| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
T Cell Involvement1
,
,
*
Department of Microbiology Pontificia Universidade Catolica do Rio Grande do SulAv, Porto Alegre, Brazil;
Department of Pediatrics, Division of Basic Sciences, National Jewish Medical and Research Center, Denver, CO 80206; and
Department of Immunology, University of Colorado Health Sciences Center, Denver, CO 80262
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and 
ß T cells from unprimed mice to
proliferate in vitro, cells are not required for the strong
humoral response, which is indistinguishable in normal and T
cell-deficient mice. Thus, the unusual immunogenicity of this protein
in eliciting a humoral response appears to be due to a strong ß T
cell response with no evidence of natural priming or a T cell
involvement. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The molecular mechanisms by which hsp70 exerts immunogenicity in vivo
as well the identity of the cellular targets, however, are unknown.
Hsps have been associated with T cell responses (14, 15, 16), and in
mice, mycobacterial hsp70 stimulates intestinal ß and T
cells (17). These observations have led investigators to hypothesize
that the immune system is naturally primed by hsp70 of gut flora (18, 19). Accordingly, the apparent "adjuvanticity" of hsp70 could be
simply a consequence of stimulating previously generated memory T
cells.
To fully exploit the potential of this protein in vaccine design and to
avoid potential autoimmune complications, it is necessary to understand
the mechanisms through which hsp70 interacts with and stimulates the
immune system. In this article, we report that the strong humoral
immune response to hsp70 itself is associated with ß T cells.
T cells, although stimulated by hsp70, are not required for the
humoral response to hsp70. Finally, it appears that animals are not
naturally primed by gut floral hsp, since germfree mice are able to
mount an IgG response to hsp70 that is comparable with the one mounted
by non-germfree controls and because there is no evidence of
hsp-specific ß T cell expansions in animals that are not
deliberately immunized.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The following specific pathogen-free (SPF) mice were purchased
from The Jackson Laboratories (Bar Harbor, ME) and maintained at the
National Jewish Biological Resource Center animal facility:
BALB/c, C57BL/6, B10.BR, C3H/HeJ, BALB/c nu/nu and
+/nu, Tcrb/Tcrb (TCRß-deficient on a C57BL/6
background), and TCR-deficient on a mixed 129 x C57BL/6
background. Germfree Black Swiss mice, as well as SPF age- and
sex-matched Black Swiss controls, were purchased from Taconic Farms
(Germantown, NY) and maintained in a sterile isolator at the National
Jewish Biological Resource Center. The germfree condition of the
animals was monitored daily by culture of fecal samples on blood agar
plates. All mice were female and between 6 and 12 wk of age except for
the Tcrb/Tcrb mice and heterozygous littermates, which were
male.
Ags and mitogens
Recombinant hsp70 from M. tuberculosis was provided by the World Health Organization and also purified by us from cultures of E. coli transformed with plasmid pY3111 (a gift from Dr. Douglas Young), as described (20), followed by passage through a Detoxi Gel column (Pierce, Rockford, IL) to eliminate potential endotoxin. IFA, CFA, keyhole limpet hemocyanin (KLH), OVA, LPS, and polymyxin B were purchased from Sigma (St. Louis, MO).
Immunizations
Ag was delivered in a total volume of 200 µl. Mice were immunized by i.p. injection with 1, 5, or 20 µg of Ag, in PBS alone or emulsified with IFA or CFA. Serum was obtained from tail artery blood.
Antibodies
The following mAbs, provided by investigators from the National
Jewish Medical and Research Center and used for FACS analysis, were
purified from hybridoma culture supernatants and conjugated to biotin:
53.7 (anti-CD5) (21) (American Type Culture Collection, Manassas,
VA), 53.6 (anti-CD8) (21), RA3-6B2 (anti-B220) (22) and BET-2
(anti-IgM) (23), H57-597 (anti-TCR-ß) (24), GK1.5
(anti-CD4) (25), 145-2C11 (anti-CD3) (26), and 405A10
(anti- TCR) (27). Anti-bromodeoxyuridine (BrdUrd) mAb
conjugated to FITC was purchased from Becton Dickinson (San Jose, CA).
Allotype-specific anti-isotype mAbs, conjugated to biotin or to
alkaline phosphatase (AKP), were purchased from PharMingen (San Diego,
CA). The following mAbs, previously produced and purified by the
senior author (L.J.W.), were used as isotype controls: P65D6-3
mouse IgM, anti-p-azophenylarsonate (Ars); mAb 36-65
mouse IgG1, anti-Ars; P65D6-5 mouse IgG2a anti-Ars; and HPSuf-1
mouse IgG2b, anti-p-azophenylsulfonate (Sulf). Goat
anti-IgM and anti-IgG, not labeled or AKP conjugated, were
purchased from Sigma.
ELISA
Ninety-six-well plates (Becton Dickinson) were coated with hsp70, 2 µg/ml in PBS, overnight at 4°C. Both these and control uncoated plates were incubated with blocking buffer (PBS with 0.02% BSA, 0.01% gelatin, 0.05% Tween-20, and 0.02% thimerosal) for at least 30 min at room temperature. After washing with PBS, serial dilutions of mouse sera in blocking buffer were incubated (50 µl/well) in duplicate at room temperature for 1 h. The highest concentration of serum tested was a dilution of 1:50. Plates were washed six times with PBS and incubated for another hour at room temperature with specific anti-isotypes labeled either with biotin or AKP. When biotinylated Ab was used, an extra step of washing and incubating with streptavidin-AKP (PharMingen), diluted in blocking buffer, for 1 h at room temperature was performed. Bound Abs were developed using substrate for AKP (Sigma) diluted to 0.5% in a buffer consisting of 1 M Tris base, 0.5 mM zinc chloride, and 2 mM magnesium chloride. ODs were read after 2 h in an ELISA reader (model 2550; Bio-Rad, Richmond, CA) at 410 nm, and the last positive dilution was determined. Isotype reagents were tested with mAbs directed to Ars and Sulf listed above, using haptenated BSA-coated wells or hsp70-coated wells (control).
Cell preparation
Single-cell suspensions of spleen or lymph node cells were obtained by disruption of the organs between frosted microscope slides in RPMI supplemented with FCS (5%). Bone marrow was obtained from femurs by flushing with the same medium using a needle and syringe. After two washes, red cells from spleen and bone marrow were lysed with ammonium chloride. Peritoneal cells were obtained by injecting ice-cold PBS with 2% FCS and recovering the injected volume with a Pasteur pipette. All cells were washed three additional times with medium and counted in a Coulter Counter (Hialeah, FL). T cells were purified by two passages through nylon wool (28), with purity determined by FACS after each passage. Percentages of CD3+ cells were approximately 35–45% before nylon wool purification, 80–85% after the first passage, and 90–95% after the second passage.
Enzyme-linked immunospot (ELISPOT) assay
An ELISPOT assay was performed as described (29), with the following modifications. Ninety-six-well flat-bottom plates (Fisher, Pittsburgh, PA) were coated with Ag, 5 µg/ml in PBS overnight at 4°C, and blocked for 30 min at 37°C with PBS made 5% in powdered skim milk. After washing with PBS, cells isolated as described above were plated in serial dilutions in RPMI without FCS. After incubation for 3–5 h at 37°C with 5% CO2 in air, the cells were removed, and the plates were washed extensively with PBS and incubated for 1 h at room temperature with specific anti-isotype Abs conjugated to AKP diluted in PBS made 1% in powdered skim milk. After extensive washing with PBS, the bound Abs were developed with BCIP (5-bromo-4-chloro-3-indolyl-phosphate; Sigma) diluted to 1 mg/ml in 50 ml AMP buffer (Sigma), with incubation for 1–3 h at 37°C. The plates were then washed with water and air dried, and the spots were counted using an inverted microscope. The frequency of Ab-forming cells in 106 total cells was calculated through a linear regression. Reagents were tested using the anti-Ars and anti-Sulf hybridomas listed above on haptenated BSA-coated wells or hsp70-coated wells (controls).
Adoptive transfers
BALB/c mice (Igha) were irradiated with 560 rads and used as recipients of single-cell suspensions of spleen and/or peritoneal cells isolated from other BALB/c mice or from C.B17 mice (Ighb). Recipients had 2 x 107 spleen cells/individual delivered by tail vein injection, in 200 µl of RPMI without sera, and/or 2 x 106 peritoneal cells injected i.p.
Proliferation assays
Spleen cells of mice with or without immunization were resuspended in RPMI with 10% FCS at a concentration of 1.25 x 106 cells/ml and cultivated for 72 h at 37°C in 5% CO2 with 12.8 µg/ml of hsp70 or molar equivalents of control Ag. Cells destined for quantification of [3H]thymidine incorporation were plated in triplicate. After 72 h, 1 µCi of [3H]thymidine (ICN, Irvine, CA) was added to each well; the cells were harvested 8 h later in a printed Filtermat A (Wallac, Turku, Finland), and the incorporation was determined in a Microbeta 1450 (Wallac). Cells destined for flow cytometry were plated in petri dishes, 10 ml/plate, 1.25 x 106 cells/ml, with Ag as described above. For BrdUrd incorporation assays, 20 µg/ml BrdUrd (Sigma) was added after 72 h of culture. The cells were then incubated for 8 h, centrifuged in Ficoll-Hypaque (Pharmacia, Piscataway, NJ), resuspended to 5 x 105 cells/ml in T cell medium, and stimulated with 50 U of recombinant IL-2 (Becton Dickinson) for 12 h before harvest and analysis by FACS.
In some assays, T cells were purified as described above, resuspended to 4 x 106/ml in RPMI without sera, and plated in 96-well tissue culture plates (Fisher) (100 µl/well) with an equal number of irradiated (3000 rads) spleen cells (100 µl/well) serving as APC. Ag was added in PBS in a volume of 20 µl/well. After 24 h of culture at 37°C in 5% CO2, 20 µl/well of FCS was added. One µCi of [3H]thymidine (ICN) was added to each well 3 days later, and incorporation was measured 24 h later as described above.
Flow cytometry
Cells (5 x 106 cells/ml) prepared as described above were washed three times with staining buffer (cold PBS with 2% FCS, 0.1% NaN3) and incubated with mAbs conjugated to biotin or FITC for 20 min on ice. They were then washed and incubated with streptavidin-phycoerythrin (Becton Dickinson) for another 20 min. After three more washes, the cells were analyzed on a Coulter 751 flow cytometer (Hialeah, FL) with CICERO acquisition software (Cytomation, Ft. Collins, CO).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
BALB/c mice that were immunized i.p. with 20 µg of hsp70 in IFA
produced high titers of IgG anti-hsp70 as early as 7 days after
immunization (Fig. 1
). Titers commonly
reached 4,000 at day 7 before climbing to 
20,000 on day 28 (Fig. 2). The response was T cell dependent, as
deduced from its absence in nude mice and in TCR
ß-deficient mice (Fig. 1). Contrary to what was observed upon
immunization with conventional Ag (OVA or KLH), IgM was below detection
in this assay, in which sera were titered starting at a dilution of
1/50 (Fig. 1). This strong day 7 IgG response occurred reproducibly in
other mouse strains. In particular, its occurrence in C3H/HeJ mice,
which are insensitive to LPS, argues against a potential role for
endotoxin (Fig. 3). The strength of
response was immunogen dose dependent, at least up to 20 µg, when
delivered in IFA (Fig. 2). If the Ag was delivered i.v., titers were
lower and more variable, but the day 7 IgG response could still be
detected in some animals. Western blotting analysis confirmed that sera
of animals immunized with hsp70 reacted exclusively with a 70-kDa
protein (data not shown).
|
|
|
Contrary to what would be expected if the response were
anamnestic, there was no detectable anti-hsp70 Ab in preimmune
serum (Fig. 1
), and none was observed until day 7 after immunization,
when the Ab was already IgG. In spite of this, cells producing
IgM that apparently binds hsp70 were detected by ELISPOT assay before
immunization. They were more abundant in the peritoneum than in the
spleen (Table I). Their frequency
increased upon immunization. Immunization also resulted in the
appearance of cells secreting IgG anti-hsp70 in the spleen but not
in the peritoneum. The discrepancy between IgM-secreting cells and the
apparent absence of anti-hsp70 IgM in the serum of both preimmune
and immunized animals is unknown but could be due to abortive
activation; rapid isotype switching; or secretion of low-affinity,
polyreactive IgM by B1 cells. In this regard, we have noted that the
IgM spots tend to be very small. If the serum contained any
anti-hsp70 IgM, however, it was present only at low levels, because
we could not detect it at a dilution of 1:50.
|
). Variations of this experiment were
performed with similar outcomes six times, including one experiment in
which splenic and peritoneal cells were distinguished by Ig allotype
differences (data not shown).
|
isotype in unimmunized
mice and the lack of an Ab response in nude and TCR
ß-deficient mice suggested that the unusual character of the hsp70
response might be attributed to Th cells. This was supported by an
experiment in which the strong response was elicited to a new epitope
introduced to hsp70. Mice were immunized with hapten conjugates (Ars)
of either hsp70 or a control carrier (KLH). The rapid IgG response with
no detectable accompanying IgM response was elicited to Ars by
immunization with Ars-hsp70 (Fig. 5A). In contrast, mice
immunized with Ars-KLH (Fig. 5B) showed a typical primary
anti-Ars response of predominantly IgM. While titers of
anti-Ars and anti-hsp IgG were not as high in mice immunized
with Ars-hsp70 as Ab titers to hsp70 in preceding experiments, this may
well be due to damage to the carrier hsp70 during the hapten
conjugation, since the titers of both Abs were depressed to a
comparable degree. Imparting strong immunogenicity to the Ars hapten by
conjugating it to the hsp70 carrier implies that the peculiarities of
the anti-hsp70 humoral response are most likely a function of Th
cells.
|
We next examined the effect of hsp70 on splenic T cells in vitro.
T cells purified from spleen of unprimed BALB/c mice were incubated
with different concentrations of hsp70, using irradiated total spleen
cells as a source of APC. A dose-dependent proliferation measured by
incorporation of [3H]thymidine was detected (Fig. 6). Unexpectedly, proliferation was also
observed in cultures consisting of T cells and Ag, without the
deliberate addition of APC. However, in these cultures 90–95% of the
cells were CD3+, leaving open the possibility that residual
APC contributed to some or all of the observed proliferation. To
address the possibility that proliferation might be due to
contaminating LPS, we cultured total spleen cells from unimmunized
BALB/c and C3H/HeJ mice with hsp70 alone, hsp70 plus polymyxin B, or a
control Ag (OVA). In BALB/c mice, in which LPS-induced proliferation
was almost completely inhibited by polymyxin B (Fig. 7A), the addition of polymyxin
B to the hsp70-stimulated cultures reduced [3H]thymidine
incorporation by approximately 50% (Fig. 7B). On the other
hand, in C3H/HeJ mice, which are hyporesponsive to endotoxin, hsp70
still induced proliferation of spleen cells from unprimed mice, and the
addition of polymyxin B to the cultures did not have an effect on
[3H]thymidine incorporation (Fig. 7C). These
results indicate that a significant amount of proliferation occurs that
cannot be attributed to contamination by endotoxin. They also suggest
that some of this proliferation may be APC independent, although this
is not crucial to any subsequent argument we wish to make.
|
|
T
cells and B cells (data not shown). Given these results and previous
reports implicating a role for T cells in immunity to hsps, we
sought to determine whether T cells might be playing a role in
the development of the humoral immune response in vivo. To this end, we
immunized TCR -deficient (Tcrd) mice with hsp70. The
results indicate that T cells are not required for the strong
rapid anti-hsp70 humoral response, because the deficient mice and
the heterozygote littermates responded similarly (Fig. 8). Concurrently, proliferation in
response to hsp70 by cells derived from Tcrd mice was
reduced by approximately 44% relative to heterozygote littermate
controls (Fig. 9). This reduction is
close to the proportion of BrdUrd incorporation attributed to T
cell division in culture assays, as seen in the top half of Table II.
|
|
|
), suggested that the
strong Ab response to hsp70 is dependent upon ß T cells. If so,
then hsp70 should induce a strong proliferation of ß T cells taken
from hsp70-primed mice. To test this prediction, we set up an assay to
measure [3H]thymidine incorporation. To be sure that
actual proliferation was being measured, as opposed to preferential
survival in vitro, a staining assay for surface markers was also
conducted in concert with BrdUrd incorporation. Total spleen cells from
unimmunized BALB/c mice or from mice immunized with hsp70 14 days
previously were cultured with either hsp70 or OVA (Table II). In this
experiment, hsp70 stimulated proliferation of both T cells and
B220+ cells as suggested by preceding cell recovery
experiments (data not shown). However, the proliferation index was
virtually the same regardless of whether these cells were derived from
immunized or unimmunized mice. On the other hand, relative
proliferation of ß T cells to hsp70 was significant only for cells
derived from hsp70-immunized mice (Table II). These data confirm the
results of the previous frequency analysis, and they suggest that the
stimulating effect of hsp70 on cells and B cells in vitro was
not necessarily related to the humoral response. Importantly, the
results also indicate that previous natural priming was probably not
the explanation for the strong, rapid IgG response to hsp70. If priming
had occurred, no difference in ß T cell proliferation would be
expected between unimmunized and deliberately immunized mice. The anti-hsp70 response in germfree animals
In a second test of the natural priming hypothesis, we immunized
three groups of germfree black Swiss mice and sex-/age-matched SPF
controls and bled the animals 7 days later. The germfree and control
mice produced strong and comparable Ab responses to hsp70 (Fig. 10). Thus, the strong IgG response and
lack of IgM appear to be independent of the presence of natural gut
flora, in argument against the natural priming hypothesis.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß Th cells. There is no evidence that Th cells have been
naturally primed by gut floral hsp70, as revealed both in proliferation
assays and by conservation of the response in germfree animals. We
cannot formally exclude the possibility that natural priming might have
occurred orally with dietary hsp70 protein or peptide fragments
thereof. However, we consider this unlikely because the strong response
was reconstituted with splenocytes, because there was a clear
difference in the extent of ß T cell proliferation between cells
of deliberately primed and unprimed mice, and because we have been
unable to influence the response by deliberate oral administration of
hsp70 (unpublished observation). In addition, oral routes of Ag
delivery are more likely to induce tolerance rather than immunity (30, 31). The comparable responses to hsp70 observed in germfree and control mice lead us to believe that natural priming to hsp70 by gut flora has not occurred. We believe this finding is in agreement with clues present in the literature. In a recent study of spontaneously colitic mice, Brandwein et al. (32) identified major bacterial Ags recognized during the course of the disease, none of which had a mass of 70 kDa. Hsp70 and hsp65 are coordinately induced in response to the same stimuli (33). In some species of bacteria, they are encoded in the same operon and, consequently, cotranscribed (34). Natural priming by one hsp should therefore be accompanied by natural priming to the other, whether by gut flora or by dietary hsp. Yet the capacity of hsp70 to function as an exceptionally strong carrier protein is not paralleled by hsp65, despite the fact that the latter is potentially immunogenic (7, 8) and that peptides able to bind class II MHC have been identified from both of these bacterial hsps (35, 36, 37).
We expected that natural priming by hsp70 might lead to natural serum Ab or to IgG-expressing memory cells, but neither of these were detectable in unimmunized mice. B cells producing IgM that apparently binds hsp70 were observed by ELISPOT assay; however, the results of adoptive transfer experiments argue against their role in the strong response. These cells were present at highest frequency in the peritoneum, and when transferred to adoptive hosts, they did not directly contribute to the serum Ab response to hsp70. The spontaneous peritoneal Ab-forming cells are also missing in CBA/N (xid) mice that displayed the strong response to hsp70 (unpublished observation). Additional ELISPOT assays revealed a comparable frequency of peritoneal precursor B cells capable of producing Ab to conventional Ags (unpublished observation), suggesting that the majority of Ag-reactive peritoneal cells belong to the B1 subset, which have a propensity to be polyreactive (38). It is possible that we simply could not detect anti-hsp70 serum IgM because of limitations in our ELISA (we have tested serum at concentrations higher than 1:50 because of signal-to-noise problems). Furthermore, there is also a possibility that the IgM produced by cells detected by ELISPOT remains sequestered in the tissue. ELISPOT is often used to detect Ab-forming cells even when no specific serum Ab can be detected (39, 40). Based on isotype specificity controls, as well as on the experiments with CBA/N (xid) mice, however, we think that cells detected in the ELISPOT assay before immunization may be polyreactive B1 cells rather than precursors to IgG anti-hsp70 Ab-forming cells. The increased frequency of IgM cells detected in immunized mice is also puzzling. The spots generated by these cells are small, which might indicate that they are polyreactive, abortively activated and dying, or rapidly switching isotype before significant IgM secretion.
It has been demonstrated that hsp70 is a favored immunogenic target and
that hsp70 can function as an immunogenic carrier for peptide epitopes
(2, 3, 4, 5, 6, 7, 8, 9, 10). Our data are in agreement with these findings. We have
shown that the primary response to this protein has the characteristics
of a secondary response to conventional Ag, that the response to hsp70
is ß T cell dependent, and that hsp70 functions as a strong
carrier for primary anti-hapten Ab responses. We have also observed
that the response is conserved among different inbred strains of mice,
indicating that hsp70 can retain its strong stimulatory effect in the
context of different MHC class II alleles and in strains that differ in
propensity for a Th1- or Th2-type response. Finally, we have excluded
the possibility of the response being due to contaminant endotoxin,
since the characteristics of the strong response are conserved in
C3H/HeJ mice.
One of our most surprising findings was the lack of a requirement for
T cells in the strong humoral response to hsp70. Several
investigators have identified T cell populations that respond to
this protein, and results of our in vitro proliferation assays are in
agreement with these earlier reports. Moreover, Pao et al. (41) have
provided evidence that T cells may function to provide help to
Ag-specific B cells. Nevertheless, we found that mice lacking these
cells due to a targeted disruption of the chain gene (42) responded
to immunization with hsp70 equivalently to chain-intact congenic
controls. The proliferation of T cells induced by hsp70 was
significant and did not differ between unprimed and primed mice
(Table II). This, together with the significant difference in
proliferation of ß T cells derived from naive and hsp70-primed
animals, suggests that strong proliferation to hsp70 by T cells
is an innate rather than an acquired characteristic.
If the strong, rapid response to hsp70 is not due to natural
priming or innate features of T cells, what is it due to?
Epitope mapping studies for the hsp70 of M. leprae suggest
the presence of several stimulatory epitopes in the C-terminal region
of the protein for both mice and humans (36, 37, 43, 44). It is
possible that the same holds true for M. tuberculosis hsp70
and other parasitic hsp analogues. An alternative but more complex
possibility is that the biologic function of hsp70 plays a role in its
own presentation. Hsps of the 70-kDa family have been implicated in
different steps of Ag processing/presenting pathways. Hsp70 seems to be
directly involved in MHC class I loading (45, 46), as well as in
lysosomal degradation of intracellular proteins (47) and Ag processing
(48). Heat shock itself has been associated with Ag processing and
presentation (49, 50, 51). The particular immunogenicity of hsp70 could
reflect the combination of its particular structure with its role
in Ag presentation. These intriguing possibilities are currently under
investigation.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Cristina Bonorino, Department of Microbiology Pontificia Universidade Catolica do Rio Grande do SulAv, Ipiranga 6681, Sala 21290619, 900 Porto Alegre RS, Brazil. E-mail address:
3 Abbreviations used in this paper: hsp, heat shock protein; KLH, keyhole limpet hemocyanin; BrdUrd, bromodeoxyuridine; AKP, alkaline phosphatase; Ars, p-azophenylarsonate; Sulf, p-azophenylsulfonate; ELISPOT, enzyme-linked immunospot.
Received for publication November 6, 1997. Accepted for publication July 9, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
+ lymphocytes from the intestinal epithelium of weaning mice. J. Immunol. 149:2315.[Abstract]
T lymphocytes and heat shock proteins. Curr. Top. Microbiol. Immunol. 167:191.[Medline]
ß T cells. J. Immunol. 142:2736.[Abstract]
: analysis of T cells during thymic ontogeny and in peripheral lymphoid organs. Proc. Natl. Acad. Sci. USA 86:5094. T cell help of B cells is induced by repeated parasitic infection, in the absence of other T cells. Curr. Biol. 6:1317.[Medline]
and ß block thymocyte development at different stages. Nature 360:225.[Medline]
ß dimers. J. Immunol. 152:1546.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  R. Duarte, A. M. Silva, L. Q. Vieira, L. C. C. Afonso, and J. R. Nicoli Influence of normal microbiota on some aspects of the immune response during experimental infection with Trypanosoma cruzi in mice J. Med. Microbiol., August 1, 2004; 53(8): 741 - 748. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Maranon, L. Planelles, C. Alonso, and M. C. Lopez HSP70 from Trypanosoma cruzi is endowed with specific cell proliferation potential leading to apoptosis Int. Immunol., December 1, 2000; 12(12): 1685 - 1693. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Julia, S. S. McSorley, L. Malherbe, J.-P. Breittmayer, F. Girard-Pipau, A. Beck, and N. Glaichenhaus Priming by Microbial Antigens from the Intestinal Flora Determines the Ability of CD4+ T Cells to Rapidly Secrete IL-4 in BALB/c Mice Infected with Leishmania major J. Immunol., November 15, 2000; 165(10): 5637 - 5645. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Tanaka, S. Itohara, M. Sato, T. Taniguchi, and Y. Yokomizo Reduced Formation of Granulomata in {gamma}{delta} T Cell Knockout BALB/c Mice Inoculated with Mycobacterium avium subsp. paratuberculosis Vet. Pathol., September 1, 2000; 37(5): 415 - 421. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
