Tolerance Induction in Presensitized Bone Marrow Recipients by Veto CTLs: Effective Deletion of Host Anti-Donor Memory Effector Cells

Shlomit Reich-Zeliger, Esther Bachar-Lustig, Avichai Bar-Ilan and Yair Reisner
J Immunol November 15, 2007, 179 (10) 6389-6394; DOI: https://doi.org/10.4049/jimmunol.179.10.6389

Abstract

Veto cells have been defined as cells capable of inducing apoptosis of effector CD8 cells recognizing their disparate MHC Ags. Tolerance induced by donor-type veto cells is desirable, because it is restricted to depletion of anti-donor clones without depletion of other immune specificities. It has been shown that anti-third party CTLs exhibit marked veto activity with reduced capacity to induce graft-vs-host disease, when tested on naive effector cells. However, presensitized T cells could play an important role in graft rejection, and therefore, their sensitivity to veto cells could be critical to the implementation of the latter cells in bone marrow transplantation. To address this question, we compared naive and presensitized TCR transgenic effector CD8 T cells, bearing a TCR against H-2d. Both cell types exhibited similar predisposition to killing by veto CTLs in vitro, and this killing was dependent in both cell types on Fas-FasL signaling as shown by using Fas-deficient CD8 T cells from (lprx2c) F1 mice. When tested in a stringent mouse model, in which bone marrow rejection is mediated by adoptively transferred host type T cells into lethally irradiated recipients, veto CTLs were equally effective in overcoming rejection of naive or presensitized host T cells.

The establishment of donor-type chimerism under sublethal conditioning represents a most desirable goal in transplantation biology, as it is generally associated with durable tolerance toward cells or tissues from the original donor. However, the marked level of host hemopoietic and immune cells surviving mild preparatory regimens, represents a difficult barrier for the engraftment of donor cells. Several studies have shown that this challenge can be successfully addressed in rodents by using large doses of bone marrow (BM)4 cells, adequately depleted of T cells to avoid graft-vs-host disease (GVHD) (1). Alternatively, this goal could be achieved by using regular doses of T cell-depleted marrow in conjunction with one form or another of tolerance-inducing cells. In this context, several veto cells or BM facilitating cells have been described (2, 3, 4, 5, 6, 7, 8). The veto concept, originally proposed by Miller et al. (9, 10, 11) refers to an activity of alloantigen-bearing cells in which they inactivate T cells that recognize them. Several studies published by the same group between 1979 and 1983 described specific inhibition of CTL by cells bearing the histocompatibility Ags also expressed by the stimulator cells. This activity is unique in the direction of recognition, i.e., the T cell to be inactivated bears the burden of recognition of the veto cell. Cells from a variety of tissues can mediate veto activity, including BM, thymus, spleen, lymph node, and fetal liver (9, 10, 12, 13, 14). Various cell types have been isolated from these tissues and shown to mediate veto activity including T lymphocytes, NK cells, and dendritic cells. A very strong veto activity was documented for CD8+ CTL lines or clones (14, 15, 16, 17, 18, 19, 20, 21). Recently we have shown that engraftment of early progenitors could be enhanced by using host nonreactive CTLs directed against nonrelevant stimulators (anti-third party CTLs), in conjunction with nonmyeloablative rapamycin-based conditioning protocols, thereby significantly reducing the toxicity of allogeneic transplantation (22).

However, considering that host sensitization to a broad range of donor MHC Ags from multiple blood transfusions, or pregnancies, represents a significant barrier especially for mismatched BM transplants (21, 23, 24, 25, 26, 27), a major question remaining is whether veto cells can also overcome graft rejection mediated by preexisting memory T cells. Indeed, many sensitized patients are either precluded from receiving a transplant or may experience an increased rate of early rejection episodes that are irreversible or difficult to treat with current immunosuppressive agents (28, 29, 30, 31, 32).

Although many tolerance strategies, such as regimens using T cell costimulation blockade, successfully target and control naive T cell responses, they may not effectively neutralize memory T cells as the latter cells are activated faster, with a lower threshold for activation and with a reduced requirement for costimulatory signals (33, 34, 35). Thus, Valujskikh et al. (36) have shown that the presence of presensitized cells prevents the beneficial effects of anti-CD40L in prolonging allograft survival. An early study addressing the potential of veto CTLs to suppress presensitized T cells, suggested that in the context of presensitization to minor histocompatability Ags, a cytotoxic clone with a marked veto activity against naive T cells exhibited significantly less effective veto reactivity against presensitized cells (17). However this suggestion was not tested in vivo. Moreover, the clone tested expressed a CD8+CD4+ phenotype which is unusual for CTLs.

In the past, it was difficult to track memory cells directed against a particular Ag in vivo, but with the advent of TCR transgenic mice it has become possible to retrieve such memory cells and examine their sensitivity to veto cells. Thus, in the present study, we initially addressed this question by comparing in vitro the interaction of veto CTLs with naive and presensitized T cells, using effectors from TCR transgenic and Fas knockout mice. Furthermore, the capacity of the veto cells to delete presensitized T cells, was investigated in vivo, using a stringent model for T cell mediated BM allograft rejection.

Materials and Methods

Animals

Mice used were 6- to 12-wk-old females. BALB/c (H-2d), FVB (H-2q), SJL (H-2s), and C57BL/6 (H-2b) were obtained from the Weizmann Institute Animal Center (Rehovot, Israel). DBA/2 (H-2d) and C3H/HeJ (H-2k) mice were obtained from the Roscoe B. Jackson Memorial Laboratory. A breeding pair of transgenic (Tg) H-2b mice expressing the TCR from the CTL clone 2c with specificity for H-2Ld was provided by Janko Nikolic-Zugic (Sloan-Kettering, New York). Progeny of these Tg mice were bred at the Weizmann Institute Animal Breeding Center. 2c-lpr Tg mice were produced by breeding 2c Tg mice with C3H.MRL-Tnfrsf6lpr mice as previously described (37). The F1 2c/lpr mice were bred again with C3H.MRL-Tnfrsf6lpr. All mice were kept in small cages (five animals in each cage) and fed sterile food and acid water.

Preparation of host nonreactive anti-third party CTLs from donor-type spleen cells

Anti-third party CTLs were prepared as described by Bachar et al. (22). In brief, spleen cells from BALB/c or DBA/2 origin were harvested, lysed in 0.15 M cold ammonium chloride-potassium (ACK) buffer (0.5 ml/spleen) to remove RBC, and brought to a concentration of 2 × 106 cells/ml in RPMI 1640 supplemented with 25 mM HEPES (pH 7.4), 10% heat-inactivated FCS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM l-glutamine, 5 × 10–5 2-ME, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. This splenic single-cell suspension was cocultured at a ratio of 1:1 with irradiated (20 Gy) FVB (third party stimulators) splenocytes. Responders (2 × 106/ml) and stimulators (2 × 106/ml) were cultured for 6 days at 37°C in a 5% CO2/air incubator without exogenous IL-2. The IL-2 deprivation period was established based on the maximal time that mouse spleen T cells can be maintained in MLR culture without IL-2 (data not shown). However, attempts to grow large numbers of cells for the investigation of these veto cells in vivo, revealed irregularities in the cell composition of the harvested cells due to difficulties in controlling outgrowth of lymphokine-activated killer (LAK) cells and/or CD4 T cells. This problem was addressed by removal of CD4+ and NK cells at the end of the IL-2 deprivation, before the addition of IL-2. Thus, cells were fractionated on Ficoll and the lymphoid fraction was then subjected to positive selection of CD8+ cells using magnetically labeled anti-CD8 Abs and a MACS system (Miltenyi Biotec). The isolated cells (2 × 106/ml) were then re-stimulated with irradiated (20 Gy) splenocytes from the original third party donors (FVB, 2 × 106/ml), and human rIL-2 (40 U/ml; Eurocetus) was added from that day every second day to the MLR culture (day 6, 8 and 10). At day 10 the MLR cultures were harvested, fractionated on Ficoll-Paque plus (Amersham Pharmacia Biotech), analyzed by FACS for their CD8 level and tested for their veto activity at different cell ratios, as described in Results.

Generation of presensitized T cells

Irradiated (20 Gy) BALB/c splenocytes (5 × 106) were injected i.p. into naive 2c Tg hosts at day 0 and day 21. In experiments described in Figs. 1–4⇓⇓⇓⇓ the whole population of presensitized spleen cells was harvested at 45–60 days after the end of sensitization and tested for its responsiveness to the veto activity of anti-third party CTLs. In the experiment shown in Fig. 2⇓c presensitized wild-type spleen cells were obtained by immunization of C3H/Hej mice as described above. In the experiment shown in Fig. 5⇓, C3H/Hej mice were immunized as described above and CD44+ T cells were isolated from their spleens as described below.

FIGURE 1.
FIGURE 1.

Expression of CD44 and CD122 on 1B2+ effector cells in naive and presensitized 2c mice. Splenocytes from 2c Tg naive mice or from 2c Tg immunized mice were harvested, and the expression of CD44 and CD122 on 1B2+ CD8+ gated cells was determined by FACS.

FIGURE 2.
FIGURE 2.

Veto activity of anti-third party CTLs on naive and presensitized 2c cells. The inhibitory effect of 10% relevant CTLs (origin of H-2d) on naive (A) and presensitized 2c T cells (B) was measured after 72 h of culture. The percentage (a) or the total cell number (b) of 1B2+CD8+ cells is shown in the presence of 10% nonrelevant veto cells (origin of H2s, black bars), or relevant veto cells (origin of H-2d, white bar) (p < 0.001). The results represent average ± SD of three different experiments. To address the inhibitory activity of veto CTLs on naive (A) and presensitized (B) wild-type C3H/HeJ responders, relevant BALB/c (white bar) or nonrelevant SJL (black bar) veto CTLs were added to MLR in which C3H/HeJ splenocytes were stimulated against irradiated BALB/c stimulators and then tested for their cytotoxicity of BALB/c targets by the 51Cr release assay (c).

FIGURE 3.
FIGURE 3.

Veto activity of anti-third party CTLs on presensitized T cells: a representative experiment. A, FACS analysis showing the level of proliferation of naive (a, b) and presensitized (c, d) 2c T cells (1B2+CD8+) in a 72 h MLR culture in the presence (b, d) and absence (a, c) of 10% relevant veto CTLs. B, Histograms showing the level of annexin V expressed by the stimulated naive (a and b) and presensitized (c and d) 2c T cells in the MLR cultures with (b and d) and without (a and c) relevant veto CTLs that were described in A.

FIGURE 4.
FIGURE 4.

Veto activity of anti-third party CTLs on presensitized T cells: the role of Fas. Deletion of CD8+1B2+ cells in MLR cultures of naive or presensitized responder T cells from 2c or 2c-lpr mice, stimulated against irradiated BALB/c stimulators (white bar), in the presence of 10% relevant (BALB/c anti-FVB, striped bars) and nonrelevant (SJL anti-FVB, black bars) veto CTLs. Results represent average ± SD of three different experiments (p < 0.001).

FIGURE 5.
FIGURE 5.

Veto CTLs in conjunction with rapamycin overcome BM allograft rejection mediated by naive or presensitized host type T cells. Kaplan-Meier survival plot following T cell-depleted BM transplantation in supralethally irradiated recipients that were adoptively transferred with host naive T cells (A) or with presensitized host CD44+ T cells (B). Survival rate is shown for lethally irradiated mice transplanted with T-depleted BM (—•—), T-depleted BM + HTC (—▵—), T cell-depleted BM + HTC + 10 × 106 veto CTLs (—▪—), T cell-depleted BM + HTC + 5 μg/day rapamycin (—○—) or T cell-depleted BM + HTC + 10 × 106 veto CTLs + 5 μg/day rapamycin (—□—). Results are pooled from four independent experiments (in each group n ≥ 34).

Assay for veto activity in the 2c TCR Tg mouse model rusha

Spleen cells of 2c Tg H-2b mice, expressing a TCR-αβ with specificity for H-2Ld mice were used throughout as effector cells in the veto assay as previously described (38), except that, in addition to naive 2c mice, we also used presensitized 2c mice as described above. Briefly, spleen cells were harvested, lysed in cold ACK buffer to remove RBC, and brought to a concentration of 2 × 106 cells/ml in RPMI complete tissue culture medium (CTMC). The cells (2 × 106/ml) were then stimulated with irradiated (20 Gy) BALB/c splenocytes (2 × 106/ml) in the presence or absence of relevant (BALB/c origin) and nonrelevant (SJL origin) veto CTLs at two concentrations (5, 10%). Cultures were incubated for 72 h in 24-well plates. The deletion of specific effector T cells was monitored by cytofluorometric analysis, measuring the level of 2c Tg cells, specifically stained by the 1B2 Ab, directed against the clonotypic anti-H-2Ld TCR.

Cytofluorometric analysis

FACS analysis was performed using a modified FACScan (BD Biosciences, Mountain View, CA). Fluorescence data were collected using 3-decade logarithmic amplification on 25–50 × 103 viable cells, as determined by forward light scatter intensity. Cells were stained with anti-CD8α (Ly-2)-FITC, anti-CD8α (Ly-2)-CyChrome, anti-CD8α (Ly-2)-allophycocyanin, anti-CD122-PE (TM-β1), anti-CD3ε-PE, anti-CD95 (Fas)-FITC and anti-CD4-PE (BD PharMingen, San Diego, CA). Biotinylated 1B2 Abs (provided by J. Nikolic-Zugic, Sloan-Kettering Institute) were stained with R-PE streptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA).

Detection of apoptotic cells

Annexin V-Cy5 was used to detect apoptotic cells. Cells were incubated in annexin V binding buffer (1.4M NaCl, 25 mM CaCl2, 0.1M HEPES Ph 7.4) and supplemented with 5 μl of annexin V-Cy5. The cells were incubated at room temperature for 5 min in the dark, and then washed in binding buffer. Positive cells were monitored by flow cytometry.

The mouse model for T cell mediated BM allograft rejection

C3H/HeJ female mice were exposed to a single dose of 10 Gy (supralethal conditioning) TBI on day 0. The following day, the mice received i.v. 1 × 104 purified host T cells. Transplantation of 2 × 106 allogeneic T depleted BALB/c BM cells was performed on day 2 in conjunction with either rapamycin alone, veto CTLs alone or both. The survival of the mice was monitored daily. Due to changes of the radiation source from Cobalt-60 to Cesium at the Weizmann Institute of Science, we experienced some subtle differences in our graft rejection mouse model which required minor adjustments. Thus, to reproduce our previous data on the synergistic role of veto CTLs and Rapamycin in this model (22), we introduced the following changes: 10 GY instead of 11 GY TBI was used for conditioning, 1 × 104 host T cells instead of 1.5 × 104 T cells were used for induction of rejection and half dose of rapamycin (5 μg/mouse/day for 5 days) was used for immune suppression. As in our previous study, under these conditions rapamycin alone, or veto cells alone, could only marginally overcome graft rejection mediated by naive host T cells, but the two agents together led to marked engraftment and survival (data not shown). Once this was reproduced we could begin comparing the effect of veto CTLs on naive vs presensitized host T cells.

Purified naive and presensitized host T cells were prepared by selection with magnetic beads. Briefly, host (C3H/HeJ) splenocytes were obtained from naive or from mice presensitized with irradiated BALB/c spleen cells, as described above. The cells were fractionated on Ficoll/Paque and the isolated mononuclear cells were subjected to a negative selection of T cells, removing myeloid (CD11b), NK (DX5 (CD49b)) and B cells (CD45R (B220)) and sparing CD4+ and CD8+ cells, by magnetic cell sorting (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany). The cells from presensitized mice were then incubated with anti-CD44–PE Ab (Pharmingen) and anti PE Magnetic beads, and isolated again by MACS using positive selection. Cytofluorimetric analysis of the fractionated cells was conducted by triple immunofluorescent staining using the following directly labeled Abs (Pharmingen): FITC-CD4/L3T4 (clone H129.19), PE-CD44e (clone 145-2C11), and Cy-Chrome-CD8a/Ly-2 (clone 53-6.7).

Results

Inhibition of presensitized CD8 effector T cells by veto CTLs in vitro

The 2c TCR transgenic mouse model.

The 2c TCR transgenic mouse, in which the CD8+ T cells express a TCR transgene against H2d (stained by the clonotypic Ab 1B2) enables monitoring the fate of anti-H2d clones upon interaction with relevant veto cells of H2d origin, as opposed to nonrelevant veto cells of a different H2 origin. Thus, by using this model we were able previously to demonstrate that CTLs of H2d origin induce deletion of primary effector cells via a Fas-FasL mechanism (37, 39). In the present study, to define the role of veto CTLs in the context of presensitized effectors, the same 2c mice were used except that the mice were presensitized by infusion of irradiated BALB/c spleen cells, as previously described (40).

Following presensitization, the lymphoid compartment in the spleens of presensitized mice was compared by FACS analysis to spleen cells obtained from un-sensitized mice. As can be seen in Fig. 1⇑, IB2+ cells in immunized mice exhibit a memory phenotype, namely a high level of CD44 (68%) or CD122 (60%) in contrast to primary 1B2+ cells which exhibit reduced expression of these markers (18% and 6%, respectively).

As explained above, the 2c model enables FACS monitoring of veto activity, either by measuring the inhibition of proliferation or the induction of apoptosis (Annexin staining) in the effector cells. In the present study we used both assays to test whether veto CTLs suppress the allo-immune response mediated by memory CD8+ T cells. Thus, when measuring the proliferation and expansion of 2c cells by their cognate MHC stimulators, in the presence or absence of veto cells, marked reduction of total 2c cell number, as well as the percentage of CD8+1B2+ cells, was recorded by FACS after 72 h. As can be seen in Fig. 2⇑, the average total cell number in 3 different experiments of naive or presensitized 1B2+CD8+ cells, in the presence of nonrelevant veto CTLs (SJL background, H2s), were 63,500 ± 1650 and 48000 ± 1224 respectively. These high levels of 1B2+ cells were significantly reduced upon the addition of relevant anti-third party CTLs from the DBA (H2d) background (3310 ± 381 and 1573 ± 360, respectively). A similar trend was also found by recording the percentages of surviving 1B2+CD8+ cells at the end of the 72 h MLR in the presence of nonrelevant or relevant veto CTLs.

It could be argued that the veto sensitivity exhibited by presensitized 2c TCR transgenic T cells, might not be relevant to wild type T cells, due to the artificial expression of TCR on the transgenic cells. In particular, this is important in light of the early study of Fink et al. suggesting that a veto CTL clone which was effective against naive effectors was not effective in blocking presensitized effectors stimulated in vivo against minor histocompatibility Ags (17). To address potential discrepancy between TCR transgenic and wild type effectors, we also tested the inhibitory activity of anti-third party veto CTLs upon addition to MLR in which in vivo presensitized wild type C3H/HeJ splenocytes were re-stimulated in vitro against irradiated BALB/c stimulators. As can be seen in Fig. 2c⇑, the killing of BALB/c targets by CTLs generated from both naive and presensitized wild type C3H splenocytes, as measured by the Cr51 release assay (used by Fink et al.), was effectively inhibited by the veto CTLs. Thus, there is no significant difference (p > 0.15) in the sensitivity of wild type naive and presensitized effectors to inhibition by anti-third party veto CTLs.

Considering that veto CTLs were previously shown to kill naive effector T cells by apoptosis, it was of interest to test the induction of annexin V on presenstitized effectors upon their interaction with veto CTLs.

As shown in Fig. 3⇑ presensitized CD8 cells (Fig. 3⇑, c-d) similarly to naive CD8 cells (Fig. 3⇑, a-b), undergo deletion in the presence of 10% veto CTLs. The annexin V level on presensitized or naive effector cells in the presence of veto CTLs was markedly higher (62%–75% respectively) compared with the expression of annexin V on naive or presensitized 2c cells in the absence of veto cells (6%–11% respectively), indicating that deletion of presensitized cells, similarly to naive cells, is mediated by apoptosis.

The role of Fas-FasL apoptosis in the deletion of presensitized effectors by veto CTLs

Previous studies demonstrated that the activation of the 2c effectors is associated with up-regulation of the cell surface Fas receptor (CD95), leading to apoptosis by interaction with FasL on the veto CTLs (37, 39). To further establish the role of Fas in the apoptosis of presensitized CD8 T cells, 2c cells were harvested from 2c-lpr mice following presensitization with 1 × 106 irradiated splenocytes from BALB/c mice. Thus, Fas negative presensitized 2c-lpr cells were tested for their sensitivity to veto CTLs in comparison to Fas positive presensitized 2c splenocytes. As can be seen in Fig. 4⇑, similarly to previous results with naive effector 2c cells, (37) deletion by veto cells was markedly reduced when testing the Fas deficient 2c-lpr presensitized cells compared with Fas positive 2c presensitized cells. Thus, presensitized CD8 T cells, similarly to naive cells, are likely deleted by veto CTLs via a Fas-FasL mediated mechanism.

Enhancement of engraftment of T cell-depleted BM in presensitized mice by veto CTLs

It has been shown in a stringent mouse model for T cell mediated BM allograft rejection, that optimal enhancement of BM allografting by veto CTLs is attained in combination with rapamycin based conditioning (22). In this model, recipient mice are conditioned by supralethal TBI and radio protected with T cell-depleted BM. Rejection is induced by adoptively transferred purified host T cells (HTC). To evaluate the potential of veto CTLs to overcome rejection in presensitized mice, we used the same model except that host type CD44+ T cells, harvested from presensitized mice, were used instead of naive T cells. The CD44+ T cells were isolated by a two step procedure, comprising an initial negative selection of HTC followed by positive selection of CD44+ cells. As can be expected, the rejection mediated by 5 × 103 adoptively transferred presensitized HTC is more vigorous compared with that mediated by naive HTC. Thus, in 4 independent experiments average survival time in recipients infused with presensitized HTC was 19.2 ± 1.2 days compared with 30 ± 3 days in recipients of naive HTC. Upon infusion of 1 × 104 HTC, the difference in the rate of survival between recipients of presensitized or naive HTC is less pronounced (21 ± 2 days and 23 ± 1 respectively) presumably due to the strong antigenic stimulus mediated by fully allogeneic BM cells. Considering that we have previously found that veto cells are markedly enhanced by short treatment with rapamycin in this in vivo model, we have now compared their efficacy to neutralize naive and presensitized HTC, in the presence or absence of rapamycin. As can be seen in Fig. 5⇑, while rejection mediated both by naive and presensitized HTC was not effectively overcome by rapamycin alone (18% and 23% survival, respectively), marked survival was attained when veto CTLs were administrated in conjunction with rapamycin (69% and 82%, respectively. p < 0.0001 and p < 0.0001 compared with survival of mice adoptively transferred only with naive or presensitized HTC (Log-Rank and Wilcoxon test)). Thus, in accordance with the in vitro studies, the presensitized HTC are as prone as the naive HTC (p > 0.05 Log-Rank and Wilcoxon test) to the inhibitory reactivity of veto CTLs in vivo.

Interestingly, when veto CTLs were infused alone, enhancement of engraftment was more effective in mice adoptively transferred with presensitized HTC compared with naive HTC (35% and 54% survival, respectively.) However, this trend suggesting potential higher sensitivity of presensitized HTC was not significant (p > 0.05 Log-Rank and Wilcoxon test).

As can be expected for BM recipients conditioned with supralethal TBI, full donor-type chimerism (96–100%) was exhibited at 60 days post transplant in both naive and presensitized groups treated with veto CTLs and rapamycin. Splenocytes recovered from the chimeric mice of both groups exhibited significant capacity to generate alloreactive CTLs against nonrelevant SJL stimulators (30% and 66% cytotoxic index, respectively) while diminished responses were recorded against host (2.3% and 6.5%, respectively) or donor-type stimulators (0.6% and 3%, respectively).

Discussion

Immune memory is an indispensable component of the immune response protecting the host from future challenges of the same pathogen or even in certain settings of unrelated pathogens. As a consequence, anti-viral effector T cells capable of directly recognizing foreign MHC might be generated, inducing long-lived alloreactive memory cells (41). The use of immunosuppressive drug treatment predisposes graft recipients to viral infections which, in turn, sensitize alloreactive immune cells to induce graft rejection. In addition to such alloreactive memory cells, many patients undergoing BM transplantation are presensitized to a broad range of donor MHC Ags from multiple blood transfusions. Consequently, many presensitized patients are either precluded from receiving a transplant or may experience an increased rate of early rejection episodes that are irreversible or difficult to treat with current immunosuppressive agents. This has led to the emerging concept that environmental exposures and large memory pools may constitute a more formidable barrier to tolerance induction. Furthermore, it was shown that memory T cells which exhibit selective resistance to therapeutic depletion, are resistant to several immunosuppressants and are less dependent on costimulatory signals. Thus, although in vivo depletion decreases the allospecific precursor frequency, the remaining cells are less susceptible to therapies that successfully interrupt naive cell activation.

Our previous data clearly have shown that, anti-third-party CTLs generated under IL-2 deprivation, afford a suitable source for effective veto cells that can enhance BM allografting without GVHD in lethally irradiated mice, adoptively transferred with graduated numbers of purified naive host-type T cells. This model, which has been designed to distinguish between T cell mediated rejection as opposed to rejection mediated by non-T cell mechanisms, including failure to engraft due to stem cell competition, has also been used recently to evaluate the role of Treg cells (42).

An early study has suggested that veto CTLs might not be capable of effectively inhibiting in MLR, effector T cells presensitized in vivo (17).

Considering that with the advent of TCR transgenic models it has become possible to follow and isolate memory T cells after presensitization in vivo, we reinvestigated this important question in the context of BM allograft rejection, and we demonstrate both in vitro and in vivo that presensitized effectors are as sensitive as their primary counterparts to the inhibition of veto CTLs. Furthermore, by using Fas knockout 2c mice, we were able to show that similarly to naive effector cells, deletion of presensitized effectors is mediated through Fas-FasL apoptosis.

It could be argued that the conclusion raised by our in vitro studies differ from the early study of Fink et al. (17), due in part to the use in the present study of artificial TCR transgenic effectors. However, the same efficacy of the veto CTLs was also demonstrated against wild type effectors presentitized in vivo, using the same chromium release cytotoxic assay used in the earlier study. Clearly, such assays measuring functional inhibition of CTLp frequency, do not allow to monitor directly the fate of the specific effector cells, in contrast to the 2c model in which effectors can be decorated with an appropriate clonotypic Ab.

The discrepancy between the earlier study and our present results might be attributed to the clone used in the former, which bears an unusual phenotype and is likely not representative of the anti-third party CTL preparation from which CD4+ cells are removed at the end of the culture.

Our results seem to be also in line with tolerance induction by veto CTLs out of the context of BM allografting, namely for CTLs presensitized against viral Ags which were found to be as susceptible in vitro to veto cell inhibition as their naive counterparts (43). However, this veto activity on presenstized effectors directed against viral Ags has to be confirmed in vivo.

The sensitivity of presensitized effectors to veto CTLs is clearly important for BM transplantation, however an obstacle remaining is their reduced reactivity in vivo compared with their remarkable ability to delete anti-donor clones in vitro. The enhancement mediated by short term treatment with rapamycin is compatible with the recent demonstration that veto CTLs operate via Fas-FasL triggering, upstream of rapamycin inhibition of IL-2R signaling. Thus, effector T cells that might have escaped deletion by the veto cells could still be eliminated by rapamycin. Moreover, Anderson et al. (43) suggested that rapamycin prevents the responding cells from differentiating into fully activated CTLs and that the point of inhibition precedes the loss of a veto sensitive phenotype.

One attractive explanation could be offered by the suggestion that up-regulation of L-selectin on veto CTLs by rapmycin, (44, 45, 46) might direct them more effectively to lymph nodes, where the deletion and inhibition of host T cells by veto cells likely occurs. However, considering the broad spectrum of rapamycin cellular reactivities, other pathways and mechanisms must be considered.

Recently, it has been suggested that anti-CD70 treatment could prevent CD8 T cell-mediated rejection (47), and it was further demonstrated that it could inhibit the proliferation and activation of effector CD8 T cells, and diminish the expansion of effector and memory CD8 T cells in vivo (48). Thus, the C27-CD70 pathway seems critical for CD28-independent effector/memory CD8+ alloreactive T cell activation in vivo. Considering that Anderson et. al (43) showed recently that addition of rapamycin extends the window during which responding cells are susceptible to deletion by veto cells, it would be interesting to test the possibility that blockade of the CD27–CD70 costimulatory pathway might be synergistic with veto CTLs, so as to potentially substitute the need for treatment with rapamycin.

In conclusion, our results suggest that allograft rejection mediated by naive or presensitized T cells can be equally overcome by combining rapamycin and veto CTLs, and that the deletion of the presensitized effector CD8 cells is dependent on Fas/FasL interaction, as shown for naive T cells. This conclusion is highly relevant to the prospect of clinical application of anti-third party veto cells in leukemic recipients of BM transplantation, in whom rejection is often mediated by residual presensitized T cells. A protocol for effective large scale production of human veto CTLs has been described recently (49) and clinical trials in leukemia patients are in the final stages of preparation. The present data showing the effective deletion of presensitized T cells, suggest that if successful in leukemic BM recipients, this approach could be further extended to induction of tolerance for organ transplantation or in autoimmunity, in which presensitized T cells present a major obstacle.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported in part by National Institutes of Health Grant PO1CA100265-01A1, (project 5 of Dana Farber PO1); European Commission LSHB-CT-2004-503319 (Allostem Project); the Gabriella Rich Center for Transplantation Biology Research; and by E. Drake.

  • ↵2 S.R.Z. and E.B.L. contributed to research design and performance and the writing of the paper. A.B. contributed to research design and performance and Y.R. to research design and writing of the paper.

  • ↵3 Address correspondence and reprint requests to Prof. Yair Reisner, Chairman, Department of Immunology, The Henry H. Drake Professorial Chair of Immunology, Yair Reisner, Weizmann Institute of Science, Department of Immunology, Rehovot, Israel. E-mail address: yair.reisner{at}weizmann.ac.il

  • ↵4 Abbreviations used in this paper: GVHD, graft-vs-host disease.

  • Received February 22, 2007.
  • Accepted August 12, 2007.

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