3 Gy TBI/anti-CD154, with or with-
out depleting anti-CD8 mAb. A, MHC class I and II
expression on blood B cells (B220?). Representative
B63B6 and CIITA?/?KbDb?/?3B6 chimeras are
shown (top and bottom, respectively) prior to allogeneic
BMT. B, Incidence of granulocyte chimerism at indi-
cated times (four to six animals/group). C, Mean per-
cent of granulocyte and CD4 cell chimerism (?SEM)
Evidence for a role for host MHC class
168ALLOSPECIFIC CD8 TOLERANCE REQUIRES HOST MHC II?APCs
(Fig. 7A). B6 mice reconstituted with either DTR tg or B6 BM
received DT three times per week on days ?1 through ?17 with
respect to B10.A BMT with 3Gy TBI and anti-CD154. One group
of DTR tg BM-reconstituted mice was also depleted of CD8 T
cells. In B6-reconstituted mice, no difference in chimerism was
observed between DT-treated and untreated groups. In contrast,
DT treatment in DTR tg BM-reconstituted mice completely pre-
vented chimerism induction (Fig. 7, B–D). However, CD8
depletion allowed achievement of robust mixed chimerism in DC-
depleted mice. Because CD4 cells in DT-treated DTR tg BM-re-
constituted recipients were able to reject allogeneic BM given
without anti-CD154 treatment (data not shown), the studies to-
gether indicate that recipient CD11c?DCs are required for toler-
ance induction of CD8, but not CD4 T cells.
The studies presented here demonstrate that host MHC class II
recognition by CD4 T cells is required for tolerization of directly
alloreactive peripheral CD8 T cells in recipients of allogeneic
BMT with anti-CD154. Surprisingly, both recipient B cells and
DCs are required in the pathway leading to peripheral CD8 T cell
Alloimmune responses are unique in that they involve two path-
ways of alloantigen recognition, termed direct and indirect (15).
Direct recognition denotes recognition of an intact foreign MHC
molecule presented on donor cells and is characterized by a high
precursor frequency of alloreactive T cells (16, 17) and vigorous
mixed lymphocyte and cytotoxic responses. The indirect pathway
of alloantigen presentation requires uptake and processing of do-
nor Ags by recipient APCs and presentation of donor peptides on
recipient MHC molecules (18, 19). The precursor frequency of
indirectly alloreactive T cells is ?100-fold lower than that of di-
rectly alloreactive T cells (17). The role of indirectly vs directly
alloreactive CD4 T cells for tolerance induction using BMT and
anti-CD154 and the APC populations involved in this process are
unknown. One possible explanation for our results is that indirectly
alloreactive CD4 cells promote the tolerance of directly alloreac-
tive CD8 cells. An important role for the MHC class II indirect
pathway for successful heart allograft tolerance induction was pre-
viously suggested (20, 21), but the role of indirect CD4 recognition
in tolerizing CD4 vs CD8 cells was not dissected. Because, in
contrast to bone marrow rejection, heart allograft rejection in the
mouse is highly dependent on CD4 cells, these observations were
dition of IL-2 only if recipient CD4 T cells are depleted. B6 mice received
allogeneic B10.A BMT with 3 Gy TBI/anti-CD154 (A and B). One group
also received depleting anti-CD4 mAb (C and D). Four days post-BMT,
splenocytes were restimulated in vitro with donor B10.A or third party
B10.RIII cells, in the absence (A and C) or presence (B and D) of lL-2.
After 5 days, cytotoxicity against donor-type or third party target cells was
measured. Each line represents one mouse.
Early donor-specific unresponsiveness is overcome by ad-
cipient B cells for CD8 T cell toler-
ance induction. A, B cell-deficient
?MTand WT B6 mice received T
cell-depleted B10.A BMT after 3 Gy
TBI/anti-CD154. The incidence of
shown at indicated times. One of two
similar experiments is shown (seven
to eight animals/group/experiment).
B, WBC granulocyte and CD4 chi-
merism (?SEM) for groups in A. C,
Mice treated as in A also received de-
pleting anti-CD8 mAb. Incidence of
granulocyte chimerism is shown at in-
dicated times. One of two similar ex-
periments is shown (seven to eight
animals/group/experiment). D, WBC
granulocyte and CD4 chimerism lev-
els (?SEM) for groups in C.
Requirement for re-
169The Journal of Immunology
consistent with a role for indirectly alloreactive CD4 cells in toler-
izing directly alloreactive CD4 cells. A study in a skin graft model
showed that indirect class II allorecognition promotes tolerance of
directly alloreactive CD4 cells with costimulatory blockade (22).
In our model, no cross-talk is needed between the two CD4 path-
ways, because the presence of only directly alloreactive CD4 T
cells in MHC class II-deficient recipients allowed achievement of
stable mixed chimerism when CD8 T cells were depleted. Surpris-
ingly, however, our studies show that recipients lacking the ca-
pacity for indirect presentation not only on MHC class II but also
on class I fail to achieve chimerism. Thus, if indirectly alloreactive
CD4 T cells are involved, these data suggest the existence of a
pathway allowing cross-talk between indirectly alloreactive CD4
cells and directly alloreactive CD8 T cells, as it was recently pro-
posed in a model of fully mismatched tracheal allograft rejection
An alternative explanation for the requirement for recipient he-
matopoietic class II in tolerizing directly alloreactive CD8 cells
and for other studies implicating the indirect pathway in tolerance
models would be that the ability to activate mature CD4 T cells is
altered in the absence of peripheral MHC class II expression (24),
so that they are incapable of delivering tolerogenic signals to crit-
ical host APCs that are needed for CD8 tolerance. In fact, CD4
cells adoptively transferred from WT mice to an MHC class II-
deficient environment become hyperreactive to self, lose regula-
tory T cell function, and reject third-party grafts with accelerated
kinetics (25). Thus, CD4 cells may be generally hyperreactive in
the absence of peripheral host MHC class II. We do not favor a
general lack of “Treg” function as an explanation for our results,
because the regulatory CD4 cells required for CD8 cell tolerance
in this model do not have characteristics of natural Tregs (8). Fur-
thermore, a general requirement for class II for regulatory function
would not help us to understand the results using mice deficient
only in B cells or DCs, because in these models MHC class II
signal is still delivered by other professional APCs. However, it is
possible that tonic interactions of CD4 T cells with MHC class II
are needed to make the CD4 cells capable of conditioning host
and/or donor APCs in a manner that renders them tolerogenic for
Our studies demonstrate that recipient B cells and CD11c?DCs
are both necessary to tolerize peripheral alloreactive CD8 cells. To
our knowledge, neither cell population has been previously impli-
cated in tolerizing alloreactive CD8 cells in vivo, and the require-
ment for both cell types is rather surprising. Direct interaction
between B cells and DCs has been reported to play an important
role for CTL priming (26–28), and such an interaction might also
play a role in CTL tolerance. Immature DCs and those differenti-
ated under special conditions in vitro (29, 30), which may have
low expression of MHC class II and costimulatory molecules
(reviewed in Ref. 31), have been shown to suppress immunity
in various models. Our findings are consistent with the possi-
bility that recipient DCs present donor-specific peptides to in-
directly alloreactive CD4 cells, and that these DCs modify the
microenvironment in a manner that leads to tolerance of directly
alloreactive CD8 cells encountering Ag on donor APCs. This
might occur via secretion of soluble mediators that inhibit T cell
received T cell-depleted B10.A BMT following 3 Gy TBI/anti-CD154. An additional group of B cell-deficient ?MTmice also received 20–23 ? 106purified
B cells from WT B6 mice on day 0. The incidence of WBC granulocyte chimerism is shown at indicated times. One of two similar experiments is shown
(three to seven animals/group/experiment). B, WBC granulocyte and CD4 chimerism levels (?SEM) for the groups in A. Circles, Granulocyte chimerism.
Diamonds, CD4 T cell chimerism. Open symbols, WT B6 recipients. Closed symbols with continuous line, ?MTrecipients. Closed symbols with dashed
lines, ?MTrecipients of WT B6 B cells. C, Chimeras shown in A and B received donor-type B10.A and third-party B10.RIII skin grafts 5 wk after BMT.
Graft survival is shown over time. D, B cell-deficient ?MTand WT B6 mice received T cell-depleted B10.A BMT after 3 Gy TBI/anti-CD154 conditioning.
Some ?MTmice also received 20–23 ? 106purified B cells from WT B6 mice or CIITA?/?(MHC class II-deficient) mice on day 0. The incidence of
WBC granulocyte chimerism is shown at indicated times (three to seven animals/group/experiment).
Adoptive transfer of WT or MHC class II-deficient B cells restores chimerism and tolerance. A, B cell-deficient ?MTand WT B6 mice
170ALLOSPECIFIC CD8 TOLERANCE REQUIRES HOST MHC II?APCs
Small resting B cells can tolerize naive T cells recognizing Ags
presented by the B cells (33–35). However, the concept that B cells
not expressing the donor alloantigens recognized by a CD8 T cell
population can tolerize that population is novel. If, in our model,
B cells were merely required as a source of host class II MHC
to down-modulate CD4 activation levels, adoptive transfer of B
cells at the time of transplant would be unlikely to correct the
deficiency rapidly enough to allow achievement of chimerism
and tolerance. Moreover, recipient B cells lacking class II MHC
expression, when given at the time of allogeneic BMT, pro-
moted chimerism and tolerance in B cell-deficient recipients.
This observation confirms that B cells are not simply required
as a source of recipient class II and argues for a more specific
role for recipient B cells in the induction of CD8 tolerance to
B cells might promote tolerance by inducing regulatory T cell
function and IL-10 production as described in experimental auto-
immune encephalitis (36) and in a graft-vs-host disease model
(37). B cells play a role when anti-CD45RB mAb is used for tol-
erance induction to heart allografts (38). This model is dependent
on Tregs (39), but the T cell population tolerized by these B cells
was not identified and the role of DCs was not investigated. B cells
may also produce TGF-?. Indeed, such regulatory B cells have
been described in several models of chronic inflammation (40),
and TGF-? production by LPS activation has been shown to toler-
ize resting CD8 T cells (41). B cells might also provide critical
tolerogenic signals to CD8 cells, such as PD-L1, which we have
recently shown to play an essential role in CD8, but not CD4 cell
tolerance in this model (47). Of considerable significance, up-reg-
ulation of PD-L1 on both DCs and B cells of the recipient requires
host CD4 cells in animals rendered tolerant with our regimen (47).
Thus, our data could be explained by a model in which naive
alloreactive CD4 cells are required to up-regulate PD-L1 in the
presence of anti-CD154 on these APC populations that then inter-
act with alloreactive PD-1-positive CD8 cells, rendering them tol-
erant. Pick-up and presentation of donor-derived MHC class I mol-
ecules to directly alloreactive CD8 cells by recipient B cells (and
DCs) could be invoked to explain a role for these cell types in
tolerizing directly alloreactive CD8 cells. Donor alloantigen
pick-up by recipient DCs has been described as an alternative path-
way of alloantigen presentation to directly alloreactive recipient T
cells (42–44). Interestingly, BM-resident CD11c?DCs promote
the presence of recirculating mature B cells in BM niches (45), an
interaction that may have immunological consequences in the con-
text of bone marrow transplantation.
Our previous studies showed that CD8 tolerance measured in
vitro at 2 wk in CML assays correlated with the achievement of
initial and durable mixed allogeneic chimerism in mice receiving
allogeneic BMT with 3 Gy TBI and anti-CD154 mAb (10). We
have now shown that this tolerance is evident as early as 4 days
post-BMT and cannot be overcome by the addition of exogenous
IL-2. However, when CD4 cells are depleted from recipients, tol-
erance can be broken by the addition of IL-2. Thus, in these ani-
mals, induction of initial mixed chimerism reflects successful
tolerization of pre-existing peripheral CD8 T cells, which is fol-
lowed later by central deletional tolerance of T cells that develop
de novo following the transplant (5). The CTL results presented in
this study confirm in vitro the requirement for CD4 T cells to fully
regulate donor-reactive CD8 cells early post-transplant, consistent
with our previous in vivo results (8). Consistently, donor-reactive
T cell tolerance induction. Lethally irradiated congenic
CD45.1 B6 mice were reconstituted with either WT B6
(B6) or DTR-transgenic (DTR tg) B6 BM. Eight weeks
later they received fully MHC-mismatched B10.A BMT
with 3 Gy TBI/anti-CD154. Some animals received
diphtheria toxin to deplete DCs (see Materials and
Methods), with or without depleting anti-CD8 mAb. A,
One mouse from each group was euthanized on day 18.
The spleen was harvested, digested with collagenase,
and thenstained with
CD8?-allophycocyanin and analyzed by FCM. The per-
centage of CD11c?DCs among total lymphocytes is
shown. Efficiency of DC depletion was 90–95%. The
incidence of B cell chimerism (B) and percent donor B
and CD4 cell chimerism (?SEM) (C and D) is shown.
One of two experiments is presented (seven to eight
animals/group/experiment). The repeat experiment in-
cluded T cell depletion of the allogeneic BM, with sim-
Requirement for recipient DCs for CD8
171The Journal of Immunology
CD8 cells are not deleted by day 4 and have an activated pheno-
type, with up-regulation of CD69, CD25, and CD44 (47). Donor-
reactive CD8 cells are fully deleted by day 10 post-BMT, when
CD4 cells are no longer required for their tolerance (8). It is of
interest that the CTL “anergy” on day 4 was not overcome by the
addition of exogenous IL-2 unless CD4 cells were depleted from
the recipient, in which case anti-donor responses were revealed by
IL-2. CD4 cells in this system may promote the induction in CD8
cells of a previously reported form of anergy that cannot be over-
come by IL-2 (46). In combination, our data suggest that donor-
reactive CD8 cells rapidly undergo anergy followed by death
within 2 wk of BMT in this model. It is possible that the death
phase occurred in vitro in CD8 cells from animals in which CD4
cells were not depleted, thus explaining the failure to reverse un-
responsiveness by the addition of exogenous IL-2.
Taken together, our studies show the following: 1) host MHC
class II recognition by CD4 T cells is critical for peripheral CD8
tolerance; 2) CD8 T cell tolerance requires recipient B cells and
DCs; and 3) interactions with recipient class II MHC by recipient
CD4 T cells are required to tolerize directly alloreactive CD8 T
cells, suggesting cross-talk between host MHC-restricted CD4
cells and donor MHC-restricted CD8 cells. These results provide
new mechanistic insights into allograft tolerance. We believe that
an in-depth understanding of the cellular interactions involved in
tolerance induction will be essential to the development of reliable
protocols for clinical tolerance.
We thank Prof. Rolf Zinkernagel, Dr. Thomas Wekerle, and Anne-Lau-
rence Blanc for critical review of this manuscript, Orlando Moreno for
technical assistance, and Kelly Walsh for expert assistance with the
The authors have no financial conflict of interest.
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