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MHC Class II-Dependent NK1.1⁺ T Cells Are Induced in Mice by Salmonella Infection¹

Hitoshi Nishimura^2,*, Junji Washizu^*, Yoshikazu Naiki^*, Toru Hara^*, Yoshinori Fukui, Takehiko Sasazuki and Yasunobu Yoshikai^*

^* Laboratory of Host Defense and Germfree Life, Research Institute of Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya, Japan; and Department of Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We observed the emergence of a novel population of T cells expressing NK1.1 Ag in the peritoneal cavity of mice infected with Salmonella choleraesuis. The NK1.1⁺ T cells accounted for approximately 20% of all T cells emerging in the peritoneal cavity of C57BL/6 mice and expressed preferentially rearranged V4-J1 and V6.3-D1-D2-J1 genes with N diversity. The T cells proliferated vigorously in response to PHA-treated spleen cells and produced IFN- in the culture supernatant. However, spleen cells from Aß^b-deficient mice were unable to stimulate the T cells. Furthermore, the NK1.1⁺ T cells were stimulated not only by Chinese hamster ovary (CHO) cells expressing wild-type IA^b but also by those expressing IA^b/E52-68 or IA^b/pigeon cytochrome c-derived analogue peptide complex. These proliferation activities were inhibited by mAb specific for IA^b chain. Consistent with these findings, the emergence of NK1.1⁺ T cells was reduced in the peritoneal cavity of Aß^b-deficient mice after Salmonella infection, whereas NK1.1⁺ T cells were rather abundant in the peritoneal cavity of Salmonella-infected ß₂m-deficient mice. Moreover, the NK1.1⁺ T cells were easily identified in the thymus of ß₂m-deficient but not Aß^b-deficient mice. Our results indicated that MHC class II expression is essential for development and activation of NK1.1⁺ T cells in the thymus and the periphery.

	Introduction

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Major histocompatibility complex class I and class II molecules are essential for the intrathymic maturation of a normal repertoire of ß T cells and serve as Ag-presenting molecules for mature ß T cells in the periphery (1, 2, 3, 4, 5, 6). Development of CD4⁺ T cells depends on MHC class II expression by the thymic cortical epithelium, whereas the development of mature CD8⁺ T cells requires intrathymic exposure to MHC class I molecules (5, 7, 8). Mature CD4⁺ or CD8⁺ T cells are thus markedly reduced in MHC class II- or MHC class I-deficient mice, respectively (9, 10). In contrast to the ß T cells, most T cells can develop normally in the absence of MHC class I and II molecules (11, 12). Furthermore, there are several lines of evidence that T cells recognize Ag in the absence of MHC molecules (13, 14). These results suggest that MHC molecules are not essential for development and recognition of T cells. However, a number of T cells have been shown to be specific for either classical MHC molecules and MHC-related gene products such as TL, Qa, and CD1c (15, 16). Therefore, it appears that at least a significant fraction of T cells may not develop or be activated in MHC class I- or class II-deficient mice.

A particular subset of ß T cells expressing NK1.1 Ag are found in the thymus and most peripheral tissues in mice. These consist of CD4⁺ and CD4^-8^- double-negative cells and express invariant TCR V14 and skewed Vßs such as Vß8, -7, or -2 (17, 18, 19, 20). NK1.1⁺ß T cells are almost completely absent in ß₂-microglobulin (ß₂m)-deficient mice, and a large fraction of NK1.1⁺ß T cells are thought to recognize the nonpolymorphic MHC class I-like surface protein CD1 (21). NK1.1⁺ß T cells not only lyse NK-sensitive and Fas-expressing targets but also secrete large amounts of cytokines, especially IL-4, upon primary stimulation through their ß TCRs (22, 23). Yoshimoto et al. showed that this cell population was essential for switching to IgE in response to injection of Abs to IgD (24). We have recently reported that NK1.1⁺ß T cells inhibit the generation of Th1 cells during the course of Salmonella infection via excessive IL-4 production (25). Thus, NK1.1⁺ß T cells have been suggested to play an immune regulatory role. On the other hand, the expression of NK1.1 Ag by thymic TCR⁺ T cells has recently been reported (26, 27). NK1.1⁺ thymocytes produce IL-4 in response to anti-TCR Ab, similarly to NK1.1⁺ß T cells (26, 27). In contrast to NK1.1⁺ß T cells, these cells are present in ß₂m-deficient mice (26), suggesting that development of the NK1.1⁺ T cells in the thymus is independent of MHC class I-related genes, including nonclassical MHC genes such as those encoding CD1, TL, or Qa. However, the ligands and selection molecules of NK1.1⁺ T cells remain unknown.

The dominant T cell response to infection with various microbial pathogens suggests that at least a significant fraction of T cells represent a first line of host defense (1, 28, 29). We have previously reported that the number of T cells significantly increases during primary infection with Listeria monocytogenes or Salmonella choleraesuis in mice (30, 31). These T cells produce Th1-type cytokines, particularly IFN-, and a study of mice depleted of T cells by in vivo treatment with TCR- mAb revealed that the T cells play a protective role at least at an early stage in bacterial infection (32, 33). This view was strengthened by the findings of a recent study using TCR gene-targeted mice (34). On the other hand, during infection with the helminth Nippostrongylus brasiliensis, T cells preferentially produce Th2-type cytokines, mostly IL-4 (35). TCR-deficient mice showed exaggerated intestinal damage after oral infection with Eimmeria verformis, suggesting that T cells play an important role in resolution of the inflammatory process (36). Thus, T cells may be heterogeneous in function during the course of infectious diseases.

In this study, we observed a novel population of T cells expressing NK1.1 Ag emerging in the peritoneal cavity of mice infected with S. choleraesuis. The T cells proliferated in response to PHA-treated spleen cells from ß₂m-deficient mice but not to those from Aß^b-deficient mice. Furthermore, the NK1.1⁺ T cells could be stimulated by IA^b-transfected CHO cells. The emergence of NK1.1⁺ T cells was impaired in the peritoneal cavity of Aß^b-deficient mice on day 6 after Salmonella infection, whereas the NK1.1⁺ T cells were rather abundant in the peritoneal cavity of Salmonella-infected, ß₂m-deficient mice. The NK1.1⁺ T cells were easily identified in the thymus of ß₂m-deficient but not in Aß^b-deficient mice. Our results indicate that MHC class II expression is essential for development and activation of NK1.1⁺ T cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals

All mice used in this study were bred in the Nagoya University School of Medicine (Nagoya, Japan) animal barrier facility under specific pathogen-free conditions. Mice genetically deficient in ß₂m and Aß^b gene expression bred to the C57BL/6 (B6) background were obtained from Taconic (Germantown, NY). Age- and sex-matched B6 mice obtained from Japan SLC (Hamamatsu, Japan) were used as controls.

Microorganisms

Salmonella subspecies choleraesuis serovar choleraesuis strain 31N-1 (37) was maintained by several passages through BALB/c mice. The approximate LD₅₀ was 10⁷ CFU in BALB/c mice inoculated i.p. Heat-killed Salmonella (HKS) was prepared by incubating viable S. choleraesuis at 74°C for 120 min.

Ab and reagents

Phycoerythrin (PE)³-conjugated anti-TCR, anti-TCRß, and anti-CD4; FITC-conjugated anti-CD3 and anti-CD8, and biotin-conjugated purified anti-NK1.1 mAb (PK136, mouse IgG2a), anti-IA^b mAb (AF6-12.1, mouse IgG2a), and anti-H-2K^b/D^b (28-8-6, mouse IgG2a) were purchased from PharMingen (San Diego, CA). Red-613-conjugated streptavidin was obtained from Life Technologies (Gaithersburg, MD). Anti-TCR-ß mAb (H57-597) was a gift from Dr. R. Kubo (National Jewish Center of Immunology and Respiratory Medicine, Denver, CO). Anti-TCR- mAb (UC7-13D5) was a gift from Dr. J. A. Bluestone (The Ben May Institute, the University of Chicago, Chicago, IL).

Cell preparation

T cells on day 6 after infection with S. choleraesuis were enriched according to the procedure described previously (38). Briefly, mice were killed 6 days after i.p. inoculation with 2 x 10⁶ CFU of avirulent strain 31N-1. Peritoneal exudate cells (PEC) were prepared by centrifugation at 100 x g for 10 min and suspended in RPMI 1640 medium supplemented with L-glutamine (4 mM) and 10% heat-inactivated FCS. The cells were plated in 100-mm tissue culture dishes and allowed to adhere for 1 h at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Nonadherent cells were separated out by passage through a nylon wool column (Wako Pure Chemical Industries, Osaka, Japan). T cells were enriched from the nylon wool-passed cells by the panning method using anti-TCR mAb. Briefly, tissue culture dishes were incubated overnight at 4°C with a solution of 100 µg/ml anti-TCR- mAb (UC7-13D5) in PBS. The dishes were washed thoroughly and incubated for 1 h at 37°C with RPMI 1640 medium containing 10% FCS. Aliquots of 2 x 10⁷ nylon wool-passed cells were added to the dishes and incubated for 1 h at 37°C. After nonadherent cells were discarded by washing twice with HBSS, adherent cells (6 x 10⁵) were collected by vigorous pipetting and used as T cells. The TCR-positive cells were enriched to greater than 95% as assessed by FACScan analysis (Becton Dickinson, San Jose, CA).

CHO-K1 cells, CHO-expressing wild-type IA^b or IA^b covalently bound to E52-68 (39, 40) or pigeon cytochrome-c-derived analogue peptide (50V) (41), were cultured in RPMI 1640 medium. CHO transfectants were generated as described previously (42, 43). Briefly, the nucleotide sequence corresponding to E52-68 or 50V and a flexible linker was introduced into the sequence encoding the third and the fourth amino acid residues of the I-Aß^b chain by PCR using cDNA obtained from B6 mouse spleen cells. The cDNA or the chimeric gene was subcloned into the vector and transfected into CHO cells by electroporation. Expression of IA^b on the transfectants was confirmed by flow cytometry using Y3p (anti-IA^b, a gift from Dr. M. Kimoto, Saga Medical School, Saga, Japan) (44).

Flow cytometry

T cells were stained with PE-, FITC-, or biotin-conjugated mAbs. To block FcR-mediated binding of the mAb, 2.4G2 (anti-FcR mAb) was added. All incubation steps were performed at 4°C for 30 min. To detect biotin-conjugated mAb, cells were stained with Red-613-conjugated streptavidin after incubation with primary mAb. The stained cells were analyzed with a FACScan flow cytometer (Becton Dickinson). Small lymphocytes were gated by forward and side scattering. Separation of NK1.1⁺- or NK1.1^- T cells from nonadherent PEC of mice infected with Salmonella was performed with Coulter EPICS ESP (Coulter, Miami, FL).

Cell culture

The enriched T cells (10⁴ cells) from PEC were cultured in 200 µl of complete culture medium in 96-well flat-bottom plates (Falcon, Becton Dickinson, Oxford, U.K.) at a density of 10⁴ cells with mitomycin (MMC)-treated spleen cells (1 x 10⁵ cells) from uninfected B6 mice or ß₂m- or Aß^b-deficient mice, or with MMC-treated CHO-K1, CHO-expressing IA^b or IA^b covalently bound to E52-68 or 50V (1 x 10⁵ cells). In some experiments, spleen cells from B6 mice were cultured with PHA 10 µg/ml or LPS 10 ng/ml for 12 h at 37°C. After culture, these cells were washed four times with HBSS for removing the mitogens from spleen cells and treated with MMC. After treatment with MMC, the cells were washed four times with HBSS and used as APCs. The cells were cultured for 2 days at 37°C under 5% CO₂ in air and pulsed with [³H]TdR 6 h before harvesting. [³H]TdR incorporation was then determined by liquid scintillation counting. In some experiments, after culturing, the supernatants were collected for cytokine ELISA.

TCR V repertoire of T cells

Total RNA was extracted from nonadherent PEC of Aß^b-deficient mice or B6 mice on day 6 after infection with Salmonella. In some experiments, NK1.1⁺ T cells and NK1.1^- T cells, isolated from PEC of B6 mice on day 6 after infection with Salmonella by sorting using a Coulter EPICS ESP, were applied. Total RNA was extracted according to the method of Chomczynski and Sacchi (45). First, strand cDNA was synthesized from 2 mg of RNA using reverse transcriptase (SuperScript II RT, Life Technologies) and 20 pmol of C (5'-CTTATGGAGATTTGTTTCAGC-3') or C (5'-CTTGGTCAGTATGGAGATC-3') primer in 21-ml reaction mixtures, according to the manufacturer’s instructions. The synthesized first strand cDNA was diluted to a total volume of 20 µl with distilled water. An aliquot of first strand cDNA was Ampli-Taq (Perkin-Elmer/Cetus, Norwalk, CT) in a total volume of 100 µl of reaction buffer consisting of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.001% gelatin, and 0.2 µM dNTP. One PCR cycle consisted of denaturation at 94°C for 1 min, annealing at 54°C for 1 min, and extension at 72°C for 0.5 min. Before the first cycle, an initial denaturation step of 3 min at 94°C was included, and, after 23 to 35 cycles, the extension reaction was prolonged for 4 min at 72°C. After amplification, the PCR products were separated by electrophoresis through 1.8% agarose gels, then transferred on to GeneScreeen Plus membranes (New England Nuclear, Boston, MA), then hybridized with ³²P-labeled MNG6 cDNA containing the C2 gene or oligo probe J1 (5'-TTGGTTCCACAGTCACTTGG-3') or J2 (5-CTCCACAAAGAGCTCTATGCCA-3'). Following 16 h at 60°C in 1 M NaCl, 10% dextran sulfate, and 100 µg/ml heat-denatured salmon sperm DNA, the filters were washed for 3 min in 2x SSC, 1% SDS, at 60°C, and exposed to a PhosphorImaging plate for visualization on a Fuji BAS-2000 PhosphorImaging system (Fuji Photo Film, Tokyo, Japan).

Cloning of junctional region between V-J and nucleotide sequencing

To clone the junctional region between V and J gene segments, cDNA from NK1.1⁺- or NK1.1^- T cells in the peritoneal cavity of mice infected with Salmonella was amplified by PCR using primers for V and C gene segments as described above. The RT-PCR products were resolved in low-melting agarose gels, isolated, and cloned in to TA vector PCR II (Invitrogen, San Diego, CA). Purified dsDNAs were sequenced using the Taq Dye primer Cycle Sequencing Kit and an ABI 373A DNA sequencer (Applied Biosystems, Foster City, CA).

Cytokine ELISA

The cell-free culture supernatants were collected at the indicated times. The cytokine activity in the culture supernatant was assayed by an ELISA using mouse IFN- DuoSet ELISA Development Systems (Genzyme Diagnostics, Cambridge, MA).

Statistics

The data were analyzed by Student’s t test, and a p value of less than 0.05 was taken as significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Appearance of NK1.1⁺ T cells in the peritoneal cavity of B6 mice after Salmonella infection

We have previously reported that T cells emerge in the peritoneal cavity after Salmonella infection in mice (31). To examine the presence of NK1.1⁺ T cells in the peritoneal cavity of B6 mice on day 6 after Salmonella infection, the T cells were enriched from nylon wool-passed peritoneal cells of mice inoculated with S. choleraesuis 31N-1 6 days previously by the panning method using anti-TCR mAb and were examined for the expression of the NK1.1 Ag. T cells were highly purified to more than 95% after panning (Fig. 1A). Fig. 1A shows representative flow cytometry results regarding the expression of NK1.1 and TCR- by enriched T cells induced by Salmonella infection. In accordance with our previous report, T cells appeared in the peritoneal cavity at an early stage after i.p. infection of C57BL/6 mice with S. choleraesuis. NK1.1⁺ T cells constituted 14.5 ± 4.3% of the total number of T cells.

View larger version (49K): [in this window] [in a new window]   FIGURE 1. A, Flow cytometric analysis of the enriched T cells from C57BL/6 mice on day 6 after an i.p. challenge with S. choleraesuis 31N-1 2 x 106 CFU. T cells were stained with FITC-anti TCR mAb (GL3), PE-anti NK1.1 mAb (PK136), and analyzed by EPICS Elite ESP. B, V or V usages of NK1.1+ T cells or NK1.1- T cells from C57BL/6 mice on day 6 after an i.p. challenge with S. choleraesuis 31N-1 2 x 106 CFU. Total RNA extracted from EPICS-sorted NK1.1+ T cells or NK1.1- T cells were reverse transcribed into cDNA and amplified by PCR using primers for C or C and various V or V segments, respectively. The Southern blot of -PCR products was hybridized with MNG6. The Southern blot of -PCR products was hybridized with J1 or J2 oligonucleotide probe.

V repertoire of NK1.1⁺ T cells in the peritoneal cavity of B6 mice infected with Salmonella

Most NK1.1⁺ß T cells express an invariant V14-J281 TCR -chain, together with a diverse TCR ß-chain repertoire that is biased toward the use of Vß8.2, Vß7, and Vß2 (17, 18, 19, 20). To assess whether the V repertoire of NK1.1⁺ T cells was biased, we examined the TCR V repertoire of NK1.1⁺- or NK1.1^- T cells isolated by EPICS sorting. After this procedure, >99% of the sorted cells were NK1.1⁺ T cells and NK1.1^- T cells. As shown in Fig. 1B, NK1.1⁺ T cells predominantly expressed V4, while NK1.1^- T cells expressed V1/2. Although both T cell subsets expressed V4, -5, or -7 gene segments, the V6 gene segment was predominantly expressed in the NK1.1⁺ T cells but not in the NK1.1^- T cells. Thus, the TCR encoded by V4 and V6 gene segments is unique to NK1.1⁺ T cells in the peritoneal cavity of mice infected with Salmonella.

To examine the junctional diversity of rearranged V or V genes in the NK1.1⁺ T cells in the peritoneal cavity of infected mice, RT-PCR products amplified by 5'V- and 3'C-specific primers were cloned into the TA vector, and the nucleotide sequences were determined. All clones of V4 or V6 rearranged to J1 or J1 gene segments among 11 or 21 random clones, respectively. The nucleotide sequences of V4-J1 or V6-J1 junctional regions of the NK1.1⁺ T cells are shown in Fig. 2. Most of the TCR- V6⁺ T cells utilized the V6.3 gene, and the junctional regions of rearranged V4-J1 or V6-J1 genes showed N diversity. These results suggest that the V repertoire of NK1.1⁺ T cells in the peritoneal cavity of B6 mice is diversified although they preferentially used V4 and V6.3.

View larger version (43K): [in this window] [in a new window]   FIGURE 2. The nucleotide sequences of V-J junctions of cDNA from the NK1.1+ T cells in C57BL/6 mice infected with S. choleraesuis 31N-1 2 x 106 CFU. A, Rearrangement of V4 to J1. B, Rearrangement of V6 to J1. The rearrangements are designated as productive (+) or nonproductive (-) as determined by the reading frame of the J segment. N and P, Denote nucleotide not present present in germline sequences.

NK1.1⁺ T cells respond to PHA-treated spleen cells

Various subsets of T cells appear to be specialized to recognize either classical MHC molecules and MHC-related gene products, such as TL, Qa, and CD1c, or highly conserved Ags such as heat-shock proteins (HSP) and thymidine-containing nucleotide conjugates (15, 16). To determine the possible ligands for the NK1.1⁺ T cells, we examined the proliferative response of the NK1.1⁺ T cells to spleen cells treated with various reagents, including LPS or PHA. When the enriched T cells were cultured with PHA-treated spleen cells from C57BL/6 mice, T cells significantly proliferated as assessed by [³H]thymidine incorporation, while LPS- or nontreated self spleen cells did not stimulate the T cells (Fig. 3A). After 48 h of culture, the proliferating cells were analyzed by two-color staining with anti-TCR- mAb and anti-NK1.1 mAb. The NK1.1⁺ T cells were increased from 14.2% to 41.2% in TCR⁺ cells after stimulation with PHA-treated spleen cells (Fig. 3B). The number of total viable cells was increased 1.5-fold from the input cell number at the end of the culture period. To further investigate the response of NK1.1⁺ T cells to PHA-treated spleen cells, we examined the effects of NK1.1⁺ T cell depletion by treatment with anti-NK1.1 mAb and complement on the proliferation of T cells in response to PHA-treated spleen cells. As shown in Fig. 4A, NK1.1⁺ cells were almost completely depleted by NK1.1 mAb (PK136). The proliferative activity of the NK1.1-depleted T cell fraction was significantly decreased as compared with that of the nondepleted fraction, although it was not completely abolished (Fig. 4B).

View larger version (26K): [in this window] [in a new window]   FIGURE 3. Proliferative response of Salmonella-induced T cells to PHA-treated self spleen cells. Purified T cells (1 x 104 cells) from C57BL/6 mice on day 6 after an i.p. challenge with S. choleraesuis 31N-1 2 x 106 CFU were cultured with PHA-treated self spleen cells (1 x 105 cells) from uninfected C57BL/6 mice for 48 h at 37°C. A, After the incubation period, proliferative responses were assayed for [3H]thymidine incorporation during the final 6 h of incubation. B, Before or after the incubation period, the T cells were stained with FITC-anti CD3 mAb (145-2C11), PE-anti- TCR mAb (GL3), and biotin-anti NK1.1 mAb (PK136), followed by streptavidin-RED 613TM and analyzed by FACScan, and the analysis gate was set on CD3+, TCR+ cells. Data were representative of three independent experiments and shown as typical single histogram of NK1.1 staining.

View larger version (27K): [in this window] [in a new window]   FIGURE 4. Effect of in vitro depletion of NK1.1+ cells on proliferative response of T cells to PHA-treated self spleen cells from C57BL/6 mice. Purified T cells were incubated with anti-NK1.1 mAb (PK136) or isotype control Ab (mouse IgG) and guinea pig complement and then cultured with PHA-treated self spleen cells from C57BL/6 mice for 48 h at 37°C. Before incubation, the treated T cells were stained purified-anti NK1.1 mAb (PK136) followed by FITC anti-mouse IgG Ab and analyzed by FACScan. Data were representative of three independent experiments and shown as typical single histogram of NK1.1 staining. B, After the incubation period, proliferative responses were assayed for [3H]thymidine incorporation during the final 6 h of incubation. The data are representative of three independent experiments using pooled cells from ten mice and are shown as the mean of triplicate determinations ± SD. Statistical analysis was performed with Student’s t test. Significant differences are shown. *, p < 0.05).

MHC class II molecules are involved in the proliferation of NK1.1⁺ T cells

To examine the involvement of MHC molecules in the recognition of PHA-treated self spleen cells by NK1.1⁺ T cells, we used PHA-treated spleen cells from MHC class I (ß₂m)- or MHC class II (Aß^b)- deficient mice. As shown in Fig. 5A, T cells responded to the PHA-treated spleen cells from naive C57BL/6 mice and ß₂m-deficient mice. However, those from Aß^b-deficient mice were unable to stimulate the T cells. The proliferative activity to PHA-spleen cells from B6 mice was significantly inhibited by mAb specific for IA^b but not H-2K^b/D^b. To provide further direct evidence for the involvement of MHC class II molecules in recognition of NK1.1⁺ T cells, we examined the cytokine production of NK1.1⁺ T cells cultured with IA^b gene-transfected CHO cells. As shown in Fig. 5B, the CHO cells transfected with wild-type IA^b gene significantly stimulated T cells induced by Salmonella infection. Surface expression of IA^b on the CHO cells was detected by mAb Y3P specific for IA^b (data not shown). To investigate whether the T cells require a specific peptide-MHC complex for recognition, we further examined the cytokine production of T cells in response to CHO cells expressing a single peptide derived from MHC class II E52-68 or pigeon cytochrome-c-derived analogue peptide (50V)/MHC class II IA^b complex. To demonstrate that all IA^b molecules were actually loaded with E52-68 or 50V peptide, we examined the expression of IA^b/E52-68 or IA^b/50V complex in these transfectants using mAb Y3P, which is specific for IA^b, irrespective of binding peptides (44) and YAe, which is specific for IA^b bound to E52-68 (39, 40) or anti-50V peptide mAb (41). There was no difference in the expression level of IA^b molecules among IA^b transfectants (data not shown). Staining the transfectants with Y3P was completely inhibited to the level of the background by preincubation of the cells with YAe or anti-50V mAb, respectively. These results indicate that IA^b molecules are almost completely occupied with each peptide and are expressed on the cell surface. As shown in Fig. 5B, these transfectants also stimulated the T cells to produce IFN-. Furthermore, the IFN- production was significantly inhibited by mAb specific for IA^b chain. Taken together, MHC class II molecules are involved in the recognition by NK1.1⁺ T cells. Specific peptides may not be required for recognition by the T cells.

View larger version (24K): [in this window] [in a new window]   FIGURE 5. Proliferative response of Salmonella-induced T cells to PHA-treated spleen cells from MHC-deficient mice (A) or CHO cell-transfected MHC class II genes (B). Purified T cells (1 x 104 cells) from C57BL/6 mice on day 6 after an i.p. challenge with S. choleraesuis 31N-1 2 x 106 CFU were cultured with PHA-treated spleen cells (1 x 105 cells) from uninfected C57BL/6 mice or Aßb- or ß2m-deficient mice (A) or CHO cells (1 x 105 cells) transfected with IAb gene, IAb/E52-68 gene, or IAb/50V gene (B), with or without mAb (10 µg) specific for IAb or H-2Kb/Db molecules for 48 h at 37°C. A, After incubation period, proliferative responses were assayed for [3H]thymidine incorporation during the final 6 h of incubation. B, The supernatants of T cells cultured with CHO transfectant were collected and assayed production of IFN- by cytokine ELISA. The data are representative of three independent experiments using pooled cells from ten mice and are shown as the mean of triplicate stimulation index ± SD. Statistical significance was determined by the Student’s t test. (*, p < 0.001).

NK1.1⁺ T cell numbers are diminished in MHC class II-deficient mice

To examine whether NK1.1⁺ T cells are present in the peritoneal cavity of MHC class II-deficient mice infected with S. choleraesuis, we inoculated Salmonella i.p. into ß₂m- or Aß^b-deficient mice and analyzed expression of the NK1.1 Ag on T cells in the peritoneal cavity by flow cytometry. Typical results are shown in Fig. 6, and mean ± SE based on the absolute and relative cell number of NK1.1⁺ T cells obtained from three to nine mice were summarized in Table I. A significant number of NK1.1⁺ T cells appeared in the peritoneal cavity of ß₂m-deficient mice after infection with S. choleraesuis, whereas the appearance of NK1.1⁺ T cells was reduced in the peritoneal cavity of Aß^b-deficient mice infected with Salmonella. Thus, MHC class II molecules were required for the appearance of large numbers of NK1.1⁺ T cells in the peritoneal cavity following Salmonella infection. We next compared the TCR V repertoire of T cells in B6 and Aß^b-deficient mice infected with Salmonella. As shown in Fig. 7, the T cells in the peritoneal cavity of B6 mice infected with Salmonella expressed V1, -2, -4, or -6 and V1, -2, -4, -5, -6, -7, or -8 whereas those in MHC class II-deficient mice infected with Salmonella showed expression of a skewed TCR V repertoire encoded by V1, -2, or -6 and V1 or -8 gene segments. These results suggest that MHC class II-dependent T cells preferentially express V4 and V4, -5, -6, and -7. As shown in Fig. 1B, NK1.1⁺ T cells expressed V4 and V5, -6, and -7. Taken together, it thus appears that NK1.1⁺ T cells represent MHC class II-dependent T cells.

View larger version (42K): [in this window] [in a new window]   FIGURE 6. Flow cytometric analysis of NK1.1+ T cells from MHC-deficient mice or C57BL/6 mice on day 6 after an i.p. challenge with S. choleraesuis 31N-1 2 x 106 CFU. Nonadherent PEC were stained with FITC anti-CD3 mAb (145-2C11), PE anti-CD8 mAb (53-6.7), RED anti-CD4 mAb (RM4-5), or FITC anti-CD3 mAb (145-2C11), PE anti- TCR mAb (GL3). The cells were also stained with FITC anti-CD3 mAb (145-2C11), PE anti-ß TCR mAb (H57-597), and biotin anti-NK1.1 mAb (PK136), followed by streptavidin-RED 613TM and analyzed by FACScan. Analysis gate was set on CD3+ ß TCR- cells, and expression of NK1.1 was displayed as a single histogram. Data were representative of three independent experiments and shown as typical profiles.

View this table: [in this window] [in a new window]   Table I. Proportion and absolute number of NK1.1+ T cells in the peritoneal cavity or thymus of MHC-deficient mice1

View larger version (28K): [in this window] [in a new window]   FIGURE 7. V or V usages of T cells from Aßb-deficient mice on day 6 after an i.p. challenge with S. choleraesuis 31N-1 2 x 106 CFU. Total RNA extracted from T cells from Aßb-deficient mice were reverse transcribed into cDNA and amplified by PCR using primers for C or C and various V or V segments, respectively. The Southern blot of -PCR products was hybridized with MNG6. The Southern blot of -PCR products was hybridized with J1 or J2 oligonucleotide probe.

View larger version (44K): [in this window] [in a new window]   FIGURE 8. Flow cytometric analysis of the NK1.1 expression on CD4-CD8- TCR+ or CD4-CD8-ß TCR+ thymocytes from naive MHC-deficient mice or C57BL/6 mice. The thymocytes were stained with FITC-anti TCR mAb (GL3), PE anti-NK1.1 mAb (PK136), biotin-CD8 mAb (53-6.7), and biotin anti-CD4 mAb (RM4-5) followed by streptavidin-RED 613TM and analyzed by FACScan. Analysis gates were set on CD4-CD8- TCR+ or CD4-CD8-ß TCR+ cells, and expression of NK1.1 was displayed as a single histogram. Data were representative of three independent experiments and shown as typical profiles.

	Discussion

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NK1.1⁺ß T cells are known to express invariant TCR V14 and a preferential set of Vß genes, mainly Vß8, -2, or -7 (17, 18, 19, 20). We showed that NK1.1⁺ T cells preferentially used V4 and V6.3 gene segments, although the V repertoire was diversified and functionally rearranged and genes had diverse V-D-J and V-J junctions. O’Brien et al. reported reactivity of T cell hybridomas to the mycobacterial hsp 60 Ag, in which PCR analysis of hybridoma mRNA revealed that nearly 60% of responders expressed either the V6.3⁺ or the V4⁺ TCR (46). Greater than 90% of all the V6⁺ hybridomas responded to hsp 60. Furthermore, heat shock syngeneic resident pulmonary lymphocytes stimulated T cells bearing V4 and V6 (47). It is thus possible that the NK1.1⁺ T cells recognized a self Ag presented by MHC class II molecules that is induced by the stress of inflammation or infection. However, we showed here that the T cells recognized not only CHO cells expressing wild-type IA^b but also those expressing IA^b covalently bound to E52-68 or 50V peptide, suggesting that peptides do not confer specificity. Matis et al. (48) reported that T cell hybridomas expressing V2 and V6 are specific for IE^k,ds, or IA^d. These T cells show a broad cross-reactivity that is not seen for ß T-alloreactive T cells. Furthermore, Schild et al. (49) demonstrated that the recognition of the T cell hybridomas does not require a class II Ag-processing pathway nor presumably peptides and that T cells recognize an epitope distal to the peptide-binding site, completely different from that of ß T cells. Similarly, it is possible that NK1.1⁺ T cells may recognize MHC class II molecules independently of Ag processing and binding peptides. However, since our T cells consisted of heterogeneous populations, the possibility cannot be excluded that different T cells may recognize MHC class II molecules in a different manner. Additional experiments using single T cell clones may clarify the nature of MHC class II recognition by NK1.1⁺ T cells.

The NK1.1⁺ T cells proliferated vigorously in response to PHA-treated spleen cells but not to naive or LPS-stimulated spleen cells. Although the effects of PHA treatment on Ag recognition of NK1.1⁺ T cells are unknown, Ag presentation by PHA-treated spleen cells is an important method of activation of T cells. The marked ability of PHA-treated spleen cells to stimulate NK1.1⁺ T cells may be cause that Ag expression level is higher on spleen cells treated with PHA. In fact, MHC class II expression on B220⁺ cells is much higher in PHA-blast than naive or LPS-stimulated cells. It is possible that PHA-blastoid cells may enhance the MHC class II expression on B cells via cytokine production or CD40/CD40 ligand interaction, which in turn stimulate the T cells. There may be the effect of cytokines derived from PHA-treated spleen cells, especially MHC class II-restricted CD4⁺ T cells. However, the response of T cells was inhibited by anti-IA^b mAb, indicating that MHC class II is major a factor in the response of T cells to PHA-treated spleen cells. Another possibility is that unique costimulatory molecules on activated ß T cells may activate the NK1.1⁺ T cells. Cross-linking of CD28 on T cells is essential for their activation (50, 51). An ß T cell line prolonged activation expresses B7-1 molecule. Hathcock et al. (52) observed low levels of B7-2 on activated ß T cells. Herpes simplex virus-stimulated human T cells preferentially expanded in response to the HSV-infected autologous PHA blasts (53, 54). NK1.1⁺ T cells induced by culturing T cells from influenza-infected mice with IL-2 exhibited PHA-dependent cytolytic activity against virus-infected cells (55). Spaner et al. (56) reported that KN6⁺ T cells responded very strongly to mitogen-activated ß T cells. It is necessary to examine the possible existence of other costimulatory systems between NK1.1⁺ T cells and PHA-treated self spleen cells.

There have been several reports concerning the expression of NK1.1 on T cells (26, 27, 55, 57). NK1.1⁺ T cell number were increased following in vitro stimulation with anti-CD3 mAb using bronchoalveolar lavage (BAL) populations from mice infected with influenza virus (55). The NK1.1⁺ T cells in BAL may express TCR encoded by V4 gene segment, because the phenotype is considered to be characteristic of the resident T cell population in the lung. IL-2 stimulation of cultured CD4^-CD8^- T cells from the thymus and spleen results in the generation of TCR⁺CD16⁺NK1.1⁺B220⁺CD5^- large granular lymphocytes (57). These results suggest that NK1.1 may be preferentially expressed on the activated T cells. However, we showed that TCR repertoires differed between NK1.1⁺- and NK1.1^- T cells. Although the possibility of preferential NK1.1 expression on the activated T cells remains to be determined, NK1.1⁺- and NK1.1^- T cells in the peritoneal cavity of infected mice are derived from different lineages.

NK1.1⁺ß T cells are almost completely absent in the thymus of ß₂m-deficient mice, whereas NK1.1⁺ T cells are reduced in the thymus of Aß^b-deficient mice. The emergence of NK1.1⁺ T cells was severely impaired in the peritoneal cavity of Aß^b-deficient mice after Salmonella infection. These results indicated that thymic and peripheral NK1.1⁺ T cells required the presence of MHC class II molecules for their generation. Azuara et al. (27) recently reported that approximately half of Thy-1^dull thymocytes expressed the NK1.1, and these cells expressed a restricted TCR V1 and TCR V6 repertoire without V-D-J junctional diversity. The repertoire of the Thy-1^dull thymocytes including NK1.1⁺ thymocytes in DBA/2 mice seems to be different from NK1.1⁺ T cells in the peritoneal cavity of B6 mice infected with Salmonella. It is likely that different haplotypes of MHC class II molecules select the different TCR V repertoire of NK1.1⁺ T cells. In fact, the NK1.1⁺ T cells in the thymus preferentially expressed the V4 gene segment (data not shown). We speculate that the NK1.1⁺ T cells in the peritoneal cavity are derived from thymus. However, a significant fraction of T cells are thought to develop extrathymically. Therefore, the possibility cannot be excluded that NK1.1⁺ T cells in the periphery are derived from progenitors different from those of thymic NK1.1⁺ T cells. Additional experiments are required to clarify the positive selection of NK1.1⁺ T cells by MHC class II in the thymus and periphery.

In conclusion, we observed a unique population of T cells in the peritoneal cavity of mice infected with Salmonella, which express NK1.1 Ag and TCR preferentially encoded by V4 and V6.3 gene segments. NK1.1⁺ T cells recognized MHC class II, and thymic and peripheral NK1.1⁺ T cells were diminished in MHC class II-deficient mice. In contrast to NK1.1⁺ß T cells specific for CD1, MHC class II molecules are involved in generation of thymic and peripheral NK1.1⁺ T cells.

	Acknowledgments

 
We thank Drs. R. Kubo (National Jewish Center), J. A. Bluestone (The University of Chicago), and M. Kimoto (Saga Medical School) for providing H57-597, UC7-13D5 hybridomas, or Y3p Ab and Mr. Yamakawa for technical assistance with the EPICS sorting.

	Footnotes

 
¹ This work was supported in part by grants to H.N. from the Ministry of Education, Science and Culture of the Japanese Government, the Ohyama Health Foundation, and the Inoue Foundation for Science, by a Searle Scientific Research Fellowship, and by the Ciba-Geigy Foundation (Japan) for the Promotion of Science. This work was also supported in part by grants to Y.Y. from the Ministry of Education, Science and Culture and the Ministry of Health Welfare of the Japanese Government.

² Address correspondence and reprint requests to Dr. Hitoshi Nishimura, Laboratory of Host Defense and Germfree Life, Research Institute of Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya 466, Japan. E-mail address:

³ Abbreviations used in this paper: PE, phycoerythrin; PEC, peritoneal exudate cells; MMC, mitomycin; ß₂m, ß₂-microglobulin; CHO, Chinese hamster ovary; B6, C57BL/6.

Received for publication June 8, 1998. Accepted for publication September 29, 1998.

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