1 Division of Cancer Biology Research, Sunnybrook Health Science Centre, Toronto, Ontario M4N 3MS, Canada
2 Department of Medical Biophysics, University of Toronto, and Ontario Cancer Institute, Toronto, Ontario M56 2M9, Canada
Correspondence to: D. Spaner, Division of Cancer Biology Research, Sunnybrook Health Science Centre, Research Building, S-Wing, Rm S-218, 2075 Bayview Avenue, Toronto, Ontario M4N 3M5, Canada
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Abstract |
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Keywords: graft versus host disease, immunosenescence, in vivoanimal model, superantigens, tolerance/suppression
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Introduction |
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Development of GVHD appears to require the injection of at least 106 donor T cells/kg (3). Are T cell numbers below this threshold simply rejected by host defense mechanisms that survive the conditioning regimen? If small numbers of mature donor T cells survive, it is not clear why GVHD is prevented. Donor cells expand to reconstitute the peripheral T cell compartment (4,5) and, a priori, alloreactive cells should be preferentially selected. In principle, a single alloreactive cell should be able to expand and cause GVHD.
To study the fate of mature alloreactive donor T cells and the effect of initial cell number on GVHD, we have used a previously characterized model in which enriched T cells from the spleens of C57BL/6J mice (H-2b) are injected into sublethally irradiated C.B-17-SCID mice (H-2d) (6,7). This model is especially useful for tracking the fate of donor T cells in vivo because the lack of host T cells precludes host versus donor lymphocyte interactions and donor marrow is not required for survival. In addition, the specific pathogen-free conditions required for the maintenance of SCID mice partially control for the effects of exogenous infections. In this paper, the behavior of low numbers (<3x104) of alloreactive B6 T cells in sublethally irradiated SCID hosts was compared with that of higher numbers (106) that were previously shown to cause acute GVHD (7). Peripheral lymph node cells (PLNC) that do not contain hematopoietic stem cells (8) were used to ensure that the peripheral T cell compartment was not reconstituted by thymic regeneration. Low numbers of allogeneic B6 T cells failed to cause GVHD but repopulated the peripheral T cell compartment to the same extent as an initial injection of 50-fold higher cell numbers. Moreover, these cells differentiated into a long-lived, IL-2-unresponsive state with a characteristic surface phenotype.
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Methods |
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Antibodies, reagents, and cell lines
Anti-CD3 (145-2C11) (10) was purified from hybridoma culture supernatants by Protein G column chromatography. Phycoerythrin (PE)- or FITC-labeled CD4 and CD8 antibodies, 7-AAD, propidium iodine, and streptavidinPE were purchased from Sigma (St Louis, MO). The anti-FcRIII-
antibody (2.4G2) (11) and the anti-heat stable antigen (HSA) antibody (J11d) were obtained from ATCC (Rockville, MD) and used as culture supernatants. PE- and FITC-labeled antibodies against Thy-1.1, Thy-1.2, Mac-1, H-2b, H-2d, HSA, IFN-
, IL-2, CD3 and CD44, and biotinylated antibodies against Vß3 and CD25 were purchased from PharMingen (San Francisco, CA). Anti-CD4 and anti-CD8TriColor antibodies were purchased from Caltag (Burlingame, CA). The hamster anti-murine CD28 hybridoma (37.51) was obtained from Dr James Allison (University of California, Berkeley, CA) (12) and antibodies were purified from culture supernatants by Protein G-affinity chromatography (Pharmacia, Uppsala, Sweden) in our laboratory.
Phorbol myristate acetate (PMA) and Brefeldin A were purchased from Sigma, and ionomycin was purchased from Calbiochem (La Jolla, CA). Murine IL-2 and IL-4 cDNA-transfected X63Ag8-653 cells were a generous gift of H. Karasuyama (13). CT.4S and P-815 lines were obtained from ATCC and maintained in exponential growth by serial passage in complete medium (CM) (-MEM, 10% FCS, 5x105 2-mercaptoethanol, 15 mM HEPES, 2 mM glutamine) at 37°C in an atmosphere of 5% CO2.
Cell preparations
Where multiple sites were to be studied for the presence of donor T cells, mice were anesthetized in vaporous Enflurane USP (Abbott Laboratories, Montreal, Canada) and exsanguinated by cutting the right axillary artery. Blood was collected in CM with 100 U/ml of heparin. Cell suspensions of spleen, thymus and PLNC were made by passage through metal screens. Bone marrow cell suspensions were made from the femurs and tibiae of each mouse by injecting ~610 ml of CM via a 25-gauge needle and a 3-cm3 syringe. Peritoneal washings were collected by infusion of approximately 6 ml of cold PBS via a 10- cm3 syringe and an 18-gauge needle. A small cut was made in the peritoneal membrane and the infusate was collected using a sterile Pasteur pipette. The process was repeated once.
To purify donor T cells from chimeric spleens by negative selection, red cells were first depleted by ammonium chloride treatment. To remove J11d+ erythroid cells, spleen cells were washed and incubated for 30 min at 4°C in the presence of J11d hybridoma supernatant at a 1:4 dilution. Cells were then washed and incubated with rabbit complement (Cedarlane, Hornsby, Ontario, Canada) at a 1:10 dilution in cytotoxicity medium (Cedarlane) at 37°C for 45 min. Viable cells were then harvested over Lympholyte columns (Cedarlane).
DNA analysis
T cells (~1x106) were washed and fixed in 70% ethanol at 20°C for several days at 106 cells/ml. The cells were then washed and resuspended in 1 ml of Ca+2, Mg+2-free PBS to which 0.1% Triton X-100, 0.1 mM EDTA and 50 µg/ml RNase were added, and incubated for 1 h at 37°C. This incubation period allows low mol. wt DNA to escape through the permeabilized membranes (14). Cells were then washed and resuspended in staining buffer (0.1 mM EDTA, 0.1% Triton X-100 and 50 µg/ml of propidium iodide) at room temperature in the dark for 412 h. Cells were then filtered through nylon mesh and analyzed on a Becton Dickinson (Mountain View, CA) FACScan flow cytometer using Lysys II software.
Proliferation assays and mixed lymphocyte responses
In some assays, responder T cells were diluted to 2x105 cells/ml in CM and cultured with syngeneic irradiated (2000 cGy) spleen cells as filler cells and 0.1 µg/ml of soluble anti-CD3 antibody for 72 h. Then 1 µCi of [3H]thymidine was added to each culture for 18 h and the amount of incorporated thymidine was measured in a ß-scintillation counter. In other assays, donor T cells were stimulated with plate-bound anti-CD3 plus or minus anti-CD28 antibodies as previously described (15). For mixed lymphocyte reactions, responder spleen cells were diluted to a concentration of 2.5x105 T cells/ml in CM. Irradiated spleen cell stimulators (2000 cGy) were from BALB/c and BALB.K or C3H mice at 5x106 cells/ml. The mixed lymphocyte reactions were incubated for 72 h and then 1 µCi of [3H]thymidine was added to the cultures for a subsequent 18 h. The cells were then harvested and the amount of thymidine incorporation measured in a -scintillation counter.
Redirected lysis assays
P815 tumor targets in exponential growth phase were collected by centrifugation, resuspended in two drops of 100% FCS and radiolabeled with 50 µl of sodium chromate (7.14 mCi/ml) (Dupont, NEN, Boston, MA) for 1 h. Effector cells, purified from spleen cells using Lympholyte separation medium, were added at varying effector:target ratios in 100 µl of CM to individual wells of a U-bottom plate. Anti-CD3 antibody (0.2 µg) was then added at a final concentration of 1 µg/ml (10). Chromium-labeled targets were washed 3 times with -MEM + 1% FCS and 100 µl of target cells (2x104/ml in CM) were added to each well. The plates were centrifuged at 600 r.p.m. for 3 min and then incubated at 37°C for 4 h. Plates were then centrifuged at 800 r.p.m. for 5 min and 100 µl of the supernatant transferred to Fisherbrand flint glass tubes (Fisher Scientific, Pittsburgh, PA) and counted in a
-counter (CompuGamma Model 1282; LKB, Stockholm, Sweden). Total release (TR) was measured by lysis of tumor targets with 1% acetic acid and spontaneous release (SR) was measured in the absence of effector cells. Percent cytotoxicity was determined by the ratio (c.p.m. SR)/ (TR SR)x100%.
Immunofluorescence
Cell staining was performed after first blocking non-specific binding with a 10 min incubation at room temperature with 10 µl of mouse serum (Cedarlane) and 40 µl of 2.4G2 culture supernatant. Cells (5x105) were then allowed to react with pre-titrated doses of antibodies for 20 min, washed, incubated with 7-AAD to exclude dead cells and then analyzed on a FACScan flow cytometer (Becton Dickinson) using Lysys II software.
Intracellular cytokine staining
The method of Ferrick et al. (16) was mainly followed. Cells were suspended at a concentration of 5x106/ml in CM and incubated at 37°C for 4 h in the presence of 5 µg/ml Brefeldin A (unstimulated) or Brefeldin A plus 10 ng/ml PMA and 500 ng/ml ionomycin (stimulated). Cells were then washed and non-specific binding blocked with 2.4G2 and mouse serum in a total volume of 90 µl, and fixed in 75 µl of Solution A (Caltag) for 30 min at room temperature. After washing in Ca+2, Mg+2-free PBS, cells were stained at room temperature with 0.2 µg of CD4 and CD8TriColor, and IL-4PE (0.2 µg) and IFN-FITC (0.1 µg) in 75 µl of solution-B (Caltag) for 30 min. Cells were then washed and analyzed as described above.
Production of GVHD
C.B-17-SCID mice were irradiated with 275 cGy from a source (137Cs) (Gammacell 40 Exactor, Nordion International, Kanata, Ontario, Canada) on the same day as the injection. Inguinal lymph node cells were obtained from donor mice and varying numbers were injected into the tail veins of SCID hosts. Mice were examined daily and sacrificed if moribund.
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Results |
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Samples of 3x103 PLNC behaved either the same as 3x104 PLNC and reached the plateau level after a couple of weeks or else were not found from the first determination at around days 57. This behavior suggested that 3x103 PLNC was near the limit below which injected cells could be rejected by radioresistant mechanisms in SCID mice. Consequently, the rest of the experiments described in this paper were performed using 3x104 B6 PLNC as the initial donor inoculum.
When 106 PLNC were injected, cell numbers also increased during the first week but surprisingly reached approximately the same plateau level as an initial injection of 3x104 PLNC. They could not be followed any longer than 23 weeks due to the morbidity of the injected animals.
In summary, Fig. 3 shows that, by ~2 weeks after injection, the load of donor T cells was the same in animals initially injected with 3x104 or 106 PLNC. However, in the former case the animals survived without evidence of disease, while, in the latter, they became moribund.
Similar expansion of potentially alloreactive T cells in F1 SCID mice
To determine the effect of host H-2b expression on donor T cell accumulation, B6 T cells were injected into sublethally irradiated (B6xC.B-17) F1 SCID mice. At least for the first 2 months after injection, B6 T cells behaved much the same in F1 SCID mice as they did in fully MHC disparate C.B-17-SCID mice (Fig. 3). In contrast, syngeneic BALB/c T cells expanded to reach a plateau level in sublethally irradiated C.B-17-SCID mice that was higher than for allogeneic cells (Fig. 3
). Moreover the ratio of CD4+ to CD8+ cells was skewed towards CD4, whereas it was inverted for B6 cells (legend to Fig. 3
).
Destruction of host hematopoietic cells by 106 but not by 3x104 B6 PLNC
The number of host spleen cells was also determined as a function of time after irradiation and injection of varying numbers of donor T cells (Fig. 4). The number of mononuclear spleen cells in an unirradiated SCID mouse is ~107. Shortly after a radiation dose of 275 cGy, this number fell to ~105 cells but recovered by the end of the second week to the level of an otherwise untreated mouse (Fig. 4
).
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In contrast, the recovery of host cells from radiation was virtually unimpeded in animals injected with low numbers of allogeneic T cells. If anything, there was an initial overshoot of host cells, possibly in response to secretion of growth factors such as IL-3 or granulocyte macrophage colony stimulating factor from the proliferating donor cells. The majority of host cells were erythroid (by examination of Wright's-stained cytospins). Approximately 10% were Mac-1+ and many were in cycle (not shown). After several months, the total number of host cells declined regardless of whether donor cells were injected. The explanation for this decline was not clear but may be related to exhaustion of SCID hematopoietic precursors. Cells homozygous for the SCID defect are known to be more radiosensitive than wild-type cells (19).
The results of Figs 3 and 4 showed that allogeneic T cells could persist in a sea of host antigen for many months after an initial injection of low numbers of cells. For the rest of this paper, such animals will be called low-dose chimeras (LDC) and arbitrarily divided into short-term (<3 months after injection) and long-term (>3 months after injection) LDC.
Characterization of donor T cells in GVHD: persisting cells develop an intrinsic proliferative defect that cannot be reversed by IL-2
Figures 3 and 4 demonstrated the simultaneous presence of donor cells and normal numbers of host hematopoietic cells in LDC. Despite this potentially inflammatory situation, there was no overt GVHD suggesting that the donor cells were functionally tolerant of host antigens.
To study the in vitro proliferative responses of donor T cells from short-term LDC was technically difficult due to their low numbers. Two strategies that gave equivalent results were employed. The first was to sort H-2b+Thy-1.2+ cells or Thy-1.1+ cells, when Thy-1.1 was used as a marker for donor cells, on a cell sorter and to use similarly purified naive B6 PLNC as controls in proliferation assays. Purity of the sorted T cells was >95% (unpublished results). The second strategy used the HSA-specific mAb, J11d, to deplete HSA+ cells by complement-mediated cytotoxicity. Preliminary studies had revealed that the majority of host cells in the spleens of SCID mice and LDC were J11d+ while the donor cells were J11d. This negative selection procedure could only enrich the percentage of donor cells several fold, to ~10% (unpublished results). The percentage of donor T cells in the spleens of long-term LDC was usually high enough (1020%) that functional assays could be performed without need for further enrichment procedures.
The functional properties of B6 donor T cells from long-term LDC were compared to B6 T cells, from normal donors and after injection into C.B-17- and B6-SCID mice, and to BALB/c T cells, after injection into C.B-17-SCID mice. Naïve B6 cells were used to compare the behavior of donor T cells after adoptive transfer to their initial behavior. The most important control is the study of B6 T cells in irradiated B6-SCID mice in the absence of alloantigenic stimuli. Unfortunately, B6-SCID mice are inherently `leaky' (6) so that, without a genetic marker, it is difficult to distinguish adoptively transferred B6 T cells from cells that undergo thymic development. We have used B6-Thy-1.1 T cells in order to distinguish exogenous from endogenous T cells in B6-SCID (Thy-1.2+) mice. However the thy-1 polymorphism provides an antigenic stimulus for transferred Thy-1.1 T cells (7). Indeed, at low doses, we found evidence for activation of Thy-1.1+ donor cells [i.e. inversion of the CD4/CD8 ratio and in vitro anergy (see below)]. Irradiated adult C.B-17-SCID mice are much less leaky. Therefore, the adoptive transfer of syngeneic BALB/c T cells into sublethally irradiated C.B-17-SCID mice was used to ensure that the behavior of long-term surviving T cells was not due to some non-specific, poorly understood mechanism.
Observations on the in vitro functions of T cells after adoptive transfer into allogeneic and syngeneic hosts as a function of initial cell number are summarized in Table 1. Cells from long-term LDC could not respond to host or third-party antigens (Table 1
, rows 7 and 16; column 4) and this lack of response could not be rescued by IL-2 (Table 1
, row 8, column 4). The response to a mitogenic stimulus (anti-CD3) was depressed compared with fresh B6 T cells and only minimally increased by provision of co-stimulation by simultaneous cross-linking of CD28 molecules (Fig. 5
). T cells taken shortly after injection (<31 days) into sublethally irradiated SCID mice also showed decreased proliferative responses after re-stimulation with host and third-party antigens or mitogens (Fig. 5
and Table 1
, rows 912 and 17, column 4). In contrast to cells from long-term LDC, the response to exogenous IL-2 was strong (Fig. 5
and Table 1
, rows 34, column 4). Syngeneic donor T cells studied early after injection into irradiated SCID mice did not undergo growth arrest upon reactivation in vitro (Fig. 5
and Table 1
, row 1314, column 4).
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To determine if the proliferative defect in T cells from long-term LDC was due to active suppression, they were mixed with fresh B6 T cells, and reacted against host and third-party stimulators in mixed lymphocyte reactions (Table 1, rows 1819). Neither specific or non-specific suppression was observed, suggesting that the observed proliferative defect was intrinsic to the cells.
Attempts to derive cell lines from these cultures were relatively unsuccessful but did show that donor cells could persist in culture, in the presence of 20 U/ml of IL-2, for more than 4 weeks without recovering their proliferative capability (unpublished results).
Donor cells persisting in LDC are able to produce Interferon-
Intracellular cytokine production was measured using flow cytometry. Figure 6 shows that donor T cells taken from LDC did not directly produce IFN-
ex-vivo but could produce some IFN-
after a 4 h stimulation with PMA and ionomycin. In comparison, T cells actively mediating GVHD directly produced IFN-
ex-vivo and the level of production was significantly enhanced by stimulation (Fig. 6
). Note that if cells are not making a cytokine directly but do so after stimulation, this is thought to mean that they previously produced the cytokine (20).
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Donor cells reside in multiple organs
Donor cells could be found in multiple organs of host mice as shown in Fig. 7. The majority of donor cells were found in the spleen as described in the figure legend. Low percentages of donor cells were also found in the thymus, bone marrow and blood. Note that animals were exsanguinated before collection of thymuses and bone marrow to avoid contamination with peripheral blood. A significant increase in the percentage of donor cells was found in the lymph nodes and the peritoneal cavity. Despite the increased proportion, the total number of donor cells was low at these sites because of their low total cellularity (legend to Fig. 7
).
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The cell cycle status of donor cells was determined by flow cytometric analysis of propidium iodine staining of DNA. Figure 7(g and i) shows that the number of donor T cells from long-term LDC in the G2/S phase of the cell cycle was little higher than the number for T cells from naive B6 mice. In contrast, significantly higher numbers of donor cells were in cycle within the first few weeks after injection (Fig. 7h
).
The surface phenotype of donor cells in LDC is consistent with prior activation
Cell size and surface expression of CD25, CD3 and CD44 on donor T cells from LDC were determined by flow cytometry (Fig. 8). As described for memory T cells (24), CD44 was up-regulated in LDC (MFI = 1485) and in SCID mice given initial injections of high numbers of donor T cells (MFI = 1993) when compared with donor B6 mice (MFI = 675) (Fig. 8
). Despite this phenotypic change, donor T cells in LDC over 100 days after initial injection were only slightly larger on forward scatter (mean = 120) than naive T cells (mean = 112) (Fig. 8
) although, shortly after the injection of high numbers of donor T cells, the majority were significantly larger (mean = 147) (Fig. 8
, right column). The TCR receptor density on persisting donor T cells in LDC was also significantly less (MFI = 45) than on both naive cells (MFI = 167) and cells actively mediating GVHD in vivo (MFI = 160) (Figs 8 and 10b
). TCR down-regulation is also a marker for recent activation (25). CD25 expression on T cells from long-term LDC was about the same as naïve donor T cells and not as high as on donor T cells 6 days after initiation of GVHD with high doses of cells (Fig. 8
).
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Discussion |
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Radiation was presumably required to prevent the immediate rejection of donor PLNC by NK cells (18,26) that recognize the full MHC mismatch between B6- and C.B-17-SCID mice (Fig. 3). Prolonged persistence of allogeneic cells suggests that the host NK cells that recover from irradiation are tolerant of donor cells (27) or that another cell type required for allogeneic resistance does not survive sublethal irradiation.
Despite the clear relationship to clinical outcome, the early behavior of the donor T cells after injection did not seem to depend on their initial number. In both cases, the total number of donor T cells reached a plateau level of ~3x105 cells/spleen (Fig. 3). Even with initially low numbers of donor cells, host superantigen-reactive T cells, represented by CD4+Vß3+ T cells stimulated by the open reading frame protein of the mtv-6 provirus in the SCID genome, expanded up to 10-fold within the first month (unpublished results). This result suggests that failure of low doses of T cells to cause GVHD was not because the number of transferred immunocompetent precursors was below the frequency required to respond to host antigens. Significant numbers of donor T cells could not be found in other sites (Fig. 7
).
Superficially, the apparent expansion of initially high numbers of donor T cells (Fig. 3), followed by a phase of deletion, is similar to other in vivo experiments that lead to peripheral tolerance, such as the response of T cells following superantigen injection or transgenic T cells after their injection into mice expressing their specific antigen (2830). However, the decrease in donor T cell numbers may simply reflect the morbidity of mice with GVHD at this time. In contrast to normal superantigen responses (31), CD4+Vß3+ T cells were not deleted at the time that total numbers of donor T cells were decreasing (unpublished results and manuscript in preparation).
Within the first month after injection, both LD and HD T cells secreted IFN- directly ex vivo (Fig. 6
and unpublished results), and HD T cells could mediate perforin-dependent cellular cytotoxicity (Table 1
, row 4, column 6). Also within the first month, HD and LD T cells proliferated strongly in response to exogenous IL-2 (Table 1
) but were growth inhibited after reactivation through the TCR in vitro (Table 1
and Fig. 5
). We and others have previously shown that this growth arrest reflects activation-induced T cell death, and is mediated by multiple factors including perforin, tumor necrosis factor-
and IFN-
(7,32,33).
Subsequently, LD T cells entered a long-lived non-functional state, characterized by small size (Fig. 8), unresponsiveness to exogenous IL-2 or allogeneic stimuli (Table 1
), lack of direct ex-vivo secretion of IFN-
(Fig. 6
), or killing in a redirected lysis assay (Table 1
). Failure of third-party killing could possibly be due to an alteration in frequency of antigen-reactive cells, following the expansion phase of the response to BALB/c antigens, rather than to a tolerance process. However, third-party responses were decreased early (Table 1
), even before the expansion process was finished. Moreover, the persisting cells showed evidence of prior activation indicated by up-regulation of CD44 and down-regulation of the TCR cell surface density (Fig. 8
), and the failure to respond to mitogens suggested a global defect in the persisting T cells. The fate of HD T cells could not be determined after several weeks because of the morbidity of host mice.
Further evidence for the proliferative arrest of T cells in LDC was provided by their behavior after transfer into a freshly irradiated secondary host (data not shown). Secondary transfer of 3x104 T cells from LDC caused no apparent morbidity. Several weeks after passage, H-2b+ T cells could only be found in the peritoneal cavity. The total number was 5x103, suggesting that donor T cells from the LDC did not proliferate although they could persist in vivo. In contrast, the same number of donor T cells expanded 50- to 100-fold after primary transfer, although clinical GVHD was not observed (Fig. 3).
The finding that polyclonal, alloreactive T cells eventually became unresponsive after chronic stimulation in vivo is consistent with, and extends, recent observations with TCR transgenic T cells that encounter persistent antigen. Lanoue et al. (34) showed that transgenic CD4+ T cells, specific for an influenza hemmagglutinin epitope, initially proliferated after transfer into hemmagglutinin transgenic hosts. However, by day 30 after transfer, the majority of transgenic CD4+ T cells had been deleted and the remainder were anergic. Tanchot et al. (35) showed that transgenic CD8+ T cells specific for the HY male antigen became anergic after adoptive transfer under conditions of male antigen excess. This state of proliferative anergy was not restored by ionomycin and PMA, which restored IL-2 and IFN- production and IL-2 R expression, and was associated with increased IL-10 production. In contrast, TCR down regulation (Fig. 8
), rather than production of Th2 cytokines, was associated with the tolerant state of chronically in vivo allo-activated CD4+ and CD8+ T cells.
Previous attempts to follow the fate of mature donor T cells in GVHD were performed in irradiated hosts given donor stem cells and peripheral T cells that could be distinguished by congenic markers (36). The results of such studies suggested that the mature T cells disappeared within several months after the initiation of GVHD and that chronic GVHD was maintained by T cells of donor origin that developed in the host thymus. Although these findings may apply to pediatric bone marrow transplantation, thymic reconstitution is limited in adult recipients of stem cell transplants (37) and may be non-existent for the first 6 months (38). The model of GVHD used in the studies presented here, involving sublethally irradiated SCID hosts and donor T cells from peripheral lymph nodes, may be more representative of BMT in adults where T cell reconstitution is dominated by adoptively transferred peripheral T cells (39).
The long-term survival of tolerant donor T cells described in this paper may depend on the absence of thymic development in SCID mice. Anergic autoreactive transgenic B cells persist in the absence of B cells with a normal Ig repertoire (40) but, in their presence, do not compete successfully for access to survival niches in the germinal centers of secondary lymphoid organs. A similar competition for T cell survival niches between functional and tolerant donor T cells, probably in the parafollicular areas of secondary lymphoid organs, may `dilute out' the latter and account for their disappearance in other models of GVHD. Indeed, Tanchot and Rocha (41,42) have recently shown that virgin and tolerant CD8+ TCR transgenic T cells persist for prolonged times in the absence of thymic output and that thymic emigrants cause the deletion of tolerant, but not memory, cells.
Note that syngeneic T cells appeared to expand more than allogeneic cells (Fig. 2) after adoptive transfer into sublethally irradiated SCID mice. This somewhat paradoxical finding may be related to different TCR affinities for host antigens. Contact with host antigens may be required for the prolonged survival of donor cells as has been recently reported for endogenous peripheral T cells (43,44). All BALB/c T cells that were positively selected in the thymus would have weak binding affinities for host H-2d molecules (45). The antigen reactivity of the initially injected allogeneic cells may be considered in three groups: (i) superantigen reactive cells [Vß3 and Vß5 cells reactive against mtv-6 (~5% of the TCR repertoire)] (46), (ii) alloreactive cells (12% of the TCR repertoire) (47) and (iii) the remaining cells which, although not host reactive, must have been positively selected on H-2b molecules. The latter cells should not persist in host mice if antigen is required as a survival signal (48). This population might have been expected to survive in F1 SCID mice where the selecting molecules are present on host hematopoietic cells. However, the expansion of donor T cells was no higher than in fully allogeneic SCID mice (Fig. 3
, open squares), possibly suggesting their suppression by the alloreactive cells. Based on their increased CD44 and down-regulated TCR expression, possibly representing recent contact with antigen (24,25), the remaining T cells may represent alloreactive cells. The lower plateau level reached by proliferating allogeneic T cells may be related to their exhaustive differentiation (29) in response to alloantigens. In contrast, the affinity of survival signals for syngeneic cells may be low enough that they can expand without being driven to exhaustion.
We speculate that the behavior of donor T cells is independent of their initial number and that chronic activation causes them to enter a globally unresponsive state. These results are consistent with the `geographical' view of immune responses espoused by Zinkernagel and co-workers (49). Because host antigens are `everywhere' in lymphoid organs in GVHD, the fate of host reactive cells is to become `exhausted'. However, before exhaustion they may cause significant immunopathology. In this case, immunopathology is GVHD caused by the resulting `cytokine storm' (1). Low doses of T cells also become exhausted but do not cause clinically significant immunopathology simply because the `cytokine storm' is not large enough. Our ability to identify them in sublethally irradiated SCID hosts suggests that `exhausted' cells are small, their surface phenotype is CD44hi, TCRlo, and they are unresponsive to exogenous IL-2 and activation in vitro (Fig. 8 and Table 1
). Previous observations regarding the difficulty of adoptively transferring GVHD (50) plus our own failure to cause GVHD in secondary sublethally irradiated SCID hosts by transferring donor T cells from mice at least 10 days after initiation of lethal GVHD (unpublished observations) are compatible with this view.
`Exhausted' cells may simply be senescent (51). Replicative senescence is a property of the Hayflick limit that was discovered >30 years ago from studies on human diploid fibroblasts (52). Concepts of replicative senescence have not been prominently applied to T cell biology and GVHD (53). Features of the `exhausted' cells consistent with senescence include their restriction to the G1 phase of the cell cycle, failure to proliferate in response to multiple mitogens, decreased TCR density (54) and failure to die in cell culture (51). Mathematical models (55) suggest that a Hayflick limit for reactive T cells is probably not important for an immune response to extracellular pathogens, which can be cleared long before it is reached and where senescent cells can be replaced by thymic emigrants. However, a Hayflick limit may be extremely important for GVHD where thymic reconstitution is limited and the response to host antigens is prolonged. Here, mathematical models predict that the immune response will `exhaust' as antigen reactive T cells become senescent (55). Our findings in the SCID model support these theoretical results.
Current approaches to the clinical management of GVHD (56) may actually interfere with disease resolution if the natural history of mature alloreactive T cells is to enter a senescent-like state. GVHD is prevented and treated with drugs that block TCR signal transduction (cyclosporin A) or T cell proliferation (methotrexate) which are toxic, contribute to the morbidity of BMT and may even prevent the induction of the suppressed state (57). If alloreactive T cells eventually become unresponsive on their own, then therapies may be better directed to blocking the effects of their cytokine products, assuming that such cytokine blockade will not interfere with senescence. The development of replicative senescence of chronically activated T cells in a setting of limited thymic replacement is a form of tolerance that is also relevant to understanding the immunobiology of chronic viral infections and even cancer in adult patients.
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Acknowledgments |
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Abbreviations |
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BMT | bone marrow transplantation |
C57BL/6J | B6 |
CM | complete medium |
GVHD | graft versus host disease |
HD | high dose |
HSA | heat-stable antigen |
LD | low dose |
LDC | low-dose chimera |
mtv | mammary tumor virus |
PE | phycoerythrin |
PLNC | peripheral lymph node cells |
PMA | phorbol myristate acetate |
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Notes |
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Received 20 August 1998, accepted 3 June 1999.
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References |
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