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Department of Pathology, University of California, San Francisco, CA 94143
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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 the dramatic phenotype of the knockout, restricting the specificity of CTLA-4-/- cells in TCR-transgenic mice reveals a surprisingly mild alteration in their response to immunization with protein in adjuvant (7). Furthermore, the response of CTLA-4-deficient T cells to viral infection closely mimics that of wild-type T cells in mixed bone marrow chimeras (8). Taken together, these data suggest that the predominant effect of CTLA-4 deficiency or blockade appears to be to unleash T cell responsiveness to "tolerogenic" Ag (e.g., soluble peptide i.v.) (7, 9) or self Ag (5, 6) rather than to modulate responses to immunogenic Ag (e.g., peptide plus adjuvant or microbes). One caveat to the use of i.v. soluble protein to mimic self Ag is that it is unclear whether the former is subject to the same processing and presentation events as a tissue-expressed self protein. To further dissect the types of antigenic encounter that are controlled by CTLA-4, we have developed a model in which a known protein (hen egg lysozyme; HEL3) is transgenically expressed as a tissue self Ag and can also be administered exogenously by immunization in adjuvant. By introducing naive HEL-specific T cells from class II-restricted TCR transgenic wild-type or CTLA-4-/- mice, we have been able to assess the relative ability of CTLA-4 to regulate responsiveness to either source of protein. We show that when T cells are exposed to HEL both as a tissue Ag and an administered immunogen, CTLA-4 dramatically limits the local expansion to tissue Ag, but only marginally affects the T cell response to immunizing Ag. In the absence of the CTLA-4 pathway, failure to limit the local accumulation of Ag-specific T cells is associated with the onset of aggressive tissue destruction. We discuss the implications of these results for the pathogenesis of tissue autoimmunity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3A9 TCR-transgenic mice (wild type or CTLA4-/-) and rat insulin promoter (RIP)-HEL mice (obtained from C. Goodnow, John Curtin School of Medical Research, Canberra, Australia) were maintained on an MRL background. Animals were housed in the University of California (San Francisco, CA) animal facility in accordance with university guidelines and used between 6 and 12 wk of age. Mice were genotyped using PCR and flow cytometry.
T cell transfers
Combined lymph nodes (LN) (axilliary, inguinal, brachial, and mesenteric) from 3A9/wild-type or 3A9/CTLA-4-/- mice were stained with the clonotypic Ab, 1G12, and the number of T cells expressing the transgenic TCR was assessed by flow cytometry. The indicated number of 1G12-positive cells was transferred into recipient mice by tail vein injection. Where indicated, cells were incubated before transfer with 1 µM CFSE (Molecular Probes, Eugene, OR) for 10 min at room temperature followed by two washes with RPMI supplemented as below.
Immunization
HEL protein (Sigma-Aldrich, St. Louis, MO) was prepared emulsified in IFA or CFA (Difco, Detroit, MI) or alum-precipitated or in buffered saline and 100 µg were administered i.p. 24 h following adoptive transfer of 3A9 T cells.
Flow cytometry
At the indicated time point, mice were sacrificed and cells isolated from the relevant lymphoid tissue for analysis. Spleen samples were treated with Tris ammonium chloride to lyse RBCs. Cells were preincubated with anti-CD16/CD32 (mouse FcR) before staining with the clonotypic Ab to the 3A9 TCR (1G12-biotin; purified from cell culture supernatant), streptavidin-PE, and CD4-PerCP. All Abs were purchased from BD PharMingen (San Diego, CA) unless otherwise indicated. For analysis of CFSE profiles, samples were pooled from two to six mice.
Blood glucose
Blood glucose levels were measured every 3–4 days (Glucometer Elite XL; Bayer, Elkhart, IN) and mice were considered diabetic following two consecutive readings of >250 mg/dl.
Histology
H&E staining of sections from paraffin-embedded tissues was performed according to standard procedures. Pancreatic islets were scored by eye based on infiltration (none, peri-islet, or invasive) with at least 15 islets being scored per individual mouse. Results are expressed as percentage of total islets counted.
In vitro restimulation
Pooled pancreatic LN, or draining (axilliary and inguinal) LN from recipients of 3A9/wild-type or 3A9/CTLA-4-/- cells that were immunized s.c. with 100 µg alum-precipitated HEL were stained with 1G12 and CD4 Abs and purified by high speed cell sorting (MoFlow; Cytomation, Fort Collins, CO) (5–15 mice/group). 1G12+CD4+ cells (4 x 103) were then cultured with 1.2 x 105 spleen cells from nontransgenic mice in 0.2 ml of RPMI 1640 supplemented with 1 mM L-glutamine, penicillin, streptomycin, nonessential amino acids, sodium pyruvate, HEPES (all from Life Technologies, Grand Island, NY), 5 x 10-5 M 2-ME, and 10% FBS (Sigma-Aldrich) containing the indicated concentration of HEL46–61 peptide. Proliferation assays were pulsed with 1 µCi [3H]thymidine (NEN, Boston, MA) for the final 7–8 h of the 72-h period, and incorporated radioactivity was measured in a Betaplate scintillation counter (Amersham Pharmacia Biotech, Piscataway, NJ). Supernatants were removed at day 3 for the assessment of cytokine production by ELISA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We hypothesized that the way in which Ag is processed and
presented might influence the ability of CTLA-4 to regulate T cell
responses to the Ag. Therefore, we set out to compare the role of
CTLA-4 in controlling T cell proliferation to HEL protein expressed as
a tissue Ag vs HEL protein administered exogenously in adjuvant. To
analyze T cell proliferation to endogenous tissue protein, mice
expressing HEL in pancreatic 
cells under the control of the RIP
(RIP-HEL mice) were adoptively transferred with CFSE-labeled
HEL-specific (3A9) CD4 T cells that were either wild type or
CTLA-4-/-. Both 3A9/wild-type and
3A9/CTLA-4-/- cells were phenotypically naive
before transfer as evidenced by high CD62L expression (data not
shown). On day 10 after adoptive transfer, mice were sacrificed and the
CFSE profiles of the 3A9 T cells isolated from the pancreatic LN or
control (inguinal) LN were assessed. A proportion of the 3A9 cells
isolated from the pancreatic LN had divided as evidenced by a loss of
CFSE staining, while those isolated from the inguinal LN remained
undivided (Fig. 1
A). These
data support the notion that pancreas-derived self proteins are brought
to the draining LN, presumably by APCs providing surveillance in the
pancreas, and presented to naive T cells (10, 11).
Strikingly, there was a clear increase in cell division in the
pancreatic LN if the transferred 3A9 cells were CTLA-4-deficient. This
suggests that CTLA-4 engagement negatively regulates T cell
proliferation in response to tissue-derived Ag. To assess the role of
CTLA-4 in controlling T cell proliferation to exogenous Ag,
3A9/wild-type or 3A9/CTLA-4-/- cells were
transferred to nontransgenic recipients that were either left untreated
or immunized i.p. 24 h later with alum-precipitated HEL protein.
By day 10 after adoptive transfer, all the 3A9 T cells had become CFSE
negative (data not shown). Therefore, we examined the response at day 3
when individual peaks of CFSE could still be discerned. Although there
was a slight increase in cell division if the transferred cells lacked
CTLA-4, the difference in CFSE profiles between 3A9/wild-type and
3A9/CTLA-4-/- cells was not as marked as in the
response to tissue Ag. Importantly, even extensive titration of the
dose of HEL protein used for immunization failed to reveal a larger
difference between the response of 3A9/wild-type and
3A9/CTLA-4-/- cells (data not shown). This
suggests that the regulation of T cell cycling by CTLA-4 is more
profound during T cell responses to tissue Ag than during T cell
responses to immunizing Ag.
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We next analyzed the ability of CTLA-4 to control the responses of
T cells exposed to both an endogenous and exogenous source of Ag in the
same mouse. 3A9/wild-type or 3A9/CTLA-4-/- T
cells were transferred to RIP-HEL mice that were immunized 24 h
later with alum-precipitated HEL protein. Similar to the data presented
in Fig. 1
, the clearest difference between the CFSE profiles of
3A9/wild-type cells compared with 3A9/CTLA-4-/-
cells was in the pancreatic LN, where the cell cycle progression of the
CTLA-4-/- cells was markedly augmented compared
with the wild-type cells (data not shown). Consistent with this
difference in cell cycling, 3A9/CTLA-4-/- T
cells accumulated in the pancreatic LN to a much greater extent than
3A9/wild-type cells, both in terms of percentage (Fig. 2, A and B) and
absolute number (Fig. 2C). In the absence of immunization,
although the cells divided in response to pancreatic Ag, they did not
accumulate in the pancreatic LN, suggesting that they either die in
situ or migrate to distal sites (Fig. 2, triangles). The percentage
(Fig. 2B) and absolute number (Fig. 2C) of 3A9 T
cells in the spleen was increased in i.p. immunized mice compared with
unimmunized mice, consistent with the spleen being a target site for
immunization by this route. However, the difference between
accumulation of 3A9/wild-type cells and
3A9/CTLA-4-/- cells in the spleen was marginal.
Thus, CTLA-4-mediated regulation of cell cycling and T cell
accumulation is most marked in the LN draining the site of tissue Ag
and appears minimal in the site responding to administered
immunogenic Ag.
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To explore further the differential role of CTLA-4 in controlling
responses to Ags processed as self vs those administered as foreign, we
performed a kinetic analysis of the T cell response. Again the
adoptively transferred 3A9 T cells were provided with both an
endogenous (pancreas-expressed) and exogenous (immunizing) source of
HEL protein, and the T cell accumulation at the responding sites was
assessed. To exclude the possibility that the minimal difference
between wild-type and CTLA-4-/- cells observed
in response to i.p. immunization was a specific feature of splenic
responses, we performed this set of experiments using s.c. immunization
and assessed T cell numbers in the draining LN. Although the number of
3A9 T cells in the draining nodes increased at both days 4 and 7
in response to immunization, the difference in expansion between
wild-type and CTLA-4-/- cells was small (Fig. 3, middle panels). In
comparison there was a clear difference in the number of 3A9/wild-type
cells and the number of 3A9/CTLA-4-/- T cells
that accumulated at both time points in the pancreatic LN (Fig. 3, left panels). The observation that
CTLA-4-/- cells did not show increased
expansion in response to the s.c. immunization could not be attributed
to preferential migration of primed cells toward the second source of
Ag in the pancreas, because equivalent data were obtained after
adoptive transfer to nontransgenic recipients (Fig. 3, right
panels). Taken together these data indicate a minimal role for
CTLA-4 in controlling T cell expansion in response to foreign Ag
administered as an immunogen, but a more profound role for regulating
accumulation of T cells at the site of tissue Ag expression.
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We were next interested to assess whether the accumulation of T
cells in the absence of CTLA-4 would have functional consequences for
the maintenance of tolerance to tissue Ags. Because the tissue
expressing HEL protein in this system is the pancreas, we were able to
assess loss of tolerance to the HEL-expressing pancreatic cells by
monitoring the ability of the mice to regulate their blood glucose
levels. RIP-HEL mice were adoptively transferred with 3A9/wild-type or
3A9/CTLA-4-/- T cells, and where indicated,
were then immunized i.p. with alum-precipitated HEL protein (as in Fig. 2). In the absence of immunization, the recipient mice maintained
normal blood glucose for at least 6 wk (Fig. 4A, triangles, and data not
shown) regardless of whether they received 3A9/wild-type or
3A9/CTLA-4-/- cells. Examination of blood
glucose levels in mice that were immunized after adoptive transfer
revealed that diabetes was rapidly induced in mice that received
3A9/CTLA-4-/- T cells, but not in recipients of
3A9/wild-type T cells (Fig. 4A). Transfers of 3A9/wild-type
or 3A9/CTLA-4-/- cells into normal mice not
expressing HEL, followed by immunization, did not result in
dysregulation of blood glucose levels (data not shown). The onset of
diabetes in RIP-HEL recipients of 3A9/CTLA-4-/-
cells after immunization was associated with aggressive lymphocytic
infiltration of the pancreatic islets as demonstrated by H&E staining
(representative micrographs, Fig. 4B, quantitation, Fig. 4C). A greater number of islets were infiltrated in
recipients of 3A9/CTLA-4-/- cells compared with
recipients of 3A9/wild-type cells, and in addition, the infiltrate was
of a more invasive nature. In contrast, mice that received wild-type
cells showed only modest peri-islet infiltration after immunization.
Thus, in the absence of CTLA-4, the local accumulation of activated T
cells is associated with invasion and destruction of the Ag-bearing
tissue.
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The fact that the accumulation of
CTLA-4-/- T cells in the pancreatic LN and
subsequent diabetes induction only occurred after immunization suggests
that T cells that have become activated in the periphery expand upon
reencountering tissue-derived Ag in the draining LN, and that this
expansion is regulated by CTLA-4. Failure to control this expansion,
due to loss of CTLA-4-function, is associated with the initiation of
tissue injury. To address whether the effect of CTLA-4 on the T cells
responding to tissue Ag was simply to regulate T cell numbers or
whether T cell effector differentiation was also altered, we analyzed
the functional properties of the 3A9/wild-type and
3A9/CTLA-4-/- cells in the pancreatic LN. To
compare the same numbers of 3A9/wild-type and
3A9/CTLA-4-/- cells, we used high-speed cell
sorting to isolate 3A9 T cells from the pancreatic LNs of mice that had
received 3A9/wild-type or 3A9/CTLA-4-/- cells
and had been immunized (HEL/alum s.c.), and analyzed their
responsiveness in vitro. We were particularly interested in the ability
of the T cells to produce IFN-
because most studies have indicated a
key role for this cytokine (12, 13, 14) and its receptor
(15, 16, 17) in the pathogenesis of diabetes. Despite the
striking difference in the ability of wild-type and
CTLA-4-/- 3A9 T cells to induce diabetes in
RIP-HEL mice, when isolated from the pancreatic LN and analyzed on a
per cell basis, both cell types showed comparable proliferative
responses to HEL peptide and comparable levels of IFN- production
(Fig. 5). IL-4 production was below
levels of detection in all cases (data not shown). The response of
sorted 3A9 T cells isolated from the LN draining the site of
immunization confirmed that both wild-type and
CTLA-4-/- cells had responded to the
immunization, as evidenced by enhanced proliferation and cytokine
production compared with unimmunized mice. However, there was no
obvious difference in in vitro responsiveness between wild-type and
CTLA-4-/- cells. The major effect of the CTLA-4
pathway in T cells responding to tissue Ag thus appears to be to limit
T cell expansion and accumulation rather than to alter T cell effector
differentiation.
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In our experimental model, 3A9 T cells failed to accumulate in the
pancreatic LN of RIP-HEL mice in the absence of immunization. When a
peripheral activation stimulus was provided by immunization,
3A9/CTLA-4-/- cells accumulated in the
pancreatic LN and the mice became diabetic, while 3A9/wild-type cells
failed to accumulate in the pancreatic LN and did not induce diabetes.
These data suggested that the interaction of the T cells with Ag at a
peripheral site could affect the subsequent response to encounter with
pancreas-derived HEL in the pancreatic LN. We were interested to
address whether the nature of the encounter with exogenously provided
Ag in the periphery influenced the subsequent response to tissue Ag.
Therefore, we altered the immunization conditions by preparing the HEL
protein in different adjuvants and examined the effect on accumulation
of Ag-specific T cells in the pancreatic LN. As described earlier
(Figs. 2 and 3), after immunization with alum-precipitated HEL, there
was increased accumulation of CTLA-4-/- T cells
compared with wild-type cells in the pancreatic LN of RIP-HEL mice,
implying that CTLA-4 expressed by wild-type cells was negatively
regulating T cell numbers. This was also the case if HEL was
administered without adjuvant in buffered saline (Fig. 6A). However, if HEL was
prepared in IFA or CFA, no difference was seen between the number of
3A9/CTLA-4-/- cells and 3A9/wild-type cells in
the pancreatic LN. The overall expansion in the LN draining the site of
immunization was equivalent between mice primed with HEL/alum compared
with HEL/IFA or HEL/CFA at this time point (data not shown), suggesting
that the differences observed in the pancreatic LN could not be
attributed to the production of an increased number of effector cells
in response to immunization in Freund’s adjuvant. Instead, the data
implied that T cells primed by interaction with HEL administered in
Freund’s adjuvant were less susceptible to regulation through CTLA-4.
Accumulation of 3A9 T cells in the pancreatic LN correlated with
induction of diabetes (Fig. 6B). Accordingly, after
immunization with HEL/PBS or HEL/alum, only
CTLA-4-/- cells accumulated in the pancreatic
LN and caused diabetes, whereas after immunization with HEL/IFA or
HEL/CFA both 3A9/wild-type and 3A9/CTLA-4-/-
cells accumulated (Fig. 6A), and both went on to induce
diabetes as evidenced by increased blood glucose levels (Fig. 6B). These data suggest that the context of T cell priming
in the periphery may influence the ability of CTLA-4 to regulate
subsequent responses to tissue Ags.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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),
are diminished in the presence of strong adjuvants. Thus, while CTLA-4
can clearly function to some extent in the presence of CFA (19, 20), our data suggest that regulation by CTLA-4 is maximal in
the absence of adjuvant, for example, during the T cell response to
tissue protein. Our experiments suggest that the chief effect of CTLA-4 in regulating T cell responses to tissue Ag is to control T cell numbers rather than to regulate T cell effector differentiation. CTLA-4 has been shown to regulate expression of the cell cycle proteins cyclin D3, cyclin-dependent kinase 4, and cyclin-dependent kinase 6 (21) and its role in controlling cell cycle progression is now well supported (22, 23). Mathematical studies of T cell proliferation have shown that costimulatory signals causing relatively small effects on the kinetic parameters regulating cell division can result in a large net increase in cell number (24). Thus, a relatively small effect of CTLA-4 at each division could over time result in a large increase in T cell numbers. The cumulative effects of CTLA-4 ligation would be reinforced by the increased levels of CTLA-4 protein expressed with each round of cell cycling (25). The consequences of CTLA-4 engagement are ultimately functionally significant since the presence or absence of this molecule in the adoptively transferred T cells can determine diabetes induction. Thus, the critical function of CTLA-4 may be to restrict the frequency of T cells after activation such that they fail to reach the threshold number required to cause pathogenicity.
In our study, the quantitative effect of CTLA-4 in controlling T cell numbers in the pancreatic LN translates into a qualitative effect on the aggressiveness of pancreatic islet infiltration, and ultimately leads to the dysregulation of glucose homeostasis. The observation that quantitative differences can trigger qualitative effects is reminiscent of observations made when bone marrow chimeras were used to dilute the number of TCR transgenic CD4 T cells specific for a pancreatic islet Ag (26): a high T cell precursor frequency triggered invasive islet infiltration, while at a low precursor frequency only peri-islet infiltration was seen. It also parallels recent studies on CD8 T cells specific for pancreatic-expressed Ag that demonstrate that quantitative differences in CTL precursors can affect qualitative parameters of a response, for example, the dependence on CD4 help (27). The conversion of the relatively innocuous peri-islet infiltration into aggressive infiltration by eliminating CTLA-4 function is consistent with previously published work using another TCR transgenic system (28).
The adoptive transfer of naive HEL-specific CD4 T cells into RIP-HEL recipients can be used to model the thymic release of self-reactive T cells that have escaped central deletion. The fact that such transfers do not induce diabetes suggests that the mere presence of self-reactive T cells in the peripheral repertoire is not in itself sufficient to trigger autoimmune pathology. Such a finding is consistent with the demonstration that T cells specific for self proteins exist in healthy individuals without detrimental consequences (29, 30). In fact, many animal models demonstrate a surprising absence of autoimmune pathology despite a high frequency of T cells specific for a self Ag (31, 32). This suggests that in the absence of an initiating trigger, such as infection or immunization, the frequency of self-reactive CD4 T cells is uncoupled from the induction of autoimmune tissue injury. In some cases, the lack of pathology may reflect immunological ignorance of the self protein. However, in other models, T cell responsiveness has clearly been modulated by exposure to pancreas-derived self Ag. For example, double transgenic mice bearing MHC class II-restricted hemagglutinin (HA)-specific TCR transgenic T cells in addition to pancreatic HA are resistant to lethal shock following injection of HA peptide, unlike their single transgenic TCR-HA counterparts (32). Despite this indication that the T cells have encountered pancreatic HA, progression to diabetes is rare in these double transgenic mice (32). Similarly, our results with RIP-HEL mice show that the absence of diabetes following transfer of naive T cells is not attributable to a lack of exposure to self Ag, since the transferred T cells undergo local proliferation in the pancreatic LN of RIP-HEL recipients.
The role of CTLA-4 in self tolerance was first highlighted by the
autoimmune phenotype of the CTLA-4 knockout mouse. Subsequent studies
have shown that blocking CTLA-4 Abs enhance autoimmune disease in
several experimental models (3, 4). When comparing data
derived from different systems, it is useful to remember that injecting
anti-CTLA-4 mAb in vivo could potentially modify the function of
CD4+CD25+ regulatory T
cells in addition to its effects on disease-inducing T cells. One
informative model uses BDC2.5 TCR transgenic mice in which the T cells
recognize an unknown pancreatic islet Ag (4, 28). In this
system, anti-CTLA-4 Abs or transfer of
CTLA-4-/- T cells result in accelerated
diabetes. Analysis of the V4+ population of T
cells, that contain the transferred BDC2.5 cells, failed to show
effects on cell division in the pancreatic LN after injection of a
blocking anti-CTLA-4 Ab (28). However, these
experiments were done by transferring T cells into lymphocyte-deficient
hosts; therefore, homeostatic proliferation may have masked the
responses to the self Ag. By varying the timing of anti-CTLA-4
injection, the authors showed that functional engagement of CTLA-4
occurs before the initiation of insulitis (4), consistent
with the early regulation of Ag-specific T cells in the pancreatic LN
revealed in our study.
It is noteworthy that ablation of CTLA-4 alone in T cells specific for
a pancreas-expressed protein fails to trigger diabetes in our model,
revealing a requirement for an initiating stimulus to tip the balance
between tolerance and pathogenicity. The idea that an infectious cue
might trigger autoimmunity receives support from a number of models.
Diabetes induction in the RIP-lymphocytic choriomeningitis
virus-gp P14 class I-restricted TCR-transgenic mice after
peptide injection is dependent on an additional trigger such as
injection of agonistic anti-CD40 Ab or LPS (33).
Similarly, there are many examples of diabetes being associated with
viral infection both in mice (34, 35) and humans
(36, 37, 38). In fact, our data suggest that depending on the
nature of the inciting trigger, even a functional CTLA-4 pathway may be
insufficient to prevent diabetes (Fig. 6).
In summary, this study implicates the CTLA-4 pathway in preferentially controlling T cell responses to endogenous tissue-expressed Ag rather than to immunized Ag. In combination with an inciting trigger such as immunization, the inability of CTLA-4-deficient T cells to regulate responses to tissue Ag results in a dramatic accumulation of T cells in the LN draining the site of tissue Ag expression, and ultimately leads to tissue invasion and injury. Postulated pathogenic mechanisms in autoimmunity are a susceptible genetic background and infections (39). Susceptibility genes may well turn out to encode proteins that participate in peripheral T cell tolerance, exemplified by CTLA-4 in the model we have used. Infection may incite autoimmunity by molecular mimicry or through bystander activation: in our system, this is paralleled by peripheral immunization. Therefore, our model can be used to dissect the complex interplay of endogenous control mechanisms and exogenous inciting triggers in the development of CD4 T cell-dependent organ-specific autoimmunity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Lucy Walker, Department of Pathology, University of California, Box 0511, Room Health Sciences West 518, 513 Parnassus Avenue, San Francisco, CA 94143. E-mail address: walkerl{at}itsa.ucsf.edu
3 Abbreviations used in this paper: HEL, hen egg lysozyme; RIP, rat insulin promoter; HA, hemagglutinin; LN, lymph node.
Received for publication August 9, 2002. Accepted for publication October 1, 2002.
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References |
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is essential for destruction of cells and development of insulin-dependent diabetes mellitus. J. Exp. Med. 185:531.-cells die through inflammatory cytokines and not perforin from autoreactive (anti-viral) cytotoxic T-lymphocytes. Diabetes 49:1801.[Abstract]
impacts at multiple points during the progression of autoimmune diabetes. Proc. Natl. Acad. Sci. USA 94:13844. affects homing of diabetogenic T cells. J. Immunol. 167:6637. cell neo-antigen. Immunity 2:573.[Medline]
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