# MHC Disparate Resting B Cells Are Tolerogenic in the Absence of  Alloantigen-Expressing Dendritic Cells

Authors: Hugh I. McFarland; Kazuhide Tsuji; Karen P. Mason; Amy S. Rosenberg
Journal: ISRN Transplantation
 (2013)

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## Abstract

Resting B cell (rB) populations have been shown to tolerize to soluble proteins and to minor-H but not to MHC alloantigens. We speculated that the reason for failing to tolerize to MHC alloantigen is that the few remaining dendritic cells (DCs) contaminating purified rB cell populations efficiently activate MHC allospecific T cells which are present at a higher frequency than T cells specific for  minor-H alloantigen and soluble proteins. We established that MHC disparate rB cells are indeed tolerogenic when devoid of DC populations, as parental strain mice showed delayedDd skin graft rejection when infused with rB cells from mice in which MHC class I Dd alloantigen was specifically targeted to T and B cells (CD2-Dd transgenic mice). In contrast, treatment of parental strain mice with allogeneic rB cells purified from MHC-Dd transgenic mice, in which Dd is ubiquitously expressed, including DCs, induced accelerated Dd graft rejection. We also showed that adding only 5,000 Dd expressing DCs to CD2-Dd rB cells abrogated the tolerogenic effect. Surprisingly, allogeneic rB cells prolonged graft survival in Dd-primed mice. Thus, MHC disparate rB cells are tolerogenic and their failure to delay graft rejection can be explained by contaminating allogeneic DCs.


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## Body

## 1. Introduction

Resting B cells have been suggested to be potent inducers of specific unresponsiveness bothin vitro and in vivo [1, 2] and have been thought to mediate such effects by directly presenting Ag (signal 1) in the absence of productive costimulatory interactions, principally mediated by B7-CD28 and CD40-CD40L interactions (signal 2) [3–6]. This hypothesis, however, has been challenged by studies demonstrating that rB cells not only express CD40, but also upregulate expression of B7 family members in a timely fashion in vivo [7]. Moreover, it has become increasingly appreciated that indirect antigen presentation, in which antigen shed from donor cells is presented via immature host DC, may be crucial for tolerance induction [8–11]. Regardless of the mechanism, rB cells clearly have tolerogenic effects on T cells specific for some types of antigen, but not others. For instance, rB cells induce tolerance to soluble proteins [2, 12] and to minor-H mismatched transplants [1], but not to transplants discrepant in MHC alloantigens [7].That MHC disparate grafts present a higher bar for tolerance induction in general is emphasized by studies showing that MHC-expressing allografts are rejected even in the absence of the “danger” signals that appear requisite in generation of immune responses to other antigens, presumably by enhancement of APC activity [13]. One explanation for the ability of rB cells to induce tolerance to minor-H but not MHC disparate grafts is that the few DCs associated with rB cells purified from normal mice effectively activate the far more frequent and high affinity MHC allospecific T cells. To investigate this possibility, we assessed the ability of allogeneic rB cells, in which alloantigen expression is genetically restricted to T and B cells, to thwart allograft rejection.


## 2. Results

### 2.1. Allogeneic rB Cells from Gene-Targeted Mice Induce Hyporesponsiveness

To assess the effects of rB cell presentation of class I MHC alloantigen on rejection responsesin vivo, we utilized lymphocyte populations from two lines of transgenic mice, because of their differential expression of Dd alloantigen on cellular populations. Dd is ubiquitously expressed in MHC-Dd transgenic mice (FVB background, H-2q), with DC expressing low to moderate levels, while in CD2-Dd transgenic mice, (FVB)Dd is expressed on T and B cells, but not on CD11c+ DC [14] (Figure 1). rB cells recovered from the spleens of the two transgenic lines after extensive depletion of T cells, macrophages, and DC populations were comparable with respect to DC content (<1%), as well as expression of B lineage and costimulatory markers (Figure 2). Naive and Dd-primed FVB mice were pretreated with 107rB cells from MHC-Dd or CD2-Dd mice. Though B cells purified from these two transgenics were highly comparable in terms of DC content and other markers (Figure 2), they induced dramatically different effects on Dd graft rejection when infused into host FVB mice: MHC-Dd B cells induced accelerated graft rejection median survival time (MST) 9 days versus 12 days for control mice, whereas CD2-Dd B cells caused delayed graft rejection (MST 18 days) (Figure 3(a)). Surprisingly, infusing either B cell population into primed mice consistently abrogated accelerated rejection (MST 12 days versus 8.5 days for control) and converted response kinetics to a primary rejection rate (Figure 3(b)). Thus, MHC disparate rB cells from gene-targeted mice induced hyporesponsiveness in both naïve and memory allospecific CD8+ T cells while paradoxically, MHC Dd rB cells containing small numbers of alloantigen-expressing DC, amidst a plethora of rB cells, efficiently primed naïve CD8+ T cells, but failed to significantly activate memory class I allospecific T cells.Figure 1
Dd expression on splenic T cells, B cells, and DC from FVB, MHC-Dd, and CD2-Dd transgenic mice.Figure 2
B cell profiles after depletion of T cell, macrophages, and DC from spleen cells of MHC-Dd and CD2-Dd transgenic mice. Isotype-matched controls are shown with dotted lines.Rejection of Dd skin grafts in mice infused with resting B cells. (a) Naive mice received no injection, 107 purified MHC-Dd B cells, or 107 purified CD2-Dd B cells intravenously: (filled triangle) = MHC-Dd; (open circle) = CD2-Dd; and the dashed line = none. Mice were grafted with MHC-Dd tail skin 2 weeks later. MST of skin grafts: naive mice = 12 days (n=16); naive plus CD2-Dd B cell = 18 days (P<0.001 versus naive, by log-rank) (n=10); naive plus MHC-Dd B cell = 9 days (P<0.001 versus naive) (n=8). (b) Dd-primed mice received no injection, 107 purified MHC-Dd B cells, or 107 purified CD2-Dd B cells intravenously: filled triangle = MHC-Dd; open circle = CD2-Dd; and the dashed line = none. Mice were grafted with MHC-Dd tail skin 2 wks later. MST of skin grafts: primed mice = 8.5 d (n=20); priming plus CD2-Dd B cell = 12 d (P=0.021 versus primed; P=0.87 versus naive) (n=10); priming plus MHC-Dd B cell = 11.5 days (P=0.012 versus primed; P=0.42 versus naive) (n=8).
(a)
(b)Similar results were observed in naïve thymectomized mice infused with CD2-Dd cells (MST 17 days versus 12 days for control) or MHC-Dd B (MST 8 days), indicating that rejection did not depend on the activity of lymphocytes newly emigrated from the thymus that had not been initially exposed to alloantigen. Furthermore, we do not think that the difference in response is attributable to higher Dd expression on MHC-Dd versus CD2-Dd B cells because in our CD2-Dd transgenic mouse lines, with line-dependent Dd expression levels, we have observed that tolerance, as assessed by skin allograft rejection, correlated directly with the Dd expression level [14], suggesting that extensive TCR cross-linking by the rB cells is of importance in tolerance induction [15]. These data suggest that if Dd expression was as high on CD2-Dd B cell as on MHC-Dd rB cells, the CD2-Dd cells would be able to induce more prolonged hyporesponsiveness, or even tolerance.


### 2.2. Primary and Secondary Graft Rejection Responses Mediated by CD8+ T Cells Are Abrogated by Genetically Targeted Dd-Expressing rB Cells

To better explore the effects of rB cells on the T cells mediating rejection, we utilized an adoptive transfer model in which T-depleted FVB host mice (thymectomized and irradiated with 500 cGy [16]) were reconstituted with limiting numbers of CD8+ T responder cells.  CD8+ T cells are both necessary and sufficient to reject MHC class I disparate Dd skin allografts in naïve mice and for accelerated rejection in antigen primed-mice, whereas CD4+ T cells are neither necessary nor sufficient for allograft rejection [17, 18].Ddskin allograft rejection was substantially delayed in naïve CD8+ T cell-reconstituted mice that were infused with CD2-Dd rB cells (MST 35.5 days versus 18.5 days) (Figure 4). Even more striking was the substantially prolonged graft survival in mice reconstituted with Dd-primed CD8+ T cells and infused with CD2-Dd B cells (MST 17.5 versus 7.5 days) (Figure 4). These data confirm that both naïve and memory CD8+ allospecific T cells are rendered hyporesponsive in vivo by alloantigen expressed on nonprofessional APCs, when contaminating allogeneic DCs are eliminated.Rejection of Dd skin grafts in adoptively transferred mice. T cells (a) or purified CD8+ T cells (b) from Dd primed or naive FVBs were transferred into ATX FVB mice 1 week after irradiation (500 cGy). Mice were infused with CD2-Dd B cells 1 wk after T cell transfer, and were grafted with MHC-Dd tail skin 2 wks later. MST of skin grafts: naive CD8+ T cell-infused mice (open square) = 18.5 days (n=4); naive CD8+ T cell plus CD2-Dd B cell-treated mice (filled square) = 35.5 days (n=4); primed CD8+ T cell plus CD2-Dd B cell-treated mice (filled circle) = 17.5 days (n=4); primed CD8+ T cell-infused mice (open circle) = 7.5 days (n=4); irradiation alone (x) = 32.5 days (n=4).
(a)
(b)


### 2.3. Effects of Number of Dendritic Cells on Priming versus Hyporesponsiveness

Since contaminating DC in purified populations of rB cells are likely responsible for priming naïve class I allospecific lymphocytes, we wished to quantitate the minimum number required. Accordingly, we added graded numbers of MHC-Dd DCs (positively selected by cell sorting with anti-CD11c mAb and identified as 97% pure) to 107 purified CD2-Dd B cells and infused the cell mixture into naive FVB mice. Remarkably, as few as 5,000 DCs (0.005%) abrogated the tolerogenic effect of the rB cells, and partially primed naive allospecific T cells with full priming were achieved with as few as 10,000 DCs (Table 1). Clearly, MHC- Dd DC can accelerate Dd skin graft rejection via direct antigen presentation. In contrast, cross-presentation of Dd peptides by DC from CD2-Dd (4906) mice might rather be expected to tolerize to Dd [19], in which case the delayed skin graft rejection observed on infusion of CD2-Dd rB cells could theoretically be attributable to the DC contaminating the CD2-Dd rB cell preparation, rather than to the rB cells themselves. To test this possibility, we purified CD11c+ DC by MACS from FVB, MHC-Dd (3604), and CD2-Dd (4906) mice. Log doses of these cells were transferred i.v. into FVB recipients. Two weeks later, the mice were given Dd (3604) skin grafts. The MHC-Dd DC accelerated graft rejection at the 105 and 106 DC doses (Figure 5). While DC from the CD2-Dd mice failed to delay graft rejection at any dose tested remarkably, rejection was accelerated at the highest (106) DC dose. Thus, the infused DCs from CD2-Dd mice were not responsible for the induction of hyporesponsiveness to allogeneic skin grafts. Moreover, even in genetic targeting strategies in which alloantigen is not directly expressed by DC, but rather may be acquired from neighboring cells, high doses of DC may prime rather than tolerize, an important caveat for this tolerance strategy.Table 1
Effects of number of dendritic cells (DCs) on priming versus hyporesponsivenessa.
Dendritic cell dose
Percentage of infused cells
Graft survival (days)
MST (days)
1
×
10
5
1
8, 8, 9
8
5
×
10
4
0.5
8, 9, 9
9
1
×
10
4
0.01
8, 9, 14
9
5
×
10
3
0.005
13, 17, 23
17
1
×
10
3
0.0001
21, 22, 22
22
0
0
20, 20, 21
20
aGraded numbers of MHC-Dd DCs were added to 1×107 purified CD2-Dd B cells and infused into naive FVB mice.  After 2 weeks, mice were engrafted with MHC-Dd tail skin grafts and monitored daily for rejection.CD2-Dd DC accelerate Dd skin graft rejection. CD11c+ cells from the indicated strains were isolated by MACS magnetic separation and administered i.v. to FVB recipients. Two weeks later, recipient mice were engrafted with MHC-Dd tail skin. Plots A-D represent log doses of DC administered: (a) 103 DC, (b) 104 DC, (c) 105 DC, and (d) 106 DC.
(a)
(b)
(c)
(d)


## 2.1. Allogeneic rB Cells from Gene-Targeted Mice Induce Hyporesponsiveness

To assess the effects of rB cell presentation of class I MHC alloantigen on rejection responsesin vivo, we utilized lymphocyte populations from two lines of transgenic mice, because of their differential expression of Dd alloantigen on cellular populations. Dd is ubiquitously expressed in MHC-Dd transgenic mice (FVB background, H-2q), with DC expressing low to moderate levels, while in CD2-Dd transgenic mice, (FVB)Dd is expressed on T and B cells, but not on CD11c+ DC [14] (Figure 1). rB cells recovered from the spleens of the two transgenic lines after extensive depletion of T cells, macrophages, and DC populations were comparable with respect to DC content (<1%), as well as expression of B lineage and costimulatory markers (Figure 2). Naive and Dd-primed FVB mice were pretreated with 107rB cells from MHC-Dd or CD2-Dd mice. Though B cells purified from these two transgenics were highly comparable in terms of DC content and other markers (Figure 2), they induced dramatically different effects on Dd graft rejection when infused into host FVB mice: MHC-Dd B cells induced accelerated graft rejection median survival time (MST) 9 days versus 12 days for control mice, whereas CD2-Dd B cells caused delayed graft rejection (MST 18 days) (Figure 3(a)). Surprisingly, infusing either B cell population into primed mice consistently abrogated accelerated rejection (MST 12 days versus 8.5 days for control) and converted response kinetics to a primary rejection rate (Figure 3(b)). Thus, MHC disparate rB cells from gene-targeted mice induced hyporesponsiveness in both naïve and memory allospecific CD8+ T cells while paradoxically, MHC Dd rB cells containing small numbers of alloantigen-expressing DC, amidst a plethora of rB cells, efficiently primed naïve CD8+ T cells, but failed to significantly activate memory class I allospecific T cells.Figure 1
Dd expression on splenic T cells, B cells, and DC from FVB, MHC-Dd, and CD2-Dd transgenic mice.Figure 2
B cell profiles after depletion of T cell, macrophages, and DC from spleen cells of MHC-Dd and CD2-Dd transgenic mice. Isotype-matched controls are shown with dotted lines.Rejection of Dd skin grafts in mice infused with resting B cells. (a) Naive mice received no injection, 107 purified MHC-Dd B cells, or 107 purified CD2-Dd B cells intravenously: (filled triangle) = MHC-Dd; (open circle) = CD2-Dd; and the dashed line = none. Mice were grafted with MHC-Dd tail skin 2 weeks later. MST of skin grafts: naive mice = 12 days (n=16); naive plus CD2-Dd B cell = 18 days (P<0.001 versus naive, by log-rank) (n=10); naive plus MHC-Dd B cell = 9 days (P<0.001 versus naive) (n=8). (b) Dd-primed mice received no injection, 107 purified MHC-Dd B cells, or 107 purified CD2-Dd B cells intravenously: filled triangle = MHC-Dd; open circle = CD2-Dd; and the dashed line = none. Mice were grafted with MHC-Dd tail skin 2 wks later. MST of skin grafts: primed mice = 8.5 d (n=20); priming plus CD2-Dd B cell = 12 d (P=0.021 versus primed; P=0.87 versus naive) (n=10); priming plus MHC-Dd B cell = 11.5 days (P=0.012 versus primed; P=0.42 versus naive) (n=8).
(a)
(b)Similar results were observed in naïve thymectomized mice infused with CD2-Dd cells (MST 17 days versus 12 days for control) or MHC-Dd B (MST 8 days), indicating that rejection did not depend on the activity of lymphocytes newly emigrated from the thymus that had not been initially exposed to alloantigen. Furthermore, we do not think that the difference in response is attributable to higher Dd expression on MHC-Dd versus CD2-Dd B cells because in our CD2-Dd transgenic mouse lines, with line-dependent Dd expression levels, we have observed that tolerance, as assessed by skin allograft rejection, correlated directly with the Dd expression level [14], suggesting that extensive TCR cross-linking by the rB cells is of importance in tolerance induction [15]. These data suggest that if Dd expression was as high on CD2-Dd B cell as on MHC-Dd rB cells, the CD2-Dd cells would be able to induce more prolonged hyporesponsiveness, or even tolerance.


## 2.2. Primary and Secondary Graft Rejection Responses Mediated by CD8+ T Cells Are Abrogated by Genetically Targeted Dd-Expressing rB Cells

To better explore the effects of rB cells on the T cells mediating rejection, we utilized an adoptive transfer model in which T-depleted FVB host mice (thymectomized and irradiated with 500 cGy [16]) were reconstituted with limiting numbers of CD8+ T responder cells.  CD8+ T cells are both necessary and sufficient to reject MHC class I disparate Dd skin allografts in naïve mice and for accelerated rejection in antigen primed-mice, whereas CD4+ T cells are neither necessary nor sufficient for allograft rejection [17, 18].Ddskin allograft rejection was substantially delayed in naïve CD8+ T cell-reconstituted mice that were infused with CD2-Dd rB cells (MST 35.5 days versus 18.5 days) (Figure 4). Even more striking was the substantially prolonged graft survival in mice reconstituted with Dd-primed CD8+ T cells and infused with CD2-Dd B cells (MST 17.5 versus 7.5 days) (Figure 4). These data confirm that both naïve and memory CD8+ allospecific T cells are rendered hyporesponsive in vivo by alloantigen expressed on nonprofessional APCs, when contaminating allogeneic DCs are eliminated.Rejection of Dd skin grafts in adoptively transferred mice. T cells (a) or purified CD8+ T cells (b) from Dd primed or naive FVBs were transferred into ATX FVB mice 1 week after irradiation (500 cGy). Mice were infused with CD2-Dd B cells 1 wk after T cell transfer, and were grafted with MHC-Dd tail skin 2 wks later. MST of skin grafts: naive CD8+ T cell-infused mice (open square) = 18.5 days (n=4); naive CD8+ T cell plus CD2-Dd B cell-treated mice (filled square) = 35.5 days (n=4); primed CD8+ T cell plus CD2-Dd B cell-treated mice (filled circle) = 17.5 days (n=4); primed CD8+ T cell-infused mice (open circle) = 7.5 days (n=4); irradiation alone (x) = 32.5 days (n=4).
(a)
(b)


## 2.3. Effects of Number of Dendritic Cells on Priming versus Hyporesponsiveness

Since contaminating DC in purified populations of rB cells are likely responsible for priming naïve class I allospecific lymphocytes, we wished to quantitate the minimum number required. Accordingly, we added graded numbers of MHC-Dd DCs (positively selected by cell sorting with anti-CD11c mAb and identified as 97% pure) to 107 purified CD2-Dd B cells and infused the cell mixture into naive FVB mice. Remarkably, as few as 5,000 DCs (0.005%) abrogated the tolerogenic effect of the rB cells, and partially primed naive allospecific T cells with full priming were achieved with as few as 10,000 DCs (Table 1). Clearly, MHC- Dd DC can accelerate Dd skin graft rejection via direct antigen presentation. In contrast, cross-presentation of Dd peptides by DC from CD2-Dd (4906) mice might rather be expected to tolerize to Dd [19], in which case the delayed skin graft rejection observed on infusion of CD2-Dd rB cells could theoretically be attributable to the DC contaminating the CD2-Dd rB cell preparation, rather than to the rB cells themselves. To test this possibility, we purified CD11c+ DC by MACS from FVB, MHC-Dd (3604), and CD2-Dd (4906) mice. Log doses of these cells were transferred i.v. into FVB recipients. Two weeks later, the mice were given Dd (3604) skin grafts. The MHC-Dd DC accelerated graft rejection at the 105 and 106 DC doses (Figure 5). While DC from the CD2-Dd mice failed to delay graft rejection at any dose tested remarkably, rejection was accelerated at the highest (106) DC dose. Thus, the infused DCs from CD2-Dd mice were not responsible for the induction of hyporesponsiveness to allogeneic skin grafts. Moreover, even in genetic targeting strategies in which alloantigen is not directly expressed by DC, but rather may be acquired from neighboring cells, high doses of DC may prime rather than tolerize, an important caveat for this tolerance strategy.Table 1
Effects of number of dendritic cells (DCs) on priming versus hyporesponsivenessa.
Dendritic cell dose
Percentage of infused cells
Graft survival (days)
MST (days)
1
×
10
5
1
8, 8, 9
8
5
×
10
4
0.5
8, 9, 9
9
1
×
10
4
0.01
8, 9, 14
9
5
×
10
3
0.005
13, 17, 23
17
1
×
10
3
0.0001
21, 22, 22
22
0
0
20, 20, 21
20
aGraded numbers of MHC-Dd DCs were added to 1×107 purified CD2-Dd B cells and infused into naive FVB mice.  After 2 weeks, mice were engrafted with MHC-Dd tail skin grafts and monitored daily for rejection.CD2-Dd DC accelerate Dd skin graft rejection. CD11c+ cells from the indicated strains were isolated by MACS magnetic separation and administered i.v. to FVB recipients. Two weeks later, recipient mice were engrafted with MHC-Dd tail skin. Plots A-D represent log doses of DC administered: (a) 103 DC, (b) 104 DC, (c) 105 DC, and (d) 106 DC.
(a)
(b)
(c)
(d)


## 3. Discussion

Our studies indicate that MHC disparate rB cells are tolerogenic, when alloantigen-expressing DCs are completely eliminated, implicating contaminating DC as a source of allostimulation in B lymphocyte populations isolated from normal mice. Thus, in an ideal world, where all potential DC can be depleted, one could anticipate that purified populations of rB cells would be tolerogenic, even for MHC alloantigenic disparities. The likelihood that current technologies can reduce the DC content of a cellular population to <0.005%, the percentage of DCs that gave rise to partial priming for a single class I MHC mismatch is low. A gene therapy approach [20] with B cell-specific transduction of alloantigen may hold more promise in facilitating alloengraftment in human clinical trials. However, our data indicate that the success of this approach requires limitations in the numbers of nontargeted DC in the infusate, as expression of allogeneic peptides on nontargeted DC, which acquire allopeptide via cross-presentation mechanisms, provokes immunity.Quite perplexing was the finding that the memory (accelerated) rejection response, mediated by CD8+ T memory cells, was eliminated by the same population of rB cells that primed naïve CD8+ T cells (i.e., by MHC-Dd rB cells), suggesting that memory CD8+ T cells are even more sensitive than naïve T cells to tolerogenic effects of quiescently presented antigen and require either larger numbers of DC than were provided in purified MHC-Dd B cell populations, or require more mature and activated DCs, such as those provided by skin grafts. Indeed, skin-grafting Dd-primed mice have never failed to elicit a memory response. However perplexing, the finding that rB cell populations delayed the memory response in vivo is consistent with recent studies demonstrating that CD8+ memory T cells can be tolerized to the same extent as naïve populations [21], and with earlier studies demonstrating that CD8+ T cell clones, representative of memory populations, exhibit split anergy, that is, mediate CTL activity, but fail to proliferate and clonally expand unless supplied with exogenous IL-2 [22, 23] when presented with antigen by quiescent APC.Although our findings appear to be at odds with other reports in the literature showing that memory T cells can be activated to alloantigen expressed on rB cells [1], such is not the case. As with memory responses to minor-H alloantigen, we too saw preservation of memory CTL responses in mice exposed to alloantigen on rB cells in vivo (data not shown). However, skin graft rejection in minor-H primed mice exposed to minor-H disparate rB cells was not tested [1], so it is possible that exposure of minor-H primed mice to minor-H-expressing rB cells, prior to skin grafting, would also have abrogated accelerated rejection.In conclusion, we have demonstrated that MHC disparate rB cell populations are tolerogenic and that the reason that MHC disparate rB cells derived from normal mice are immunogenic is the presence of small numbers of contaminating dendritic cells. Lastly, we have confirmed the susceptibility of memory CD8+ T cell responses to disruption via exposure to tolerogenic lymphocyte populations.


## 4. Materials and Methods

### 4.1. Transgenic Mice

CD2-Dd transgenic mice were generated by injecting FVB embryos linking the CD2 promoter with Dd cDNA construct (a kind gift of Dr. Randy Ribaudo) coding sequence in pDd SELFIX.34 and an appropriate enhancer expression cassette p29Δ2(sal-) [14]. The CD2-Dd mice used were from the 4906 line, which showed the highest level of Dd expression [14]. MHC-Dd transgenic mice were generated by injecting FVB embryos with the genomic Dd gene, including the MHC class I promoter isolated from pDd1 (a kind gift of Dr. Gilbert Jay) [14].  Mouse studies were performed in accordance with Institutional Animal Care and Use Committee guidelines.


### 4.2. Mice and Priming

FVB/N mice and thymectomized (ATX) FVB/N mice, an inbred H-2q strain, were purchased from Taconic Farms (Germantown, NY, USA). For priming experiments, mice were given a single injection (i.p.) of 2×107 spleen cells from MHC-Dd mice at least 3 weeks before treatment.


### 4.3. Cytofluorometry Analysis

1 or 2 color analysis was performed by incubating cells with the following PE or FITC-conjugated mAbs: anti-CD11c (PE) (09705B), anti-CD86 (B7.2) (PE) (09275B), anti-CD40 (PE) (09665B), anti-CD4 (L3T4) (PE) (01065A), and anti-Dd (FITC) (06134D) (PharMingen Co, CA Becton Dickinson).


### 4.4. Preparation of Resting B Cell Populations

Splenocytes from MHC-Dd mice and CD2-Dd mice were depleted of erythrocytes with ACK lysis buffer (Biosource) and centrifuged over a discontinuous Percoll gradient (Pharmacia) containing 50–60% and 70% layers as described previously [1].  Cells at the 60%–70% interface were collected and depleted of T cells, macrophages, and DC by incubating with biotinylated mAbs (PharMingen) directed against CD3 (01082D), CD4 (09422D), CD8 (01042D), Thy1.1 (22212D), CD11b (Mac-1) (01712D), and CD11c (09702D), then mixed with streptavidin-coated microbeads (Miltenyi Biotec GmbH, Germany) and passed over a MACS separation column (Miltenyi Biotec GmbH). CD2-Dd T cells were prepared by depleting CD2-Dd B cells from the spleen cells of CD2-Dd mice by incubating with biotinylated mAbs directed against B220 and I-Aq, and then adding streptavidin-coated microbeads (Miltenyi Biotec GmbH, Germany) and passage over an MACS separation column (Miltenyi Biotec GmbH).


### 4.5. Isolation of DC by FACS

CD11c+ cells from MHC-Dd mice were isolated by cell sorting, using positive selection, as described previously [24]. Briefly, low-density spleen cells from MHC-Dd were incubated with PE-CD11c mAb (PharMingen). The suspensions, at 10 × 106 cells/mL of phosphate-buffered saline with 2% FBS and 0.5 mM EDTA, were sorted in an FACStar Plus instrument (Becton Dickinson). Recovered cells contained 91% CD11c+ DC.


### 4.6. MACS DC Isolation

Briefly, spleens were digested with 1 mL of complete RPMI containing collagenase D (Roche), 331 Mandl U/1 mL and DNase I, and bovine pancreas grade II (Roche) 16.5 ug/mL injected into the spleen and incubated at 37°C for 30 min. Spleens were then minced and passed 5x through a 19 ga needle, passed through a cell strainer (Becton Dickinson), treated with ACK lysing buffer (Biousource), and washed once with PBS containing 0.5% BSA and 2.5 mM EDTA. MACS separations were performed as per manufacturers recommendations using LS columns (Miltenyi Biotec) and anti-CD11c (N418) microbeads (Miltenyi Biotec).


### 4.7. T Cell Adoptive Transfer

Naive or Dd-primed T cells were prepared by negative selection from spleen cells of naive or primed FVB as described in [25]. Briefly, spleen cell suspensions from naive or primed FVB mice were incubated for 30 minutes on ice with a cocktail of mAbs directed against B220, I-Aq, CD11b, and CD11c mAbs, then were washed, incubated with streptavidin-coated microbeads for 15 min, and depleted by MACS. 3 × 107 of these cells were infused into naive ATX FVB mice that had been irradiated with 500 cGy 1 week previously. For purification of CD8+ T cells by negative selection with MACS, spleen cells from naive or primed FVB mice were depleted of CD4-, CD11b-, CD11c-, B220-, and I-Aq-positive cells using similar techniques, with a resulting CD8+ cell purity of >91.9%. 2 × 107 of these CD8+ cells were infused into naive ATX FVB mice that had been irradiated with 500 cGy 1 week previously.


### 4.8. Transplant Recipient Treatment

In vivo depletion of CD4+ and/or CD8+ T cells was performed by mAb injection. ATX FVB mice were injected i.p. with GK1.5 (150 μg/mouse) mAb and/or 53.6.7 (200 μg/mouse) mAb on days −6, −5, −1 and +5, with respect to transplantation. Mice were checked for depletion on the day prior to skin grafting [14, 26]. Depletion of CD4+ T cells or CD8+ T cells, or both, from the peripheral blood was >98% in all mice, as assessed by flow cytometry (FACScan, Becton Dickinson). In studies evaluating the role of CD40-CD40L interactions, MR1 (anti-CD40L mAb, 250 μg/mouse) was injected into ATX FVB mice on days –14, −10, −7, −3, 0, 4, and 7 and twice weekly thereafter, with respect to skin transplantation [15]. Anti-Dd sera were prepared by injecting MHC-Dd splenocytes into naive FVB mice i.p. (5×106 cells per mouse). The mice received three injections of cells at weekly intervals and 7 days after the last injection they were bled for the purpose of preparing a single pool of antisera. Mice were injected i.v. with 0.2 mL of anti-Dd sera 7, 9, or 12 days after receiving MHC-Dd skin graft.


### 4.9. Skin Grafting

Tail skin from donor mice was engrafted onto the recipient’s flank as described previously [27]. Grafts were scored daily or every other day until they were rejected (>80% loss of graft tissue).


## 4.1. Transgenic Mice

CD2-Dd transgenic mice were generated by injecting FVB embryos linking the CD2 promoter with Dd cDNA construct (a kind gift of Dr. Randy Ribaudo) coding sequence in pDd SELFIX.34 and an appropriate enhancer expression cassette p29Δ2(sal-) [14]. The CD2-Dd mice used were from the 4906 line, which showed the highest level of Dd expression [14]. MHC-Dd transgenic mice were generated by injecting FVB embryos with the genomic Dd gene, including the MHC class I promoter isolated from pDd1 (a kind gift of Dr. Gilbert Jay) [14].  Mouse studies were performed in accordance with Institutional Animal Care and Use Committee guidelines.


## 4.2. Mice and Priming

FVB/N mice and thymectomized (ATX) FVB/N mice, an inbred H-2q strain, were purchased from Taconic Farms (Germantown, NY, USA). For priming experiments, mice were given a single injection (i.p.) of 2×107 spleen cells from MHC-Dd mice at least 3 weeks before treatment.


## 4.3. Cytofluorometry Analysis

1 or 2 color analysis was performed by incubating cells with the following PE or FITC-conjugated mAbs: anti-CD11c (PE) (09705B), anti-CD86 (B7.2) (PE) (09275B), anti-CD40 (PE) (09665B), anti-CD4 (L3T4) (PE) (01065A), and anti-Dd (FITC) (06134D) (PharMingen Co, CA Becton Dickinson).


## 4.4. Preparation of Resting B Cell Populations

Splenocytes from MHC-Dd mice and CD2-Dd mice were depleted of erythrocytes with ACK lysis buffer (Biosource) and centrifuged over a discontinuous Percoll gradient (Pharmacia) containing 50–60% and 70% layers as described previously [1].  Cells at the 60%–70% interface were collected and depleted of T cells, macrophages, and DC by incubating with biotinylated mAbs (PharMingen) directed against CD3 (01082D), CD4 (09422D), CD8 (01042D), Thy1.1 (22212D), CD11b (Mac-1) (01712D), and CD11c (09702D), then mixed with streptavidin-coated microbeads (Miltenyi Biotec GmbH, Germany) and passed over a MACS separation column (Miltenyi Biotec GmbH). CD2-Dd T cells were prepared by depleting CD2-Dd B cells from the spleen cells of CD2-Dd mice by incubating with biotinylated mAbs directed against B220 and I-Aq, and then adding streptavidin-coated microbeads (Miltenyi Biotec GmbH, Germany) and passage over an MACS separation column (Miltenyi Biotec GmbH).


## 4.5. Isolation of DC by FACS

CD11c+ cells from MHC-Dd mice were isolated by cell sorting, using positive selection, as described previously [24]. Briefly, low-density spleen cells from MHC-Dd were incubated with PE-CD11c mAb (PharMingen). The suspensions, at 10 × 106 cells/mL of phosphate-buffered saline with 2% FBS and 0.5 mM EDTA, were sorted in an FACStar Plus instrument (Becton Dickinson). Recovered cells contained 91% CD11c+ DC.


## 4.6. MACS DC Isolation

Briefly, spleens were digested with 1 mL of complete RPMI containing collagenase D (Roche), 331 Mandl U/1 mL and DNase I, and bovine pancreas grade II (Roche) 16.5 ug/mL injected into the spleen and incubated at 37°C for 30 min. Spleens were then minced and passed 5x through a 19 ga needle, passed through a cell strainer (Becton Dickinson), treated with ACK lysing buffer (Biousource), and washed once with PBS containing 0.5% BSA and 2.5 mM EDTA. MACS separations were performed as per manufacturers recommendations using LS columns (Miltenyi Biotec) and anti-CD11c (N418) microbeads (Miltenyi Biotec).


## 4.7. T Cell Adoptive Transfer

Naive or Dd-primed T cells were prepared by negative selection from spleen cells of naive or primed FVB as described in [25]. Briefly, spleen cell suspensions from naive or primed FVB mice were incubated for 30 minutes on ice with a cocktail of mAbs directed against B220, I-Aq, CD11b, and CD11c mAbs, then were washed, incubated with streptavidin-coated microbeads for 15 min, and depleted by MACS. 3 × 107 of these cells were infused into naive ATX FVB mice that had been irradiated with 500 cGy 1 week previously. For purification of CD8+ T cells by negative selection with MACS, spleen cells from naive or primed FVB mice were depleted of CD4-, CD11b-, CD11c-, B220-, and I-Aq-positive cells using similar techniques, with a resulting CD8+ cell purity of >91.9%. 2 × 107 of these CD8+ cells were infused into naive ATX FVB mice that had been irradiated with 500 cGy 1 week previously.


## 4.8. Transplant Recipient Treatment

In vivo depletion of CD4+ and/or CD8+ T cells was performed by mAb injection. ATX FVB mice were injected i.p. with GK1.5 (150 μg/mouse) mAb and/or 53.6.7 (200 μg/mouse) mAb on days −6, −5, −1 and +5, with respect to transplantation. Mice were checked for depletion on the day prior to skin grafting [14, 26]. Depletion of CD4+ T cells or CD8+ T cells, or both, from the peripheral blood was >98% in all mice, as assessed by flow cytometry (FACScan, Becton Dickinson). In studies evaluating the role of CD40-CD40L interactions, MR1 (anti-CD40L mAb, 250 μg/mouse) was injected into ATX FVB mice on days –14, −10, −7, −3, 0, 4, and 7 and twice weekly thereafter, with respect to skin transplantation [15]. Anti-Dd sera were prepared by injecting MHC-Dd splenocytes into naive FVB mice i.p. (5×106 cells per mouse). The mice received three injections of cells at weekly intervals and 7 days after the last injection they were bled for the purpose of preparing a single pool of antisera. Mice were injected i.v. with 0.2 mL of anti-Dd sera 7, 9, or 12 days after receiving MHC-Dd skin graft.


## 4.9. Skin Grafting

Tail skin from donor mice was engrafted onto the recipient’s flank as described previously [27]. Grafts were scored daily or every other day until they were rejected (>80% loss of graft tissue).



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*Source: 701051-2012-11-28.xml*