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Institut für Medizinische Mikrobiologie und Hygiene, Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Mannheim, Germany, and
Lehrstuhl für Mikrobiologie, Theodor-Boveri-Institut für Biowissenschaften, Würzburg, Germany
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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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The murine Listeria monocytogenes infection is one standard model system for the analysis of the mechanisms governing specificity, induction, and expansion of T cells during microbial infection (reviewed in Ref. 10). The murine L. monocytogenes infection is characterized by the development of a protective T cell-dependent immunity mediated by both CD4 and CD8 T cells. Most of the previous work on the antilisterial T cell response focused on the specificity and function of L. monocytogenes-specific CD8 T cells. The identification of the Kd-restricted CD8 T cell epitopes listeriolysin O (LLO)3 91–99 (11), p60 217–225 (12), and p60 449–457 (13) from L. monocytogenes-infected BALB/c mice was catalytic for the further analysis of the antilisterial CD8 T cell response and revealed a distinct hierarchy of peptide-specific CD8 T cells (14). Significantly less information is available concerning the function and specificity of L. monocytogenes-specific CD4 T cells. Both the LLO and the secreted p60 protein of L. monocytogenes are targets for CD4 T cells, but until now only the H-2Ek-restricted peptide LLO 215–234 (15, 16) and the H-2Ad-restricted peptide p60 301–312 have been studied in some detail (17).
We used a novel approach for the direct ex vivo identification and characterization of T cell epitopes based on the screening of peptide spot libraries with ex vivo isolated lymphocytes in a highly sensitive enzyme-linked immunospot (ELISPOT) assay. This approach was applied for the analysis of LLO- and p60-specific T cells during L. monocytogenes infection. The data presented in the current report show that this approach rapidly identified L. monocytogenes-specific CD4 and CD8 T cell epitopes.
Making use of sequence data available for a variety of pathogenic microorganisms and also target structures relevant in tumor or autoimmune diseases ELISPOT-based screening of peptide spot libraries could represent a general approach for the rapid identification and characterization of T cells during various disease states.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 (H-2b) and BALB/cOlaHsd (H-2d) mice were purchased (Harlan-Winkelmann, Borchen, Germany), kept under conventional conditions, and used at 8–10 wk of age. Mice were immunized by i.p. injection of 1 x 103 CFU L. monocytogenes serovar 1/2a EGD in 0.2 ml PBS and boosted i.p. with 1 x 106 CFU 2 wk later. Generally, spleens were removed and tested in the ELISPOT assay 10–14 days postinfection (p.i.). Bacteria used for infection were in the logarithmic growth phase. The bacterial concentration was estimated by determination of the OD at 600 nm.
Cell lines
Transfected cell lines expressing specific MHC class I molecules were used for the restriction analysis of CD8 T cell epitopes. Transfected L fibroblasts (H-2k) expressing the MHC class I molecules Kd (18), Dd, or Ld (19) and nontransfected parental L-tk- cells were obtained from M. Reddehase (University of Mainz, Mainz, Germany). Nontransfected 1T22-6 cells (H-2q) and transfected 1T-Kb and 1T-Db cells expressing the MHC class I molecules Kb and Db, respectively, were kindly provided by B. Arnold (German Cancer Research Center, Heidelberg, Germany) (20).
Peptides and peptide spot libraries
Cellulose-adsorbed peptide spot libraries were prepared commercially by automated spot synthesis on cellulose membranes (Jerini Biotools, Berlin, Germany) using a modified standard protocol (5, 21). The amino acid sequences of LLO (22) and p60 (23) were represented by peptide spot libraries with 247 and 229 dodecamer peptides, respectively. The offset between consecutive peptides was two residues. Fine mapping of regions antigenic for CD4 or CD8 T cells was performed with dodecamer or nonamer sublibraries, respectively. Sublibraries were synthesized with a single residue offset between consecutive peptides. After the final dry state cleavage by ammonia vapor peptides remain adsorbed to the cellulose membrane and carry an additional glycineamid at the carboxyl terminus. The peptide spots with adsorbed peptides were punched out, transferred to 96-well microtiter plates, and were eluted with 200 µl PBS for 30 min at room temperature to yield a 2.5 x 10-4 M peptide stock solution, which was stored at -80°C. Bulk peptides were commercially synthesized using standard Fmoc chemistry (Jerini Biotools) and purified by HPLC. Bulk peptides were synthesized with a free carboxyl terminus and thus did not contain an additional carboxyl-terminal glycineamid. Bulk peptides were diluted to a concentration of 1 x 10-3 M in 30% acetonitril in distilled water and stored at -80°C.
T cell separation
CD4 and CD8 T cells were purified by immunomagnetic separation using Minimacs (Miltenyi Biotec, Bergisch Gladbach, Germany) equipment. Spleen cells were stained either with FITC anti-CD4 (clone RM4-5; PharMingen, San Diego, CA) or FITC anti-CD8 (clone 53-6.7; PharMingen) mAbs and separated after secondary labeling with paramagnetic microbeads coupled with monoclonal mouse anti-FITC isomer 1 Ab (Miltenyi Biotec) applying the standard positive selection protocol provided by the manufacturer. Purity of separated T cell populations was generally between 80 and 90%.
ELISPOT
The frequency of T lymphocytes was determined with an
IFN-
-specific ELISPOT assay 10–14 days post booster infection
(14, 24). In some experiments splenocytes were tested 6 wk
after booster infection when most L. monocytogenes-specific
T cells show a memory T cell phenotype (25). Assays were
performed in nitrocellulose-backed 96-well microtiter plates (Nunc,
Wiesbaden, Germany) coated with rat anti-mouse IFN- mAb (RMMG-1;
Biosource, Camarilla, CA). For the testing of peptide spot libraries,
the 2.5 x 10-4 M peptide stock solution
was further diluted 25-fold with PBS to yield a 1 x
10-5 M diluted peptide solution. In round-bottom
96-well microtiter plates per well 6 x 105
unseparated splenocytes in 135 µl culture medium (
modification of
Eagle’s medium (Life Technologies, Eggenstein, Germany) supplemented
with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 x
10-5 M 2-ME, and 2 mM glutamine) were mixed with
15 µl prediluted peptide to yield a final peptide concentration of
1 x 10-6 M. After 6 h of incubation
at 37°C, cells were resuspended by vigorous pipetting, and 100 µl
or 10 µl of cell suspension (4x105/well or
4 x 104/well, respectively) was transferred
to Ab-coated ELISPOT plates and incubated overnight at 37°C. In the
ELISPOT plates, the final volume was adjusted to 150 µl to ensure
homogenous distribution of cells. Bulk peptides were tested similarly
at a final concentration of 10-6 M peptide.
Purified CD4+ or CD8+ T
cells were tested in a modified assay as follows: 15 µl prediluted
peptide (1 x 10-5 M) was directly added to
Ab-coated ELISPOT plates and mixed with 4 x
105 splenocytes from nonimmune animals as APC to
yield a final volume of 100 µl. After 4 h of preincubation of
APC at 37°C, 1 x 105
CD4+ or CD8+ cells purified
from L. monocytogenes-immune mice were added per well in a
volume of 50 µl and plates were incubated overnight at 37°C. The
ELISPOT-based ex vivo MHC restriction analysis was performed after
loading of cell lines expressing specific MHC class I molecules with
1 x 10-6 M peptide for 2 h at 37°C.
Subsequently, unbound peptides were washed off (four times) to
prevent binding of peptides to responder splenocytes. Per well of the
ELISPOT plate, 1 x 105 peptide-loaded
APC were mixed with 4 x 105 or 4 x
104 responder splenocytes in a final volume of
150 µl. After overnight incubation at 37°C ELISPOT plates were
developed with biotin-labeled rat anti-mouse IFN- mAb (clone
XMG1.2; PharMingen), HRP streptavidin conjugate (Dianova, Hamburg,
Germany), and aminoethylcarbazole dye solution. The frequency of
Ag-specific cells was calculated as the number of spots per splenocytes
seeded. The specificity and sensitivity of the ELISPOT assay was
controlled with IFN- secreting CD8 T cell lines specific for p60
217–225, p60 449–457, and LLO 91–99. Recovery of seeded CD8 T cells
was higher than 90% for all cell lines.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The identification of antigenic peptides generally depends on the analysis of T cell lines selected by repeated rounds of in vitro restimulation. We used a different approach for the direct ex vivo identification of T cell epitopes based on the screening of peptide spot libraries with ex vivo isolated lymphocytes in a highly sensitive ELISPOT assay.
Peptide spot libraries with dodecamer peptides and two residues offset
between neighboring peptides were synthesized according to the known
sequences of LLO (22) and p60 (23) of
L. monocytogenes. These proteins were chosen as
representative model Ag for the study of this experimental approach
because both are secreted abundantly by L. monocytogenes,
both are known targets for CD4 and CD8 T cells, and for both proteins a
number of T cell epitopes has been defined (11, 12, 13, 15, 17). These peptide libraries were screened with
splenocytes from L. monocytogenes-infected BALB/c and
C57BL/6 mice in an IFN--specific ELISPOT assay. Fig. 1
shows the frequency of IFN-
secreting cells among splenocytes incubated with individual peptides
from the LLO (Fig. 1A) and p60 (Fig. 1B) peptide
spot libraries, respectively. Peptides that induced significant
activity are indicated with the peptide number and the corresponding
sequences are listed in Table I.
Antigenicity of peptides was considered significant if the induced
activity was more than three SDs above the mean background activity in
the absence of peptides, which was 1.98 ± 1.2, and 3.3 ±
1.6 per 4 x 105 spleen cells for BALB/c and
C57BL/6 splenocytes, respectively. Splenocytes from nonimmune mice were
not significantly activated by any peptide tested (data not shown).
Peptides that contain one of the known CD8 T cell epitopes of L.
monocytogenes (i.e., LLO 91–99, p60 217–225, and p60 449–457)
were clearly positive and also peptide p60 no. 135, which contains most
of the sequence of the p60 epitope p60 301–312, was antigenic in the
ELISPOT assay (Fig. 1). In addition to these known T cell epitopes, 12
new antigenic regions of LLO and p60 were identified (Table I). Six of
these new antigenic regions were defined in BALB/c and six in
C57BL/6 mice.
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To determine the MHC class restriction of the newly identified
epitopes from LLO and p60, reactive peptides from the peptide spot
libraries were tested in an ELISPOT assay with separated
CD4+ and CD8+ cells from
L. monocytogenes-infected BALB/c (Fig. 2, A and B) and
C57BL/6 mice (Fig. 2, C and D). Because the
absolute number of T cells reactive with different peptides varies over
a wide range, the frequencies of cells reactive with a specific peptide
are presented as the percentage of CD4+ or
CD8+ cells in relation to the indicated total
number of cells reactive with an individual peptide per 1 x
105 CD4+ and
CD8+ cells, which was defined as 100%. This
ELISPOT analysis clearly separated putatively MHC class II-restricted
peptides recognized by CD4 T cells from putatively MHC class
I-restricted peptides recognized by CD8 T cells. The assay confirmed
the restriction of peptides containing the known CD8 T cell epitopes
LLO 91–99, p60 217–225, and p60 449–457 and the known CD4 T cell
epitope p60 301–312 (summarized in Table I
). In the absence of
synthetic peptide, the spontaneous activity of sorted T cells was less
than one spot per 1 x 105 cells (not
shown). Among the 12 newly identified antigenic regions, peptides from
10 regions stimulated CD4 T cells and peptides from 2 regions
stimulated CD8 T cells (Table I).
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Screening of LLO and p60 peptide libraries revealed 12 antigenic
regions that could not be attributed to any known T cell epitope of
L. monocytogenes. For the fine mapping of the newly
identified antigenic regions, the putative MHC class restriction of
these peptides as summarized in Table I was taken into consideration.
Antigenic regions recognized by CD4 T cells were mapped with dodecamer
sublibraries with a single residue offset between consecutive peptides.
Regions antigenic for CD8 T cells were mapped similarly with nonamer
sublibraries with a single residue offset between consecutive peptides.
These sublibraries of the antigenic regions of LLO (Fig. 3, A and C) and p60
(Fig. 3, B and D) were screened with freshly
isolated splenocytes from L. monocytogenes-immune BALB/c
(Fig. 3, A and B) or C57BL/6 mice (Fig. 3, C and D) as appropriate. As an internal control,
also a sublibrary of the antigenic region p60 no. 135, which covers the
Ad-restricted CD4 T cell epitope p60 301–312 was
included. The fine mapping of the two CD8 T cell epitopes revealed
single peptides with an antigenicity significantly above the
antigenicity of related peptides. Peptides LLO 296–304 (VAYGRQVYL,
Fig. 3C) and p60 476–484 (KYLVGFGRV, Fig. 3B)
were identified as CD8 T cell epitopes from the antigenic regions LLO
no. 135/no. 136 and p60 no. 224, respectively. The mapping of CD4 T
cell epitopes generally revealed broader antigenic plateaus as evident
for the LLO antigenic regions LLO no. 81/no. 82, LLO no. 93/no. 94, LLO
no. 96/no. 97 (Fig. 3A), LLO no. 83/no. 84, LLO no. 114/no.
115, and LLO no. 146/no. 147 (Fig. 3C), and also the p60
antigenic regions p60 no. 135, p60 no. 170/no. 171, p60 no. 195/no. 196
(Fig. 3B), p60 no. 75/no. 76, and p60 no. 186/no. 187 (Fig. 3D). Peptides with a central position in the sublibrary and
also a strong antigenic activity were considered as core CD4 T cell
epitopes and are marked in Fig. 3. These peptides were synthesized as
bulk peptides with free acid at the carboxyl terminus and used for
further confirmatory experiments.
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BALB/c and C57BL/6 mice express multiple MHC class I and class II
molecules. To define the MHC restriction of the two new CD8 T cell
epitopes, the recognition by freshly isolated splenocytes of
peptide-loaded transfected cells that express specific MHC molecules
was tested in an IFN--specific ELISPOT assay. Due to the limited
availability of MHC class II-transfected cell lines this approach was
only possible for the analysis of CD8 T cell epitopes. The restriction
analysis of the new H-2d-restricted CD8 T cell
epitope p60 476–484 was performed with Kd-,
Dd-, and Ld-expressing
transfected L cells. The known Kd-restricted CD8
T cell epitope p60 217–225 was also included as a control. The
restriction of LLO 296–304 was tested similarly with
Kb- and Db-expressing
transfected 1T cells. As shown in Fig. 4A, the majority of p60
217–225- and p60 476–484-specific cells reacted in the context of the
Kd molecule. The majority of LLO
296–304-specific cells responded in the context of the
Kb molecule (Fig. 4B). Nontransfected
cells and cells expressing the wrong MHC class I mol-ecule also induced
some T cell activation. However, this activity was not enhanced by the
addition of peptide, which indicates that it was triggered by the
allogeneic MHC of the parental cell line.
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To confirm the T cell epitopes identified by the screening of
peptide spot libraries and to determine the hierarchy of responding T
cells, peptides were newly synthesized with free acid at the carboxyl
terminus. This was necessary to exclude any influence of the
glycineamid, which remained at the carboxyl terminus after cleavage of
spot-synthesized peptides. Peptides were tested in an IFN--specific
ELISPOT assay with splenocytes from L.
monocytogenes-infected BALB/c and C57BL/6 mice (Table II). Splenocytes were tested 10–14 days
and 6 wk p.i. when most responding T cells exhibit an effector and
memory T cell phenotype, respectively (25). In BALB/c mice
10–14 days p.i. the dominant T cell populations were CD8 T cells
specific for the well known Kd-restricted CD8 T
cell epitopes LLO 91–99 and p60 217–225. The new CD8 T cell epitope
p60 476–484 and the new CD4 T cell epitope LLO 189–200 showed
significantly lower frequencies between 20 and 15 positive cells per
1 x 105 splenocytes. The remainder of the
H-2d-restricted epitopes were recognized with
frequencies between 10 and 1 positive cells per 1 x
105 splenocytes. Interestingly, in C57BL/6 mice
10–14 days p.i. the most dominant cell population with 125 positive
cells per 1 x 105 spleen cells reacted with
the CD4 T cell epitope LLO 190–201. The CD4 T cell epitopes LLO
318–329, LLO 253–264, p60 401–412, and p60 177–188, and the CD8 T
cell epitope LLO 296–304 showed a weaker reaction in the range between
25 and 5 positive cells per 1 x 105
splenocytes. We tested also the octamer peptide LLO 297–304 because it
is known that Kb molecules preferentially bind
octamer peptides (7). Interestingly, compared with LLO
297–304, significantly more cells reacted against the nonamer peptide
LLO 296–304. In the absence of peptide, the background activity was
less than one IFN--positive cell per 1 x
105 splenocytes for both BALB/c and C57BL/6 mice.
The general hierarchy of strong and weak epitopes did not change
significantly if splenocytes were tested in the memory phase 6 wk p.i.
(Table II). Minor changes in the hierarchy occurred only within the
group of weak epitopes, which induced <10 spots per 1 x
105 splenocytes (p60 367–378 and LLO 211–222;
p60 401–412 and p60 177–188). These data obtained with bulk
synthesized peptides confirmed the T cell epitopes identified by
screening of spot-synthesized peptide libraries. Furthermore, a
distinct hierarchy between immunodominant and subdominant CD4 and CD8 T
cell epitopes is shown.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ex vivo analysis identified a number of new CD4 T cell epitopes from LLO and p60 in BALB/c and C57BL/6 mice. As C67BL/6 mice solely express the MHC class II molecule Ab, it can be assumed that CD4 T cell epitopes identified in this mouse strain are Ab-restricted. An ex vivo MHC restriction analysis for the H-2d-restricted CD4 T cell epitopes in BALB/c mice, could not be performed because appropriate MHC class II-transfected cell lines were not available.
The T lymphocyte response to pathogenic microorganisms focuses on a limited number of epitopes. Immunodominant epitopes generate a strong T cell response, while subdominant epitopes induce a comparatively weak response (32). According to this definition the epitopes LLO 91–99 and p60 217–225 of L. monocytogenes are considered immunodominant and p60 449–457 and mpl 84–92 are considered subdominant (14, 25). Similarly, comparison of the frequencies of H-2b-restricted CD4 T cells revealed a distinct hierarchy of dominant and subdominant epitopes. This hierarchy of CD4 T cell epitopes was principally conserved in the early effector and late memory phase of the antilisterial T cell response confirming previous studies with CD8 T cell epitopes (14, 25). Interestingly, in C57BL/6 mice the dominant T cell epitope, LLO 190–201, which reacted with 5-fold more T cells than the second strong peptide, is a CD4 T cell epitope. This antigenic region was conserved in BALB/c mice, where LLO 189–200 was also the strongest CD4 T cell epitope. The existence of a dominant LLO-specific CD4 T cell response is remarkable in the light of the immunoevasive function of LLO (33). A number of detailed in vitro studies show that LLO inhibits the MHC class II-restricted recognition of LLO itself (16), of p60 (24) and also of a model Ag as OVA (34). A recent study that monitored the expansion of adoptively transferred lymphocytes from Listeria-immune mice shows that during L. monocytogenes infection, in addition to a strong CD8 T cell response, also a significant CD4T cell response occurs (35). The identification of an immunodominant MHC class II-restricted T cell epitope directly demonstrates the existence of a strong L. monocytogenes-specific CD4 T cell response in vivo and allows the direct ex vivo analysis of these CD4 T cells.
The ex vivo ELISPOT analysis identified two new CD8 T cell epitopes of L. monocytogenes. The MHC class I-restricted epitopes p60 476–484 and LLO 296–304 were identified from BALB/c and C57BL/6 mice, respectively. As the other known CD8 T cell epitopes from p60, p60 217–225, and p60 449–457, also the new epitope p60 476–484 is Kd restricted. This antigenic peptide was not detected in previous detailed studies of possible epitopes recognized by p60-specific CD8 T cells (12, 13). The p60 476–484 epitope has a high antigenicity score and ranks at position five among the possible Kd-binding nonamer peptides (36). It exhibits the typical Kd-peptide binding motif with a tyrosine at position two and valine at the carboxyl terminus (9). Compared with the frequency of CD8 T cells specific for the immunodominant epitope p60 217–225 and the subdominant epitope p60 449–457, the third CD8 T cell epitope of p60 revealed an intermediate strength. An interesting feature of p60 476–484 is that it represents the carboxyl-terminal nine amino acids of the intact p60 protein.
The peptide LLO 296–304 is the first H-2b-restricted CD8 T cell epitope identified of L. monocytogenes. It is known that Kb generally binds octamer peptides (7) suggesting that the octamer LLO 297–304 could be the naturally processed epitope in vivo. However, it was consistently found that more splenocytes react against the nonamer LLO 296–304 than against the octamer LLO 297–304. Recent studies that tested the peptide length preference of Kb show that Kb can also accommodate longer peptides suggesting that both antigenic peptides are possible candidates for this epitope. The antigenic peptide LLO 296–304 is difficult to predict from the Kb-binding motif and the known LLO sequence. The peptide exhibits the hydrophobic anchor at the carboxyl terminus but misses the typical octamer Kb-binding motif with a tyrosine or phenylalanine residue at position five (7), which results in the low Kb-binding score of this peptide (36). A previous study has already shown that LLO is a target for protective H-2b-restricted CD8 T cells (37). LLO 296–304 and LLO 297–304 are possible targets of these CD8 T cells for which an epitope has not yet been defined.
Compared with the analysis of T cell lines or clones the identification of T cell epitopes with freshly isolated splenocytes is not prone to any selective pressure exerted during extended in vitro selection and expansion of T cell lines and allows the direct identification of strong T cell activities. A previous study of p60-specific CD4 T cell clones identified p60 301–312 as a CD4 T cell epitope (17). The ex vivo ELISPOT analysis with Listeria-immune BALB/c mice showed that this epitope is just the weakest of four p60 CD4 T cell epitopes. The different results obtained from the ex vivo analysis are likely due to the selection of specific T cell clones during in vitro restimulation. CD8 T cells have specific demands for the concentration of antigenic peptide necessary for successful in vitro expansion. Although the requirement of a minimal peptide concentration for T cell stimulation seems obvious, detailed studies of the in vitro expansion of CD8 T cell lines specific for L. monocytogenes and the murine CMV showed that, remarkably, also concentrations above a defined optimum can prevent the expansion of peptide specific CD8 T cells (38, 39). This would explain the loss of some T cell specificities during in vitro expansion of T cell lines.
Recent genome sequencing programs generated a wealth of sequence information of the human genome and a number of microorganisms including pathogenic viruses, bacteria and protozoa (40). The current report demonstrates that ELISPOT-based screening of peptide spot libraries with freshly isolated lymphocytes can rapidly identify target structures of murine antilisterial T cells. Thus it can be anticipated, that with this approach also the T cell response in other pathologic immune responses could be diagnosed as well. Importantly, the use of peptide libraries as an Ag source also overcomes MHC restriction of CD4 and CD8 T cell epitopes, which means that a single peptide library could be used for the study of the T cell response in an outbred population. Thus, ELISPOT-based screening of peptide spot libraries representing single or multiple proteins or even whole genomes of small viruses could be a general approach for the rapid identification and monitoring of relevant T cell populations during various disease states.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gernot Geginat, Institut für Medizinische Mikrobiologie und Hygiene, Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Theodor-Kutzer-Ufer 1–3, 68167 Mannheim, Germany.
3 Abbreviations used in this paper: LLO, listeriolysin O; ELISPOT, enzyme-linked immunospot; p.i., postinfection.
Received for publication August 28, 2000. Accepted for publication November 1, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 S. E.F. D'Orazio, C. A. Shaw, and M. N. Starnbach H2-M3-restricted CD8+ T cells are not required for MHC class Ib-restricted immunity against Listeria monocytogenes J. Exp. Med., February 21, 2006; 203(2): 383 - 391. [Abstract] [Full Text] [PDF]
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H. Xu, T. Chun, H.-J. Choi, B. Wang, and C.-R. Wang Impaired response to Listeria in H2-M3-deficient mice reveals a nonredundant role of MHC class Ib-specific T cells in host defense J. Exp. Med., February 21, 2006; 203(2): 449 - 459. [Abstract] [Full Text] [PDF]
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B. Abel, S. Freigang, M. F. Bachmann, U. Boschert, and M. Kopf Osteopontin Is Not Required for the Development of Th1 Responses and Viral Immunity J. Immunol., November 1, 2005; 175(9): 6006 - 6013. [Abstract] [Full Text] [PDF]
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J. S. Haring, V. P. Badovinac, M. R. Olson, S. M. Varga, and J. T. Harty In Vivo Generation of Pathogen-Specific Th1 Cells in the Absence of the IFN-{gamma} Receptor J. Immunol., September 1, 2005; 175(5 |
) and CD8+
(
) cells in relation to the indicated total number of reactive cells
per 1 x 105 CD4+ and CD8+
cells (total), which was defined as 100%.
), and after loading with p60 476–484 (