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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Am J Transplant. Author manuscript; available in PMC 2015 February 1.
Published in final edited form as:
PMCID: PMC4018729

Alloprimed CD8+ T cells Regulate Alloantibody and Eliminate Alloprimed B cells through Perforin- and FasL-dependent mechanisms


While it is well known that CD4+ T cells and B cells collaborate for antibody production, our group previously reported that CD8+ T cells downregulate alloantibody responses following transplantation. However, the exact mechanism involved in CD8+ T cell-mediated downregulation of alloantibody remains unclear. We also reported that alloantibody production is enhanced when either perforin or FasL is deficient in transplant recipients. Here, we report that CD8+ T cell-deficient transplant recipient mice (high alloantibody producers) exhibit an increased number of primed B cells compared to wild-type transplant recipients. Furthermore, CD8+ T cells require FasL, perforin, and allospecificity to downregulate posttransplant alloantibody production. In vivo CD8-mediated clearance of alloprimed B cells was also FasL- and perforin-dependent. In vitro data demonstrated that recipient CD8+ T cells directly induce apoptosis of alloprimed IgG1+ B cells in co-culture in an allospecific and MHC class I-dependent fashion. Altogether these data are consistent with the interpretation that CD8+ T cells downregulate posttransplant alloantibody production by FasL- and perforin-dependent direct elimination of alloprimed IgG1+ B cells.

Keywords: Alloantibody, CD8+ T cells, B cells, FasL, Perforin


Transplantation has become the treatment of choice for end stage liver, renal, cardiac, and pulmonary disease. This modality of treatment can be life saving, and in the case of renal transplantation, vastly improves the quality of life and prolongs survival. Despite the improvement in short-term graft survival, this has not translated into an increase in the half-life of transplants which has remained the same due to chronic rejection, the main cause of long-term graft failure (13). Clinical and experimental data indicate that MHC-directed alloantibodies play a critical role in acute and chronic rejection after solid-organ, as well as after cellular transplant (1,2,410). Antibody-mediated allograft rejection, as well as conditions that promote humoral immunity posttransplant, are not well understood despite their critical impact on transplant outcomes.

A conceptual barrier to progress in modulating posttransplant humoral alloimmunity has been the dominant focus on CD4+ T cells as regulators of antibody responses (11,12). In contrast, we recently provided the first evidence supporting a pivotal role played by IFN-γ+CD8+ T cells to inhibit the magnitude of IL-4-dependent, IgG1 alloantibody produced following transplant (13). In these studies we reported that one mechanism by which CD8+ T cells regulate alloantibody production is through cytokine (IFN-γ)-mediated skewing of CD4+ T cell maturation away from an IL-4 (Th2)-dominant pathway (IL-4 promotes IgG1 alloantibody production). However, in other studies, we also noted that perforin- or FasL-deficient transplant recipient mice produced increased amounts of alloantibody compared to wild-type controls (14). Alloprimed CD8+ T cells develop multiple cytotoxic effector mechanisms including FasL, perforin, and TNF-α (1517). The current studies were conducted to test the hypothesis that CD8+ T cells might also downregulate alloantibody production through other mechanisms such as FasL-, perforin-, and/or TNF-α-mediated cytotoxic clearance of alloprimed IgG1+ B cells.

Materials and Methods

Experimental animals

FVB/N (H-2q MHC haplotype, Taconic), C57BL/6 (wild-type), CD8 KO, TNF-α KO, Beta-2 microglobulin KO, Perforin KO, and FasL mutant (all H-2b; Jackson Labs) mouse strains (all 6–10 weeks of age) were used in this study. Transgenic FVB/N mice expressing human alpha-1 antitrypsin (hA1AT) were the source of “donor” hepatocytes, as previously described (18). All experiments were performed in compliance with the guidelines of the Institutional Laboratory Animal Care and Use Committee of The Ohio State University (Protocol 2008A0068-R1).

Hepatocyte isolation, purification, and transplantation

Hepatocyte isolation, purification, and transplantation were performed, as reported (18). Graft survival was determined by detection of the secreted hA1AT in serial recipient serum samples by ELISA (18,19).

CD8+ T cell isolation

Isolation of CD8+ T cells was performed by negative selection columns as per the manufacturer’s recommendations (R&D Systems, Minneapolis, MN; purity routinely >90%).

B cell isolation

B cells were purified from splenocytes using anti-mouse B220+ magnetic beads to isolate B cells from untransplanted mice (naïve B cells) or anti-mouse IgG1+ magnetic beads to isolate alloprimed IgG1+ B cells obtained from recipient mice following the manufacturer’s instructions (Miltenyi Biotech, Auburn, CA; purity routinely >95%). B cells were collected in complete media (RPMI 1640, 10% FBS, 20 mM Hepes, 2 mM L-glutamine, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, 1% non-essential amino acids, and 1% pen-strep antibiotics).


Analysis of B cells for IgG1 production was analyzed as previously described (20). Briefly, PVDF MultiScreen-IP plates (Millipore, Billerica, MA) were coated overnight (4°C) with anti-IgG1 antibody. 5×105 splenocytes (from naïve or recipient mice) were plated and serial diluted (down to 976 cells/well) with media, incubated overnight (37°C and 5% CO2), and then lysed with diH2O. Wells were incubated (4°C, overnight) with alkaline phosphatase conjugated anti-IgG1 (Southern Biotechnology, Birmingham, AL). Plates were developed with BCIP/NBT phosphatase substrate (Sigma), dried, and analyzed by computer-assisted image analysis using a KS ELISPOT Automated Reader using KS ELISPOT software 4.2 (Carl Zeiss Inc, Thornwood, NY). Data is reported as Relative Spot Forming Cells (SFC) per 1×106 splenocytes. A side-by-side ELISA was run in a similar fashion and performed as previously described (21). Notably, IgG1 standard (Southern Biotechnology) and colormetric analysis (PNPP substrate, Sigma) were utilized to quantitate in vitro antibody production by a Spectramax Plus microplate reader (Molecular Devices, Sunnyvale, CA).

Assay of allospecific antibody

Antibody IgG isotypes from recipient serum was tested for allospecificity by incubation with allogeneic FVB/N target splenocytes, as previously described (13). Total lymphocytes were utilized for gating. Alloantibody levels are represented as the percentage of target cells labeled by secondary fluorescent antibody (14).

In vivo cytotoxicity assay

Detection of in vivo cytolytic elimination of alloprimed IgG1+ B cells was modified from published methods (22,23). Control target B220+ B cells were isolated from naïve wild-type C57BL/6 mice and were stained with 0.2μM Carboxyfluorescein Diacetate Succinimidyl Ester (CFSElow, Molecular Probes, Eugene, OR). Allohepatocyte primed IgG1+ target B cells were isolated from CD8 KO recipient mice and stained with 2.0μM CFSE (CFSEhigh). Allograft recipient mice and control naïve mice received 10×106 CFSE-labeled naïve B220+ B cells and 10×106 alloprimed IgG1+ B cells by tail vein injection. Eighteen hours following adoptive transfer, B cells were retrieved from the spleen and analyzed by flow cytometry (CFSE gating). Percentage of allospecific cytotoxicity was calculated using a published formula (24).

In vitro cytotoxicity assay

Alloprimed IgG1+ B cells (CD8 KO recipients) and bulk CD8+ T cells (wild-type recipients) were purified from recipient spleens on day 7 posttransplant. Naïve B cells and CD8+ T cells were utilized as controls. Cytotoxicity was measured using a LIVE/DEAD cell-mediated cytotoxicity kit (Invitrogen, Eugene, OR) and performed according to the manufacturer’s instructions. In brief, target B cells were stained with 3,3′-dioctadecyloxacarbocyanine (DiOC18(3)), a green fluorescent membrane stain. CD8+ T cells and B cells were co-cultured at a 10:1 ratio for 4 hours. In some experimental groups, a transwell membrane was utilized to separate CD8+ T cells and B cells. Cells were stained with propidium iodide (PI) to assess cell death and uptake was immediately analyzed by flow cytometry.

Statistical analysis

Statistical calculations were performed using a one-tailed Student’s t test to analyze differences between experimental groups. P<0.05 was considered significant. To demonstrate the distribution of the data, results are listed as the mean plus or minus the standard error.


The number of alloprimed IgG1-producing B cells is higher in the absence of CD8+ T cells

To determine the number of B cells that produced IgG1 alloantibody posttransplant within wild-type and CD8-deficient recipient mice, we utilized ELISPOT technology on days 4, 7, 11, and 14. Wild-type recipients exhibited significant induction of IgG1-producing cells (spot forming cells, SFC) per 106 splenocytes analyzed on days 4 and 7 posttransplant compared to naïve (day 0) wild-type mice. CD8 KO recipients exhibited a significantly higher number of IgG1-producing cells per 106 cells on days 4 and 7 posttransplant compared to wild-type recipients (Figure 1A). In vitro antibody production, as demonstrated in a side-by-side ELISA, was higher in CD8 KO splenocytes versus wild-type splenocytes (Figure 1B). Of note, splenocytes from CD8 KO mice and wild-type recipient mice have similar percentages of B cells prior to transplant (B220+; 37.9±1.2% versus 42.1±2.4%, p=0.066). These findings clarify our previous results that CD8 KO recipients have elevated serum alloantibody levels compared to wild-type recipients based on development of a higher number of alloantibody producing B cells, rather than production of a higher amount of alloantibody per cell by a similar number of alloprimed B cells (13).

Figure 1Figure 1
CD8+ T cell deficient recipients have increased number of antibody producing cells

We also addressed the possibility that these observed differences were due to CD8+ T cell-mediated interference with B cell expansion posttransplant. In studies where CD8 KO splenocytes were CFSE stained and adoptively transferred into CD8 KO or WT hepatocyte recipient mice on day 0, we found that on day 4, in CFSE dilution studies gating on B220+ B cells, there was no difference in B cell proliferation within CD8-sufficient and CD8-deficient recipients (data not shown). Collectively these data suggest that the difference in the number of IgG1-producing cells between CD8-sufficient and CD8-deficient recipients (Figure 1A) is due to CD8-mediated cytotoxicity of alloprimed IgG1+ B cells, rather than due to CD8-mediated suppression of B cell expansion or the amount of antibody secreted per B cell.

CD8-mediated inhibition of alloantibody production is dependent on allospecific activation of CD8+ T cells

To determine if CD8+ T cells require allospecific activation to inhibit posttransplant alloantibody production, we transplanted CD8 KO mice (H-2b MHC haplotype) with FVB/N hepatocytes (H-2q MHC haplotype), and then adoptively transferred CD8+ T cells from either naïve C57BL/6 mice (H-2b MHC haplotype), FVB/N hepatocyte recipient C57BL/6 mice (bulk alloprimed, anti-H-2q CD8+ T cells), or B10.BR hepatocyte recipient C57BL/6 mice (bulk third-party primed, anti-H-2k CD8+ T cells). Serum alloantibody levels were measured by flow cytometry on day 14 posttransplant. As previously reported (13), CD8 KO mice in the present study exhibited maximal levels of serum alloantibody on day 14 posttransplant (Figure 2). When naïve CD8+ T cells were adoptively transferred on day 0, CD8+ T cells induced a significant reduction in alloantibody production. However, if naïve CD8+ T cells were adoptively transferred into CD8-deficient recipients on day 7 following transplantation, no significant reduction in alloantibody occurred. Presumably, these results reflect the requirement for CD8+ T cell priming to affect downregulation of posttransplant alloantibody. In order to address this possibility, CD8-deficient recipient mice were adoptively transferred on day 7 posttransplant with bulk alloprimed CD8+ T cells. These recipients exhibited significantly reduced serum alloantibody when tested on day 14 posttransplant. Third party-primed CD8+ T cells that were adoptively transferred into CD8 KO recipients on day 7 posttransplant had no effect on alloantibody levels. These findings suggest that primed allospecific CD8+ T cells mediate downregulation of alloantibody production posttransplant.

Figure 2
CD8+ T cell-mediated downregulation of alloantibody production is dependent on allospecificity

CD8+ T cell perforin and FasL are required for downregulation of posttransplant alloantibody

To determine if FasL and perforin expression in CD8+ T cells play a critical role in the regulation of posttransplant alloantibody, we adoptively transferred FasL- or perforin-deficient CD8+ T cells into CD8 KO hepatocyte recipients on the day of transplant. A cohort of CD8 KO mice were adoptively transferred with wild-type CD8+ T cells as a positive control. Serum alloantibody levels were measured on day 14 posttransplant. CD8 KO recipients that were adoptively transferred with wild-type (WT) CD8+ T cells had significantly less serum alloantibody compared to CD8 KO recipients without adoptive transfer of CD8+ T cells 14 days posttransplant (Figure 3). CD8 KO recipients reconstituted with either FasL mutant CD8+ T cells or perforin KO CD8+ T cells had significantly higher serum alloantibody levels compared to recipients receiving WT CD8+ T cells. However, both CD8 KO recipient mice adoptively transferred with FasL- or perforin-deficient CD8+ T cells exhibited significantly reduced alloantibody production compared to CD8 KO recipient mice without adoptive transfer of CD8+ T cells. Reconstitution of CD8 KO recipients with TNF-α KO CD8+ T cells resulted in serum alloantibody levels that were not significantly different than levels in recipients reconstituted with WT CD8+ T cells. These data indicate that CD8+ T cells require both FasL and perforin but not TNF-α for maximal downregulation of posttransplant alloantibody production. Despite alloantibody downregulation following bulk CD8+ T cell adoptive transfer to CD8 KO recipient mice, transplant survival was no different in CD8 KO recipients with or without adoptive transfer (day 14 for both). This is not surprising since bulk CD8+ T cells also reject hepatocytes (17,2527).

Figure 3
CD8+ T cell-mediated downregulation of alloantibody production is dependent on perforin and FasL

In vivo survival of alloprimed IgG1+ B cells is decreased by CD8+ T cells through a FasL- and perforin-dependent mechanism

To investigate whether alloprimed IgG1+ B cells were preferentially depleted in vivo by CD8+ T cell-dependent mechanisms, an in vivo cytotoxicity assay was used as previously described (24). We tested the recovery rate of alloprimed IgG1+ B cells (CFSEhigh) compared to naïve B cells (B220+; CFSElow) following adoptive transfer of these target B cells into recipient mice on day 7 posttransplant. Day 7 posttransplant was chosen because CD8+ T cell-mediated in vivo cytotoxicity was found to be maximal on that day (24). Naïve mice also received target B cells as a control. The relative recovery of alloprimed IgG1+ B cells, as compared to naïve B cells, from these recipient mice was calculated as previously described (24). To investigate this, we utilized CD8 KO recipient mice and adoptively transferred them with wild-type, FasL mutant, or perforin KO CD8+ T cells. CD8 KO recipient mice exhibited low cytotoxicity to alloprimed IgG1+ B cell targets. The adoptive transfer of wild-type naïve CD8+ T cells (day 0 posttransplant) resulted in significant alloprimed IgG1+ B cell cytotoxicity (Figure 4), whereas FasL- and perforin-deficient CD8+ T cells exhibited reduced cytotoxicity compared to wild-type CD8+ T cells. These findings suggest that alloprimed CD8+ T cells mediate alloprimed B cell clearance through FasL- and perforin-dependent cytotoxic mechanisms.

Figure 4
CD8-mediated in vivo cytotoxicity of alloprimed IgG1+ B cells is dependent on FasL and perforin

Alloprimed CD8+ T cells directly induce apoptosis of alloprimed IgG1+ B cells which is dependent on target B cell expression of MHC I

To determine if alloprimed IgG1+ B cells were being eliminated directly by allospecific CD8+ T cells, we performed an in vitro cytotoxicity assay utilizing a two-color fluorescence assay (see methods). Alloprimed CD8+ T cells and alloprimed IgG1+ B cells were harvested from spleens of FVB/N (H-2q) hepatocyte recipients on day 7 (wild-type and MHC I-deficient mice). Naïve CD8+ T cells and naïve B220+ B cells from untransplanted mice and third party-primed CD8+ T cells from B10.BR transplanted wild-type mice were used as controls in the cytotoxicity assay. CD8+ T cells and B cells were combined at a 10:1 target:effector ratio and co-incubated for 4 hours. Following co-incubation, the level of B cell-induced apoptosis was analyzed. As shown in Figure 5A, when alloprimed CD8+ T cells were co-incubated with alloprimed IgG1+ B cells, the level of target B cell apoptosis was significantly higher than that observed in co-cultures with naïve CD8+ T cells and bulk third party primed CD8+ T cells. Bulk alloprimed CD8+ T cells did not induce specific cytotoxicity of naïve B220+ B cells beyond baseline cytotoxicity observed in co-cultures with naïve CD8+ T cells or with bulk third party primed CD8+ T cells. Alloprimed CD8+ T cells did not induce apoptosis of alloprimed IgG1+ B cells from beta-2-microglobulin KO mice (MHC I-deficient) beyond baseline cytotoxicity observed in co-cultures with naïve CD8+ T cells. These findings are consistent with the interpretation that alloprimed CD8+ T cells mediate cytotoxicity of alloprimed IgG1+ B cells through recognition of allopeptide presentation by MHC I. Since our prior studies demonstrate that the in vitro cytotoxicity assay preferentially reflects perforin-mediated cytotoxicity (17), we limit the interpretation of these results of allospecificity and MHC I/TCR cell-cell contact interactions to CD8+ T cell perforin-mediated cytotoxicity. In addition, we performed a similar side-by-side in vitro cytotoxicity assay for CD8 and B cell co-cultures using transwell membranes. In all conditions, using transwell membranes, cytotoxicity was not observed indicating that the observed in vitro cytotoxicity is contact-dependent (Figure 5B).

Figure 5Figure 5
In vivo alloprimed CD8+ T cells induce in vitro apoptosis of alloprimed IgG1+ B cells in an MHC class I dependent fashion


Clinical and experimental studies highlight the barrier that both alloantibody-mediated acute and chronic rejection pose to successful allograft survival (2833). Acute antibody-mediated rejection occurs despite the use of maintenance immunosuppression to prevent rejection and is associated with worse graft outcome than T cell-mediated rejection (34). Many current immunosuppressive treatments effectively inhibit alloreactive T cell responses. Therefore, it is somewhat surprising that de novo acute antibody-mediated rejection occurs in the setting of effective regulation of CD4+ T cells known to be critical to antibody production. We and others have previously reported that posttransplant production of alloantibody is markedly enhanced in the absence of CD8+ T cells (14,3537). Thus it is possible that immunosuppressive agents or other conditions that impair or deplete CD8+ T cell function might inadvertently promote alloantibody production posttransplant by inhibiting this natural mechanism of immune regulation.

Based on our observations that transplant recipients, which are deficient in either CD8+ T cells or the cytotoxic molecules perforin and FasL, are high alloantibody producers (13,14), we postulated that CD8+ T cell-mediated regulation of posttransplant alloantibody production might be perforin- and FasL-dependent. We show herein, for the first time, a novel mechanism in which CD8+ T cells inhibit alloantibody production by directly killing antibody-producing B cells through both FasL- and perforin-mediated cytotoxicity. From these data, we presumed that perforin and FasL are equally important to regulation of antibody. However, we noted in subsequent in vivo studies that while FasL-deficient CD8+ T cells exhibited impaired cytotoxicity of alloprimed IgG1+ B cells, as compared to wild-type CD8+ T cells, perforin-deficient CD8+ T cells exhibited moderate levels of cytotoxicity. Our results are consistent with a recent report indicating that perforin-mediated CD8+ T cell cytotoxicity is inhibited in the absence of FasL, suggesting that perforin-mediated cytotoxicity requires the presence of FasL for efficient killing (38). Alternatively, CD8+ T cells may utilize perforin to downregulate antibody production in other ways, in addition to killing B cells.

CD8+ cytotoxic effector T cells could also potentially influence humoral immunity by killing antigen presenting cells, such as dendritic cells, as reported by others as a means of restoring homeostasis. For example, Guarda et al. found that primed effector and memory OT-I CD8+ T cells infiltrate lymph nodes and eliminate antigen-presenting dendritic cells (39). In other studies, primed CD8+ T cells have been shown to eliminate antigen-carrying (viral or bacterial antigen) dendritic cells (4043) and macrophages in a perforin- and FasL-dependent mechanism (4446). However, while these studies have reported CD8+ T cell and dendritic cell interactions as a mechanism to restore immune homeostasis (39,42,43), we do not think this mechanism significantly influences alloantibody levels in our model. Our view is based on evidence that the kinetics of antigen presenting cell (APC) involvement in antibody production is early, while CD8-mediated inhibition of posttransplant alloantibody occurs late with respect to antigen stimulation. The primary function of APCs (e.g., dendritic cells) in antibody production is to activate CD4+ T cells (4749), which has been documented to occur within 1 to 2 days (5052). In our in vivo model system, posttransplant alloantibody is first detectable at day 7 and peaks at day 14 (13), and is highly regulated by CD8+ T cells, since adoptive transfer of alloprimed CD8+ T cells on day 7 reduces ongoing alloantibody production (Figure 2). This shows that CD8+ T cells can inhibit alloantibody relatively late, which is concurrent with the development of CD8+ T cell cytotoxic potential which we have reported to be maximal level between 5 and 7 days posttransplant (24). With these kinetics in mind, we hypothesize that while antigen presentation is required early for antibody production (between days 1 and 2), CD8+CTL inhibition of alloantibody production occurs later (maximal cytotoxic function after day 5) and largely targets elimination of IgG1+ B cells. Furthermore, Wong et al. showed that CD8+ T cell killing of APCs does not begin to occur until after 3 days (42) which would be after CD4+ T cell activation occurs in our model system. Therefore, while CD8+CTL’s have been reported to kill APCs, we do not believe this mechanism significantly influences alloantibody levels acutely in our model. However, this homeostatic mechanism may influence alloantibody levels at a later time-point as we have reported peak alloantibody levels plateau between days 14–21 and then progressively decline (13). Further studies are underway to determine the extent to which CD8+ T cell-mediated killing of APCs regulates alloantibody production.

In CD8+ T cell-mediated transplant rejection, CD8+ T cells are recognizing alloMHC class I on transplanted cells. In our studies, we found that CD8+ T cells regulated posttransplant antibody production in an antigen-specific fashion and required self-MHC I expression on target B cells. This suggested that CD8+ T cells recognize allopeptide presented by alloprimed IgG1+ B cells. This antigen presentation, or cross-presentation, of extracellular antigen through self-MHC I is predominantly found in dendritic cells and macrophages (53). However, it is reported that B cells can cross-present extracellular antigen through MHC I (5457). There is also the potential that since B cells can process antigen and release it extracellularly (58), degraded peptide may bind to MHC I molecules on a B cell that are devoid of peptide in the MHC groove, as has been documented for “empty” MHC II molecules (59). Another possibility is that B cells could present whole alloMHC I, using a non-degradative pathway as has been described for dendritic cells (60,61). However, our analysis of alloprimed IgG1+ B cells in recipient mice shows no detectable expression of whole allogeneic MHC I (data not shown). A recent publication by Kim et al. shows that regulatory CD8+ T cells mediate suppression of autoantibody by inhibiting Qa-1+TFH cells (62). However, we have not been able to detect Qa-1 expression on recipient alloprimed IgG1+ B cells (data not shown). Our data show that alloprimed CD8+ T cells do not kill MHC I-deficient (beta-2 microglobulin KO) IgG1+ B cells. Furthermore, an in vivo study utilizing RAG1 KO mice, shows that co-transfer of alloprimed CD8+ T cells inhibits alloantibody production by alloprimed IgG1+ B cells (Figure S1). These data are consistent with the interpretation that cognate interactions between alloprimed CD8+ T cells and alloprimed IgG1+ B cells via TCR and self-MHC I are required for CD8+ T cell-mediated B cell killing and regulation of posttransplant alloantibody production. Additional data using differentially labeled CD8+ T cells and B cells shows that alloprimed CD8+ T cells colocalize in the spleen with alloprimed IgG1+ B cells in vivo (Figure S2). B cells are not efficient in inducing the activation of CD8+ T cell responses (63) and may even induce transplant tolerance (64). Therefore, we predict that CD8+ T cells are initially cross-primed by dendritic cells (allopeptide presented by self-MHC I) and later target alloprimed IgG1+ B cells for removal, thus downregulating the posttransplant alloantibody response.

In summary, our studies provide the first evidence that CD8+ T cells use perforin and FasL to mediate downregulation of alloantibody by eliminating alloprimed IgG1+ B cells. This is an interesting paradox because although alloreactive CD8+ T cells initiate graft rejection through perforin- and FasL-mediated cytotoxicity (6573), these data suggest that a subset of CD8+ T cells (restricted to self-MHC I presenting allopeptide) inhibit the humoral immune response by the same cytotoxic molecules. Clinical identification of a specific subset of CD8+ CTLs that recognize primed B cells through allopeptide/self-MHC I complexes could translate into clinical strategies to induce antibody suppression without promoting allograft rejection. Experimental approaches to distinguish between “rejector” and “antibody-inhibiting” alloreactive CD8+ T cells are in progress. We expect that “rejector” CD8+ T cells directly recognize alloantigen on transplanted hepatocytes; whereas “antibody-inhibiting” CD8+ T cells, which kill alloprimed B cells, are self-restricted and indirectly primed to MHC class I allopeptide. Understanding the mechanisms involved in the regulation of posttransplant alloantibody production is necessary for the development of novel diagnostic tools and targeted immunotherapeutic strategies that can be used to avoid alloantibody-mediated graft damage. While our transplant model produces IgG1-dominant humoral responses, other laboratories studying antibody production in response to platelets, allergens, bacteria or viruses report increased levels of IgG1, IgG2, IgG3, and IgE following CD8-depletion, suggesting that CD8-mediated inhibition of alloantibody is not limited to transplantation nor restricted to IgG1 (14,3537,7478). Thus, results in the current study may have relevance to in vivo regulation of antibody production in other circumstances such as in response to autoimmune or infectious antigenic stimuli.

Supplementary Material

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Supp Fig S1-S2

Figure S1: Alloprimed CD8+ T cells are sufficient to inhibit alloantibody produced by alloprimed IgG1+ B cells. Wild-type (WT) and CD8 KO mice were transplanted with hepatocytes. Five days later, splenocytes were isolated. Bulk alloprimed CD8+ T cells were purified from WT recipients and alloprimed IgG1+ B cells were purified from CD8 KO mice. Naïve B220+ B cells were utilized as a negative control. Pooled CD8+ T cells (10*106 cells) and B cells (25*106 cells) were adoptively transferred into naïve RAG1 KO mice (T and B cell deficient). In order to stimulate alloprimed B cells, RAG1 KO mice received FVB/N splenocytes (10*106 cells) delivered by intraperitoneal injection (i.p.; day 0 with respect to adoptive transfer) as well as anti-CD40 mAb (0.2 mg, i.p.; day 1 post-adoptive transfer; FGK4.5; BioXCell, West Lebanon, NH) and IL-4 (0.625 μg, i.p.; day 1 post-adoptive transfer; eBioscience, San Diego, CA). RAG1 KO mice were then observed for alloantibody production on day 14 postadoptive transfer. The adoptive transfer of naïve B220+B cells did not initiate alloantibody production (3.6±0.3%; n=4). The adoptive transfer of alloprimed IgG1+ B cells induced significant levels of alloantibody production (56.1±2.9%; n=4; p=0.0001) as compared to control naïve B cells. The adoptive transfer of bulk alloprimed CD8+ T cells along with alloprimed IgG1+ B cells significantly reduced alloantibody production by IgG1+ B cells (2.0±0.3%; n=5; p=0.0001).

Figure S2: Alloprimed CD8+ T cells colocalize with alloprimed IgG1+ B cells. In order to investigate the colocalization of alloprimed CD8+ T cells and alloprimed IgG1+ B cells, we transplanted wild-type and CD8 KO mice. On day 5 posttransplant, CD8+ T cells and IgG1+ B cells were isolated from recipient splenocytes. Naïve CD8+ T cells were used as controls. Following isolation, CD8+ T cells were stained with PKH26 (red) (Sigma-Aldrich, St. Louis, MO) and B cells were stained with CFSE (green) (Molecular Probes, Eugene, OR) by manufacturers’ recommendations. Pooled CD8+ T cells and B cells were then adoptively transferred into CD8 KO recipient mice (day 5 posttransplant; n=2 for all conditions). Spleen, lymph node, and liver were harvested 4 hours and 18 hours postadoptive transfer of fluorescent CD8+ T cells and B cells. Tissues were fixed with paraformaldehyde and cryoprotected by incubating overnight with an isotonic 20% sucrose solution. A,B) Spleens harvested after 4 hours were analyzed by a Olympus FV1000 MPE Multiphoton Laser Scanning Confocal microscope (Olympus, Center Valley, PA). C,D) Spleen samples which were frozen and cryosectioned (20 μm) were mounted onto slides and evaluated by spectral confocal Olympus FV 1000 Spectral Confocal microscope (Olympus). Representative 2-dimentional (A,C) and 3-dimentional (Z-stack; B,D) images were created of colocalized CD8+ T cells and B cells. Only when alloprimed CD8+ T cells were adoptively transferred with alloprimed IgG1+ B cells was colocalization observed (4 hour time point). Of note, colocalization of alloprimed CD8+ T cells and IgG1+ B cells was a rare event (less than 1% of cells were colocalized). In mice adoptively transferred with alloprimed CD8+ T cells, less IgG1+ B cells were observed in the spleen at the 18 hour time point, which correlates with our data that CD8+ T cells eliminate IgG1+ B cell targets. No colocalization was observed when naïve CD8+ T cells were co-adoptively transferred with alloprimed IgG1+ B cells. While adoptively transferred, fluorescent-tagged alloprimed CD8+ T cells and alloprimed IgG1+ B cells were observed in the lymph node, a high amount of auto-fluorescence prevented clear visualization of individual cells. In addition, fluorescent-tagged CD8+ T cells and B cells, while present, were not prevalent in the liver.


Support: This work was supported in part by grants from the Roche Organ Transplantation Research Foundation (to G.L.B.), the ASTS-NKF (National Kidney Foundation) Folkert Belzer, MD, Research Award (to T.A.P.), and National Institutes of Health grants AI083456 (to G.L.B.) and F32 DK082148 (NIDDK; to J.M.Z.).

We would like to thank Caroline Padro (Ohio State University) for informative discussions and assistance with the isolation and ELISPOT analysis of B cells.

Nonstandard Abbreviations

Adoptive transfer
human alpha-1 antitrypsin



The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Diabetes and Digestive and Kidney Diseases or the National Institutes of Health.

Supporting Information

Additional Supporting Information may be found in the online version of this article.


1. Ponticelli C. Progression of renal damage in chronic rejection. Kidney Int Suppl. 2000;75:S62–70. [PubMed]
2. Tilney NL, Paul LC. Antigen-independent events leading to chronic graft dysfunction. In: Tilney NL, Strom TB, Paul LC, editors. Transplantation biology: cellular and molecular aspects. Philadelphia: Lippincott-Raven; 1996. pp. 629–637.
3. Baluja P, Haragsim L, Laszik Z. Chronic allograft nephropathy. Advances in chronic kidney disease. 2006;13(1):56–61. [PubMed]
4. Trpkov K, Campbell P, Pazderka F, Cockfield S, Solez K, Halloran PF. Pathologic features of acute renal allograft rejection associated with donor-specific antibody, Analysis using the Banff grading schema. Transplantation. 1996;61(11):1586–1592. [PubMed]
5. McKenna RM, Takemoto SK, Terasaki PI. Anti-HLA antibodies after solid organ transplantation. Transplantation. 2000;69(3):319–326. [PubMed]
6. Lobo PI, Spencer CE, Stevenson WC, Pruett TL. Evidence demonstrating poor kidney graft survival when acute rejections are associated with IgG donor-specific lymphocytotoxin. Transplantation. 1995;59(3):357–360. [PubMed]
7. Halloran PF, Schlaut J, Solez K, Srinivasa NS. The significance of the anti-class I response. II. Clinical and pathologic features of renal transplants with anti-class I-like antibody. Transplantation. 1992;53(3):550–555. [PubMed]
8. Cecka J, Terasaki P, editors. Clinical Transplants: 1997. Los Angeles: 1998. UCLA Tissue Typing Laboratory; pp. 1–14.
9. Cecka JM. The UNOS Scientific Renal Transplant Registry. Clin Transpl. 1996:1–14. [PubMed]
10. Puttarajappa C, Shapiro R, Tan HP. Antibody-mediated rejection in kidney transplantation: a review. Journal of transplantation. 2012;2012:193724. [PMC free article] [PubMed]
11. Blanchard D, Gaillard C, Hermann P, Banchereau J. Role of CD40 antigen and interleukin-2 in T cell-dependent human B lymphocyte growth. European journal of immunology. 1994;24(2):330–335. [PubMed]
12. Steele DJ, Laufer TM, Smiley ST, Ando Y, Grusby MJ, Glimcher LH, et al. Two levels of help for B cell alloantibody production. The Journal of experimental medicine. 1996;183(2):699–703. [PMC free article] [PubMed]
13. Zimmerer JM, Pham TA, Sanders VM, Bumgardner GL. CD8+ T Cells Negatively Regulate IL-4-Dependent, IgG1-Dominant Posttransplant Alloantibody Production. J Immunol. 2010;185(12):7285–7292. [PMC free article] [PubMed]
14. Horne PH, Lunsford KE, Eiring AM, Wang Y, Gao D, Bumgardner GL. CD4+ T-Cell-Dependent Immune Damage of Liver Parenchymal Cells Is Mediated by Alloantibody. Transplantation. 2005;80(4):514–521. [PubMed]
15. Russell JH, Ley TJ. Lymphocyte-mediated cytotoxicity. Annual review of immunology. 2002;20:323–370. Epub 2001 Oct 2004. [PubMed]
16. Heusel JW, Wesselschmidt RL, Shresta S, Russell JH, Ley TJ. Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells. Cell. 1994;76(6):977–987. [PubMed]
17. Zimmerer JM, Horne PH, Fiessinger LA, Fisher MG, Pham TA, Saklayen SL, et al. Cytotoxic Effector Function of CD4-Independent, CD8+ T Cells Is Mediated by TNF-alpha/TNFR. Transplantation. 2012;94(11):1103–1110. [PMC free article] [PubMed]
18. Bumgardner GL, Heininger M, Li J, Xia D, Parker-Thornburg J, Ferguson RM, et al. A Functional Model of Hepatocyte Transplantation for in Vivo Immunologic Studies. Transplantation. 1998;65(1):53–61. [PubMed]
19. Bumgardner GL, Gao D, Li J, Baskin J, Heininger M, Orosz CG. Rejection responses to allogeneic hepatocytes by reconstituted SCID mice, CD4 KO, and CD8 KO mice. Transplantation. 2000;70(12):1771–1780. [PubMed]
20. Benson JM, Campbell KA, Guan Z, Gienapp IE, Stuckman SS, Forsthuber T, et al. T-cell activation and receptor downmodulation precede deletion induced by mucosally administered antigen. The Journal of clinical investigation. 2000;106(8):1031–1038. [PMC free article] [PubMed]
21. Kin NW, Sanders VM. CD86 regulates IgG1 production via a CD19-dependent mechanism. J Immunol. 2007;179(3):1516–1523. [PubMed]
22. van Stipdonk MJ, Hardenberg G, Bijker MS, Lemmens EE, Droin NM, Green DR, et al. Dynamic programming of CD8+ T lymphocyte responses. Nature immunology. 2003;4(4):361–365. [PubMed]
23. Oehen S, Brduscha-Riem K. Differentiation of naive CTL to effector and memory CTL: correlation of effector function with phenotype and cell division. J Immunol. 1998;161(10):5338–5346. [PubMed]
24. Horne PH, Koester MA, Jayashankar K, Lunsford KE, Dziema HL, Bumgardner GL. Disparate Primary and Secondary Allospecific CD8+ T Cell Cytolytic Effector Function in the Presence or Absence of Host CD4+ T Cells. J Immunol. 2007;179(1):80–88. [PubMed]
25. Bumgardner GL, Li J, Prologo JD, Heininger M, Orosz CG. Patterns of immune responses evoked by allogeneic hepatocytes: evidence for independent co-dominant roles for CD4+ and CD8+ T-cell responses in acute rejection. Transplantation. 1999;68(4):555–562. [PubMed]
26. Bumgardner GL, Gao D, Li J, Baskin JH, Heininger M, Orosz CG. Rejection responses to allogeneic hepatocytes by reconstituted SCID mice, CD4, KO, and CD8 KO mice. Transplantation. 2000;70(12):1771–1780. [PubMed]
27. Wang Y, Gao D, Lunsford KE, Frankel WL, Bumgardner GL. Targeting LFA-1 synergizes with CD40/CD40L blockade for suppression of both CD4-dependent and CD8-dependent rejection. Am J Transplant. 2003;3(10):1251–1258. [PubMed]
28. Terasaki PI, Cai J. Humoral theory of transplantation: further evidence. Curr Opin Immunol. 2005;17(5):541–545. [PubMed]
29. Moll S, Pascual M. Humoral rejection of organ allografts. Am J Transplant. 2005;5(11):2611–2618. [PubMed]
30. Colvin RB, Smith RN. Antibody-mediated organ-allograft rejection. Nat Rev Immunol. 2005;5(10):807–817. [PubMed]
31. Xu H, Chilton PM, Tanner MK, Huang Y, Schanie CL, Dy-Liacco M, et al. Humoral immunity is the dominant barrier for allogeneic bone marrow engraftment in sensitized recipients. Blood. 2006;108(10):3611–3619. Epub 2006 Aug 3613. [PubMed]
32. Shimizu A, Colvin RB. Pathological features of antibody-mediated rejection. Curr Drug Targets Cardiovasc Haematol Disord. 2005;5(3):199–214. [PubMed]
33. Vongwiwatana A, Tasanarong A, Hidalgo LG, Halloran PF. The role of B cells and alloantibody in the host response to human organ allografts. Immunol Rev. 2003;196:197–218. [PubMed]
34. Lorenz M, Regele H, Schillinger M, Exner M, Rasoul-Rockenschaub S, Wahrmann M, et al. Risk factors for capillary C4d deposition in kidney allografts: evaluation of a large study cohort. Transplantation. 2004;78(3):447–452. [PubMed]
35. Chan SY, DeBruyne LA, Goodman RE, Eichwald EJ, Bishop DK. In vivo depletion of CD8+ T cells results in Th2 cytokine production and alternate mechanisms of allograft rejection. Transplantation. 1995;59(8):1155–1161. [PubMed]
36. Ensminger SM, Spriewald BM, Sorensen HV, Witzke O, Flashman EG, Bushell A, et al. Critical role for IL-4 in the development of transplant arteriosclerosis in the absence of CD40-CD154 costimulation. J Immunol. 2001;167(1):532–541. [PubMed]
37. Ensminger SM, Spriewald BM, Witzke O, Morrison K, van Maurik A, Morris PJ, et al. Intragraft interleukin-4 mRNA expression after short-term CD154 blockade may trigger delayed development of transplant arteriosclerosis in the absence of CD8+ T cells. Transplantation. 2000;70(6):955–963. [PubMed]
38. Hassin D, Garber OG, Meiraz A, Schiffenbauer YS, Berke G. Cytotoxic T lymphocyte perforin and Fas ligand working in concert even when Fas ligand lytic action is still not detectable. Immunology. 2011;133(2):190–196. [PubMed]
39. Guarda G, Hons M, Soriano SF, Huang AY, Polley R, Martin-Fontecha A, et al. L-selectin-negative CCR7− effector and memory CD8+ T cells enter reactive lymph nodes and kill dendritic cells. Nature immunology. 2007;8(7):743–752. [PubMed]
40. Hermans IF, Ritchie DS, Yang J, Roberts JM, Ronchese F. CD8+ T cell-dependent elimination of dendritic cells in vivo limits the induction of antitumor immunity. J Immunol. 2000;164(6):3095–3101. [PubMed]
41. Mueller SN, Jones CM, Stock AT, Suter M, Heath WR, Carbone FR. CD4+ T cells can protect APC from CTL-mediated elimination. J Immunol. 2006;176(12):7379–7384. [PubMed]
42. Wong P, Pamer EG. Feedback regulation of pathogen-specific T cell priming. Immunity. 2003;18(4):499–511. [PubMed]
43. Hafalla JC, Morrot A, Sano G, Milon G, Lafaille JJ, Zavala F. Early self-regulatory mechanisms control the magnitude of CD8+ T cell responses against liver stages of murine malaria. J Immunol. 2003;171(2):964–970. [PubMed]
44. Yang J, Huck SP, McHugh RS, Hermans IF, Ronchese F. Perforin-dependent elimination of dendritic cells regulates the expansion of antigen-specific CD8+ T cells in vivo. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(1):147–152. [PubMed]
45. Chen M, Felix K, Wang J. Critical role for perforin and Fas-dependent killing of dendritic cells in the control of inflammation. Blood. 2012;119(1):127–136. [PubMed]
46. Lazarevic V, Flynn J. CD8+ T cells in tuberculosis. American journal of respiratory and critical care medicine. 2002;166(8):1116–1121. [PubMed]
47. Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, et al. Immunobiology of dendritic cells. Annual review of immunology. 2000;18:767–811. [PubMed]
48. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392(6673):245–252. [PubMed]
49. Koyamada N, Sato A, Takayama J, Usuda M, Kawagishi N, Doi H, et al. Macrophage depletion prevents anti-graft antibody production and results in long-term survival in xenotransplantation. Transplantation proceedings. 2005;37(1):514–515. [PubMed]
50. Iezzi G, Karjalainen K, Lanzavecchia A. The duration of antigenic stimulation determines the fate of naive and effector T cells. Immunity. 1998;8(1):89–95. [PubMed]
51. Miller MJ, Safrina O, Parker I, Cahalan MD. Imaging the single cell dynamics of CD4+ T cell activation by dendritic cells in lymph nodes. The Journal of experimental medicine. 2004;200(7):847–856. [PMC free article] [PubMed]
52. Kirschner DE, Chang ST, Riggs TW, Perry N, Linderman JJ. Toward a multiscale model of antigen presentation in immunity. Immunological reviews. 2007;216:93–118. [PubMed]
53. Belizaire R, Unanue ER. Targeting proteins to distinct subcellular compartments reveals unique requirements for MHC class I and II presentation. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(41):17463–17468. [PubMed]
54. Hon H, Oran A, Brocker T, Jacob J. B lymphocytes participate in cross-presentation of antigen following gene gun vaccination. J Immunol. 2005;174(9):5233–5242. [PubMed]
55. Gromme M, Neefjes J. Antigen degradation or presentation by MHC class I molecules via classical and non-classical pathways. Molecular immunology. 2002;39(3–4):181–202. [PubMed]
56. Basta S, Alatery A. The cross-priming pathway: a portrait of an intricate immune system. Scandinavian journal of immunology. 2007;65(4):311–319. [PubMed]
57. McPherson M, Wei B, Turovskaya O, Fujiwara D, Brewer S, Braun J. Colitis immunoregulation by CD8+ T cell requires T cell cytotoxicity and B cell peptide antigen presentation. American journal of physiology Gastrointestinal and liver physiology. 2008;295(3):G485–492. [PubMed]
58. Vitetta ES, Fernandez-Botran R, Myers CD, Sanders VM. Cellular interactions in the humoral immune response. Advances in immunology. 1989;45:1–105. [PubMed]
59. Chesnut RW, Grey HM. Studies on the capacity of B cells to serve as antigen-presenting cells. J Immunol. 1981;126(3):1075–1079. [PubMed]
60. Curry AJ, Pettigrew GJ, Negus MC, Easterfield AJ, Young JL, Bolton EM, et al. Dendritic cells internalise and re-present conformationally intact soluble MHC class I alloantigen for generation of alloantibody. European journal of immunology. 2007;37(3):696–705. [PubMed]
61. Russo V, Zhou D, Sartirana C, Rovere P, Villa A, Rossini S, et al. Acquisition of intact allogeneic human leukocyte antigen molecules by human dendritic cells. Blood. 2000;95(11):3473–3477. [PubMed]
62. Kim HJ, Verbinnen B, Tang X, Lu L, Cantor H. Inhibition of follicular T-helper cells by CD8(+) regulatory T cells is essential for self tolerance. Nature. 2010;467(7313):328–332. [PMC free article] [PubMed]
63. Keller SA, von Allmen CE, Hinton HJ, Bauer M, Muntwiler S, Dietmeier K, et al. Follicular and marginal zone B cells fail to cross-present MHC class I-restricted epitopes derived from viral particles. J Immunol. 2009;182(10):6261–6266. [PubMed]
64. Lee KM, Kim JI, Stott R, Soohoo J, O’Connor MR, Yeh H, et al. Anti-CD45RB/Anti-TIM-1-Induced Tolerance Requires Regulatory B Cells. Am J Transplant. 2012 [PMC free article] [PubMed]
65. Barber DL, Wherry EJ, Ahmed R. Cutting edge: rapid in vivo killing by memory CD8 T cells. J Immunol. 2003;171(1):27–31. [PubMed]
66. Strehlau J, Pavlakis M, Lipman M, Shapiro M, Vasconcellos L, Harmon W, et al. Quantitative detection of immune activation transcripts as a diagnostic tool in kidney transplantation. Proc Natl Acad Sci U S A. 1997;94(2):695–700. [PubMed]
67. Halloran PF, Urmson J, Ramassar V, Melk A, Zhu LF, Halloran BP, et al. Lesions of T-cell-mediated kidney allograft rejection in mice do not require perforin or granzymes A and B. Am J Transplant. 2004;4(5):705–712. [PubMed]
68. O’Connell PJ, Pacheco-Silva A, Nickerson PW, Muggia RA, Bastos M, Kelley VR, et al. Unmodified pancreatic islet allograft rejection results in the preferential expression of certain T cell activation transcripts. J Immunol. 1993;150(3):1093–1104. [PubMed]
69. Han D, Xu X, Baidal D, Leith J, Ricordi C, Alejandro R, et al. Assessment of cytotoxic lymphocyte gene expression in the peripheral blood of human islet allograft recipients: elevation precedes clinical evidence of rejection. Diabetes. 2004;53(9):2281–2290. [PubMed]
70. Sleater M, Diamond AS, Gill RG. Islet allograft rejection by contact-dependent CD8+ T cells: perforin and FasL play alternate but obligatory roles. Am J Transplant. 2007;7(8):1927–1933. [PubMed]
71. Niederkorn JY, Stevens C, Mellon J, Mayhew E. Differential roles of CD8+ and CD8− T lymphocytes in corneal allograft rejection in ‘high-risk’ hosts. Am J Transplant. 2006;6(4):705–713. [PubMed]
72. Meiraz A, Garber OG, Harari S, Hassin D, Berke G. Switch from perforin-expressing to perforin-deficient CD8(+) T cells accounts for two distinct types of effector cytotoxic T lymphocytes in vivo. Immunology. 2009;128(1):69–82. [PubMed]
73. Martin PJ, Akatsuka Y, Hahne M, Sale G. Involvement of donor T-cell cytotoxic effector mechanisms in preventing allogeneic marrow graft rejection. Blood. 1998;92(6):2177–2181. [PubMed]
74. Coutelier JP. Enhancement of IgG production elicited in mice by treatment with anti-CD8 antibody. Eur J Immunol. 1991;21(10):2617–2620. [PubMed]
75. Byrom B, Barbet AF, Obwolo M, Mahan SM. CD8(+) T cell knockout mice are less susceptible to Cowdria ruminantium infection than athymic, CD4(+) T cell knockout, and normal C57BL/6 mice. Veterinary parasitology. 2000;93(2):159–172. [PubMed]
76. Sayeh E, Sterling K, Speck E, Freedman J, Semple JW. IgG antiplatelet immunity is dependent on an early innate natural killer cell-derived interferon-gamma response that is regulated by CD8+ T cells. Blood. 2004;103(7):2705–2709. Epub 2003 Dec 2704. [PubMed]
77. Thomas MJ, Noble A, Sawicka E, Askenase PW, Kemeny DM. CD8 T cells inhibit IgE via dendritic cell IL-12 induction that promotes Th1 T cell counter-regulation. J Immunol. 2002;168(1):216–223. [PubMed]
78. Salagianni M, Wong KL, Thomas MJ, Noble A, Kemeny DM. An essential role for IL-18 in CD8 T cell-mediated suppression of IgE responses. J Immunol. 2007;178(8):4771–4778. [PubMed]