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Departments of
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Microbiology and Immunology,
Surgery, and
Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite their widespread acceptance as tools to monitor CD8 responses, tetramers have not been used as triggers of cell activation. In principle, they should be far superior to peptides in engaging TCRs and should have a longer life span in the recipient. Thus, they could be used in many situations where peptides have been used to induce or block immune responses in intact animals. It is known that multiple exposures or high doses of Ag induce CD8+ T cell unresponsiveness in vivo.
Many approaches have been made to specifically "silence" alloreactive or autoreactive T cells, mainly through the induction of anergy (9) and deletion of T cells by overstimulation with high concentrations of specific Ag (10). These experiments have normally been performed in transgenic systems and few reports exist in which tolerance to alloantigen or autoantigen has been achieved in wild-type mice. The mechanisms responsible for such tolerance are poorly understood. In TCR-transgenic models, physical deletion of T cells (11, 12) has been observed as well as down-regulation of TCR surface levels (13) after tolerogenic exposure to exogenous Ag.
To test whether providing such a strong signal 1 to specific CD8+ T cells in vivo yields a form of "high zone tolerance," we constructed soluble MHC class I tetramers loaded with a peptide described as a highly immunogenic epitope for an H-2Db-restricted anti-H-Y (anti-male) response (14). In naive female B6 mice, we used this tetramer to detect male-specific T cells in the normal spleen. Furthermore, these naive cells were activated by injection of soluble MHC class I tetramer in both HY-TCR-transgenic and normal B6 females. Such injections primed female B6 mice for second-set skin graft rejection as effectively as injection of male spleen cells. Interestingly, multiple injections of relevant tetramer reversed the priming effect, suggesting elimination of the tetramer-reactive T cells by either anergy induction or activation-induced cell death, as demonstrated by surface annexin V staining (our unpublished observations).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HY TCR
-transgenic mice (15) that
carry a transgene specific for male HY Ag were obtained from the
National Institute of Arthritis and Infectious Diseases via Taconic
Farms (C57BL6, TgN(TcrHY); Germantown, NY). These mice are referred to
as HY-TCR mice. Normal B6 mice were purchased from (Charles River
Breeding Laboratories, Wilmington, MA). All animals used in this study
were maintained under specific pathogen-free conditions in the American
Association of Laboratory Animal Care-accredited University of North
Carolina, Department of Laboratory Animal Medicine Facilities, and were
routinely used at 8 wk of age.
Peptides
HY peptide (KCSRNRQYL) (14) and gp33 peptide (KAVYNFATM) were synthesized by the University of North Carolina Microchemical Facility, purified by HPLC, and tested for purity by mass spectroscopy.
Tetramer preparation and injection
Recombinant protein was prepared as previously described by Wang et al. (16). For in vivo tetramer injection experiments, HY-Db was prepared in sterile PBS and 30 µg/mouse in 150 µl was injected directly into the peritoneal cavity.
Flow cytometric analysis
The directly conjugated anti-mouse Abs used for cell surface staining in this study were anti-CD8 (53-6.7), anti-B220 (RA3-6B2), and anti-MHC class II (I-Ab, 25-9-17) purchased from BD PharMingen (San Diego, CA). Two- and three-color staining was performed using standard methods. List mode data were collected on a FACScan (BD Biosciences, Mountain View, CA) and analyzed using Summit software (Cytomation, Ft. Collins, CO).
Purification of CD8+ TCR-transgenic T cells from spleen
Cell suspensions were prepared from the spleens of TCR-transgenic mice. Cells were incubated at 37°C for 1 h in tissue culture dishes (Nunc, Naperville, IL) to eliminate adherent cells before purification. CD8+ T cells were negatively selected by depletion of CD4+, MHC class II+, and CD11b+ cells (16) using the MACS magnetic separation system according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA).
Preparation of irradiated splenocytes
Splenocytes were prepared from male B6 mice and resuspended at 2 x 106 cells/ml in RPMI 1640 medium. Cells were then irradiated by exposure to 3000 rad emitted from a Gammacell 40 Cesium-137 source (Atomic Energy of Canada Limited, Ottawa, Canada).
Proliferation assay
Purified CD8+ T cells (4 x 105/well) were stimulated with Ags or tetramer at different concentrations in 200 µl of complete RPMI 1640 in 96-well flat-bottom plates. The cultures were incubated for 48 h, and 1 µCi [3H]thymidine was added to each well for the final 10 h of culture. Cells were harvested using a multiple sample harvester (Otto Hiller, Madison, WI), and incorporation of [3H]thymidine was measured by scintillation counting using a Beckman LS5000 counter (Palo Alto, CA). All data represent the average counts per minute of triplicate determinations. All proliferation experiments were repeated at least three times.
Tail grafting
Tail grafting was performed as previously described (17). Each female recipient mouse received a male allograft and a female isograft as a control. Glass tubes were placed over the grafted area for 3 days to prevent removal of the graft by the mouse. Grafts that had failed to vascularize properly with apparent rejection at 3 days were classed as "technical failures" and removed from the analysis. Remaining grafts were scored daily. Fully intact grafts were scored as 100% and when <30% of the graft remained, it was considered rejected.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the ability of HY-Db-reactive
T cells to stain with PE-labeled HY-Db tetramer,
we used the HY-TCR-transgenic mouse strain (14). Fig. 1
illustrates peptide-specific staining
of CD8high (male-reactive) T cells with
HY-Db tetramer in female HY-TCR splenocytes. To
demonstrate the ability of MHC class HY-Db
tetramers to identify the presence of HY-reactive T cells in naive,
unmanipulated nontransgenic mice, splenocytes from 8-wk-old female B6
mice were stained with PE-conjugated labeled
HY-Db and counterstained according to the figure
legend. Fig. 2, a and
b, shows that HY-Db tetramer staining
revealed the presence of a surprisingly large population of HY-specific
CD8+ T cells, which represent 1.5–2.0% of
splenic CD8+ T cells. A control
Db tetramer, assembled with lymphocytic
choriomeningitis virus
(LCMV)3
gp33 peptide stained <0.6% of CD8+
T cells. Furthermore, after adoptive (i.v.) transfer of irradiated male
splenocytes into female B6 mice, the HY-Db
tetramer staining population had expanded 
2.5 fold at 5 days after
transfer (Fig. 2d). Control female B6 mice receiving
irradiated female splenocytes did not expand the HY-reactive
CD8+ T cells (Fig. 2c). Fig. 2e illustrates the summary of the data (three mice per
group). There was significant (Student’s t test,
p < 0.005) priming with male but not with female
splenocytes. These observations demonstrate that naive female B6 have
detectable male-specific T cells in the spleen.
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In our previous studies (16), using soluble MHC class
I tetramer alone to stimulate naive CD8+ T cells
into effector cells, we showed that the response to LCMV in vitro was
blocked by addition of Abs to the TCR coreceptor CD8. Since the
LCMV-transgenic TCR has a relatively high affinity to the
immunodominant gp33 peptide (18) we wanted to be certain
that the same argument pertained to the relatively low-affinity HY-TCR
(our unpublished observations). Here, we stimulated purified
CD8+ HY-TCR T cells with
HY-Db tetramer in the presence of 20 µg/ml
anti-CD8 Ab (53-6.7) or control Ig. This concentration of
anti-CD8 Ab was found to completely block HY-TCR T cell
proliferation in dose-response experiments (data not shown). As in our
LCMV study, treatment with anti-CD8 Ab blocked the T cell
proliferative response to HY-Db tetramer (Fig. 3), suggesting an important role for the
CD8 coreceptor in the activation of HY-TCR T cells by specific
HY-Db tetramer.
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We have previously shown that soluble MHC class I tetramer alone
is sufficient for in vitro activation and differentiation into effector
cells of naive CD8+ T cells from transgenic
HY-TCR mice (16). We wished to determine whether direct
injection of HY-Db into female B6 mice would
result in functional priming of a specific immune response in vivo,
measured here by accelerated rejection of male skin grafts. One group
of mice was injected with 1 x 107 male
splenocytes as a positive control. Twenty days later groups of naive
female mice were treated with either 30 µg/mouse
HY-Db tetramer or PBS (unprimed control). Three
days after HY-Db injection, we grafted tail skin
grafts from unmanipulated male B6 donors onto all the female
recipients. Fig. 4a depicts
the data from a representative (one of three) experiment. A single dose
of HY-Db tetramer to female B6 mice caused
significant priming and earlier rejection of graft tissue (Wilcoxon
rank order, p < 0.0001). Indeed, this response had
identical kinetics to that of male splenocyte-primed female mice,
demonstrating that tetramer injection had primed normal female B6
recipients. This also provides further evidence that the HY (KCSRNRQYL)
peptide is an important epitope recognized in the anti-male response by
female mice in vivo.
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To further examine the effects of tetramer injection into female
mice, 30 µg HY-Db or irrelevant
gp33-Db tetramer was injected i.p. into female
HY-TCR mice. CD8+ T cells were purified from the
spleens of HY-TCR female mice harvested 72 h after tetramer
injection and restimulated in vitro with HY-Db
tetramer or irrelevant gp33-Db tetramer. Fig. 4
b shows that CD8+ T cells purified
from tetramer injected female mice responded in an Ag-specific manner,
with an increased proliferative response to HY-Db
and not to gp33-Db. Additionally, splenocytes
from transgenic mice treated with HY-Db exhibited
an increased proliferative response to HY-Db
tetramer than gp33-Db-treated littermates. It was
apparent that in mice 48 h after HY-Db
tetramer injection, very few cells stained with either anti-CD8 or
anti-V8.2 but had a phenotype consistent with T cell activation
(data not shown). We believe that this represents down-regulation of
TCR and CD8, as observed in other studies with peptide stimulation
(19, 20). These data demonstrate that administration of
HY-Db tetramer in vivo has the ability to prime
transgenic mice to HY peptide.
To examine the ability of the in vivo-primed T cells to respond to
natural HY Ag, we stimulated them with B6 male spleen cells. Fig. 4c illustrates the restimulation response of
tetramer-treated CD8+ T cells to irradiated male
B6 splenocytes. Again, CD8+ T cells from
tetramer-treated female mice responded much more vigorously than cells
from gp33-Db-treated controls. The lack of an
apparent response of CD8+ T cells from
gp33-Db-treated female mice to male splenocytes
is due to the short assay time (48 h), typical of primed T cells.
Unprimed HY-TCR shows a peak response at 72 h at this cell
density. These data demonstrate that soluble
HY-Db tetramer can prime the female HY-TCR mouse
to respond to normally processed male Ag as well as
HY-Db tetramer.
In vivo administration of multiple doses of HY-Db tetramer resulted in nonresponsive CD8low T cells
In studies of peptide-induced T cell nonresponsiveness, multiple doses of Ag often had to be administered to observe Ag-specific tolerance (21, 22, 23). We wanted to examine whether the priming observed with a single tetramer dose could be changed to exhaustion using multiple tetramer doses.
We injected one, two, or three doses of tetramer or PBS (i.p.) into
female HY-TCR mice, each dose separated by 2-day intervals.
CD8+ T cells were purified from the spleens and
stimulated in vitro with HY-Db tetramer, HY
peptide, or irrelevant gp33. Fig. 5
summarizes these data. Mice injected with a single dose of relevant
HY-Db tetramer (all PBS-treated groups exhibited
high responses to HY-Db tetramer and HY peptide,
but no response to gp33 peptide, as in untreated mice; data not shown)
exhibited a primed response to HY peptide and tetramer (Fig. 5a). In contrast, if two injections were administered, then
the proliferative response to peptide and tetramer is greatly
diminished (Fig. 5b) and after three injections becomes
undetectable (Fig. 5c).
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8.2; data not shown) 3 wk after the final tetramer injection.
Fig. 6a shows that in HY-TCR
mice treated with three doses of PBS, the majority of
HY-Db tetramer staining cells reside in the
CD8high compartment. However, with increasing
tetramer doses, the majority of tetramer-positive T cells are
CD8low. This is a result of a loss of
tetramer-reactive CD8high T cells and an increase
in CD8low numbers. Importantly, if the same
experiment was repeated in a group of naive B6 female mice, a very
similar pattern of tetramer and CD8 staining emerged (Fig. 6b), an increase in
CD8lowtetramer+ cells and a
decrease in
CD8hightetramer+ cells.
This suggests that sustained T cell unresponsiveness associated with
multiple tetramer doses is due to sustained decreased expression levels
of CD8. Treatment with anti-CD8 Ab blocked an in vitro T cell
proliferative response to HY-Db tetramer (data
not shown), further confirming an important role for CD8 coreceptor in
the activation of HY-TCR T cells by specific
HY-Db tetramer.
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Experiments with B6 females (Fig. 4c) suggested that a
single dose of HY-Db tetramer induced priming to
HY peptide and earlier rejection of male skin grafts. However, the
above data demonstrated that multiple doses of specific
HY-Db tetramer ablated an in vitro proliferative
response to HY Ag. We therefore asked whether multiple tetramer
exposures would change rejection of male skin grafts. One group of B6
female mice was injected with 1 x 107 male
splenocytes as a positive control. Twenty days after priming this
group, three groups (six mice) of naive female mice were injected with
one, two, or three doses of 30 µg/mouse HY-Db
tetramer or PBS. Three days after tetramer administration, tail skin
grafts from naive male B6 donors were grafted onto all the B6 female
recipients. Fig. 7 illustrates the data
from a representative experiment; a single dose of
HY-Db tetramer to female B6 mice caused priming
and rapid rejection of skin grafts. However, two or three doses of
tetramer reversed this effect; grafts on mice receiving three doses of
tetramer surviving significantly (Wilcoxon rank order,
p < 0.001) longer than the control unprimed mice.
Indeed, 25% of treated mice showed long-term graft survival. Thus,
although there is at least one other defined HY epitope (15, 24, 25), induction of unresponsiveness to the KCSRNRQYL epitope
resulted in prolonged graft survival.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1.5–2.0% of the splenic CD8+ T cell
population, the first time such an estimate has been made. This is
consistent with the strong in vivo response to HY Ag. We have previously demonstrated that for naive CD8+ T cells from female HY-TCR mice, soluble MHC class I tetramer alone (signal 1) is sufficient for activation and differentiation into effector cells (16). Two other studies have used tetramers to stimulate T cell clones or hybrids (26, 27). Soluble MHC class I tetramers have also been used to stimulate calcium flux in Ag-specific T cell clones (27), and some evidence exists that MHC class I monomers were able to activate CD8+ T cells in vivo (28). Here, we describe the use of soluble MHC class I tetramers to induce and modulate an Ag-specific immune response in vivo. Injection of the HY-Db tetramer (but not the irrelevant Db tetramer) into female HY-TCR mice induced a very rapid but transient activation, resulting in expansion of HY-specific CD8+ T cells. This response primed female HY-TCR mice to more rapid responses to both the HY peptide and irradiated male splenocytes. Reinforcing the notion that HY tetramers could influence a T cell response in normal mice, HY-Db-treated B6 mice rejected male tail grafts more rapidly than untreated mice. In addition, our data show that after multiple in vivo doses of the HY-Db tetramer, recovered CD8low T cells proliferate poorly in vitro to HY Ag stimulation. In treated B6 mice, this results in longer survival times of male skin grafts.
Several studies investigating peripheral tolerance in the HY-transgenic mouse have involved injection of female-transgenic mice with syngeneic B6 male (expressing the HY Ag)-activated CD8+ lymphocytes found a similar initial expansion of female HY-reactive CD8+ T cells followed by a decline to below the starting number (29). Transfer of female B6 CD8+ T cells did not mediate this effect, suggesting an Ag-specific effect. Remaining recipient transgenic CD8high T cells were responsive, but there was a large increase in the number of recipient transgenic CD8low T cells that were nonresponsive to both male Ag and anti-CD3 Abs (29). No accompanying TCR down-regulation was observed. Similarly, other groups have also found that activated CD8+ CTL lines can inactivate T cells that recognize them, again possibly mediated by CD8 down-regulation on the recognizing T cell (13, 30, 31). Earlier studies have postulated that immune recognition of HLA molecules specifically down-modulates CD8 expression on CD8+ T cells (30).
Results from our study allow us to investigate this mechanism further. Injection with soluble MHC class I tetramer complexed with specific HY peptide into female HY-TCR mice showed a very similar effect as seen in other studies, but without the confounding signals provided by injected activated male T cells or other APC. It argues that a strong "signal 1" delivered to reactive T cell populations does indeed cause a similar initial expansion of female HY-reactive CD8+ T cells followed by a decline in numbers, likely due to activation-induced cell death as demonstrated by surface annexin V staining (our unpublished observations). Although we have shown that the CD8+ T cell response to HY peptide is not dependent on known costimulatory molecules (16), the response is CD8 dependent. This is possibly a reflection of a TCR with a low affinity to the HY epitope (our unpublished observations). Surviving CD8 HY-reactive T cells after multiple tetramer doses possessed a sustained lower expression of CD8, which may act as a mechanism of their nonresponsiveness. It will be interesting to determine whether this model of nonresponsiveness applies to T cells specific for a higher affinity epitope such as LCMV gp33. In such cases, the threshold for T cell activation might not be so dependent on levels of coreceptor molecules.
Although most other previous studies have been performed in transgenic mouse models, the tetramer-based system described here is applicable to other models, as demonstrated by the ability to stain scarce CD8+ T cells of known specificity with MHC class I tetramer and show that tetramer injection can influence the antimale rejection response in naive wild-type mice.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert Maile, Department of Microbiology and Immunology, University of North Carolina, CB 7290, Chapel Hill, NC 27599. E-mail address: robmaile{at}bellsouth.net
3 Abbreviation used in this paper: LCMV, lymphocytic choriomeningitis virus.
Received for publication May 24, 2001. Accepted for publication July 23, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2M tetramers to quantitate CD8+ cytotoxic T lymphocytes specific for dominant and nondominant viral epitopes in simian-human immunodeficiency virus-infected rhesus monkeys. J. Virol. 73:5466.This article has been cited by other articles:
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  S. H. Apte, A. Baz, P. Groves, A. Kelso, and N. Kienzle Interferon-{gamma} and interleukin-4 reciprocally regulate CD8 expression in CD8+ T cells PNAS, November 11, 2008; 105(45): 17475 - 17480. [Abstract] [Full Text] [PDF]
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 Z. Xiao, M. F. Mescher, and S. C. Jameson Detuning CD8 T cells: down-regulation of CD8 expression, tetramer binding, and response during CTL activation J. Exp. Med., October 29, 2007; 204(11): 2667 - 2677. [Abstract] [Full Text] [PDF]
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 P. R. Hess, C. Barnes, M. D. Woolard, M. D. L. Johnson, J. M. Cullen, E. J. Collins, and J. A. Frelinger Selective deletion of antigen-specific CD8+ T cells by MHC class I tetramers coupled to the type I ribosome-inactivating protein saporin Blood, April 15, 2007; 109(8): 3300 - 3307. [Abstract] [Full Text] [PDF]
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 A. F. M. Maree, P. Santamaria, and L. Edelstein-Keshet Modeling competition among autoreactive CD8+ T cells in autoimmune diabetes: implications for antigen-specific therapy Int. Immunol., July 1, 2006; 18(7): 1067 - 1077. [Abstract] [Full Text] [PDF]
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 R. Maile, C. M. Barnes, A. I. Nielsen, A. A. Meyer, J. A. Frelinger, and B. A. Cairns Lymphopenia-Induced Homeostatic Proliferation of CD8+ T Cells Is a Mechanism for Effective Allogeneic Skin Graft Rejection following Burn Injury. J. Immunol., June 1, 2006; 176(11): 6717 - 6726. [Abstract] [Full Text] [PDF]
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J. D. Stone and L. J. Stern CD8 T Cells, Like CD4 T Cells, Are Triggered by Multivalent Engagement of TCRs by MHC-Peptide Ligands but Not by Monovalent Engagement J. Immunol., February 1, 2006; 176(3): 1498 - 1505. [Abstract] [Full Text] [PDF]
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N. J. Felix, A. Suri, J. J. Walters, S. Horvath, M. L. Gross, and P. M. Allen I-Ep-Bound Self-Peptides: Identification, Characterization, and Role in Alloreactivity J. Immunol., January 15, 2006; 176(2): 1062 - 1071. [Abstract] [Full Text] [PDF]
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R. Maile, C. A. Siler, S. E. Kerry, K. E. Midkiff, E. J. Collins, and J. A. Frelinger Peripheral "CD8 Tuning" Dynamically Modulates the Size and Responsiveness of an Antigen-Specific T Cell Pool In Vivo J. Immunol., January 15, 2005; 174(2): 619 - 627. [Abstract] [Full Text] [PDF]
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T. J. Fry, M. Sinha, M. Milliron, Y.-W. Chu, V. Kapoor, R. E. Gress, E. Thomas, and C. L. Mackall Flt3 ligand enhances thymic-dependent and thymic-independent immune reconstitution Blood, November 1, 2004; 104(9): 2794 - 2800. [Abstract] [Full Text] [PDF]
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R. R. Yuan, P. Wong, M. R. McDevitt, E. Doubrovina, I. Leiner, W. Bornmann, R. O'Reilly, E. G. Pamer, and D. A. Scheinberg Targeted deletion of T-cell clones using alpha-emitting suicide MHC tetramers Blood, October 15, 2004; 104(8): 2397 - 2402. [Abstract] [Full Text] [PDF]
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K. L. Mueller, M. A. Daniels, A. Felthauser, C. Kao, S. C. Jameson, and Y. Shimizu Cutting Edge: LFA-1 Integrin-Dependent T Cell Adhesion Is Regulated by Both Ag Specificity and Sensitivity J. Immunol., August 15, 2004; 173(4): 2222 - 2226. [Abstract] [Full Text] [PDF]
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J. Stebbing, B. Gazzard, S. Patterson, M. Bower, D. Perumal, M. Nelson, A. McMichael, G. Ogg, A. Epenetos, F. Gotch, et al. Antibody-targeted MHC complex-directed expansion of HIV-1- and KSHV-specific CD8+ lymphocytes: a new approach to therapeutic vaccination Blood, March 1, 2004; 103(5): 1791 - 1795. [Abstract] [Full Text] [PDF]
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S. E. Kerry, J. Buslepp, L. A. Cramer, R. Maile, L. L. Hensley, A. I. Nielsen, P. Kavathas, B. J. Vilen, E. J. Collins, and J. A. Frelinger Interplay between TCR Affinity and Necessity of Coreceptor Ligation: High-Affinity Peptide-MHC/TCR Interaction Overcomes Lack of CD8 Engagement J. Immunol., November 1, 2003; 171(9): 4493 - 4503. [Abstract] [Full Text] [PDF]
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