Acute graft-versus-host disease (GVHD) after allogeneic stem cell transplantation is associated with impaired deletion and anergy of host-reactive T cells. To elucidate the immunoregulatory events that may contribute to such dysregulated T-cell responses in GVHD, we studied superantigen (SAg) responses after adoptive T-cell transfer into severe combined immunodeficient (SCID) mice. SAg responses are normally regulated by mechanisms involving deletion and anergy, with SAg-reactive T cells typically being deleted rapidly in vivo. In a SCID mouse model of GVHD, however, allogeneic host SAg-reactive T cells were not deleted rapidly, but rather persisted in increased numbers for several months. Moreover, depending on the timing of SAg stimulation and the numbers of T cells transferred, dysregulation (impaired deletion and anergy) of SAg responses could be demonstrated following the adoptive transfer of syngeneic T cells into SCID mice as well. Transgenic T-cell receptor-bearing KJ1-26.1+ T cells were then used to determine the fate of weakly reactive T cells after adoptive transfer and SAg stimulation. When transferred alone, KJ1-26.1+ T cells demonstrated impaired deletion and anergy. In the presence of more strongly staphylococcal enterotoxin B (SEB)–reactive T cells, however, KJ1-26.1+ T cells were regulated normally, in a manner that could be prevented by inhibiting the effects of more strongly SEB-reactive cells or by increasing the level of activation of the KJ1-26.1+ T cells themselves. We suggest that the control mechanisms that normally regulate strongly activated T cells in immunocompetent animals are lost following adoptive transfer into immunodeficient hosts, and that this impairment contributes to the development of GVHD.

Graft-versus-host disease (GVHD) is the major complication of allogeneic stem cell transplantation1(allo-SCT) and prevents this potentially curative treatment2 from being offered to a wider range of patients.3,4 GVHD is caused by T cells in the stem cell graft that recognize the antigenic differences that result from genetic disparities between the donor and host.1 Such antigens are distributed systemically on host antigen-presenting cells (APCs), and their numbers likely exceed by far those of the transplanted donor T cells. In other situations in which T cells encounter antigens in such excess (eg, overwhelming viral infections), the resultant immune responses rapidly become exhausted.5Accordingly, one might also expect that the antihost immune responses that mediate GVHD should exhaust rather quickly as well. That they do not suggests that the rules that govern T-cell responses in the immunocompetent state may be distinct from those that regulate T-cell behavior following their transfer into lymphopenic hosts.

In this paper, we have used superantigen (SAg) responses as a model to study T-cell immunoregulation after adoptive transfer. Immunoregulation of T-cell responses to SAgs has been well characterized7,8 and comprises 2 major phenomena—deletion and anergy. The SAgs stimulate subsets of T cells that use a common gene to form their T-cell receptor (TCR). After several days of rapid proliferation, a proportion of SAg-activated CD4+ T cells becomes deleted by Fas-mediated apoptosis.9 By 10 days after initial stimulation, the percentage and absolute numbers of SAg-reactive cells have decreased to around 50% of starting values and remain at this level for several months, in the absence of new thymic emigrants.7,10 The remaining cells are anergic because they do not proliferate or secrete interleukin-2 (IL-2) in response to restimulation by the SAg or the cognate antigen,12,13 either in vitro8 or in vivo.11 Such anergy may be cell intrinsic or may be maintained by multiple extrinsic mechanisms involving regulatory or suppressor T14-17 or B cells.18-20 

In the studies described here, we have used a previously characterized model in which the transfer of major histocompatibility complex (MHC)–disparate T cells from C57BL/6J (B6; H-2b) donor mice into C.B-17-scid/scid mice (SCID; H-2d) recipients results in severe GVHD.21-23 Severe combined immunodeficient (SCID) mice cannot rearrange their B-cell receptor or TCR genes and are consequently lymphopenic, allowing host-reactive donor T cells to be tracked easily after adoptive transfer.24 Using this model, we have observed that the deletion and anergy of allogeneic T cells reactive to endogenous host SAgs are markedly impaired during GVHD and have demonstrated further that the regulation of T-cell responses to exogenous SAg following the adoptive transfer of syngeneic T cells is impaired as well.

Mice

C57BL/6J [B6] (H-2b,Mtv-6) and BALB/c (H-2d, IgHa, Mtv-6+) mice were from the Jackson Laboratories (Bar Harbor, ME). C.B-17 scid/scid[SCID] and C.B-17 OVA-SCID26 (H-2d, IgHb, Mtv-6+) mice were bred and maintained in the defined flora animal colony at the Ontario Cancer Institute (Toronto, ON, Canada). The latter bear a transgenic chicken ovalbumin-specific TCR and thus provide a source of monoclonal CD4+Vβ8+ T cells.26 

Antibodies, reagents, and cell lines

Phycoerythrin (PE)– and fluorescein isothiocyanate (FITC)–conjugated Vβ8β, Vβ6, CD3, CD4, and CD8 antibodies and biotinylated Vβ3 antibodies were purchased from Pharmingen (Mississauga, ON); staphylococcal enterotoxin B (SEB), 7-amino actinomycin D (7-AAD), streptavidin-PE, chicken ovalbumin, and complete Freund adjuvant (CFA) were purchased from Sigma Chemical (St Louis, MO). The hybridomas 2.4G2 (antimurine Fc RIII-α)27 and R4-6A2 (antimurine interferon γ [IFN-γ])28 were from the American Type Culture Collection (ATCC; Manassas, VA). Anticlonotypic antibodies from the KJ1-26.1 hybridoma12 (gift of Dr J. Kappler, Denver, CO) that recognize the OVA-SCID TCR29 (derived from the D0-11.10 helper T-cell clone30) were purified and labeled with FITC in our laboratory. Recombinant soluble human tumor necrosis factor receptor (rhuTNFR:Fc)31 and ovalbumin peptides (amino acids 323-339) were gifts of Dr M. Widmer (Immunex, Seattle, WA) and Dr J. Withers (Toronto, ON), respectively. Supernatants from mouse IL-2 cDNA transfected X63Ag8-653 cells (gift of H. Karasuyama32) were titered on CTLL-2 cells (from the ATCC) and used as the source of IL-2.

Cell preparation

Suspensions of spleen and peripheral lymph node cells (PLNCs) were prepared by passage through metal screens. Peritoneal washings were collected following 2 infusions of 6 mL cold phosphate-buffered saline (PBS) using a 10-mL 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.

Syngeneic transfers

Although BALB/c and C.B-17 mice are congenic, differing only in the Ig heavy-chain allele,24 in the context of a SCID transfer they are functionally syngeneic. Spleen and PLNCs from BALB/c and PLNCs from OVA-SCID mice were mixed in a 1:1 ratio and injected into the tail veins of unirradiated SCID hosts. Cell concentrations were adjusted so that approximately 2 × 106KJ1-26+ cells were transferred. Memory KJ1-26.1+ cells were isolated on nylon wool columns33 from spleen cell suspensions from OVA-SCID mice that had been immunized 10 days earlier with chicken ovalbumin in CFA.

Induction of GVHD

Inguinal lymph node cells (3 × 104) from B6 donor mice were injected into the tail veins of host SCID mice that had been irradiated with 275 cGy from a γ source (137Cs; Gammacell 40 Exactor, Nordion International, Kanata, ON) on the day of the injection. Major toxicities were not observed when these low numbers of donor T cells were used to initiate GVHD.34 

SEB injection protocol

Control, or reconstituted mice received intraperitoneal injections of SEB (50 μg), or as indicated in the text.

Proliferation assays

In vitro proliferation.

Spleen and PLNCs were purified with Lympholyte separation medium (Cedarlane Laboratories, Hornsby, ON), washed, and then resuspended in complete medium (α-minimal essential medium [MEM], 10% fetal calf serum [FCS], 5 × 10−5 M 2-mercaptoethanol [2-ME], 15 mM HEPES [N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid], 2 mM glutamine) at various concentrations. The percentages of CD4+, KJ1-26.1+, and Vβ8+ cells were first measured by flow cytometry to determine the number of T cells to be used subsequently in culture. Responder T cells were plated onto 2 × 105 irradiated (2000 cGy) BALB/c spleen filler cells and stimulated with OVA peptide (1 μM) or SEB (5 μg/mL) for 48 hours at 37°C in 5% CO2. These antigen doses resulted in optimal stimulation in preliminary experiments (data not shown). Proliferation was measured after 48 hours by 3H-thymidine uptake.22 IL-2 (25 U/mL) was included in some cultures.

In vivo proliferation.

Groups of SCID mice were reconstituted from a single pool of syngeneic T cells. After 10 to 12 days, individual mice were injected intraperitoneally with chicken ovalbumin (3 mg) to study the responses of KJ1-26.1+ T cells (Figure 5B) or with SEB (50 μg) to study the responses of CD4+Vβ8+KJ1-26.1 T cells (Figure 6). Mice were killed 48 hours later and the average number of KJ1-26.1+ or CD4+Vβ8+KJ1-26.1 T cells in the spleens and lymph nodes was determined by flow cytometry and manual counting with a hemocytometer. Control mice that had been subjected to the same initial experimental manipulations, but were not subsequently injected with chicken ovalbumin or SEB on days 10 to 12, were used to provide baseline preinjection numbers of KJ1-26.1+ or CD4+Vβ8+KJ1-26.1 T cells. Because total cell numbers did not change significantly following adoptive transfer (Figure 2A), the results from these mice accurately reflected cell numbers present 48 hours earlier in the rechallenged mice. The ratio of the average number of T cells in mice 48 hours after injection of chicken ovalbumin or SEB, relative to the average number from the control group, was used to calculate a proliferative index as a measure of in vivo antigen responsiveness.

Immunofluorescence

Cells were harvested, incubated with antibody, and analyzed by flow cytometry as previously described.22 

Statistical analysis

The P values, comparing experimental groups, were obtained using Student t test.

Prolonged survival of SAg-reactive T cells in GVHD

In a previous study34 we showed that although at least 5 × 105 allogeneic (H-2b) B6 T cells caused severe GVHD in sublethally irradiated C.B-17 SCID (H-2d) mice, low numbers (3 × 104) of B6 T cells did not, but rather persisted long-term in an unresponsive state. The fate of specific host-reactive T cells was not determined in that study, however. To follow host-reactive cells specifically, we took advantage of the fact that C.B-17 and B6 mice also differ by the presence and absence, respectively, of the mouse mammary tumor provirusMtv-6,25 which encodes a SAg recognized preferentially by Vβ3+ T cells. B6 CD4+Vβ3+ T cells therefore represent host-reactive T cells in this model of GVHD.35 

The numbers of CD4+Vβ3+ T cells in the spleens and peritoneal cavities of host SCID mice were determined at various times after the induction of GVHD. The spleens contained the majority of donor T cells34 and the peritoneal cavity was used to represent tertiary lymphoid organs.36 As shown in Figure 1, splenic and peritoneal CD4+Vβ3+ T cells increased strikingly early in the course of GVHD, but eventually decreased to low baseline levels, likely by deletion, consistent with other SAg models.37,38 Notably, however, the time to deletion was in the order of months, rather than of weeks, as in other systems of chronic SAg activation (Figures 1 and 2A, left panel).37 38 Some host mice demonstrated elevated numbers of CD4+Vβ3+ T cells as late as 120 days after injection (Figure 1).

Fig. 1.

Prolonged survival of SAg-reactive T cells during GVHD.

B6 PLNCs (3 × 104) were injected into sublethally irradiated C.B-17 SCID mice. The percentages of CD4+Vβ3+ T cells (responding to theMtv-6 SAg expressed by SCID cells) in the spleens (left panel) and peritoneal cavities (right panel) of host mice were determined by flow cytometry after transfer. Whereas SAg-reactive T cells are normally deleted within days to weeks, transferred allogeneic SAg-reactive cells persist in immunodeficient hosts for many months. ♦ indicates a single SCID recipient; B, baseline CD4+Vβ3+ T cells (%) in the absence of adoptive transfer (n = 24).

Fig. 1.

Prolonged survival of SAg-reactive T cells during GVHD.

B6 PLNCs (3 × 104) were injected into sublethally irradiated C.B-17 SCID mice. The percentages of CD4+Vβ3+ T cells (responding to theMtv-6 SAg expressed by SCID cells) in the spleens (left panel) and peritoneal cavities (right panel) of host mice were determined by flow cytometry after transfer. Whereas SAg-reactive T cells are normally deleted within days to weeks, transferred allogeneic SAg-reactive cells persist in immunodeficient hosts for many months. ♦ indicates a single SCID recipient; B, baseline CD4+Vβ3+ T cells (%) in the absence of adoptive transfer (n = 24).

Close modal
Fig. 2.

Aberrant immunoregulation of transferred T cells in SCID mice reconstituted with BALB/c lymphocytes and challenged with SEB.

(A) Deletion: BALB/c (left panel) or sublethally irradiated SCID mice reconstituted with 2 × 107 BALB/c spleen cells and PLNCs (right panel) were injected with SEB. The percentage CD4+Vβ8+ and CD4+Vβ6+ cells in pooled axillary, mesenteric, and inguinal lymph node preparations were then determined at various times. Whereas SEB-reactive cells increase rapidly in both BALB/c and SCID recipients, they are deleted much more rapidly in the former. In contrast, CD4+Vβ6+ T cells, which are not SAg responsive, do not respond to SAg injection. The mean ± SE from n separate animals are shown (CD4+Vβ8+ T cells in SCID mice, n = 2, 6, 18, 2, and 5 for days 3, 4, 11, 15, and 20, respectively; CD4+Vβ6+ T cells in SCID mice, n = 3 for each time point; CD4+Vβ8+ T cells in BALB/c mice, n = 10 for each time point). B: The percentage of CD4+Vβ8+ T cells in SCID mice that were reconstituted with BALB/c spleen cells but were not injected with SEB, n = 16. (B) Anergy: CD4+V,8+ PLNC T-cell percentages in BALB/c and BALB/c reconstituted SCID mice (both naive and challenged with SEB 10 days earlier), were quantified flow cytometrically and were adjusted to 2 × 105 cells/mL in complete medium. Irradiated (2000 cGy) BALB/c spleen filler cells and CD4+Vβ8+ T cells (2 × 106cells/mL each), were cultured together in the presence of 1 μg/mL SEB. Proliferation of SEB-challenged cultures (filled bars) was measured 72 hours later and compared to control cultures without added SEB (open bars). Decreased SEB responses were observed in vitro using cells from BALB/c mice previously challenged with SEB, but not if such BALB/c cells were first transferred into SCID hosts. The means ± SE of a representative experiment (n = 4 for each bar) are shown. The experiment was repeated 3 times with similar results. (C) Mortality: BALB/c mice (n = 6) and reconstituted SCID mice (n = 9; 2 × 107 BALB/c spleen cells and PLNCs transferred) were injected with SEB immediately following transfer and again 48 hours later. Mortality was scored 2 days after the second injection.

Fig. 2.

Aberrant immunoregulation of transferred T cells in SCID mice reconstituted with BALB/c lymphocytes and challenged with SEB.

(A) Deletion: BALB/c (left panel) or sublethally irradiated SCID mice reconstituted with 2 × 107 BALB/c spleen cells and PLNCs (right panel) were injected with SEB. The percentage CD4+Vβ8+ and CD4+Vβ6+ cells in pooled axillary, mesenteric, and inguinal lymph node preparations were then determined at various times. Whereas SEB-reactive cells increase rapidly in both BALB/c and SCID recipients, they are deleted much more rapidly in the former. In contrast, CD4+Vβ6+ T cells, which are not SAg responsive, do not respond to SAg injection. The mean ± SE from n separate animals are shown (CD4+Vβ8+ T cells in SCID mice, n = 2, 6, 18, 2, and 5 for days 3, 4, 11, 15, and 20, respectively; CD4+Vβ6+ T cells in SCID mice, n = 3 for each time point; CD4+Vβ8+ T cells in BALB/c mice, n = 10 for each time point). B: The percentage of CD4+Vβ8+ T cells in SCID mice that were reconstituted with BALB/c spleen cells but were not injected with SEB, n = 16. (B) Anergy: CD4+V,8+ PLNC T-cell percentages in BALB/c and BALB/c reconstituted SCID mice (both naive and challenged with SEB 10 days earlier), were quantified flow cytometrically and were adjusted to 2 × 105 cells/mL in complete medium. Irradiated (2000 cGy) BALB/c spleen filler cells and CD4+Vβ8+ T cells (2 × 106cells/mL each), were cultured together in the presence of 1 μg/mL SEB. Proliferation of SEB-challenged cultures (filled bars) was measured 72 hours later and compared to control cultures without added SEB (open bars). Decreased SEB responses were observed in vitro using cells from BALB/c mice previously challenged with SEB, but not if such BALB/c cells were first transferred into SCID hosts. The means ± SE of a representative experiment (n = 4 for each bar) are shown. The experiment was repeated 3 times with similar results. (C) Mortality: BALB/c mice (n = 6) and reconstituted SCID mice (n = 9; 2 × 107 BALB/c spleen cells and PLNCs transferred) were injected with SEB immediately following transfer and again 48 hours later. Mortality was scored 2 days after the second injection.

Close modal

Absence of SEB-induced deletion and anergy in T-cell reconstituted SCID mice

To explain the impaired deletion of SAg-reactive T cells during GVHD in SCID mice, we first considered the effects of sublethal irradiation and concomitant stimulation by host peptide alloantigens, because both of these factors are known to increase the survival of activated T cells.39,40 Syngeneic BALB/c T cells were therefore injected into unirradiated SCID mice and were then challenged with another SAg, SEB, which stimulates CD4+Vβ8+ T cells. Consistent with previous observations in immunocompetent BALB/c mice,7,8CD4+Vβ8+ T cells were reduced in number by about 40% 10 days after challenge with SEB (Figure 2A, left panel), and the remaining cells were anergic (Figure 2B). In contrast, in SCID mice that had been reconstituted with syngeneic BALB/c spleen cells and challenged immediately with SEB, the increase in CD4+Vβ8+ T cells was much greater and remained significantly elevated for at least 20 days (Figure 2A, right panel). Preferential expansion of CD4+Vβ8+ T cells during the reconstitution could not account for this result, because the percentage of CD4+Vβ8+ T cells remained constant in the absence of SEB stimulation (Figure 2A, right panel, B: baseline CD4+Vβ8+ cells). Moreover, BALB/c-derived cells surviving in SCID hosts were not anergic because they proliferated as well in vitro in response to SEB as did naive CD4+Vβ8+ T cells (Figure 2B). Impaired immunoregulation following SAg stimulation was also demonstrated by the response in vivo to a second challenge with SEB. Although serial injections of SEB into BALB/c mice had no deleterious effects, as previously reported,37 over 80% of rechallenged T cell-reconstituted SCID mice died within 24 hours (Figure 2C). Taken together, therefore, these data illustrate that SEB-induced deletion and anergy were absent in T cell–reconstituted SCID mice.

Weak SEB reactivity of KJ1-26.1+ cells relative to BALB/c T cells

Because CD4+Vβ8+ T cells in BALB/c spleens are polyclonal due to variability in TCR-α chain usage,41 it is likely that they demonstrate a range of responsiveness to SEB.42 To determine more precisely the fate of individual SEB-activated T cells after adoptive transfer into SCID mice, we therefore used a monoclonal population of KJ1-26.1+CD4+ T cells from OVA-SCID transgenic mice. KJ1-26.1+CD4+ T cells30recognize chicken ovalbumin peptides, but can also be stimulated and tolerized by the SAg SEB, because their TCR uses the Vβ8 gene product.12 

The response of KJ1-26.1+ T cells to SEB was first compared to that of syngeneic BALB/c T cells. In an in vitro assay in which irradiated BALB/c filler cells were used to ensure that observed differences were intrinsic to T cells, memory and naive KJ1-26.1+ T cells required at least 10-fold more SEB for maximal proliferation than did BALB/c T cells (Figure3). We also observed that KJ1-26.1+ T cells were always outnumbered by CD4+Vβ8+KJ1-26.1 T cells within 3 days after adoptive transfer into SCID mice and challenge with SEB, regardless of the initial starting ratio of BALB/c and OVA-SCID cells (data not shown). Taken together, these results indicated that KJ1-26.1+ T cells were relatively weakly reactive to SEB, compared to other splenic BALB/c CD4+Vβ8+ T cells.

Fig. 3.

CD4+KJ1-26.1+ T cells are weakly responsive to SEB in vitro.

Naive or memory KJ1-26.1+ cells (104), or Vβ8+ BALB/c T cells (104), were cultured together with 2 × 105 BALB/c filler cells in the presence of varying concentrations of SEB. 3H-thymidine uptake was determined 48 hours later. The response of BALB/c cells to SEB exceeded that of both naive and memory KJ1-26.1+ cells. The means ± SE of 3 replicate cultures are shown.

Fig. 3.

CD4+KJ1-26.1+ T cells are weakly responsive to SEB in vitro.

Naive or memory KJ1-26.1+ cells (104), or Vβ8+ BALB/c T cells (104), were cultured together with 2 × 105 BALB/c filler cells in the presence of varying concentrations of SEB. 3H-thymidine uptake was determined 48 hours later. The response of BALB/c cells to SEB exceeded that of both naive and memory KJ1-26.1+ cells. The means ± SE of 3 replicate cultures are shown.

Close modal

BALB/c T-cell requirement for SEB-induced deletion and anergy of KJ1-26.1+ T cells after adoptive transfer

When naive monoclonal KJ1-26.1+ T cells were transferred alone into SCID mice and stimulated with SEB, their behavior recapitulated that previously observed for similarly transferred BALB/c cells. Specifically, KJ1-26.1+ cells increased in number about 400% by 10 days (Figure4A, OVA), and cells present at this time also proliferated strongly in vitro in response to OVA peptides (Figure4B, OVA) and to the SAg SEB (not shown). Because impaired proliferation to both SEB (Figure 2B) and to cognate antigens12 are properties of anergic CD4+Vβ8+ T cells, these data suggested that the transferred KJ1-26.1+ cells were not subject to the normal control mechanisms of deletion and anergy. Similar observations were made after the transfer of memory KJ1-26.1+ CD4+ T cells into SCID mice as well (not shown).

Fig. 4.

Deletion and anergy of adoptively transferred KJ1-26.1+ T cells following stimulation with SEB is restored by the cotransfer of BALB/c cells.

(A) Deletion: Naive or memory KJ1-26.1+ PLNCs were transferred into SCID mice, either alone (OVA, n = 4), or in a 1:1 ratio with 1 × 106 BALB/c spleen cells (OVA + BALB, n = 8). Reconstituted SCID mice were then observed (open bars) or injected immediately with SEB (filled bars). Splenic KJ1-26.1+ cell numbers were determined 10 to 12 days later. The presence of BALB/c cells during adoptive transfer and SEB stimulation markedly decreased the subsequent yield of KJ1-26.1+ cells. Means ± SE are shown. The transfer of naive and memory KJ1-26.1+ T cells yielded similar results. Similar trends were observed in recipient lymph nodes. OVA − SEB, n = 4; OVA + SEB, n = 4; OVA + BALB − SEB, n = 12; OVA + BALB + SEB, n = 8. (B) Anergy: OVA: 104 KJ1-26.1+ cells from SCID mice, injected 10 days earlier with OVA-SCID cells with or without SEB (open bars, no SEB; closed bars, SEB), were plated at a concentration of 1 to 2 × 106 cells/mL (determined by the percentage of KJ1-26.1+ cells) onto 105 irradiated (2000 cGy) BALB/c filler cells. OVA + BALB: 3 × 103KJ1-26.1+ cells from SCID mice, injected 10 days earlier with a mixture of OVA-SCID and BALB/c cells with or without SEB (open bars, no SEB; closed bars, SEB), were similarly plated. KJ1-26.1+ cells were then stimulated with OVA peptide (1 μM) and proliferation (as determined by 3H-thymidine incorporation) was measured 48 hours later. The presence of BALB/c cells during adoptive transfer and SEB stimulation dramatically decreased the subsequent proliferative response of KJ1-26.1+ cells to OVA peptide. The means ± SE of 3 separate experiments, each performed in triplicate are shown.

Fig. 4.

Deletion and anergy of adoptively transferred KJ1-26.1+ T cells following stimulation with SEB is restored by the cotransfer of BALB/c cells.

(A) Deletion: Naive or memory KJ1-26.1+ PLNCs were transferred into SCID mice, either alone (OVA, n = 4), or in a 1:1 ratio with 1 × 106 BALB/c spleen cells (OVA + BALB, n = 8). Reconstituted SCID mice were then observed (open bars) or injected immediately with SEB (filled bars). Splenic KJ1-26.1+ cell numbers were determined 10 to 12 days later. The presence of BALB/c cells during adoptive transfer and SEB stimulation markedly decreased the subsequent yield of KJ1-26.1+ cells. Means ± SE are shown. The transfer of naive and memory KJ1-26.1+ T cells yielded similar results. Similar trends were observed in recipient lymph nodes. OVA − SEB, n = 4; OVA + SEB, n = 4; OVA + BALB − SEB, n = 12; OVA + BALB + SEB, n = 8. (B) Anergy: OVA: 104 KJ1-26.1+ cells from SCID mice, injected 10 days earlier with OVA-SCID cells with or without SEB (open bars, no SEB; closed bars, SEB), were plated at a concentration of 1 to 2 × 106 cells/mL (determined by the percentage of KJ1-26.1+ cells) onto 105 irradiated (2000 cGy) BALB/c filler cells. OVA + BALB: 3 × 103KJ1-26.1+ cells from SCID mice, injected 10 days earlier with a mixture of OVA-SCID and BALB/c cells with or without SEB (open bars, no SEB; closed bars, SEB), were similarly plated. KJ1-26.1+ cells were then stimulated with OVA peptide (1 μM) and proliferation (as determined by 3H-thymidine incorporation) was measured 48 hours later. The presence of BALB/c cells during adoptive transfer and SEB stimulation dramatically decreased the subsequent proliferative response of KJ1-26.1+ cells to OVA peptide. The means ± SE of 3 separate experiments, each performed in triplicate are shown.

Close modal

Notably, however, the outcome was quite different when BALB/c T cells were transferred into SCID mice together with KJ1-26.1+ cells. As before (Figure 2), BALB/c T cells (CD4+Vβ8+KJ1-26.1) were not deleted and did not become anergic 10 to 12 days after adoptive transfer and stimulation by SEB in vivo (Figure5A, right panel, control and SEB only; 5C, upper and middle right panels). However, in contrast to when transferred alone (Figure 4A, OVA), KJ1-26.1+ T cell numbers in the spleen and lymph nodes decreased about 300% (Figure 4A, OVA+BALB; Figure 5A, left panel, control and SEB only; 5C, upper and middle left panels). KJ1-26.1+ T cell numbers were also decreased in the peritoneal cavity and bone marrow (not shown), suggesting that deletion following SEB, and not altered trafficking of activated cells, accounted for the changes in the secondary lymphoid organs in the presence of BALB/c T cells. Similar results were obtained with memory KJ1-26.1+ T cells (not shown). Moreover, KJ1-26.1+ T cells from OVA-scid/+ mice behaved similarly (not shown), indicating that the scid mutation itself was not responsible for the restored deletion of KJ1-26.1+ T cells.

Fig. 5.

Effect of BALB/c cells, cytokine blockade, and cognate antigen on the deletion and anergy of transferred SEB-activated KJ1-26.1+ T cells.

(A) Deletion: Mixtures of OVA-SCID PLNCs and naive BALB/c spleen cells were transferred into SCID mice. Experimental mice were then observed (control, n = 12), or injected with SEB alone (n = 8) or in combination with rhuTNFR:Fc (anti-TNF, n = 5), anti–IFN-γ (anti-IFN, n = 3), CFA (CFA, n = 3), or CFA + ovalbumin (CFA/OVA, n = 3) (see below). SCID mice reconstituted with OVA-SCID PLNCs and about 1 × 106 SEB-tolerant BALB/c spleen cells (tolerant Vβ8, n = 3) were injected with SEB in parallel. Ten days after adoptive transfer, the percentage and absolute numbers of KJ1-26.1+ and CD4+Vβ8+KJ1-26.1 cells in the spleens and lymph nodes (the latter defined as the difference between CD4+Vβ8+ cells and KJ1-26.1+cells) were determined. Circulating TNF-α and IFN-γ were blocked by intraperitoneal injections of rhuTNFR:Fc31 (200 μg) or anti–IFN-γ ascites28 61 (250 μL) every 2 days from day −1. The effect of simultaneous stimulation with cognate antigen was determined in SCID mice immunized 24 hours earlier with CFA with or without chicken ovalbumin. Deletion of KJ1-26.1+ cells in the presence of BALB/c cells was prevented by decreasing the posttransfer SEB responsiveness of cotransferred BALB/c cells by treating donors with SEB prior to cell harvest, by decreasing overall activation levels with anticytokine antibodies, or by increasing the relative strength of activation of KJ1-26.1+ T cells by simultaneous stimulation with SEB and cognate antigen. The means ± SE of n animals are shown. The initial ratio of KJ1-26.1+/CD4+Vβ8+KJ1-26.1cells at time of transfer was about 1:1, with about 1 × 106 of each of KJ1-26.1+ and CD4+Vβ8+KJ1-26.1 cells injected. Without SEB stimulation, T-cell numbers were independent of the use of anticytokine reagents or of CFA (not shown). (B) Anergy: Mixtures of OVA-SCID PLNCs and naive BALB/c spleen cells were transferred into SCID mice. Ten days later, reconstituted control mice (control, n = 7) and animals injected with SEB alone (n = 7) or in combination with rhuTNFR:Fc (anti-TNF, n = 7), or anti–IFN-γ (anti-IFN, n = 3), were injected intraperitoneally with 3 mg chicken ovalbumin. SCID mice reconstituted with OVA-SCID PLNCs and approximately 1 × 106 SEB-tolerant BALB/c spleen cells (tolerant Vβ8, n = 4) were injected in parallel. Three days later, total numbers of spleen and lymph node KJ1-26.1+ cells were determined and used to calculate the proliferation index (defined as the ratio of the cell numbers before and 48 hours after injection of chicken ovalbumin). OVA-dependent anergy of KJ1-26.1+ cells transferred together with BALB/c cells and stimulated with SEB was decreased in the presence of SEB-tolerant BALB/c cells and by TNF-α and IFN-γ blockade. The means ± SE of n animals are shown. (C) KJ1-26.1+ T cells are rescued from deletion by stimulation with cognate antigen. BALB/c and KJ1-26.1+ cells were transferred into SCID mice, some of which had been injected subcutaneously 24 hours earlier with chicken ovalbumin in CFA (lower panels). Mice were observed (control, upper panels) or injected with SEB (SEB and SEB OVA, middle and lower panels, respectively) and secondary lymphoid organs were analyzed 10 days later. The percentages of CD4+KJ1-26.1+ T cells (left panels) and CD4+Vβ8+KJ1-26.1 T cells (right panels) are shown. The reduction in transferred CD4+KJ1-26.1+ T cells that is observed following SEB stimulation (SEB, 0.7%), is prevented by stimulation with the cognate antigen ovalbumin (OVA SEB, 13.2%). One of 6 representative experiments is shown.

Fig. 5.

Effect of BALB/c cells, cytokine blockade, and cognate antigen on the deletion and anergy of transferred SEB-activated KJ1-26.1+ T cells.

(A) Deletion: Mixtures of OVA-SCID PLNCs and naive BALB/c spleen cells were transferred into SCID mice. Experimental mice were then observed (control, n = 12), or injected with SEB alone (n = 8) or in combination with rhuTNFR:Fc (anti-TNF, n = 5), anti–IFN-γ (anti-IFN, n = 3), CFA (CFA, n = 3), or CFA + ovalbumin (CFA/OVA, n = 3) (see below). SCID mice reconstituted with OVA-SCID PLNCs and about 1 × 106 SEB-tolerant BALB/c spleen cells (tolerant Vβ8, n = 3) were injected with SEB in parallel. Ten days after adoptive transfer, the percentage and absolute numbers of KJ1-26.1+ and CD4+Vβ8+KJ1-26.1 cells in the spleens and lymph nodes (the latter defined as the difference between CD4+Vβ8+ cells and KJ1-26.1+cells) were determined. Circulating TNF-α and IFN-γ were blocked by intraperitoneal injections of rhuTNFR:Fc31 (200 μg) or anti–IFN-γ ascites28 61 (250 μL) every 2 days from day −1. The effect of simultaneous stimulation with cognate antigen was determined in SCID mice immunized 24 hours earlier with CFA with or without chicken ovalbumin. Deletion of KJ1-26.1+ cells in the presence of BALB/c cells was prevented by decreasing the posttransfer SEB responsiveness of cotransferred BALB/c cells by treating donors with SEB prior to cell harvest, by decreasing overall activation levels with anticytokine antibodies, or by increasing the relative strength of activation of KJ1-26.1+ T cells by simultaneous stimulation with SEB and cognate antigen. The means ± SE of n animals are shown. The initial ratio of KJ1-26.1+/CD4+Vβ8+KJ1-26.1cells at time of transfer was about 1:1, with about 1 × 106 of each of KJ1-26.1+ and CD4+Vβ8+KJ1-26.1 cells injected. Without SEB stimulation, T-cell numbers were independent of the use of anticytokine reagents or of CFA (not shown). (B) Anergy: Mixtures of OVA-SCID PLNCs and naive BALB/c spleen cells were transferred into SCID mice. Ten days later, reconstituted control mice (control, n = 7) and animals injected with SEB alone (n = 7) or in combination with rhuTNFR:Fc (anti-TNF, n = 7), or anti–IFN-γ (anti-IFN, n = 3), were injected intraperitoneally with 3 mg chicken ovalbumin. SCID mice reconstituted with OVA-SCID PLNCs and approximately 1 × 106 SEB-tolerant BALB/c spleen cells (tolerant Vβ8, n = 4) were injected in parallel. Three days later, total numbers of spleen and lymph node KJ1-26.1+ cells were determined and used to calculate the proliferation index (defined as the ratio of the cell numbers before and 48 hours after injection of chicken ovalbumin). OVA-dependent anergy of KJ1-26.1+ cells transferred together with BALB/c cells and stimulated with SEB was decreased in the presence of SEB-tolerant BALB/c cells and by TNF-α and IFN-γ blockade. The means ± SE of n animals are shown. (C) KJ1-26.1+ T cells are rescued from deletion by stimulation with cognate antigen. BALB/c and KJ1-26.1+ cells were transferred into SCID mice, some of which had been injected subcutaneously 24 hours earlier with chicken ovalbumin in CFA (lower panels). Mice were observed (control, upper panels) or injected with SEB (SEB and SEB OVA, middle and lower panels, respectively) and secondary lymphoid organs were analyzed 10 days later. The percentages of CD4+KJ1-26.1+ T cells (left panels) and CD4+Vβ8+KJ1-26.1 T cells (right panels) are shown. The reduction in transferred CD4+KJ1-26.1+ T cells that is observed following SEB stimulation (SEB, 0.7%), is prevented by stimulation with the cognate antigen ovalbumin (OVA SEB, 13.2%). One of 6 representative experiments is shown.

Close modal

To determine if the SEB-activated KJ1-26.1+ T cells remaining after deletion were anergic, we developed an in vivo anergy assay11 to overcome the technical problems posed by the low KJ1-26.1+ cell numbers and the overwhelming contribution of BALB/c CD4+Vβ8+KJ1-26.1 T cells to in vitro proliferation assays (Figure 2B). This assay was based on the observation that anergic SAg-reactive T cells demonstrate impaired proliferative responses to their cognate antigens as well.12,13 Consequently, anergy of KJ1-26.1+ T cells in reconstituted SCID mice could be determined by measuring the proliferative response to chicken ovalbumin. This response was unlikely to involve significant numbers of BALB/c KJ1-26.1 T cells because the frequency of naive KJ1-26.1 T cells that can respond to a cognate antigen, such as chicken ovalbumin, is less than 1:10,5 43 and should not be selectively increased after priming with SEB. Proliferation of KJ1-26.1+ T cells remaining 10 to 12 days after adoptive transfer was consequently measured by an index defined as the ratio of cell number before and 48 hours after injection of chicken ovalbumin. In this assay, KJ1-26.1+ T cell numbers increased by approximately 600% in control mice that had only been reconstituted with OVA-SCID and BALB/c cells but did not change significantly if similarly reconstituted mice had been previously injected with SEB (Figure 5B, left panel, control and SEB only). These results suggested that stimulation with SEB caused KJ1-26.1+ T cells to become anergic in the presence of BALB/c T cells that could respond strongly to SEB. Consistent with this, proliferation of KJ1-26.1+ T cells in vitro in response to antigenic peptides was also decreased (Figure 4B, OVA+BALB, filled bar) compared to transferred but otherwise unstimulated KJ1-26.1+ T cells (Figure 4B, OVA+BALB, open bar), and to KJ1-26.1+ T cells that were stimulated with SEB after adoptive transfer in the absence of BALB/c spleen cells (Figure 4B, OVA).

Effect of strongly SEB-reactive T cells on deletion and anergy of KJ1-26.1+ T cells in reconstituted SCID mice

The finding that KJ1-26.1+ T cells were less reactive to SEB than were BALB/c cells (Figure 3) led us to wonder if it was the more strongly SEB-reactive T cells in BALB/c spleens that were responsible for the restored deletion and anergy of KJ1-26.1+ T cells observed when both were transferred together into SCID mice and stimulated with SEB. If so, the removal of strongly SEB-reactive T cells from BALB/c spleens before adoptive transfer would be expected to prevent the subsequent deletion and anergy of KJ1-26.1+ T cells. Moreover, by increasing the activation state of SEB-activated KJ1-26.1+ T cells after adoptive transfer, one might allow them to escape regulation by more strongly reactive BALB/c T cells.

We therefore used 3 approaches to overcome the relative effects of the strongly SEB-reactive BALB/c T cells: the physical removal of strongly reactive cells, a decrease in the overall level of T-cell activation, and increased KJ1-26.1+ T-cell activation.

Removal of strongly SEB-reactive T cells.

Intravenous injection of SEB into BALB/c mice (1 injection of 50 μg, 10 days before harvesting, or 5 injections of 10 μg, every 2 days) was used to delete strongly SEB-reactive T cells. CD4+Vβ8+ cells remaining after deletion are known to respond weakly to SEB as assessed by proliferation and cytokine production8,44 and may represent T cells that have not been deleted due to the low affinity of their TCRs for SEB.45 BALB/c mice treated previously with SEB would therefore be “SEB tolerant” donors. SCID mice were reconstituted with a mixture of KJ1-26.1+ T cells and tolerant BALB/c spleen and PLNCs and challenged with SEB. Ten days later, rather than being deleted, KJ1-26.1+ cells had increased by about 300% compared with controls (Figure 5A, left panel, control and tolerant Vβ8 bars). Despite their presumed anergy CD4+Vβ8+ KJ1-26.1 T cells also increased by about 300% in response to SEB (Figure 5A, right panel, control and tolerant Vβ8 bars). And consistent with our prediction, the response of KJ1-26.1+ T cells to chicken ovalbumin was almost completely restored in the presence of tolerant BALB/c T cells (Figure 5B, left panel, control and tolerant Vβ8 bars).

Decreased overall immune activation.

Blockade of cytokines such as TNF-α46 47 that mediate costimulatory signals for T-cell responses in vivo after SEB stimulation and adoptive transfer would be expected to decrease the level of stimulation of strongly reactive T cells without removing them physically. Consistent with this notion, deletion of KJ1-26.1+ T cells was prevented, and proliferation in vivo was restored partially, by TNF-α blockade (Figure 5A,B, left panels, control and anti-TNF bars) even in the presence of BALB/c T cells. Similar results were obtained using blocking antibodies directed against IFN-γ (Figure 5A,B, left panels, control and anti-IFN bars).

Increased KJ1-26.1+ T-cell activation.

We reasoned that the activation state of KJ1-26.1+ T cells should be increased by simultaneous stimulation with SEB and with the cognate antigen, chicken ovalbumin, which may mediate distinct and additive signaling pathways.48 49 Accordingly, SCID mice were injected with chicken ovalbumin in CFA 1 day before adoptive transfer of a mixture of BALB/c and OVA-SCID cells. Notably, KJ1-26.1+ T cells were not deleted when stimulated simultaneously by antigen, even in the presence of nontolerant BALB/c T cells (Figure 5A, left panel, control and CFA/OVA bars; 5C, lower panel). Consistent with the notion that the deletion of KJ1-26.1+ T cells was related to their relative activation state, the injection of CFA alone also prevented deletion of KJ1-26.1+ T cells by SEB, albeit to a lesser extent (Figure5A, left panel, control and CFA bars).

Time dependence of deletion and anergy of SEB-reactive T cells after adoptive transfer

In the 2 systems described above, allogeneic (B6; Figure 1) or syngeneic (BALB/c; Figure 2) T cells were stimulated with SAgs immediately after adoptive transfer. Because the timing of stimulation by the endogenous Mtv-6 SAg could not be altered easily, the SAg SEB was injected at various times after adoptive transfer of syngeneic T cells to determine the effect on deletion and anergy (Figure 6). Consistent with our previous observations (Figure 2A, right panel), the percentage of CD4+Vβ8+ T cells was elevated significantly 10 days after adoptive transfer and immediate stimulation with SEB (Figure 6, left panel). Progressively lower numbers and percentages of CD4+Vβ8+ T cells (indicating increased deletion) were observed when T cells were allowed to reconstitute SCID hosts for 2 or 9 days before stimulation with SEB, compared to cells that were transferred but not stimulated (Figure 6, left panel).

Fig. 6.

Delayed superantigenic stimulation restores deletion and anergy of adoptively transferred cells.

(A) Deletion: SCID mice reconstituted with BALB/c cells were observed (control, n = 5) or were injected with SEB immediately (day 0 SEB, n = 25), 2 (day 2 SEB, n = 2), or 9 (day 9 SEB, n = 5) days after adoptive transfer. Ten days after SEB stimulation (or 10 days after transfer, control), the percentages of peripheral lymph node CD4+Vβ8+ T cells were determined by flow cytometry. Delayed SEB challenge restores the deletion of SEB responsive cells. The percentages of peripheral lymph node CD4+Vβ8+ T cells in BALB/c mice before (BALB, n = 4) and 10 days after (BALB + SEB, n = 19) injection of SEB are shown for comparison. The percentages plotted reflect absolute cell numbers (not shown), with means ± SE from n animals shown. Asterisk indicates statistically significant difference (P < .02) between control and adoptively transferred mice. (B) Anergy: The SEB-dependent proliferative capacity of transferred CD4+Vβ8+ T cells was determined after delayed SEB challenge. Reconstituted SCID mice were initially challenged with SEB 9 days after adoptive transfer and were then rechallenged 10 days later, and the total numbers of spleen and peripheral lymph node CD4+Vβ8+ T cells were determined by flow cytometry and manual counting 48 hours later. The total number of CD4+Vβ8+ T cells was determined in a similar manner in mice that had been reconstituted for 21 days, but had received only a single injection of SEB 12 days before, and the ratio of these numbers was used to calculate a proliferation index representing the ability of SEB-stimulated T cells to proliferate in response to a second challenge with SEB (SEB rechallenge, n = 5). The proliferation index of CD4+Vβ8+ T cells in response to a primary stimulation with SEB after adoptive transfer was determined in a similar manner (1° SEB challenge, n = 3). Delayed SEB challenge restores SEB-dependent anergy (P < .02). The means ± SE from n animals are reported.

Fig. 6.

Delayed superantigenic stimulation restores deletion and anergy of adoptively transferred cells.

(A) Deletion: SCID mice reconstituted with BALB/c cells were observed (control, n = 5) or were injected with SEB immediately (day 0 SEB, n = 25), 2 (day 2 SEB, n = 2), or 9 (day 9 SEB, n = 5) days after adoptive transfer. Ten days after SEB stimulation (or 10 days after transfer, control), the percentages of peripheral lymph node CD4+Vβ8+ T cells were determined by flow cytometry. Delayed SEB challenge restores the deletion of SEB responsive cells. The percentages of peripheral lymph node CD4+Vβ8+ T cells in BALB/c mice before (BALB, n = 4) and 10 days after (BALB + SEB, n = 19) injection of SEB are shown for comparison. The percentages plotted reflect absolute cell numbers (not shown), with means ± SE from n animals shown. Asterisk indicates statistically significant difference (P < .02) between control and adoptively transferred mice. (B) Anergy: The SEB-dependent proliferative capacity of transferred CD4+Vβ8+ T cells was determined after delayed SEB challenge. Reconstituted SCID mice were initially challenged with SEB 9 days after adoptive transfer and were then rechallenged 10 days later, and the total numbers of spleen and peripheral lymph node CD4+Vβ8+ T cells were determined by flow cytometry and manual counting 48 hours later. The total number of CD4+Vβ8+ T cells was determined in a similar manner in mice that had been reconstituted for 21 days, but had received only a single injection of SEB 12 days before, and the ratio of these numbers was used to calculate a proliferation index representing the ability of SEB-stimulated T cells to proliferate in response to a second challenge with SEB (SEB rechallenge, n = 5). The proliferation index of CD4+Vβ8+ T cells in response to a primary stimulation with SEB after adoptive transfer was determined in a similar manner (1° SEB challenge, n = 3). Delayed SEB challenge restores SEB-dependent anergy (P < .02). The means ± SE from n animals are reported.

Close modal

In parallel with cell number, anergy was measured by an in vivo proliferation assay. When the initial SEB injection was delayed by 9 days, CD4+Vβ8+ T cells proliferated less well to a second challenge with SEB in vivo compared to cells that were stimulated immediately after adoptive transfer (Figure 2B), or were transferred but not stimulated initially, with SEB (Figure 6, right panel). These results suggested that anergy was also restored partially when the primary challenge of transferred CD4+Vβ8+ T cells with SEB was delayed.

Effect of transferred T-cell number on the regulation of SAg responses

We also investigated whether the impaired anergy and deletion of SAg-reactive T cells were related to the number of transferred T cells. Varying numbers of BALB/c or B6 T cells were injected into SCID mice before stimulation with SEB or Mtv-6 SAg, respectively (Table 1). The percentages of SAg-reactive T cells in the lymph nodes were then determined 12 days later (Table 1). These percentages were decreased significantly when large numbers (1 × 108 cells) were transferred, in contrast to the impaired deletion observed with the transfer of lower cell numbers (Table 1). T cells remaining in host mice after transfer of high numbers of T cells and superantigenic stimulation also proliferated less well to in vitro restimulation with the SAg (Figure2B, and data not shown), suggesting that anergy had been restored partially.

Table 1.

Deletion of SAG-reactive T cells depends on the number of adoptively transferred cells

HostTransferred cellsSAgSAg-reactive T cells, %*Control CD4+Vβ6+ T cells, %
BALB — — 19.5 ± 0.9 10.6 ± 0.3 
BALB — SEB 16.6 ± 0.6 12.7 ± 0.4 
SCID BALB — 18.6 ± 0.1 11.5 ± 0.1 
SCID 107BALB SEB 26.0 ± 0.5 9.2 ± 0.4 
SCID 108BALB SEB 18.4 ± 0.3 11.5 ± 0.4  
SCID 2 × 108BALB SEB 14.4 ± 0.5 10.3 ± 0.5 
B6 — — 3.1 ± 0.2 ND 
SCID 107B6 mtv-6 37.8 ± 6.4 ND  
SCID 2 × 108B6 mtv-6 16.4 ± 3.9 ND 
HostTransferred cellsSAgSAg-reactive T cells, %*Control CD4+Vβ6+ T cells, %
BALB — — 19.5 ± 0.9 10.6 ± 0.3 
BALB — SEB 16.6 ± 0.6 12.7 ± 0.4 
SCID BALB — 18.6 ± 0.1 11.5 ± 0.1 
SCID 107BALB SEB 26.0 ± 0.5 9.2 ± 0.4 
SCID 108BALB SEB 18.4 ± 0.3 11.5 ± 0.4  
SCID 2 × 108BALB SEB 14.4 ± 0.5 10.3 ± 0.5 
B6 — — 3.1 ± 0.2 ND 
SCID 107B6 mtv-6 37.8 ± 6.4 ND  
SCID 2 × 108B6 mtv-6 16.4 ± 3.9 ND 

Varying numbers of allogeneic (B6) or syngeneic (BALB) cells (a mixture of spleen cells and PLNCs) were injected into SCID mice. Allogeneic T cells were stimulated by the mtv-6 SAg during GVHD and syngeneic T cells were stimulated by concomitantly injected SEB. Twelve days later, the percentage of SAg-reactive cells (CD4+Vβ3+ T cells in GVHD and CD4+Vβ8+ T cells for SEB) in the lymph nodes of host mice were determined by flow cytometry. The percentage of CD4+Vβ6+ T cells, which should not be stimulated by superantigens, was also determined as a control. The data are pooled from 4 different experiments and the average and SE is reported. (n = 2, at least, for each experiment involving adoptive transfer).

ND indicates not done.

*

CD4+Vβ8+ T cells in the case of SEB and CD4+Vβ3+ T cells in the case ofmtv-6.

For GVHD studies, SCID mice were sublethally irradiated (275 cGy).

1 × 108 cells were injected twice, 24 hours apart.

In this paper we report that (1) the normal regulation (deletion and anergy) of SAg responses is markedly impaired when T cells are transferred adoptively into immunodeficient mice immediately before stimulation; (2) the degree of this impairment of deletion and anergy is dependent on the number of T cells transferred, and the timing of SAg stimulation after adoptive transfer; and (3) whether or not T cells will undergo normal or impaired regulation following SAg stimulation is determined by the strength of their individual SAg responses, relative to those of other responding T cells in the transferred population.

These studies were prompted by the unexpectedly prolonged survival ofMtv-6 SAg–reactive allogeneic B6 CD4+Vβ3+ T cells during GVHD in sublethally irradiated SCID mice (Figure 1). This observation was surprising because the chronic administration of exogenous SAgs normally results in the almost complete deletion of reactive T cells within 2 weeks.37 We had expected therefore, that chronic activation by the host-derived endogenous Mtv-6 SAg would also lead to the rapid deletion of SAg-reactive donor T cells during GVHD.

The normal regulation of SAg responses in immunocompetent mice is multifactorial in nature and involves cell-cell contact through TNF and TNF receptor family members,50,51 regulatory CD4+ and CD8+ T cells,15-17 and B-cell–mediated idiotypic networks.18-20 The altered behavior of SAg-activated T cells in immunodeficient mice indicates that these regulatory activities are not present in SCID mice and suggests as well that they are not fully transferable either. For example, although one might expect spleen cell populations from immunocompetent mice treated with SEB to be enriched in regulatory cells, appropriate deletion and anergy were nevertheless not seen after the transfer of such populations into SCID mice (Figure 5A, right panel, control and tolerant Vβ8 bars).

The altered behavior of SAg-activated donor T cells in immunodeficient mice might also relate to differences in the cellular architecture of secondary lymphoid organs in immunocompetent and T cell-reconstituted immunodeficient mice. Cell concentrations in the paracortical T-cell areas of the spleen and lymph nodes normally exceed 108cells/mL.52 Because these concentrations are probably much lower in immunodeficient mice immediately following the adoptive transfer of T cells,53 regulatory cell-cell interactions might be diluted, thereby allowing the unregulated proliferation of responding cells. This explanation is compatible with the observed partial restoration of SAg-dependent deletion and anergy seen as early as 48 hours after adoptive transfer (Figure 6). This short time period is presumably too early for idiotypic networks and suppressor T cells to have developed in host immunodeficient mice, but possibly sufficient for cell proliferation and trafficking54 55 to lead to increased cell concentrations in the paracortical areas of lymphoid organs, thereby permitting increased cell-cell interactions. The partial restoration of deletion following the transfer of larger numbers of donor T cells (Table 1) further supports this notion.

However, if it is merely lymphoid architecture and the initial dearth of regulatory cell-cell interactions that underlie the aberrant regulation of SAg-activated T cells after adoptive transfer into SCID mice, then why are transgenic OVA-SCID KJ1-26.1+ T cells seemingly regulated properly when they are transferred into SCID recipients together with BALB/c spleen cells (Figures 4 and 5)? Our results suggest that a relationship between deletion/anergy and the reactivity of individual T cells to SEB may underlie this observation. Being monoclonal, KJ1-26.1+ T cells would be expected to be uniformly SEB reactive. In contrast, BALB/c spleen cells, being polyclonal, should comprise a spectrum of SEB responsiveness. Consistent with this, as assessed by their SEB-dependent proliferative responses in vitro and in vivo, KJ1-26.1+ T cells were more weakly responsive to SEB than were at least some BALB/c T cells (Figure 3), possibly due to SEB-binding effects of the Vα chain of the transgenic TCR or of cross-reactive peptide-MHC ligands, both of which are known to alter SEB-TCR interactions.41,56 Notably, deletion and anergy of these more weakly reactive KJ1-26.1+ T cells were prevented (1) in the absence of more strongly activated T cells (by transferring KJ1-26.1+ T cells alone; Figure 4); (2) by decreasing the posttransfer SEB responsiveness of cotransferred BALB/c cells by treating donors with SEB prior to cell harvest (Figure 5), or by decreasing overall activation levels with anti–cytokine antibodies (Figure 5); or (3) by increasing the relative strength of activation of KJ1-26.1+ T cells by simultaneous stimulation with SEB and cognate antigen (Figure 5). Thus, the outcome of transferred SAg-reactive cells appears to be related both to their absolute and relative levels of activation, with the behavior of strongly and weakly reactive T cells reflecting seemingly distinct, but possibly overlapping, regulatory mechanisms. We suspect that the impaired regulation of the former is most consistent with the absence of regulatory cells and of cell-cell interactions, as suggested above. In contrast, the latter appear to be regulated specifically in the presence of more strongly reactive cells. Why the behavior of weakly reactive T cells should be altered in the presence of more strongly reactive T cells is not clear. Possibly, strongly reactive T cells merely out-compete weakly reactive T cells for effective activation by APCs.57 Another possibility is that strongly and weakly reactive T cells may respond differentially to cytokines produced following antigenic stimulation. We were unable to demonstrate experimentally, however, that weakly and strongly SEB- reactive cells responded differentially to TNF-α (data not shown).

Although we have focused here on the regulation of SAg responses in an immunodeficiency model of GVHD, we speculate that similar phenomena may define the responses of T cells to MHC-restricted peptides as well. In this light, the observation that immune responses are regulated aberrantly after the adoptive transfer of T cells into immunodeficient hosts may therefore provide insights into the pathogenesis of acute GVHD after allo-SCT. For example, the absence of normal regulation in this clinical setting may contribute to more severe GVHD in a manner analogous to the increased mortality observed after a second SEB challenge in T-cell–reconstituted mice (Figure 2C). Moreover, because the graft-versus-leukemia effect is also mediated by T cells,58 and such tumor-reactive T cells are often only weakly reactive to tumor antigens59 60 (while other allogeneic T cells mediating GVHD may be activated more strongly), the observation that weakly reactive T-cell responses can be down-regulated in the presence of strongly reactive T cells (Figures 4 and 5) may have implications for the therapeutic effects of allo-SCT as well.

We thank J. Wither for OVA-peptides, J. Kappler for the KJ1-26.1 hybridoma, M. Widmer for rhuTNFR:Fc reagents, and R. G. Miller for OVA-SCID mice.

Supported by grants from the Sunnybrook Trust Fund (D.S.), the Canadian Institutes of Health Research (D.S.), and the National Cancer Institute of Canada (A.C.S.). D.S. is a physician scientist supported by Cancer Care Ontario.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

1
Ferrara
JL
Cooke
KR
Pan
L
Krenger
W
The immunopathophysiology of acute graft-versus-host-disease.
Stem Cells.
14
1996
473
489
2
Thomas
ED
ALL and beyond: implications for other hematologic malignancies.
Leukemia
11(suppl 4)
1997
S43
S45
3
Roy
J
McGlave
PB
Filipovich
AH
et al
Acute graft-versus-host disease following unrelated donor marrow transplantation: failure of conventional therapy.
Bone Marrow Transplant.
10
1992
77
82
4
Szydlo
R
Goldman
JM
Klein
JP
et al
Results of allogeneic bone marrow transplants for leukemia using donors other than HLA-identical siblings.
J Clin Oncol.
15
1997
1767
1777
5
Moskophidis
D
Lechner
F
Pircher
H
Zinkernagel
RM
Virus persistence in acutely infected immunocompetent mice by exhaustion of antiviral cytotoxic effector T cells.
Nature.
362
1993
758
761
6
Sercarz
EE
Lehmann
PV
Ametani
A
Benichou
G
Miller
A
Moudgil
K
Dominance and crypticity of T cell antigenic determinants.
Annu Rev Immunol.
11
1993
729
766
7
Kawabe
Y
Ochi
A
Programmed cell death and extrathymic reduction of V,8+ CD4+ T cells in mice tolerant to Staphylococcus aureus enterotoxin B.
Nature.
349
1991
245
248
8
Kawabe
Y
Ochi
A
Selective anergy of V,8+ CD4+ T cells in Staphylococcus enterotoxin B-primed mice.
J Exp Med.
172
1990
10651070
9
Strasser
A
Apoptosis. Death of a T cell.
Nature.
373
1995
385
386
10
Webb
S
Morris
C
Sprent
J
Extrathymic tolerance of mature T cells: clonal elimination as a consequence of immunity.
Cell.
63
1990
1249
1256
11
Baschieri
S
Lees
RK
Lussow
AR
MacDonald
HR
Clonal anergy to staphylococcal enterotoxin B in vivo: selective effects on T cell subsets and lymphokines.
Eur J Immunol.
23
1993
2661
2666
12
Blankson
JN
Loh
DY
Morse
SS
Superantigens and conventional antigens induce different responses in alpha beta T-cell receptor transgenic mice.
Immunology
85
1995
57
62
13
Gaur
A
Fathman
CG
Steinman
L
Brocke
S
SEB induced anergy: modulation of immune response to T cell determinants of myoglobin and myelin basic protein.
J Immunol.
150
1993
3062
3069
14
Wang
ZQ
Orlikowsky
T
Dudhane
A
et al
Staphylococcal enterotoxin B-induced T-cell anergy is mediated by regulatory T cells.
Immunology.
94
1998
331
339
15
Cauley
LS
Cauley
KA
Shub
F
Huston
G
Swain
SL
Transferable anergy: superantigen treatment induces CD4+ T cell tolerance that is reversible and requires CD4− CD8− cells and interferon gamma.
J Exp Med
186
1997
71
81
16
Jiang
H
Ware
R
Stall
A
Flaherty
L
Chess
L
Pernis
B
Murine CD8+ T cells that specifically delete autologous CD4+ T cells expressing V beta 8 TCR: a role of the Qa-1 molecule.
Immunity.
2
1995
185
194
17
Jiang
H
Kashleva
H
Xu
LX
et al
T cell vaccination induces T cell receptor Vbeta-specific Qa-1-restricted regulatory CD8(+) T cells.
Proc Natl Acad Sci U S A.
95
1998
4533
4537
18
Aroeira
LS
Mouton
CG
Toran
JL
Ward
ES
Martinez
C
Anti-Vbeta8 antibodies induce and maintain staphylococcal enterotoxin B- triggered Vbeta8+ T cell anergy.
Eur J Immunol.
29
1999
437
445
19
Pereira
P
Bandeira
A
Coutinho
A
Marcos
MA
Toribio
M
Martinez
C
V-region connectivity in T cell repertoires.
Annu Rev Immunol.
7
1989
209
249
20
Stark Aroeira
L
Williams
O
Borlado
LR
Carrera
AC
Martinez
C
Evidence for B cell participation in the in vitro and in vivo maintenance of in vivo staphylococcal enterotoxin B-induced T cell anergy.
Int Immunol.
9
1997
65
72
21
Sheng-Tanner
XF
McKerlie
C
Spaner
D
Characterization of graft-versus-host disease in SCID mice and prevention by physicochemical stressors.
Transplantation.
70
2000
1683
1693
22
Spaner
D
Raju
K
Rabinovich
B
Miller
RG
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
1999
1192
1199
23
Maloy
KJ
Burkhart
C
Freer
G
et al
Qualitative and quantitative requirements for CD4+ T cell-mediated antiviral protection.
J Immunol.
162
1999
2867
2874
24
Bosma
MJ
Carroll
AM
The SCID mouse mutant: definition, characterization, and potential uses.
Annu Rev Immunol.
9
1991
323
350
25
Acha-Orbea
H
MacDonald
HR
Superantigens of mouse mammary tumor virus.
Annu Rev Immunol.
13
1995
459
486
26
Spaner
D
Raju
K
Radvanyi
L
Lin
YP
Miller
RG
A role for perforin in activation induced cell death.
J Immunology
160
1998
2655
2664
27
Unkeless
JC
Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors.
J Exp Med.
150
1979
580
596
28
Spitalny
GL
Havell
EA
Monoclonal antibody to murine gamma interferon inhibits lymphokine-induced anti-viral and macrophage tumoricidal activities.
J Exp Med
159
1984
1560
1565
29
Haskins
K
Kubo
R
White
J
Pigeon
M
Kappler
J
Marrack
P
The major histocompatibility complex-restricted antigen receptor on T cells, I: isolation with a monoclonal antibody.
J Exp Med.
157
1983
1149
1169
30
Murphy
KM
Heimberger
AB
Loh
DY
Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo.
Science.
250
1990
1720
1723
31
Xun
CQ
Thompson
JS
Jennings
CD
Brown
SA
Widmer
MB
Effect of total body irradiation, busulfan-cyclophosphamide, or cyclophosphamide conditioning on inflammatory cytokine release and development of acute and chronic graft-versus-host disease in H-2-incompatible transplanted SCID mice.
Blood.
83
1994
2360
2367
32
Karasuyama
H
Melchers
F
Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors.
Eur J Immunol.
18
1988
97
104
33
Julius
MH
Simpson
E
Herzenberg
LA
A rapid method for the isolation of functional thymus-derived murine lymphocytes.
Eur J Immunol.
3
1973
645
648
34
Spaner
D
Sheng-Tanner
XF
Raju
K
Rabinovich
B
Messner
H
Miller
RG
Long-term persistence of IL-2 unresponsive allogeneic T cells in sublethally irradiated SCID mice.
Int Immunol.
11
1999
1601
1614
35
Roberts
JL
Abe
R
Shores
EW
Singer
A
Expression of Mls determinants in mice exhibiting the severe combined immunodeficiency (scid) mutation or X-linked immunodeficiency (xid) defect.
J Immunol.
149
1992
1577
1582
36
Picker
LJ
Butcher
EC
Physiological and molecular mechanisms of lymphocyte homing.
Annu Rev Immunol.
10
1992
561
591
37
McCormack
JE
Callahan
JE
Kappler
J
Marrack
PC
Profound deletion of mature T cells in vivo by chronic exposure to exogenous superantigen.
J Immunol.
150
1993
3785
3792
38
Miconnet
I
Roger
T
Seman
M
Bruley-Rosset
M
Critical role of endogenous Mtv in acute lethal graft-versus-host disease.
Eur J Immunol.
25
1995
364
368
39
Kundig
TM
Bachmann
MF
Oehen
S
et al
On the role of antigen in maintaining cytotoxic T-cell memory.
Proc Natl Acad Sci U S A.
93
1996
9716
9723
40
McCormack
JE
Kappler
J
Marrack
P
Stimulation with specific antigen can block superantigen-mediated deletion of T cells in vivo.
Proc Natl Acad Sci U S A.
91
1994
2086
2090
41
Blackman
MA
Smith
HP
Le
P
Woodland
DL
Influence of the T cell receptor alpha-chain on T cell reactivity and tolerance to Mls-1 in T cell receptor beta-chain transgenic mice.
J Immunol.
151
1993
556
565
42
Li
H
Llera
A
Mariuzza
RA
Structure-function studies of T-cell receptor-superantigen interactions.
Immunol Rev.
163
1998
177
186
43
Merkenschlager
M
Terry
L
Edwards
R
Beverley
PC
Limiting dilution analysis of proliferative responses in human lymphocyte populations defined by the monoclonal antibody UCHL1: implications for differential CD45 expression in T cell memory formation.
Eur J Immunol.
18
1988
1653
1661
44
Williams
O
Aroeira
LS
Martinez
C
Absence of peripheral clonal deletion and anergy in immune responses of T cell-reconstituted athymic mice.
Eur J Immunol.
24
1994
579
584
45
Kawai
K
Ohashi
PS
Immunological function of a defined T-cell population tolerized to low-affinity self antigens.
Nature.
374
1995
68
69
46
Smith
CA
Farrah
T
Goodwin
RG
The TNF receptor superfamily of cellular and viral proteins: activation, costimulation, and death.
Cell.
76
1994
959
962
47
Yokota
S
Geppert
TD
Lipsky
PE
Enhancement of antigen- and mitogen-induced human T lymphocyte proliferation by tumor necrosis factor-alpha.
J Immunol.
140
1988
531
536
48
Liu
H
Lampe
MA
Iregui
MV
Cantor
H
Conventional antigen and superantigen may be coupled to distinct and cooperative T-cell activation pathways.
Proc Natl Acad Sci U S A.
88
1991
8705
8709
49
O'Rourke
AM
Mescher
MF
Webb
SR
Activation of polyphosphoinositide hydrolysis in T cells by H-2 alloantigen but not mls determinants.
Science.
249
1990
171
174
50
Lynch
DH
Ramsdell
F
Anderson
MR
Fas and FasL in the homeostatic regulation of immune responses.
Immunol Today.
16
1995
569
574
51
Rathmell
JC
Thompson
CB
The central effectors of cell death in the immune system.
Annu Rev Immunol.
17
1999
781
828
52
Picker
LJ
Siegelman
MH
Lymphoid tissues and organs.
Fundamental Immunology.
Paul
WE
1993
145
197
Raven Press
New York, NY
53
Dilly
SA
Sloane
JP
Psalti
IS
The cellular composition of human lymph nodes after allogenic bone marrow transplantation: an immunohistological study.
J Pathol.
150
1986
213
221
54
Butcher
EC
Picker
LJ
Lymphocyte homing and homeostasis.
Science.
272
1996
60
66
55
van Ewijk
W
Nieuwenhuis
P
Compartments, domains and migration pathways of lymphoid cells in the splenic pulp.
Experientia.
41
1985
199
208
56
Wen
R
Cole
GA
Surman
S
Blackman
MA
Woodland
DL
Major histocompatibility complex class II-associated peptides control the presentation of bacterial superantigens to T cells.
J Exp Med.
183
1996
1083
1092
57
Kedl
RM
Rees
WA
Hildeman
DA
et al
T cells compete for access to antigen-bearing antigen-presenting cells.
J Exp Med.
192
2000
1105
1113
58
Kolb
HJ
Schattenberg
A
Goldman
JM
et al
Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. European Group for Blood and Marrow Transplantation Working Party Chronic Leukemia.
Blood.
86
1995
2041
2050
59
Gervois
N
Guilloux
Y
Diez
E
Jotereau
F
Suboptimal activation of melanoma infiltrating lymphocytes (TIL) due to low avidity of TCR/MHC-tumor peptide interactions.
J Exp Med.
183
1996
1083
1092
60
Morgan
DJ
Kreuwel
HT
Fleck
S
Levitsky
HI
Pardoll
DM
Sherman
LA
Activation of low avidity CTL specific for a self epitope results in tumor rejection but not autoimmunity.
J Immunol.
160
1998
2403
2407
61
Ware
CF
Donato
NJ
Dorshkind
K
Human, rat or mouse hybridomas secrete high levels of monoclonal antibodies following transplantation into mice with severe combined immunodeficiency disease (SCID).
J Immunol Methods.
85
1985
353
361

Author notes

David Spaner, Division of Molecular and Cellular Biology, Research Institute, S-116A, S-Wing, Sunnybrook and Women's College Health Sciences Center, 2075 Bayview Ave, Toronto, ON, Canada M4N 3M5; e-mail: [email protected].

Sign in via your Institution