Immunology 1999 96 600–605

A role for CD45RBlow CD38+ T cells and costimulatory pathways of T-cell activationin protection of non-obese diabetic (NOD) mice from diabetes

T. C. MARTINS* & A. P. AGUAS*† *Institute for Molecular and Cell Biology, and †Department of Anatomy of the Abel SalazarInstitute for the Biomedical Sciences, University of Porto, Portugal

SUMMARY

Non-obese diabetic (NOD) mice spontaneously develop autoimmune insulin-dependent diabetesmellitus (IDDM). Infection of the animals with mycobacteria, or immunization with mycobacteria-containing adjuvant, results in permanent protection of NOD mice from diabetes and we haverecently reported that the phenomenon is associated with increased numbers of interferon-c-producing T cells, possessing increased cytotoxic activity, and also with augmented numbers ofactivated immunoglobulin M-positive (IgM+) B cells. Here, we have investigated whetherprotection of NOD mice from IDDM was associated with changes on costimulatory pathways ofT and B cells, namely CD28/CTLA-4–B7 and CD40–CD40 ligand (CD40L) and we also furthercharacterized protective T helper (Th) cells with regards to the expression of the differentiationmarkers CD45RB and CD38. We report that Th cells involved in diabetes vaccination of NODmice by mycobacterial infection seem to belong to CD45RBlo CD38+ phenotype. The protectiveeffect of Mycobacterium avium infection is also associated with increased CD40L and CTLA-4-expressing Th cells and with the generation of a CD40− IgG+ B cells. Our data are consistentwith induction by mycobacterial infection of regulatory CD45RBlo CD38+ Th cells with theability to trigger deletion or anergy of peripheral self-reactive lymphocytes, with shutting downof IgG+ B-cell response. They also implicate a role for IgG+ B cells in the autoimmune aggressionof the endocrine pancreas of NOD mice.

INTRODUCTION which spontaneously develop autoimmune diabetes.11–15 Inaddition, it has been reported that diabetes-prone NOD mice

The physiological stimulation of T lymphocytes involves atdefectively up-regulate both cytotoxic T lymphocyte antigen-

least two activation signals: the first comprises the specific 4 (CTLA-4) and CD28 molecules upon activation and thatpresentation of antigen bound to the adequate major histocom- the Idd5 diabetes-susceptibility locus encompasses the genespatibility complex (MHC) molecule, and the second, the encoding these two molecules.16 The authors16 suggest thatso-called costimulatory signal, depends on the interaction of the aberrant expression of CD28 and CTLA-4 in NOD micenon-polymorphic proteins. Costimulatory interactions play will impair the control of T-cell activity contributing to thekey roles in the establishment of immune responses, namely autoimmune attack leading to diabetes.in the decision between tolerance and immunity, and therefore Another important molecule in T-cell activation is theare of particular interest in the expression of autoimmune CD45 surface receptor. It has been shown that expression ofdiseases.1–4 B7–CD28/CTLA-4 is believed to be the most its B isoform (CD45RB) in CD4+ T cells correlates withimportant costimulatory pathway of T-cell activation.5–7 functionally distinct T helper (Th) subpopulations that exertSimilarly, B lymphocytes also need costimulatory signals to a key role in the development of phenomena of autoagres-produce full-blown responses. The CD40–CD40 ligand sion.17 For instance, in Th1-mediated colitis, the mouse model(CD40L) pair is of particular relevance in costimulation of for the human inflammatory bowel disease, CD45RBhi Th cellsB cells.8–10 It is well established that interfering with these were identified as the subpopulation responsible for the self-interactions may either prevent or accelerate the onset of the aggression seen in this pathology, whereas CD45RBlo T cellsautoimmune disorder of non-obese diabetic (NOD) mice, corresponded to a protective phenotype.18–20 In NOD mice,

CD45RBlo CD4+ T cells were further divided into two distinctReceived 27 July 1998; revised 19 October 1998; accepted

subpopulations based on their effects on diabetes onset and27 November 1998.their pattern of cytokine production.21 Distinct subsets of

Abbreviations: CD40L, CD40 ligand; hi, high; IDDM, insulin- CD45RBlo Th cells were also identified by Powrie anddependent diabetes mellitus; lo, low; NOD, non-obese diabetic. co-workers, who postulated a correlation between CD38

expression and function of each subset (F. Powrie, personalCorrespondence: Dr T. Martins, Institute for Molecular and CellBiology, R. Campo Alegre, 823, 4150 Porto, Portugal. communication).

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T-cell activation and diabetes protection in NOD mice 601

We reported previously that insulin-dependent autoimmune Spleen cell suspensionsA standard procedure was used to prepare exhaustive celldiabetes (IDDM) of NOD mice can be prevented by infection

of the animals with Mycobacterium avium.22,23 The protective suspensions from spleen.26 Viable cells were counted by atrypan blue exclusion test.effect of the mycobacterial infection seems to be associated

with peripheral deletion of autoreactive T cells through theFas pathway, the regulatory T cells belonging to the Th1 Flow cytometric analysis of cell surface markers of splenic cellsphenotype.24 To investigate the possible contribution of altered Splenic cells (106) were incubated with 50 ml of each mAbcostimulatory signals, which may be crucial in the decision preparation for 20 min on ice, in the dark. After the cell pelletsbetween immunity and tolerance, we have decided to study has been washed three times in PBS–NaN3 0·01%–FCS 3%,whether the infection modified the expression of different their staining pattern was analysed using a Becton Dickinsoncostimulatory molecules on the cell surface of the lymphocytes FACSort flow cytometer (Mountain View, CA) interfaced toof NOD mice. We have also investigated CD45RB expression a Hewlett-Packard computer. Dead cells and erythrocytes wereon CD4+ T cells of NOD mice protected from diabetes by excluded from the analysis using a combination of forwardthe infection. light scatter and propidium iodide (Sigma Chemical Co.,

St Louis, MO) gating, as previously described.28

MATERIALS AND METHODS Statistical analysisThe numerical data were statistically compared using Student’sMicet-test. We have considered two numerical populations to beBreeding nuclei of NOD/Lt mice were established in thissignificantly different if P<0·05 or P<0·01; these differencesresearch centre from animals purchased from the Jacksonare, respectively, labelled with one or two asterisks in theLaboratory, Bar Harbor, ME. Female NOD mice were infectedfigures.at 8 weeks of age, i.e. before overt diabetes was observed. All

mice were confirmed to be negative for glucose in urine at thisage. Colorimetric strips were used to monitor glycosuria(Combur-Test, Boehringer Mannheim, Germany). RESULTS

To assess the contribution of altered costimulatory pathwayson M. avium-induced prevention of diabetes in NOD mice,Mycobacterial infection8-week-old mice were infected i.p. with mycobacteria. OneMycobacterium avium strain American Type Culture Collectionmonth after infection, when the host immune response to(ATCC) 25291, serotype 2, was grown in liquid culture at 37°mycobacteria is at its peak, as we have previously shown,22,23in Middlebrook 7H9 broth (Difco Laboratories TM, Detroit,we analysed the expression of costimulatory molecules on TMI ) containing 0·04% Tween-80. Mycobacterium avium is aand B lymphocytes of infected and age-matched controlspecies that causes human disease only in immune-deficientNOD mice.individuals; a previous report from this laboratory showed

We have previously reported the involvement of Th1-likethat NOD mice are naturally resistant to this infectious agent.22T cells in the prevention of IDDM by mycobacterial infectionThe mycobacteria were harvested from liquid culture byin these animals.24 We have now tried to characterize furthercentrifugation (6000 g) and washed three times in phosphate-that helper subpopulation, with a putative role in peripheralbuffered saline (PBS), as described before.25 The bacteria weredeletion of autoreactive lymphocytes; for that, we searchedsuspended in saline containing 0·04% Tween-80 and diluted tofor phenotypic distinctions between helper T cells of protecteda concentration of 2×108 viable bacilli of M. avium per ml.mice, based on the expression of CD45RB and CD38.Eight-week-old NOD mice were infected intraperitoneally (i.p.)

with 0·5 ml of the M. avium suspension in saline (i.e. 108 viablebacilli per mouse). The animals were killed 1 month afterinfection. Age-matched control NOD mice were inoculated Expression of CD80 (B7.1) and CD86 (B7.2) on B cellswith 0·04% Tween-80 in saline.

Full activation of T cells is dependent on costimulatory signalsprovided by activated B7.1/7.2-expressing B cells. Analysis ofB7.1 and B7.2 expression on B cells of infected NOD miceMonoclonal antibodies

Monoclonal antibodies (mAb) used in the flow cytometry was performed by flow cytometry. We found that the numbersof both IgG+ and IgM+ B7.1/B7.2-expressing cells wereanalysis of splenic lymphocytes of the mice were the following:

phycoerythrin (PE)-labelled anti-CD4, anti-CD3, anti-CD8, significantly increased in infected NOD mice than in controls(Fig. 1). However, when percentage values were compared,anti-CD80, anti-CD86, anti-CD40L and anti-CTLA-4; fluor-

escein isothiocyanate (FITC)-labelled anti-CD8, anti-CD4, the data revealed that the infection caused an increase in thepercentage of B7.2+ IgM+ B cells, whereas the percentage ofanti-immunoglobulin M (IgM), anti-CD45RB and anti-

hamster; cychrome-labelled anti-CD4 and purified hamster B7.2+ IgG+ B cells of infected mice remained similar to thatobserved in control NOD mice (Table 1). Furthermore, IgM+anti-mouse CD28 all purchased from PharMingen Inc. (San

Diego, CA); FITC-labelled anti-IgG was purchased from B cells of infected mice showed increased levels of B7.2expression, whereas IgG+ B cells presented reduced expressionSouthern Biotechnology (Birmingham, AL). A CD40L–CD8

fusion protein (kindly provided by Professor Carlos Martinez, of this costimulatory molecule (Fig. 2). The percentages ofB7.1+ B cells, either IgM+ or IgG+, were not altered inCNB, Madrid, Spain) was used to stain CD40. Isotype controls

were used for B7.1, B7.2 and CTLA-4 staining. response to the mycobacterial infection (Table 1).

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T. C. Martins & A. P. Aguas602

Table 1. Increased percentage of B7.2+ IgM+ B cells in infected NODmice

IgM+ B7.1+ IgM+ B7.2+ IgG+ B7.1+ IgG+ B7.2+Control 18·98±5·36 14·55±3·12 22·24±5·39 13·57±1·91M. avium† 17·28±3·36 41·17±7·63* 19·63±6·18 16·61±3·00

*P<0·0001 infected versus control. †See the Materials andMethods. Results are means of six samples.

100

IgG

+

70

0

Co

M. avium

IgM

+

101

102

103

104

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

Co

CoCo

M. avium M. avium

M. avium

70

0

70

0

70

0

B7.2 B7.1

Figure 2. IgM+ B cells from infected mice show increased expressionof B7.2 molecules. B7.1 and B7.2 expression on B cells from infectedand control mice was compared by flow cytometry. IgM+ B cells frominfected NOD mice had increased levels of B7.2 expression, whereason IgG+ B cells the expression of this molecule was decreased.

B7.1

IgG

+ B

cel

ls

B7.2

1,0E+09

1,0E+08

1,0E+07

1,0E+06

1,0E+08

1,0E+07

1,0E+06B7.1 B7.2

IgM

+ B

cel

ls

Control

M. avium

Figure 1. Mycobacterium avium infection induces increased numbers 100

CD

4+

50

0

Co

M. avium

101

102

103

104

100

101

102

103

104

Co

M. avium

70

0

CD28 CTLA-4

of B cells expressing B7 molecules. B7.1 and B7.2 expression on theFigure 3. CTLA-4 expression is increased on CD4+ T cells of infected

surface of IgM+ and IgG+ B cells was performed by flow cytometryNOD mice. The levels of expression of CD28 and CTLA-4 in T cells

after mycobacterial infection or 0·04% Tween-80 in saline injection asof M. avium-infected and control mice were analysed by flow cytome-

control. There was a statistically significant increase in the number oftry. Mycobacterial infection induced increased expression of CTLA-4

both IgM+ and IgG+ B cells that express costimulatory moleculeson Th cells.

from B7 family.

Table 2. Mycobacterium avium infection induces an increase inCTLA-4 and CD28 expression on T cells CD40− IgG+ B lymphocytes and a concomitant decrease in the

CD40+ subsetCD28 and CTLA-4 are the coreceptors on the T cell forB7.1/7.2. The levels of expression of CD28 in T cells were CD40+ (%) CD40− (%)similar in control and infected NOD mice. In contrast, therewas an increase in CTLA-4 expression in CD4+ T cells of Control 81·47±1·17 18·54±1·17infected mice (Fig. 3). The proportions of cells expressing M. avium† 58·56±6·22* 41·44±6·22*either of these two molecules were also not changed by

*P<0·01 infected versus control. †See the Materials and Methods.infection. As expected, the augmentation in the number ofResults are means of six samples.cells that stained for CD28 and/or CTLA-4 was due to the

general increase observed in T cells of infected mice.in IgM+ B cells or in the percentage of CD40+ IgM+B lymphocytes. The enhancement observed in the total number

Expression of CD40of CD40-expressing IgM+ cells accompanied the generalincrease seen in this subpopulation of B cells.CD40–CD40L interactions are of particular relevance in

T-cell-dependent B-cell activation. Analysis of CD40expression on B lymphocytes of infected NOD mice revealed

CD40L expression by CD4+ T cellsthe existence in these mice of a subpopulation of IgG+ B cellsthat does not express CD40, which was not present in control In addition to its crucial role in T-cell-dependent B-cell acti-

vation, CD40–CD40L interactions are also important formice (Fig. 4). There was also a decrease in the proportion ofCD40-expressing IgG+ B lymphocytes (Table 2). In contrast, T-cell responses. Therefore, we also examined the expression

of CD40L on CD4+ T cells of infected and control NODthere was no alteration in the levels of expression of CD40

© 1999 Blackwell Science Ltd, Immunology, 96, 600–605

T-cell activation and diabetes protection in NOD mice 603

CD45RBlo

CD

4+ T

cel

ls

1,0E+09

1,0E+08

1,0E+07

Control

M. avium

Figure 5. Infected NOD mice show increased numbers ofCD45RBloCD4+ T cells. Infection of NOD mice with mycobacteriaresulted in enhancement of CD45RBlo Th cells.

Table 4. Mycobacterial infection in NOD mice induces an increase inCD45RBlo CD38+ and a decrease in CD45RBlo CD38− Th cells

CD45RBloCD38+ CD45RBloCD38−(%) (%)

Control 15·84±2·36 53·1±16·27M. avium† 31·3±3·2** 16·27±5·1*10

0

IgG

+

20

0

Co

M. avium

IgM

+

101

102

103

104

100

101

102

103

104

Co

M. avium

20

0

CD40

Figure 4. Mycobacterium avium infection results in the appearance of*P<0·05; **P<0·001 infected versus control. †See the Materialsa CD40− IgG+ B-cell subset. Analysis of CD40 expression on

and Methods. Results are means of six samples.B lymphocytes of either control or infected NOD mice showed theexistence in infected animals of a subpopulation of IgG+B lymphocytes that do not express CD40. aggressive disorder that occurs spontaneously in these

animals.22,23 We have also proposed that the likely mechanismmice. We found that there is an increase in the proportion of responsible for this protection is peripheral deletion of autore-CD4+ T cells expressing the ligand for CD40 in infected active lymphocytes, through the Fas death pathway, effectedanimals (Table 3). by Th1-like T cells of enhanced cytotoxic activity.24 We report

now that infected NOD mice show altered expression ofdifferent costimulatory molecules, on B and T lymphocytes,Phenotypic characterization of Th cells based on CD45RB andand an augmented subpopulation of CD45RBlo CD38+CD38 expressionTh cells.

Different subpopulations of Th cells may be distinguished Powrie et al.17 have recently shown that CD4+ T cells maybased upon their expression of CD45RB. Different patterns be divided into two distinct subpopulations depending on theirof CD38 expression also correlate with distinct T-cell functions. expression of the isoform B of the leucocyte common antigen,Analysis of expression of CD45RB on CD4+ T cells of NOD CD45. Furthermore they showed in a murine model of humanmice showed that mycobacterial infection induced a significant inflammatory bowel disease that CD45RBhi CD4+ T cellsincrease in CD45RBlo T lymphocytes (Fig. 5), the majority were the cells responsible for the Th1 autoimmune attack,being CD38+ cells (Table 4). Conversely, there was a decrease whereas CD45RBlo Th cells were protective and prevented thein the CD45RBlo CD38− population of Th cells (Table 4). phenomenon of autoaggression, probably through trans-CD45RBhi Th cells showed no significant change (data not forming growth factor-b (TGF-b)secretion.18–20 Han andshown). co-workers28 also reported a TGF-b-producing regulatory

T-cell population, which prevents autoimmune diabetes inNOD mice. Shimada and co-workers21 reported the existenceDISCUSSIONin NOD mice of two distinct subpopulations of CD45RBlo

We have shown before that M. avium infection of NOD mice Th cells, one having beneficial and the other harmful effectsprevents development of autoimmune diabetes, an auto- on diabetes development. These two functionally distinct Th

subsets had different patterns of cytokine expression.Table 3. Mycobacterium avium infection induces an increase in the Interestingly, we found that NOD mice, made resistant to

percentage of CD40L+ Th cells diabetes by mycobacterial infection, have an increased sub-population of CD45RBlo Th cells. Although these results may

CD4+CD40L+ T cells (%)seem contradictory to the observations of Shimada et al.,21we think that our data rather complement their findings. WeControl 9·28±1·8believe that the previously described functional transition fromM. avium† 21·58±1·5*protective to aggressive CD4+ CD45RBlo T cells reflects, infact, the coexistence of two distinct subsets with distinct*P<0·05 infected versus control. †See the Materials and Methods.

Results are means of six samples. dominance, one over the other, during the development of

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T. C. Martins & A. P. Aguas604

autoimmune diabetes. The CD45RBlo Th cells described here whereas IgG+ B cells show reduced expression of thismolecule;may be TGF-b-secreting cells, and belong to the same regulat-

(2) CTLA-4 expression is increased in CD4+ T cells;ory population as independently described both by Powrie(3) There is an enhanced percentage of CD40L-expressinget al.19 and Han et al.28 Curiously, they may also produce

Th cells; andinterferon-c (IFN-c), in addition to TGF-b.28 Our previous(4) Infection induces a subpopulation of IgG+ B cells thatwork has also demonstrated the involvement of IFN-c-

does not express CD40.secreting cells in the prevention of IDDM by mycobacterialinfection.24 Signalling through CD40, concomitant with immunoglob-

Further characterization of CD45RBlo Th cells was made ulin triggering, induces the expression of survival genes onby flow cytometric analysis of CD38 expression, as B cells, such as bcl-xL.6 The lack of costimulation throughT lymphocytes that express CD38 have been shown to possess CD40 may then impair transcription of these factors, eventu-unique regulatory functions.29,30 Indeed, Powrie and ally sensitizing the cells for apoptosis. The down-regulation ofco-workers reported the existence of two subsets of CD45RBlo CD40 on IgG+ B cells may help in the prevention of diabetesTh cells, which could be phenotypically distinguished by their as these cells are potentially more aggressive than IgM+surface expression of CD38, and to which were addressed B cells. We also found that the CD40L CD4+ subpopulationdifferent functions (F. Powrie, personal communication). It was increased in response to mycobacterial infection.was postulated that among CD45RBlo Th cells, CD38+ cells Alternatively, the CD40− IgG+ B lymphocytes may also haveconstituted a T-cell population with regulatory properties, a role in T-cell tolerization. In fact, B-cell dependent T-cellwhere as CD38− cells were antigen-exposed, differentiated costimulation in the absence CD40–CD4L interaction leadsT cells, which could be inhibited by the former cells. to tolerization of T lymphocytes.4,8,34

These data, together with our previous reports,24 suggest Together, these results may indicate the preferential inter-that prevention of insulin-dependent autoimmune diabetes is action of CD4+ T cells with IgM+ B cells, suggesting a roleassociated with induction/activation of a new for IgM+ B cells in IDDM prevention in infected NOD mice.CD45RBlo CD38+ regulatory subset, distinct from Th1 and This is further supported by the observation of increased B7.2Th2 cells and similar to the one described by other research expression on IgM+ B cells, which is not observed in IgG+groups28,31 (F. Powrie, personal communication). These cells B lymphocytes; in fact IgG+ B cells showed decreased levelsmay suppress Th1-mediated autoimmune diseases by two main of expression of this costimulatory molecule. The ‘shutting-mechanisms: TGF-b production19,30 and peripheral deletion down’ of the CD40 pathway may account for the failure ofof autoreactive lymphocytes through the Fas death pathway.24 these cells to up-regulate B7.2. Since B-cell activation and

The major T-cell costimulatory pathway involves the proliferation seems to depend critically upon the presence ofCD28/CTLA-4–B7 family of costimulatory molecules. CD28 cognate T-help, absence of CD40–CD40L interaction willis expressed on the majority of naıve and memory T cells. interfere with proliferation of specific B cells, namely IgG+Activation of T cells with anti-CD28 mAb blocks the induction B cells reported here, and therefore reduce class II-associatedof anergy and synergizes with anti-CD3 stimulation to increase antigen presentation by these B cells.33 Again, IgM+ B cellsboth T-cell proliferation and lymphokine production.11,32 seem to interact preferentially with T lymphocytes, namelyThere are two natural ligands to CD28: B7.1 (CD80) and B7.2 Th cells, in this model of diabetes prevention. Even though(CD86), which are expressed on professional antigen- the up-regulation of B7.2 may seem paradoxical, once it is anpresenting cells. CTLA-4 is a cell surface molecule present on important counter-receptor for T-cell costimulation, this obser-activated CD4+ and CD8+ T cells. It has been suggested that vation further supports our postulate of tolerance mediatedCTLA-4 molecules have a crucial role in the termination of by peripheral deletion via Fas of autoreactive lymphocytes inimmune responses.33 Similarly to T cells, B cells also depend this experimental model. Indeed, Boussiotis and co-workersupon costimulatory signals. T-cell-dependent B-cell activation have recently shown that one of the outcomes of costimulationis achieved by interaction of CD40, on B cells, with its ligand, of T cells through CD28 is priming of activated T cells foron T cells. Blockade of this interaction prevents isotype activation-induced cell death via Fas–FasL interactions.35 Theswitching and germinal centre formation by B cells.4–6 up-regulation of CTLA-4 in CD4+ T cells of infected mice

There are several reports of prevention or acceleration of suggests a role for this molecule in the protective effect of M.IDDM in NOD mice by the interference with these costimu- avium infection, possibly through its well-known active role inlatory pathways. Anti-B7·2 mAb treatment was shown to the termination of immune responses.36 Our results are consist-prevent the development of the disease, whereas anti-B7.1 mAb ent with data reported by Ludher et al.,15 who showed thataccelerated the onset of diabetes.12 Also, blockade of engagement of CTLA-4 at the time when potentially diabeto-CD40–CD40L interactions led to the prevention of the dis- genic T lymphocytes are first activated may be a pivotal eventease.13 Arreaza and co-workers14 suggested a role for CD28 in protection of NOD mice from diabetes.costimulation on the prevention of diabetes. Interestingly, In conclusion, we propose that the protective effect ofprevention of diabetes in NOD mice by treatment of the mycobacterial infection against diabetes of NOD mice mayanimals with anti-CD40L13 was not associated with switching involve the shutting down of CD40–CD40L interactions onof the response from a Th1 to a Th2 profile. IgG+ B cells, inhibiting the B-cell ‘aggressive’ arm (IgG+

We report here that prevention of diabetes in NOD mice B cells) in autoimmune pathogenesis, leaving intact B-cell–T-by M. avium infection is associated with significant alterations cell costimulatory interactions, through IgM+ B cells.in the expression of costimulatory molecules: Costimulation of T lymphocytes by interaction with IgM+

B cells would enable full activation of regulatory T cells,possibly CD45RBlo CD38+, IFN-c- and TGF-b-secreting cells.(1) B7.2 expression is increased on IgM+ B lymphocytes

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T-cell activation and diabetes protection in NOD mice 605

form two distinct subpopulations, defined by their expression ofWe again suggest that the mechanisms through which theseisoforms of the leukocyte common antigen, CD45. Immunologyregulatory T cells may induce a state of tolerance in M. avium-70, 427.infected NOD mice include: (a) peripheral deletion of autoreac-

18. P F., L M.W., M S., M S., C L.B. &tive cells via Fas;24 (b) suppression of autoreactive T cellsC R.L. (1994) Inhibition of Th1 responses preventsthrough TGF-b production by CD45RBlo CD4+ T cells;inflammatory bowel disease in scid mice reconstituted with

(c) shutting-down of the B-cell aggressive arm through CD45RBhi CD4+ T cells. Immunity 1, 553.down-regulation of CD40 expression on IgG+ B cells; and 19. P F., C J., L M.W., M S. & C R.L.(d) termination of the autoimmune response by CTLA- (1996) A critical role for transforming growth factor-b but not4-mediated signals. interleukin 4 in the suppression of T helper type 1-mediated colitis

by CD45RBlow CD4+ T cells. J Exp Med 183, 2669.20. C R.J., F C.J., F J.G., D C.A., P F. &

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