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Two levels of help for B cell alloantibody production.

We have examined whether T cell stimulation by direct or indirect pathways contributes to alloantibody production by B cells after major histocompatibility complex (MHC)-disparate skin graft rejection in mice. Experiments were performed using normal mice, MHC class II-deficient mice, MHC class II-deficient mice with an intact peripheral CD4+ cell population (due to expression of class II antigens only on thymic epithelium), mice lacking the cytoplasmic tail of their MHC class II antigens, and mice depleted of CD4+ cells by anti-CD4 monoclonal antibody treatment. Depletion of recipient CD4+ cells reduced alloantibody production to barely detectable levels. Absence of donor MHC class II antigens did not affect the production of either immunoglobulin (Ig)M or IgG antibodies directed at class I alloantigens. Absence of recipient MHC class II antigens, however, led to production of only IgM but not IgG antibodies, even if the recipients had an intact CD4+ cell population. Absence of the cytoplasmic tail of the recipient's MHC class II antigens led to the production of slightly reduced amounts of IgG antibody. These findings indicate that (a) CD4+ cells are essential helper cells for B cell alloantibody production; (b) production of IgM alloantibody can occur with help from CD4+ cells, which recognize either donor class II antigens or modified recipient class II antigens; (c) isotype switching from IgM to IgG alloantibody requires help from CD4+ cells activated by antigens presented by recipient MHC class II molecules; and (d) the cytoplasmic domain of the recipient MHC class II molecules may be involved in the mechanism that leads to isotype switching by B cells. Thus, there are two levels of CD4-mediated help available for B cells responding to alloantigens: one (involving a noncognate interaction) can produce B cell activation, and a second (involving a cognate interaction) is required for differentiation and IgG alloantibody production.

T cells help B cells to produce antibody against T celldependent antigens and to switch the isotype of the antibody produced from IgM to other isotypes, including IgG. This T-B cell interaction has been characterized by studies of immune responses to hapten-carrier conjugates and shown to be MHC class II antigen restricted (1,2). In the case of allogeneic tissue transplantation, helper T cells can be activated directly by class II antigens expressed on donor APC, or indirectly by peptides of donor antigens presented by self class II molecules on recipient APC. Two different schemes have therefore been proposed by which T-B cell collaboration might occur for B cell responses to class I alloantigens expressed on foreign cells (Fig. 1). First, the B cells could be stimulated by class I antigens on donor This work was presented in part at the American Society of Nephrology meeting in Orlando, FL, October 26-29, 1994. APC, and the CD4 + helper cells could be stimulated by MHC class II antigens on the same cells. This would bring the T and B cells into physical proximity, but without an interaction between the T cell receptor and the B cell's class II antigens. Thus, this would be a "noncognate" interaction. Second, the B cells could be stimulated by class I antigens on donor cells, and the CD4 + cells could be stimulated by foreign peptides presented by class II antigens on the recipient's own APC. These CD4 + cells could then interact with the B cells in a "cognate" fashion, achieved by the class II-restricted T cell recognition of foreign peptides presented by the B cells.
Using mice that lack MHC class II antigens, as either donors or recipients of MHC-disparate skin grafts, we have examined which of these pathways of interaction leads to the production ofalloantibody. The results demonstrate the importance of the indirect pathway in allogeneic responses to provide help for B cell anti-class I alloantibody production. (A) B cells could be stimulated by class I antigens on the surface of donor APC, and CD4 + helper cells could be stimulated directly by the donor class II antigens on the same APC. This would bring the T and B cells into physical contact but would not allow a cognate interaction between the respon& ing T cell receptor with the MHC class II molecule of the B cell. (B) B cells could be stimulated by antigens on donor cells, and CD4 + cells could be stimulated indirectly by foreign peptides presented by the recipient's APC. These CD4 + cells could then provide help for the B cells by a cognate interaction with the B cell's own class lI antigens.
in vivo, and they also reveal that C D 4 + cells can provide two levels o f help for B cells, depending on the nature o f the T -B cell interaction.

Materials and Methods
Mice. Male BALB/cByJ (BALB/c) (H-2 d) and C57B1/6J (B6) (H-2 b) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice lacking M H C class II antigens (class II deficient), mice expressing M H C class II antigens only on their thymic epithelium (CD4 + class II deficient), and mice expressing class II antigens with truncated cytoplasmic domains (tailless), were bred in our laboratories.
The genotype and phenoWpe of mice lacking M H C class II antigens have been previously described (3). For these experiments, mice from the third generation backcross of(B6 • 129)F1 to B6 mice were used. Prior characterization of the class II-deficient mice has shown that no class II antigen expression can be detected. These mice also show a substantial depletion of CD4 + peripheral T cells, although 3-5% of the peripheral Thyl § cells are CD4 +. Class II-deficient mice have normal levels and development of B cells since T cell-independent B cell responses occur in vivo, and T cell-dependent B cell activation can occur in vitro, despite the absence of M H C class II antigens (4).
Mice expressing M H C class II antigens only on their thymic epithelium were generated by breeding class lI-deficient mice with a C57B1/6-transgenic strain (gift of Dr. D. Lo, Scripps Research Institute, La Jolla, CA), which expresses the EIx transgene only on thymic epithelium (5). These mice are M H C class 1I deficient on all cells other than thymic epithelium, but they do have normal numbers of peripheral CD4 + cells, which proliferate in an allo M L R in vitro (Laufer, T., unpublished observations).
Mice that express a class 1I A b molecule with a truncated cytoplasmic domain were generated by mating class II-deficient mice with mice expressing an A~ b transgene that lacks 13 of its 18 cytoplasmic domain residues. The initial characterization of these mice has been previously reported, demonstrating that they develop normal levels and functions o f C D 4 + T cells and have normal B cell responses to environmental pathogens (6).
Skin Grafting. Trunk skin grafts were placed oi1 mice according to the technique of Billingham and Medawar (8). Mice were anesthetized with chloral hydrate supplemented with ether. After rejecting their grafts, mice were then bled from the retroorbital plexus and subsequently boosted with 10 s splenocytes of the same donor type by intraperitoneal injection at weekly intervals for 3 wk. Sera used for the experiments shown were all obtained 3 wk afi:er placing the skin grafts.

Antibody-mediated Complement-dependent Cytotoxicity Assay.
These assays were performed in two stages with minor modifications from the technique previously described (9). Sera were heat inactivated at 56~ for 30 min and stored at -2 0~ until tested. 10 s 51Cr-labeled mouse splenocytes of donor type were incubated with serially diluted sera in a final volume of 50 txl in U-bottomed wells for 15 rain at 37~ Cells were washed with medium, pelleted, resuspended, and incubated with rabbit complement (C-6 Diagnostic, Inc., Mequon, WI) at a final dilution of 1:12 for 30 min at 37~ Cells were pelleted and 100 Ixl of the supernatant harvested and counted on a gamma counter (Gamma 4000; Beckman Instruments, Inc., Fullerton, CA). Percentage of lysis was calculated by comparison to the S~Cr release obtained using a rabbit anti-mouse lymphocyte serum made in our laboratory. To inactivate antibody of the IgM isotype, 50 ~xl of serum was incubated with 5 Ixl of 1 M 2-ME for 30 min at 37~ before the assay.

Antibody Staining and Flow Cytometric Analysis. Thymic T cells
were used as targets for flow cytometric analysis of alloantibody production. FACS | medium contained PBS, 1% BSA, and 0.1% sodium azide. 3 X l0 s cells were added to each well of a 96 well U-bottomed plate. Mouse FcgIItL was blocked with 20 txl of 2.4 G2, a rat anti-mouse FcglIR mAb, by incubating with the cells at 4~ for 10 rain. The cells were then washed twice with 100 I~1 of FACS | medium. 25 Ixl of serum from mice that had rejected skin grafts was then added at 1:2 and 1:4 dilutions to the test wells and incubated at 4~ for 30 min. Cells were again washed twice with 100 Ixl FACS | solution. FITC-conjugated rat anti-mouse antibodies against [gM, IgG1 (Zymed Laboratories, Inc., South San Francisco, CA), IgG2a, or IgG2b (Baxter Healthcare Corp., Mundelein, IL) were then added and incubated with the cells at 4~ for 30 rain. Cells were again washed twice with 100 Izl FACS | solution, fixed with 2% paraformaldehyde, and analyzed using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, Mountain View, CA).

Results
To generate alloantibody production, skin grafts from MHC-disparate allogeneic donors were placed on recipient mice. All skin grafts were rejected within 15 d, and neither treatment with anti-CD4 mAb nor the absence of MHC class II antigens on the donor or recipient altered the time to rejection when compared with normal controls. These findings are in keeping with previously reported data (9)(10)(11). Sera from recipient mice were tested 3 wk after grafting for the presence of alloantibody, as shown in Fig. 2. Additional sera from later time points were also tested, but the results in all groups were not different in character from those shown.
Normal Mice. Normal recipients of normal MHC-disparate skin grafts produced antidonor cytotoxic alloantibody of IgM isotype and then increasing titers of IgG isotype. The levels at 3 wk are shown in Fig. 2 A. The antibodies in these sera were shown to be directed at class I alloantigens by testing them on targets from MHC-congenic strains (data not shown).
CD4-depleted Redpients. Recipients treated with thymectomy and anti-CD4 mAb before skin grafting produced very low or undetectable levels of cytotoxic alloantibody, and there was no switch to IgG isotype, as shown in Fig.  2 B. This requirement for CD4 + cells to generate alloantibody production is in agreement with previously reported data (9).

MHC Class II-deficient Mice as Donors. When MHC class
II--deficient mice were used as skin graft donors for normal recipients, both IgG and lgM alloantibody production was equivalent to normal controls, as shown in  Figure 2. Cytotoxic aUoantibody levels in response to skin grafting. Sera were obtained 3 wk after grafting, and alloantibody levels were determined in a 5tCr release antibody-mediated complement-dependent cytotoxicity assay using mouse splenocytes of donor type as targets. IgM combined with lgG levels were measured using untreated sera. IgG levels alone were obtained after treatment of sera with 2-ME. responding to modified self class II antigens, provide help efficiently for alloantibody production. M H C Class II-deficient Mice as Recipients. M H C class IIdeficient mice that received normal skin grafts produced cytotoxic alloantibodies, but in reduced titers when compared with normal controls. The antibody produced was of IgM isotype only, and no IgG was found in these recipients, both in assays for antibody-mediated complementdependent cytotoxicity as shown in Fig. 2 D and by FITC  anti-antibody staining (data not shown).
Although the donor skin grafts in these experiments expressed class II antigens that could stimulate CD4 + cells directly, the recipients had two defects that could account for their reduced antibody production: they lacked class II antigens on their own cells for use in the indirect pathway, and they also had severely reduced numbers of CD4 + cells to provide help for B cells. Therefore, to test whether direct stimulation, in the absence of the indirect pathway, could provide help for B cells, we needed recipients without class II antigen expression but that had normal numbers o f C D 4 + cells.
Mice Expressing Class II Antigens Only on Thymic Epithelium as Recipients. Class II-deficient mice that express a class II transgene only on thymic epithelium develop normal numbers of mature CD4 + cells but still lack class II antigens on their peripheral APC. When these mice were recipients of normal skin grafts, they produced only moderate titers of total cytotoxic antibody, all of which was again of IgM isotype, as shown in Fig. 2 E. These results were consistent in antibody-mediated complement-dependent assays and in assays using FITC anti-antibody staining (data not shown). Thus, in the presence of direct but in the absence of indirect T cell stimulation, CD4 + cells can provide help for B cells, but only for a limited response.
Mice with Tailless Class 1I Molecules as Recipients.
Finally, recipients lacking the intracytoplasmic domain of their M H C class I1 antigens were found to produce alloantibody that included both IgM and IgG isotypes. However, in two separate experiments, there was a slight reduction in the IgG component of the total cytotoxic antibody response compared with normal animals (Fig. 2 F).

Discussion
The primary purpose of these experiments was to determine the answer to a long-standing question in transplantation immunology: whether donor class II antigens stimulate helper CD4 + cells for alloantibody production by direct recognition, or whether recipient class II molecules, presenting peptides of donor antigens, stimulate a helper response through what has been termed the indirect pathway. We expected that the indirect pathway would be sufficient, since this is the mechanism for a normal immune response. We also expected, based on previous studies, that both IgM and IgG alloantibody responses after skin transplantation would depend on the presence of CD4 + T cells (9). Finally, we were not surprised that stimulation of CD4 + cells only by the nonphysiologic, direct pathway 702 Two Levels of Help for B Cell was unable to stimulate IgG antibody production. These experiments, therefore, settle the issue in transplantation immunology, at least as far as the response to class I antigens on skin grafts. In doing so they also provide further evidence that the so-called indirect pathway in transplantation does play a role in the immune response to foreign tissues and that this pathway is actually critical as far as alloantibody production is concerned. The result we did not expect (which was, therefore, particularly interesting) was that CD4 + cells stimulated by the direct pathway alone would allow an intermediate B cell response, including IgM but not IgG alloantibody production. This result is of interest beyond the field of transplantation immunology since it indicates that B cells are susceptible to two levels of help for antibody responses. When CD4 + cells are stimulated in a way that links them in physical proximity to B cells, their helper factors can apparently activate the B cells partially, allowing the IgM response. However, apparently only when the CD4 + cells are stimulated in a way that provides both linkage and a cognate interaction through the class II molecules of the B cells does the helper response signal the B cells to switch to IgG production. Only a transplantation experiment, especially one involving the recently available class II knock-out mice, would allow us to see this distinction in vivo. In no other way could one bring B cells, recognizing a foreign protein antigen, into a physical but noncognate linkage with CD4 § helper cells than to stimulate the CD4 + cells with allogeneic class II molecules while eliminating the class II molecules of the recipient's own APC. Thus, these transplantation experiments provide new insight into the mechanisms of normal B cell physiology.
Our experiments have not yet proven that a physical linkage is required between the CD4 + cells, stimulated by donor class II antigens, and the B cells that respond to donor class I alloantigens. It seems likely that such linkage is necessary, given the many previous experiments indicating that linkage between T and B cells is a physiologic requirement in vivo (1,2,12). However, to demonstrate this requirement in our model we will need to determine whether B cells from mice lacking their own peripheral class II antigen expression can respond to donor alloantigens, such as Thyl, which are not expressed on the same cells that express the class II alloantigens that stimulate CD4 + helper cells.
It is also not clear from our experiments exactly why the cognate interaction between T and B cells, through recognition of the B cells' class II antigens, should be important in the switch to IgG antibody production. One explanation is that such an interaction is simply the best way to achieve the closest possible physical proximity between helper cells and B cells. The other is that the class II molecules of B cells are involved in the signaling that allows conversion to an IgG response. Previous in vitro studies with transfected cell lines have suggested that the cytoplasmic tail of class II molecules is necessary for the expression of costinmlatory molecules (13)(14)(15)(16), but subsequent studies with the same transgenic mice used in our experiments, which express only class II molecules with a truncated intracytoplasmic domain, have indicated that they produce normal IgG responses to nominal antigens, although they appear to have a quantitative defect in their ability to present peptides o f foreign proteins (6). In our experiments we found that both IgM and IgG alloantibody responses were fundamentally intact in the mice lacking their class II intracytoplasmic domain, although the IgG response appeared to be weaker in several experiments. Thus, our results are consistent either with the interpretation that slightly diminished presentation of peptides by these truncated class II molecules weakens the physical association o f T and B cells, or that the absence of the cytoplasmic portion o f the B cells' class II molecules impairs the signaling that leads to IgG conversion.