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International Immunology, Vol. 11, No. 10, 1601-1614, October 1999
© 1999 Japanese Society for Immunology

Long-term persistence of IL-2-unresponsive allogeneic T cells in sublethally irradiated SCID mice

David Spaner1,2, Xiaofang Sheng-Tanner1, Kaliannan Raju2, Brian Rabinovich2, Hans Messner2 and Richard G. Miller2

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


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Donor T cells that are activated by host alloantigens initiate graft versus host disease (GVHD) but their long-term fate is poorly understood. The behavior of alloreactive donor T cells was studied in sublethally irradiated SCID mice. Intravenous injection of 106 allogeneic lymphocytes caused a severe form of GVHD, characterized by host hematopoietic atrophy. Fifty-fold fewer donor cells did not induce disease and were not simply rejected by radioresistant host mechanisms. Instead, low numbers of allogeneic T cells expanded 20- to 50-fold and remained for >1 year without causing evidence of GVHD. Persistent non-cycling donor cells with an activated phenotype were mainly found in the spleen. Tolerance was inferred by the recovery of host hematopoiesis, despite the presence of donor allogeneic T cells, and the inability of long-term persisting donor T cells to mediate cellular cytotoxicity or proliferate in response to exogenous IL-2 or antigenic stimulation in vitro. The TCR density of long-term persisting donor T cells was down-regulated. These findings suggest that the development of GVHD depends on the magnitude of the initial anti-host response. Subsequently donor cells differentiate, over several months, into a senescent-like state. This behavior questions the rationale for current treatment approaches to GVHD and is of relevance to any clinical situation where chronic T cell activation takes place in the absence of thymic development.

Keywords: graft versus host disease, immunosenescence, in vivoanimal model, superantigens, tolerance/suppression


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Mature donor T cells initiate clinical graft versus host disease (GVHD) after allogeneic stem cell transplantation (BMT) (1). T cell-containing donor leukocyte infusions are increasingly used to provoke GVHD and treat relapsed malignancies after BMT through a poorly characterized graft versus leukemia effect (2). In both these clinical situations, the long-term fate of donor T cells is poorly understood, possibly because it has been difficult to separate them from the T cells that develop from donor hematopoietic stem cells. An important question is: do chronically activated mature host-reactive donor T cells sustain GVHD or an anti-tumor effect, or is their effect only temporary?

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.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
C57BL/6J (B6) (H-2b, Thy-1.2), B6.PL-Thy-1a/Cy (H-2b, Thy-1.1), BALB/c (H-2d) and C3H/HeJ (H-2k) mice were obtained from the Jackson Laboratories (Bar Harbor, ME). BALB.K (H-2k), B6-SCID (H-2b, Thy-1.2), (B6xC.B-17) F1 SCID (H-2b,d) and C.B-17-SCID (H-2d) mice were bred and maintained in the defined flora animal colony at the Ontario Cancer Institute (Toronto, Ontario, Canada). C.B-17 is congenic to BALB/c, differing only in the region of the Ig heavy chain allele. BALB/c are IgHa and C.B-17 are IgHb (9). Breeding pairs of B6-SCID mice were initially provided by L. Shultz of the Jackson Laboratories.

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 streptavidin–PE were purchased from Sigma (St Louis, MO). The anti-Fc{gamma}RIII-{alpha} 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-{gamma}, IL-2, CD3 and CD44, and biotinylated antibodies against Vß3 and CD25 were purchased from PharMingen (San Francisco, CA). Anti-CD4– and anti-CD8–TriColor 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) ({alpha}-MEM, 10% FCS, 5x10–5 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 ~6–10 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 4–12 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 {gamma}-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 {alpha}-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 {gamma}-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 CD8–TriColor, and IL-4–PE (0.2 µg) and IFN-{gamma}–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 {gamma} 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.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Small numbers of donor T cells do not cause acute GVHD in sublethally irradiated SCID mice
Injection of 106 B6 PLNC into sublethally irradiated SCID mice caused an aggressive form of GVHD with a mean survival time of ~15 days (6) (Fig. 1Go). Injection of 3x104 or 3x103 B6 LNC did not cause noticeable GVHD (Fig. 1Go). More than 100 mice have been injected with low numbers of donor cells and have remained healthy for up to 1 year post-injection.



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  		Fig. 1. The severity of GVHD is directly proportional to the number of injected donor cells. C.B-17-SCID mice were sublethally irradiated with 275 cGy and immediately injected with 106 (open circles) (n = 19) or 3x104 (closed circles) (n = 24) PLNC from B6 mice. Cages were inspected every 1–2 days and animals were sacrificed when they were moribund or had lost 30% of their starting body weight. Results were independent of the sex of donor or recipient mice.

 
Characteristic pathological liver changes of GVHD (17) could be found after 3 months in sublethally irradiated SCID mice injected with B6 spleen cells (Fig. 2aGo). These lesions were not found even at 4 months post-injection of low numbers of PLNC (Fig. 2bGo). Note that SCID mice injected with high numbers of PLNC could not survive for 3 months.



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  		Fig. 2. Absence of histological changes of GVHD in mice injected with low numbers of allogeneic PLNC. Liver sections were prepared and stained with hematoxylin & eosin. (A) The liver of a sublethally irradiated SCID mouse injected 3 months earlier with 106 B6 spleen cells showing lymphocytic periportal infiltrates and bile ducts demonstrating apoptosis and drop-out of duct cells. Piece-meal necrosis and fatty infiltration are found in the surrounding liver parenchyma. (B) From a mouse injected with 3x104 PLNC after sublethal irradiation 125 days earlier, the portal areas are intact with scant cellular infiltrates.

 
Low numbers of allogeneic T cells persist for long periods in sublethally irradiated SCID mice
A trivial explanation for the absence of GVHD after injection of low numbers of T cells might be clearance of donor cells by radioresistant host NK cells that recognize the complete MHC mismatch between B6 donor cells and C.B-17-SCID mice (18). Injection of graded numbers of B6 PLNC into C.B-17-SCID mice not given irradiation confirmed that 5x105 donor cells were rejected within the first few days (Fig. 3Go).



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  		Fig. 3. Expansion and long-term persistence of allogeneic T cells in irradiated SCID mice. PLNC from B6 or BALB/c (congenic with C.B-17 mice and differing at the Ig heavy chain allotype locus) donors were injected into sublethally irradiated (four groups) or unirradiated (1 group) SCID mice. At serial times thereafter, host spleen cell suspensions were stained with antibodies against Thy-1.2 and H-2b, and the percentage of H2b+ Thy-1.2+ cells was determined using flow cytometry. The number of donor cells was then calculated by multiplying this number by the total number of splenocytes. The number of cells in mice initially injected with 106 B6 PLNC could not be determined beyond 15 days because of the state of the animals. Approximately 70% of PLNC are T cells and the number of cells in the spleens on day 0 was assumed to be 20% of the injected dose (58). The average + SE of the results from at least 5 mice per data point are reported. Only two mice at 365 days were examined. The initial ratio of CD4+ to CD8+ T cells, measured by flow cytometry and the appropriate antibodies, was ~1.5:1 for B6 or BALB/c mice while the CD4/CD8 ratio at >100 days was 0.56 ± 0.03 (n = 4) for LDC mice and 3.8 ± 1.2 (n = 2) for host mice injected with syngeneic PLNC. Key: {circ}, 106 B6 LNC into irradiated C.B-17-SCID hosts; •, 3x104 B6 LNC into irradiated C.B-17-SCID hosts; {square}, 3x104 B6 LNC into irradiated F1 SCID hosts; {blacksquare}, 3x104 BALB/c LNC into irradiated C.B-17-SCID hosts; {triangleup}, 5x105 B6 LNC into unirradiated C.B-17-SCID hosts.

 
The number of donor cells in the spleens of sublethally irradiated hosts was determined in order to document their survival. As shown in Fig. 3Go, 3x104 t cells reached a plateau level of ~2x105 donor cells per spleen by the second week and remained at this level for up to 1 year.

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 5–7. 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 2–3 weeks due to the morbidity of the injected animals.

In summary, Fig. 3Go 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. 3Go). 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. 3Go). Moreover the ratio of CD4+ to CD8+ cells was skewed towards CD4, whereas it was inverted for B6 cells (legend to Fig. 3Go).

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. 4Go). 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. 4Go).



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  		Fig. 4. Host hematopoietic recovery following sublethal irradiation and injection of alloreactive cells. The number of host cells in the spleens of C.B-17-SCID mice initially injected with 106 ({circ}), 3x104 ({square}) or 0 (•) B6 PLNC was determined by subtracting the number of donor cells (calculated as described in Fig. 3Go) from the total number of splenocytes. The host origin of these cells was confirmed by positive staining with an antibody directed against H-2d (not shown).

 
After injection of 106 B6 PLNC, no recovery of host hematopoietic cells was observed. Instead, their numbers in spleen fell <105 by the second week, consistent with the interpretation that hematopoietic atrophy was the terminal event.

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 4GoGo 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 4GoGo 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 (10–20%) 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 1Go. Cells from long-term LDC could not respond to host or third-party antigens (Table 1Go, rows 7 and 16; column 4) and this lack of response could not be rescued by IL-2 (Table 1Go, 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. 5Go). 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. 5Go and Table 1Go, rows 9–12 and 17, column 4). In contrast to cells from long-term LDC, the response to exogenous IL-2 was strong (Fig. 5Go and Table 1Go, rows 3–4, column 4). Syngeneic donor T cells studied early after injection into irradiated SCID mice did not undergo growth arrest upon reactivation in vitro (Fig. 5Go and Table 1Go, row 13–14, column 4).


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  		Table 1. Functional properties of donor T cells in LDC
 



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  		Fig. 5. Proliferative unresponsiveness of T cells from long-term LDC cannot be restored by activation with anti-CD3 or anti-CD28 antibodies. (Top panel) The equivalent numbers of T cells (2x104) from the spleens of normal B6 donors (1), sublethally irradiated SCID mice injected with 106 allogeneic (B6) (2) or syngeneic (BALB/c) T cells (4) 6 days earlier, or LDC injected at least 100 days earlier with 3x104 PLNC (3) were plated on microtiter plates coated with FCS, anti-CD3 or anti-CD3 and anti-CD28 antibodies in the presence of 50 U/ml IL-2, as described in Methods. The proliferative response, determined by [3H]thymidine uptake, is reported as the average + SE of four replicate cultures. The reported results are representative of at least six separate experiments, except for the syngeneic transfer which was repeated twice. (Lower panel) The equivalent T cell numbers (2x104) from the spleens of normal B6-Thy-1.1 donors and sublethally irradiated B6-SCID mice injected 21 days earlier with 106 B6-Thy-1.1 PLNC and also from BALB/c donors or sublethally irradiated C.B-17-SCID mice injected 20 days earlier with 3x104 BALB/c PLNC were stimulated with mitogens in vitro. The average + SE of three replicate cultures is reported and is representative of two separately injected host mice.

 
Cytotoxicity of in vivo activated T cells was examined by redirected lysis assays against P815 tumor targets. T cells from an inoculum of high numbers of donor cells were strongly cytolytic within the first month after injection (Table 1Go, row 4, column 6). However, T cells from long-term LDC could not kill (Table 1Go, row 2, column 6).

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 1Go, rows 18–19). 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-{gamma}
Intracellular cytokine production was measured using flow cytometry. Figure 6Go shows that donor T cells taken from LDC did not directly produce IFN-{gamma} ex-vivo but could produce some IFN-{gamma} after a 4 h stimulation with PMA and ionomycin. In comparison, T cells actively mediating GVHD directly produced IFN-{gamma} ex-vivo and the level of production was significantly enhanced by stimulation (Fig. 6Go). 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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  		Fig. 6. Donor cells in LDC can produce limited amounts of IFN-{gamma} after mitogenic stimulation. The capability of donor T cells from long-term LDC (b) compared with donor T cells from mice 8 days after initiation of acute GVHD (a) to produce IFN-{gamma} and IL-4 was determined by intracellular cytokine staining and flow cytometric analysis. Cells were stained after a 4 h incubation in the presence of Brefeldin A with and without PMA and ionomycin.

 
IL-4 production by mitogen-activated long-term LDC spleen cultures could not be detected in a bioassay and was not measurable in cultures of mitogen activated naive T cells (data not shown). IL-4 production was also not demonstrable using intracellular cytokine staining of T cells from long-term LDC or from mice actively undergoing GVHD (Fig. 6Go). Taken together with their potential ability to produce IFN-{gamma} (Fig. 6Go), this finding suggests that donor cells have not differentiated into Tc2 or Th2 cells (21).

Donor cells reside in multiple organs
Donor cells could be found in multiple organs of host mice as shown in Fig. 7Go. 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. 7Go).




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  		Fig. 7. Non-cycling donor T cells are found at multiple sites in LDC. Mice were exsanguinated and cells from the (a) spleen, (b) thymus, (c) pooled inguinal and mesenteric lymph nodes, (d) peritoneal washings, and (e) bone marrow were harvested, stained with anti-Thy-1.2–PE and H-2b–FITC antibodies, and analyzed by flow cytometry. Spleen cells from a mouse that was only sublethally irradiated were analyzed in the same way (f) as a negative control. Shown are the results from an LDC 77 days post-initial injection but are representative of animals up to 160 days. The total cellularity at each site for this example was: spleen (7x106), bone marrow (1.8x107), thymus (5.3x105), peritoneum (2.5x105) and pooled lymph nodes (4x104). DNA content histograms from B6 control animals (g) and a day 162 LDC (i), gated on H-2b+ spleen cells that were CD4+ or CD8+, show that donor T cells in the LDC were mainly in the G1/G0 phase of the cell cycle. A significantly higher number were in cycle 6 days after induction of GVHD with high doses of donor T cells (h).

 
PLNC were used as the source of donor T cells to avoid potential reconstitution by donor hematopoietic stem cells (8). Evidence that the long-term persisting T cells derived from the initial inoculum of mature T cells is shown in Fig. 7Go. The only H-2b+ cells were also Thy-1.2+. If donor hematopoietic reconstitution had occurred, Thy-1.2–H-2b+ cells, such as B cells, should have developed. Note that a population of Thy-1.2+H-2b– cells is seen in Fig. 7Go. This population may represent NK or T cells that develop from `leaky' SCID stem cells after recovery from sublethal irradiation (22,23) but was an inconsistent finding.

The cell cycle status of donor cells was determined by flow cytometric analysis of propidium iodine staining of DNA. Figure 7Go(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. 7hGo).

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. 8Go). 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. 8Go). 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. 8Go) although, shortly after the injection of high numbers of donor T cells, the majority were significantly larger (mean = 147) (Fig. 8Go, 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 10bGo). 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. 8Go).



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  		Fig. 8. Activation markers on persisting donor T cells in LDC. Spleen cells from a B6 control (left column) and LDC (center column) mouse were harvested and, after gating on H-2b+ cells that were CD4+ or CD8+, subjected to flow cytometric analysis of the following activation markers: (a) forward scatter (cell size), (b) CD44, (c) IL-2R{alpha} (CD25), (d) TCR density and (e) isotype controls. The results shown are from a day 360 mouse but are representative of mice at least 6 weeks post-injection of allogeneic cells. For comparison, the same activation markers on HD T cells 6 days after initiation of GVHD is shown (right column).

 
Rechallenge of LDC with naive, congenic B6 T cells
A possible explanation for the behavior of donor cells in LDC was that the host environment could no longer support the proliferation and differentiation of alloreactive cells. Down-regulation of immunogenic antigen and co-stimulatory signals required to support an alloresponse or the presence of a suppressor cell population, despite evidence that donor T cells in LDC were not suppressors in vitro (Table 1Go, rows 18–19), could possibly account for this altered host environment. To evaluate these possibilities, LDC were challenged with a second injection of PLNC from B6-Thy-1.1 mice. The allelic difference at the Thy-1 locus allowed the rechallenge cells to be distinguished from the pre-existing tolerant donor cells. As shown in Fig. 9Go(a), B6-Thy1.1 PLNC increased in number and percentage compared with pre-existing Thy-1.2+ cells in the spleens of LDC, arguing that the tolerant state of the latter was not caused by suppressor cells or insufficient immunogenic host antigen. Further evidence that Thy-1.1+ T cells were responding to host antigens was that some of the rechallenged mice became moribund within 1 week, likely due to GVHD. Moreover, the number of Thy-1.1+ T cells increased to the same level as a primary challenge of sublethally irradiated SCID mice with >105 B6 T cells (Fig. 3Go). Note that the Thy-1.2+ CD3+ cells in Fig. 9Go(b) represent the donor cells remaining from the initial injection which clearly have down-regulated TCR densities compared to the responding cells from the second injection (see also Fig. 8Go).



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  		Fig. 9. Naive allogeneic donor T cells are activated normally in LDC. LDC, at various times after their initial injection with B6-Thy-1.2 PLNC, were given a second i.v. injection of 105 PLNC from B6-Thy-1.1 mice without any other preparation (a). One week later, the percentage and total numbers in the spleen of the original donor cells (H-2b+Thy-1.2+) and the secondarily injected cells (H-2b+Thy-1.1+) were determined. The dot-plot shown is representative of results from LDC >42 days after the initial injection. The numbers in the right upper and lower quadrants refer to the percentages of Thy-1.2+ and Thy-1.1+ cells in the spleen cell preparation. The Thy-1.1+ cells increased to 5.2 ± 4.3x106 (n = 2) 1 week after rechallenge (b). By staining for CD3 and Thy-1.2, the decreased TCR density on tolerant cells (CD3+Thy-1.2+) compared to cells mediating GVHD (CD3+Thy1.2–) is demonstrated in another mouse (b).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In this paper, low numbers of allogeneic B6 T cells (low dose or LD) were shown to increase ~50-fold in number after adoptive transfer into sublethally irradiated C.B-17-SCID mice and persist for up to a year without causing GVHD. In contrast, an initial injection of 50-fold higher numbers of donor T cells (high dose or HD) caused a severe form of GVHD within 2 weeks.

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. 3Go). 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. 3Go). 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. 7Go).

Superficially, the apparent expansion of initially high numbers of donor T cells (Fig. 3Go), 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 (28–30). 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-{gamma} directly ex vivo (Fig. 6Go and unpublished results), and HD T cells could mediate perforin-dependent cellular cytotoxicity (Table 1Go, row 4, column 6). Also within the first month, HD and LD T cells proliferated strongly in response to exogenous IL-2 (Table 1Go) but were growth inhibited after reactivation through the TCR in vitro (Table 1Go and Fig. 5Go). 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-{alpha} and IFN- {gamma} (7,32,33).

Subsequently, LD T cells entered a long-lived non-functional state, characterized by small size (Fig. 8Go), unresponsiveness to exogenous IL-2 or allogeneic stimuli (Table 1Go), lack of direct ex-vivo secretion of IFN- {gamma} (Fig. 6Go), or killing in a redirected lysis assay (Table 1Go). 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 1Go), 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. 8Go), 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. 3Go).

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- {gamma} production and IL-2 R expression, and was associated with increased IL-10 production. In contrast, TCR down regulation (Fig. 8Go), 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. 2Go) 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 (1–2% 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. 3Go, 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. 8Go and Table 1Go). 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.


   Acknowledgments
 
This work was supported by grants from the Medical Research Council of Canada (to R. G. M.). D. S. was supported by a special fellowship from the National Cancer Institute of Canada with funds provided by Sandoz. We thank H. Karasuyama for the gift of cytokine cDNA transfected cells and Dr J. Allison for providing the 37.51 anti-CD28 hybridoma.


   Abbreviations
 
BMTbone marrow transplantation
C57BL/6JB6
CMcomplete medium
GVHDgraft versus host disease
HDhigh dose
HSAheat-stable antigen
LDlow dose
LDClow-dose chimera
mtvmammary tumor virus
PEphycoerythrin
PLNCperipheral lymph node cells
PMAphorbol myristate acetate

   Notes
 
Transmitting editor: R. H. Schwartz

Received 20 August 1998, accepted 3 June 1999.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Ferrara, J. L., Cooke, K. R., Pan, L. and Krenger, W. 1996. The immunopathophysiology of acute graft-versus-host-disease. Stem Cells 14:473.[Web of Science][Medline]
  2. Kolb, H. J. and Holler, E. 1997. Adoptive immunotherapy with donor lymphocyte transfusions. Curr. Opin. Oncol. 9:139.[Medline]
  3. Mackinnon, S., Papadopoulos, E. B., Carabasi, M. H., Reich, L., Collins, N. H., Boulad, F., Castro-Malaspina, H., Childs, B. H., Gillio, A. P. and Kernan, N. A. 1995. Adoptive immunotherapy evaluating escalating doses of donor leukocytes for relapse of chronic myeloid leukemia after bone marrow transplantation: separation of graft-versus-leukemia responses from graft-versus-host disease. Blood 86:1261.[Abstract/Free Full Text]
  4. Rocha, B., Freitas, A. A. and Coutinho, A. A. 1983. Population dynamics of T lymphocytes. Renewal rate and expansion in the peripheral lymphoid organs. J. Immunol. 131:2158.[Abstract]
  5. Miller, R. A. and Stutman, O. 1984. T cell repopulation from functionally restricted splenic progenitors: 10,000-fold expansion documented by using limiting dilution analyses. J. Immunol. 133:2925.[Abstract]
  6. Phillips, R. A. and Spaner, D. E. 1991. The scid mouse: mutation in a DNA repair gene creates recipients useful for studies on stem cells, lymphocyte development and graft-versus-host disease. Immunol. Rev. 124:63.[Web of Science][Medline]
  7. Spaner, D., Raju, K., Rabinovich, B. and Miller, R. G. 1999. A role for perforin in activation induced T cell death in vivo: increased expansion of alloreactive T cells in SCID mice in the absence of perforin. J. Immunol. 162:1192.[Abstract/Free Full Text]
  8. Sprent, J., Schaefer, M., Hurd, M., Surh, C. D. and Ron, Y. 1991. Mature murine B and T cells transferred to SCID mice can survive indefinitely and many maintain a virgin phenotype. J. Exp. Med. 174:717.[Abstract/Free Full Text]
  9. Bosma, M. J. and Carroll, A. M. 1991. The SCID mouse mutant: definition, characterization, and potential uses. Annu. Rev. Immunol. 9:323.[Web of Science][Medline]
  10. Leo, O., Foo, M., Sachs, D. H., Samelson, L. E. and Bluestone, J. A. 1987. Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc. Natl Acad. Sci. USA 84:1374.[Abstract/Free Full Text]
  11. Unkeless, J. C. 1979. Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors. J. Exp. Med. 150:580.[Abstract/Free Full Text]
  12. Gross, J. A., Callas, E. and Allison, J. P. 1992. Identification and distribution of the costimulatory receptor CD28 in the mouse. J. Immunol. 149:380.[Abstract]
  13. Karasuyama, H., Tohyama, N. and Tada, T. 1989. Autocrine growth and tumorigenicity of interleukin 2-dependent helper T cells transfected with IL-2 gene. J. Exp. Med. 169:13.[Abstract/Free Full Text]
  14. Darzynkiewicz, Z., Bruno, S., Del Bino, G., Gorczyca, W., Hotz, M. A., Lassota, P. and Traganos, F. 1992. Features of apoptotic cells measured by flow cytometry. Cytometry 13:795.[Web of Science][Medline]
  15. Radvanyi, L. G., Shi, Y., Vaziri, H., Sharma, A., Dhala, R., Mills, G. B. and Miller, R. G. 1996. CD28 costimulation inhibits TCR-induced apoptosis during a primary T cell response. J. Immunol. 156:1788.[Abstract]
  16. Ferrick, D. A., Schrenzel, M. D., Mulvania, T., Hsieh, B., Ferlin, W. G. and Lepper, H. 1995. Differential production of interferon-gamma and interleukin-4 in response to Th1- and Th2-stimulating pathogens by gamma delta T cells in vivo. Nature 373:255.[Medline]
  17. Crawford, J. M. 1997. Graft-versus-host disease of the liver. In Ferrara, J. L. M., Deeg, H. J. and Burakoff, S. J., eds, Graft-vs.-Host Disease, p. 315. Marcel Dekker, New York.
  18. Murphy, W. J., Kumar, V. and Bennett, M. 1987. Rejection of bone marrow allografts by mice with severe combined immune deficiency (SCID). Evidence that natural killer cells can mediate the specificity of marrow graft rejection. J. Exp. Med. 165:1212.[Abstract/Free Full Text]
  19. Fulop, G. M. and Phillips, R. A. 1990. The scid mutation in mice causes a general defect in DNA repair. Nature 347:479.[Medline]
  20. Thiel, A., Schmitz, J., Miltenyi, S. and Radbruch, A. 1997. CD45RA-expressing memory/effector Th cells committed to production of interferon-gamma lack expression of CD31. Immunol. Lett. 57:189.[Web of Science][Medline]
  21. Sad, S., Marcotte, R. and Mosmann, T. R. 1995. Cytokine-induced differentiation of precursor mouse CD8+ T cells into cytotoxic CD8+ T cells secreting Th1 or Th2 cytokines. Immunity. 2:271.[Web of Science][Medline]
  22. Danska, J. S., Pflumio, F., Williams, C. J., Huner, O., Dick, J. E. and Guidos, C. J. 1994. Rescue of T cell-specific V(D)J recombination in SCID mice by DNA-damaging agents. Science 266:450.[Abstract/Free Full Text]
  23. Murphy, W. J., Durum, S. K., Anver, M. R., Ferris, D. K., McVicar, D. W., O'Shea, J. J., Ruscetti, S. K., Smith, M. R., Young, H. A. and Longo, D. L. 1994. Induction of T cell differentiation and lymphomagenesis in the thymus of mice with severe combined immune deficiency (SCID). J. Immunol. 153:1004.[Abstract]
  24. Beverley, P. C. 1996. Generation of T cell memory. Curr. Opin. Immunol. 8:327.[Web of Science][Medline]
  25. Minami, Y., Samelson, L. E. and Klausner, R. D. 1987. Internalization and cycling of the T cell antigen receptor. Role of protein kinase C. J. Biol. Chem. 262:13342.[Abstract/Free Full Text]
  26. Barry, T. S., Jones, D. M., Richter, C. B. and Haynes, B. F. 1991. Successful engraftment of human postnatal thymus in severe combined immune deficient (SCID) mice: differential engraftment of thymic components with irradiation versus anti-asialo GM-1 immunosuppressive regimens. J. Exp. Med. 173:167.[Abstract/Free Full Text]
  27. Kung, S. K. and Miller, R. G. 1997. Mouse natural killer subsets defined by their target specificity and their ability to be separately rendered unresponsive in vivo. J. Immunol. 158:2616.[Abstract]
  28. Kawabe, Y. and Ochi, A. 1991. Programmed cell death and extrathymic reduction of Vbeta8+ CD4+ T cells in mice tolerant to Staphylococcus aureus enterotoxin B. Nature 349:245.[Medline]
  29. Moskophidis, D., Lechner, F., Pircher, H. and Zinkernagel, R. M. 1993. Virus persistence in acutely infected immunocompetent mice by exhaustion of antiviral cytotoxic effector T cells. Nature 362:758.[Medline]
  30. Rocha, B., Grandien, A. and Freitas, A. A. 1995. Anergy and exhaustion are independent mechanisms of peripheral T cell tolerance. J. Exp. Med. 181:993.[Abstract/Free Full Text]
  31. Webb, S., Morris, C. and Sprent, J. 1990. Extrathymic tolerance of mature T cells: clonal elimination as a consequence of immunity. Cell 63:1249.[Web of Science][Medline]
  32. Spaner, D., Raju, K., Radvanyi, L., Lin, Y. P. and Miller, R. G. 1998. A role for perforin in activation induced cell death. J. Immunol. 160:2655.[Abstract/Free Full Text]
  33. Krenger, W., Falzarano, G., Delmonte, J., Snyder, K. M., Byon, J. C. and Ferrara, J. L. 1996. Interferon-gamma suppresses T cell proliferation to mitogen via the nitric oxide pathway during experimental acute graft-versus-host disease. Blood 88:1113.[Abstract/Free Full Text]
  34. Lanoue, A., Bona, C., von Boehmer, H. and Sarukhan, A. 1997. Conditions that induce tolerance in mature CD4+ T cells. J. Exp. Med. 185:405.[Abstract/Free Full Text]
  35. Tanchot, C., Guillaume, S., Delon, J., Bourgeois, C., Franzke, A., Sarukhan, A., Trautmann, A. and Rocha, B. 1998. Modifications of CD8+ T cell function during in vivo memory or tolerance induction. Immunity. 8:581.[Web of Science][Medline]
  36. Hollander, G. A., Widmer, B. and Burakoff, S. J. 1994. Loss of normal thymic repertoire selection and persistence of autoreactive T cells in graft vs host disease. J. Immunol. 152:1609.[Abstract]
  37. Mackall, C. L. and Gress, R. E. 1997. Pathways of T cell regeneration in mice and humans: implications for bone marrow transplantation and immunotherapy. Immunol. Rev. 157:61.[Web of Science][Medline]
  38. Dilly, S. A., Sloane, J. P. and Psalti, I. S. 1986. The cellular composition of human lymph nodes after allogenic bone marrow transplantation: an immunohistological study. J. Pathol. 150:213.[Web of Science][Medline]
  39. Roux, E., Helg, C., Dumont-Girard, F., Chapuis, B., Jeannet, M. and Roosnek, E. 1996. Analysis of T cell repopulation after allogeneic bone marrow transplantation: significant differences between recipients of T cell depleted and unmanipulated grafts. Blood 87:3984.[Abstract/Free Full Text]
  40. Cyster, J. G., Hartley, S. B. and Goodnow, C. C. 1994. Competition for follicular niches excludes self-reactive cells from the recirculating B-cell repertoire. Nature 371:389.[Medline]
  41. Tanchot, C. and Rocha, B. 1998. The organization of mature T cell pools. Immunol. Today 19:575.[Web of Science][Medline]
  42. Tanchot, C. and Rocha, B. 1997. Peripheral selection of T cell repertoires: the role of continuous thymus output. J. Exp. Med. 186:1099.[Abstract/Free Full Text]
  43. Tanchot, C., Lemonnier, F. A., Perarnau, B., Freitas, A. A. and Rocha, B. 1997. Differential requirements for survival and proliferation of CD8 naive or memory T cells. Science 276:2057.[Abstract/Free Full Text]
  44. Takeda, S., Rodewald, H. R., Arakawa, H., Bluethmann, H. and Shimizu, T. 1996. MHC class II molecules are not required for survival of newly generated CD4+ T cells, but affect their long-term life span. Immunity. 5:217.[Web of Science][Medline]
  45. von Boehmer, H. 1994. Positive selection of lymphocytes. Cell 76:219.[Web of Science][Medline]
  46. Scherer, M. T., Ignatowicz, L., Winslow, G. M., Kappler, J. W. and Marrack, P. 1993. Superantigens: bacterial and viral proteins that manipulate the immune system. Annu. Rev. Cell Biol. 9:101.[Web of Science]
  47. Matzinger, P. and Bevan, M. J. 1977. Hypothesis: why do so many lymphocytes respond to major histocompatibility antigens? Cell. Immunol. 29:1.[Web of Science][Medline]
  48. Neuberger, M. S. 1997. Antigen receptor signaling gives lymphocytes a long life. Cell 90:971.[Web of Science][Medline]
  49. Zinkernagel, R. M., Ehl, S., Aichele, P., Oehen, S., Kundig, T. and Hengartner, H. 1997. Antigen localisation regulates immune responses in a dose- and time-dependent fashion: a geographical view of immune reactivity. Immunol. Rev. 156:199.[Web of Science][Medline]
  50. Grebe, S. C. and Streilein, J. W. 1976. Graft-versus-Host reactions: a review. Adv. Immunol. 22:119.[Medline]
  51. Smith, J. R. and Pereira-Smith, O. M. 1996. Replicative senescence: implications for in vivo aging and tumor suppression. Science 273:63.[Abstract]
  52. Hayflick, L. 1965. The limited in vitro lifetime of human diploid cell strains. Exp. Cell. Res. 37:614.[Web of Science][Medline]
  53. Effros, R. B. 1998. Replicative senescence in the immune system: impact of the Hayflick limit on T cell function in the elderly. Am. J. Hum. Genet. 62:1003.[Web of Science][Medline]
  54. Wakikawa, A., Utsuyama, M. and Hirokawa, K. 1997. Altered expression of various receptors on T cells in young and old mice after mitogenic stimulation: a flow cytometric analysis. Mech. Ageing Dev. 94:113.[Web of Science][Medline]
  55. Pilyugin, S., Mittler, J. and Antia, R. 1997. Modeling T cell proliferation: an investigation of the consequences of the Hayflick limit. J. Theor. Biol. 186:117.[Web of Science][Medline]
  56. Lazarus, H. M., Vogelsang, G. B. and Rowe, J. M. 1997. Prevention and treatment of acute graft-versus-host disease: the old and the new. A report from the Eastern Cooperative Oncology Group (ECOG). Bone Marrow Transplant. 19:577.[Web of Science][Medline]
  57. Hiruma, K. and Gress, R. E. 1992. Cyclosporine A and peripheral tolerance. Inhibition of veto cell-mediated clonal deletion of postthymic precursor cytotoxic T lymphocytes. J. Immunol. 149:1539.[Abstract]
  58. Rocha, B. and von Boehmer, H. 1991. Peripheral selection of the T cell repertoire. Science 251:1225.[Abstract/Free Full Text]

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D. Spaner, X. Sheng-Tanner, and A. C. Schuh
Aberrant regulation of superantigen responses during T-cell reconstitution and graft-versus-host disease in immunodeficient mice
Blood, August 28, 2002; 100(6): 2216 - 2224.
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