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IL-4-Producing T Cells That Express a Very Restricted TCR Repertoire Are Preferentially Localized in Liver and Spleen¹

David J. Gerber^*, Véronique Azuara, Jean-Pierre Levraud, Shu Ying Huang^*, Marie-Pierre Lembezat and Pablo Pereira^2,

^* Howard Hughes Medical Institute, Center for Cancer Research, and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; and Unité du Développement des Lymphocytes, Centre National de la Recherche Scientifique, URA 1961, and Unité de Biologie Moléculaire du Gène, Institut National de la Santé et de la Recherche Medicale, Unité 277, Institut Pasteur, Paris, France

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
IL-4-producing thymocytes in normal mice belong to a distinct subset of T cells characterized by low expression of Thy-1. This thymocyte subset shares a number of phenotypic and functional properties with the NK T cell population. Thy-1^dull thymocytes in DBA/2 mice express a restricted repertoire of TCRs that are composed of the V1 gene product mainly associated with the V6.4 chain and exhibit limited junctional sequence diversity. Using mice transgenic for a rearranged V1J4C4 chain and a novel mAb (9D3) specific for the V6.3 and V6.4 murine TCR chains, we have analyzed the peripheral localization and functional properties of T cells displaying a similarly restricted TCR repertoire. In transgenic mice, IL-4 production by peripheral T cells was confined to the ⁺9D3⁺ subset, which contains cells with a TCR repertoire similar to that found in Thy-1^dull thymocytes. In normal DBA/2 mice such cells represent close to half of the T cells present in the liver and around 20% of the splenic T cells.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Most T lymphocytes in the thymus and peripheral lymphoid organs express a TCR composed of - and ß-chains (ß T cells) and either the CD4 or CD8 coreceptor. However, two distinct populations of T cells have received increased attention during the last few years. One is composed of cells that use - and -chains to form their TCR ( T cells) (1). The other is composed of ß T cells that bear markers common to NK cells and are referred to as NK T cells (2, 3, 4). Although they comprise a minor portion of T cells in the thymus and peripheral lymphoid organs, these T cell populations constitute an important fraction of T lymphocytes present in other anatomical sites. Thus, T cells are the predominant T cell population in most epithelial surfaces (1), whereas NK T cells represent around half of the T lymphocytes found in liver and bone marrow (2, 5, 6, 7).

We have recently characterized a population of thymocytes that shares a number of phenotypic and functional characteristics with NK T cells (8). In the thymus, this T cell population differs from conventional T cells in its low expression of Thy-1, and thus, we referred to it as the Thy-1^dull T cell population. Similar to NK T cells, most Thy-1^dull thymocytes express a phenotype usually associated with activated or memory T cells, and approximately half of them express the NK1.1 cell marker (a member of the NKR-P1 gene family) and/or the CD4 coreceptor (8). Upon activation in vitro, both NK T cells and Thy-1^dull T cells produce large amounts of various cytokines, including IL-4, IFN-, IL-3, IL-5, IL-10, and GM-CSF (8, 9, 10, 11, 12). Finally, both cell populations have been shown to express a highly restricted TCR repertoire (8, 13, 14, 15). The skewed TCR repertoire expressed by NK T cells is selected by the MHC class-I like molecule CD1d (16, 17, 18, 19). The identity of the putative endogenous molecule selecting the TCR repertoire of the Thy-1^dull T cell population (20) is not known.

The physiological functions of NK T cells and Thy-1^dull T cells remain unknown. However, recent experiments have shown that NK T cells mediate IL-12-induced tumor cell killing in vivo (21, 22, 23), and several independent lines of evidence suggest that NK T cells may be involved in regulating autoimmunity (24, 25, 26, 27). Despite their ability to promptly produce large amounts of IL-4 upon stimulation in vivo (28), the role of NK T cells in the induction of Th2 responses has been questioned (29, 30, 31). In contrast, a major role of T cells has been demonstrated in the early production of IL-4, which is required for the development of specific IgE responses in the periphery and for subsequent airway inflammation upon intranasal Ag challenge (32). This led to the suggestion that IL-4 production by T cells in the periphery could be important for the development of certain Th2 responses to protein Ags, and thus focused attention on the Thy-1^dull T cell population.

An important step in understanding the physiological function of the Thy-1^dull T cells is the characterization of their peripheral localization. In this report we have used mice transgenic (Tg)³ for a rearranged V1J4C4 chain and a novel mAb (9D3) specific for the V6.3 and V6.4 murine TCR chains to analyze this issue. Our results show that T cells expressing functional abilities and TCR repertoire similar to those described for the Thy-1^dull thymocytes are mainly present in liver and spleen. In normal DBA/2 mice, these T cells are found in the thymus, liver, and spleen at a level of 1–4 x 10⁵ cells/organ.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Mice

C57BL/6 (B6) mice Tg for a rearranged V1J4C4 chain (V1Tg) and TCR knockout mice were maintained in our animal facilities. DBA/2 and (B6 x DBA/2)F₁ (B6D2F₁) mice were obtained from Iffa Credo (L’Abresle, France). (DBA/2 x B6)F₁ (D2B6F₁) V1Tg were produced by mating DBA/2 females with B6 V1Tg males. F₁ mice were typed for the presence of the transgene by PCR analysis on tail DNA using primers specific for the V1 gene and for the V1J4 junction (see below). Mice were used between 7–15 wk of age.

Antibodies

Anti-CD4 (RL.174), anti-CD8 (HO-2.2), anti-heat-stable Ag (anti-HSA; J11d), anti-Cß (H57-597), anti-C (3A10), anti-V1 (2.11), and anti-V6.4/V6.3 (9D3) were prepared and used as previously described (33). PE-labeled anti-C (GL3), FITC- or PE-labeled anti-HSA, anti-Thy-1, anti-CD62 ligand (anti-CD62L), anti-CD44, anti-CD69, anti-CD3, anti-CD45RB, anti-NK1.1, anti-CD4, and anti-CD8 were purchased from PharMingen (San Diego, CA). Goat anti-mouse IgM was purchased from Sigma (St. Louis, MO). For the cytokine-specific ELISA we used the following mAb: anti-IFN- (clones R46A2, and AN18) and anti-IL-4 (clones BVD4 and BVD6; a gift from Dr. P. Minoprio, Unité d’Immunoparasitologie, Institut Pasteur, Paris, France).

Immunofluorescence staining and FACS

Cells (10⁵-10⁶) were incubated in staining buffer (PBS, 3% FCS, and 0.1%NaN₃) with the indicated labeled mAbs for 30 min on ice and washed twice. When biotin-conjugated mAbs were used, the cells were further incubated with either PE-labeled streptavidin (Southern Biotechnology Associates, Birmingham, AL) or streptavidin Tricolor (Caltag, South San Francisco, CA) for 15 min on ice. After another wash, cells were resuspended in staining buffer containing 1 µg/ml propidium iodide and analyzed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Dead cells were gated out either by their staining with PI or by their forward and angle light scatter profile. Data were analyzed using the CellQuest program.

For FACS sorting, cell suspensions were prepared as indicated above and incubated with the appropriate mAb at a concentration of 30 x 10⁶ cells/ml for 30 min on ice. After two washes, cells were resuspended in PBS-10% FCS and sorted in a FACStar^Plus cell sorter (Becton Dickinson). The purity of the sorted populations was >95%.

Cell purification, cell culture, and cytokine-specific ELISA

Single-cell suspensions were prepared from the thymus, spleen, lymph nodes, and liver according to standard procedures. CD4^-CD8^- (DN) and CD8^-HSA^- thymocytes were prepared by complement-mediated killing as previously described (8). Liver mononuclear cells were isolated by centrifugation in a discontinuous gradient of Percoll (Pharmacia, Uppsala, Sweden). Briefly, total liver cells were resuspended in 100% isotonic Percoll solution and overlaid with 70 and 40% isotonic Percoll solutions. After centrifugation for 30 min at 500 x g, cells at the 40–70% interface were collected, washed twice, and further purified from contaminating RBC by density gradient centrifugation using Lympholyte M (Cedarlane, Hornby, Canada). The cells were washed twice in medium containing 5% FCS. Splenic RBC were lysed by incubating spleen cells for 2 min in 5 ml of NH₄Cl solution. Lymph node cells and RBC-depleted spleen cells were resuspended in medium containing 10% FCS at a concentration of 2.5–10 x 10⁶ cells/ml and incubated for 90 min in petri dishes (Optilux, Falcon 1005, Oxnard, CA) previously coated with anti-Cß and anti-IgM Ab (each at 5 µg/ml in PBS), with sporadic agitation. Nonadherent cells were recovered and washed twice before use.

FACS-sorted cells (1.5 x 10⁵ cells/ml) were cultured in flat-bottom microtiter plates previously coated with 10 µg/ml anti-C mAb (3A10) in complete medium, i.e., DMEM with Glutamax-I medium (Life Technologies, Gaithersburg, MD) supplemented with sodium pyruvate, 5 x 10^-5 M 2-ME, nonessential amino acids, and antibiotics (all from Life Technologies) and 10% FCS (Boehringer Mannheim, Meylan, Germany). Mouse rIL-2 was added at a final concentration of 100 U/ml. Supernatants from 3-day cultures were tested for the presence of IL-4 and IFN- by ELISA as previously described (34).

Production of the 9D3 mAb

TCR KO mice were immunized i.p. twice at 2-wk intervals with 5 x 10⁶ DTN40 (V1/V6.4) hybridoma cells and once i.v. with 10⁶ DTN40 hybridoma cells resuspended in PBS. The DTN40 hybridoma was obtained by fusing Thy-1^dull thymocytes of DBA/2 origin with BW5147 thymoma cells (8). Three days after the last injection, spleen cells were fused with SP2/0 cells as described previously (33). The cells were then distributed in 96-well flat-bottom plates in complete medium supplemented with hypoxanthine-aminopterin-thymidine. Culture supernatants from growth-positive wells were tested for their ability to bind to the immunizing hybridoma cell but not to a TCR-negative variant of the same hybridoma. Binding of the Ab to the hybridoma cells was detected with FITC-labeled goat anti-mouse Ig (Caltag) and analyzed with a FACScan. The fine specificities of the selected Ab were determined by their ability to stain T cell hybridomas that express different TCR and TCR chains.

Oligonucleotide primers and PCR conditions

The following oligonucleotide primers were used: V6, TCTGTAGTCTTCCAGAAATCA; V6.4, GTTTTCCTTATTCGACAAACA; C, CGAATTCCACAATCTTCTTG; JS1, GTTCCTTGTCCAAAGACGAG; V1, CCGGCAAAAAGCAAAAAAGTT; and V1J4Tg junction, CCCATGATGTGCCTGACCAG. PCR was performed using a GeneAmp PCR system 9600 (Perkin-Elmer/Cetus, Norwalk, CT). Each cycle consisted of incubation at 92°C for 20 s, followed by 55°C for 30 s and 72°C for 30 s. Before the first cycle, a 2-min 94°C denaturation step was included, and after the 35th cycle the extension at 72°C was prolonged for 4 min.

Nucleic acid preparation and population analysis of TCR rearrangements

Total cellular RNA from sorted cells was extracted with RNA-B (Bioprobe Systems, Montreuil, France). Before RNA extraction, sorted cells were mixed with 10⁶ SP2/0 cells as a carrier. cDNA was synthesized with oligo(dT) (Pharmacia) using superscript reverse transcriptase (Life Technologies) according to the manufacturer’s instructions. The population analysis of TCR rearrangements has been previously described in detail (8, 20).

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
V1J4C4 Tg animals of appropriate genetic background contain a population of Thy-1^dull. thymocytes

Given the paucity of cells in the peripheral lymphoid organs in the mouse, it was difficult to study the peripheral representation of the Thy-1^dull population in normal mice. To overcome this problem we took advantage of the availability of a mouse Tg for a rearranged V1J4C4 chain that we produced previously (35). The DNA inserted to produce this Tg line consisted of a 45-kb cosmid containing the rearranged V1J4C4 gene flanked by 10 kb of sequence upstream of the V1 segment and 26 kb of sequence downstream of the last C4 exon (Fig. 1A). Thus, this construct should contain all the regulatory elements required to ensure normal expression of the transgene. The cosmid clone was isolated from a T cell hybridoma of B6 origin (T3.13.1) (33), and the V1J4 junctional sequence is identical with one of the two more common V1J4 junctional sequences found among the Thy-1^dull thymocytes in DBA/2 mice (8). ß T cell development in these mice appears quite normal, as indicated by the similar number of total thymocytes (not shown) and of the major thymocyte populations defined by double staining with CD4 and CD8 mAbs in Tg animals and littermate controls (Fig. 1B). In contrast, a 3- to 10-fold increase in the number of thymocytes is present in Tg mice compared with littermates, and most of these cells express the transgenic chain (Fig. 1C). This increase in the absolute numbers of T cells in the Tg mice is also evident in the periphery, where Tg animals contain 3- to 10-fold more T cells than control littermates; here again, most of the T cells in Tg mice express the V1 chain (Fig. 1C).

View larger version (39K): [in this window] [in a new window]   FIGURE 1. A, Restriction map of the 2F construct used to generate V1Tg mice. Variable and constant regions are indicated beneath the map. The sizes of the variable and constant regions are approximate. Not all the restriction sites are necessarily shown. A, ApaI; F, FspI; K, KpnI; N, NotI; R, EcoRI; S, SnaBI. B, Expression of CD4 and CD8 in the thymus of V1Tg mice and non-Tg littermates. Thymocytes from the indicated mice were stained with FITC-labeled anti-CD4 and PE-labeled anti-CD8 mAb and analyzed with a FACScan. Numbers indicate the percentage of positive cells in each quadrant. C, Expression of the TCR and the V1 chain in different organs of V1Tg mice and non-Tg littermates. Cells isolated from the indicated organs were stained with FITC-labeled anti-CD3 and biotin-labeled anti- mAb (left panels) or with FITC-labeled anti- and biotin-labeled anti-V1 Ab (right panels) followed by streptavidin-PE. Dot plots show the log10 of fluorescence intensity and were produced with the CellQuest program. Numbers indicate the percentages of CD3++ and CD3+- (left panels) and of +V1+ and +V1- among total cells present in the lymphocyte gate. Some 20,000 to 100,000 cells were analyzed in each panel.

View larger version (24K): [in this window] [in a new window]   FIGURE 2. V1Tg mice of D2B6F1 genetic background contain a major population of Thy-1dull thymocytes. DN thymocytes from the indicated V1Tg mice were stained with PE-labeled anti-Thy-1, biotin-labeled anti-, and FITC-labeled anti-HSA, anti-CD62L, or anti CD44 mAb, followed by streptavidin-Tricolor and analyzed in a FACScan. A, Dot plots of vs Thy-1 staining in B6 and D2B6F1 V1Tg mice. Numbers indicate the percentage of Thy-1dull thymocytes among total + cells (mean ± 1 SD of 10 animals analyzed individually). B, Profiles of expression of the indicated marker on +Thy-1bright (upper panels) and +Thy-1dull (lower panels) thymocytes from D2B6F1 V1Tg mice.

The 9D3 mAb recognizes V6.3 and V6.4 chains

To produce such an mAb we immunized -deficient mice with a Thy-1^dull T cell hybridoma of DBA/2 origin. Spleen cells from immunized mice were fused with SP2/0 cells, and the resulting hybridomas were tested for their ability to bind to the immunizing cells but not to a TCR-negative variant of the same hybridoma. The fine specificities of the selected mAbs were analyzed by staining a large panel of T cell hybridomas of B6 and DBA/2 origin previously characterized for their V and V usage (P. Pereira, unpublished observations). One hybridoma, termed 9D3, specifically stained some T cell hybridomas expressing a V6 chain but did not stain T cell hybridomas expressing other V chains, including V2, V4, V5, and V7 (not shown). Further analyses with T cell hybridomas expressing different members of the V6 subfamily showed that 9D3 stained all T cell hybridomas of B6 origin expressing the V6.3 chain (36) and all T cell hybridomas of DBA/2 origin expressing the V6.4 (8) chain but not hybridomas expressing any other member of the V6 subfamily, including the related B6 V6.1 (37) chain and the DBA/2 V6.5 and V6.6 chains (8) (Table I). Furthermore, 9D3 stained a sizable proportion of thymocytes, splenocytes, and intestinal intraepithelial lymphocytes in DBA/2 and B6 strains as well as in B10 and B10.D2 mice that share the same TCR/ haplotype as B6 mice (39, 40, 41), but did not stain any T cells in BALB/c, C3H, CBA, or 129/Sv strains of mice, which share a different TCR/ haplotype (39, 40, 41), suggesting that the 9D3 mAb does not recognize the V6.2 chain (37) (not shown).

View larger version (22K): [in this window] [in a new window]   FIGURE 3. Correlation between 9D3 expression and low levels of Thy-1. DN thymocytes from D2B6F1 V1Tg mice were stained with FITC-labeled anti-, PE-labeled anti-Thy-1, and biotin-labeled 9D3 mAb followed by streptavidin-Tricolor and analyzed in a FACScan. A dot plot of vs 9D3 staining in total DN cells (left) and Thy-1 expression by +9D3+ (R2) and +9D3- (R3) populations is shown. R2 and R3 denote the electronic gates used for the analysis.

Frequency of ⁺9D3⁺ cells in different organs

We next analyzed the frequency of 9D3⁺ cells among lymphocytes in different organs in D2B6F₁ Tg mice. As shown in Table II, 9D3⁺ cells represent roughly 50, 30, and 20% of the T cells in liver, spleen, and peripheral lymph nodes, respectively. In other sites, such as bone marrow and peritoneal cavity, the frequency of T cells was too low to allow accurate quantification even in Tg mice, although ⁺9D3⁺ cells were present at those sites.

View this table: [in this window] [in a new window]   Table II. Representation of +9D3+ T cells in different organs from D2B6F1 V1Tg mice

⁺9D3⁺ cells in different organs express a restricted TCR repertoire

One of the characteristics of the Thy-1^dull thymocytes is their very restricted TCR repertoire. Besides their almost exclusive use of the V1 chain and one or two members of the V6 chain subfamily, their VDJ junctional sequences show limited diversity, and the great majority of them show identical length (8, 20). To investigate the putative relationship between the Thy-1^dull thymocytes and the ⁺9D3⁺ cells in the periphery we examined the junctional length of the V6 transcripts present in sorted ⁺9D3⁺ and ⁺9D3^- cells isolated from thymus, spleen, liver, and peripheral lymph nodes as well as from a Thy-1^dull hybridoma (DTN40). RT-PCR reactions with V6- and C-specific primers on cDNA isolated from all sorted populations were performed. An aliquot of each amplification product was submitted to a run-off reaction in the presence of a 6-carboxy-fluorescein (FAM)-labeled J1 primer, and the fluorescent products were resolved on a denaturing acrylamide gel cast on an automated sequencer.

Typical of polyclonal VDJ junctions, the profiles obtained from sorted ⁺9D3^- populations isolated from different organs showed 11–15 defined peaks that form a Gaussian-type curve (Fig. 4). All adjacent peaks differ in size by three nucleotides, and thus, the profiles show mainly in-frame junctions. In contrast, the profiles obtained for the sorted ⁺9D3⁺ populations showed a prominent peak at a CDR3 length identical with that displayed by the DTN40 hybridoma cell line, although the relative representation of this peak varied depending on the organ from which the cells were isolated. Calculation of the area of this peak relative to the area of all peaks in each organ indicated that 70, 35, 50, and 25% of the V6(D)J1 rearrangements present in ⁺9D3⁺ populations isolated from thymus, spleen, liver, and lymph nodes, respectively, showed this particular CDR3 size.

View larger version (34K): [in this window] [in a new window]   FIGURE 4. Population analysis of V6(D)J1 rearrangements expressed by +9D3+ and +9D3- populations isolated from different anatomical sites. Total RNA samples isolated from sorted +9D3+ and +9D3- populations isolated from the indicated anatomical sites and from the DTN40 hybridoma cells were converted to cDNA and amplified with V6- and C-specific primers. An aliquot of the amplified products was submitted to a run-off reaction with a FAM-labeled J1 primer, and the final products were resolved with an automated sequencer. Plots show the profile of fluorescence intensity vs the length of the fragments. LN, lymph node cells

⁺9D3⁺ cells in the periphery produce IL-4 and IFN- upon activation with anti- mAbs and IL-2

We have previously shown that Thy-1^dull thymocytes secrete high levels of IL-4 and IFN- upon activation in vitro with coated anti- mAbs and IL-2 (8). To investigate whether ⁺9D3⁺ cells in the periphery have the same functional abilities as the Thy-1^dull thymocyte population, sorted ⁺9D3⁺ and ⁺9D3^- cells from thymus, spleen, lymph nodes, and liver of D2B6F₁ Tg mice were cultured in anti- mAb-coated plates in the presence of IL-2, and the levels of IL-4 and IFN- present in the culture supernatants were quantitated (Table III). Regardless of the organ from which they were isolated, T cells secreted substantial amounts of IFN-, although 2- to 4-fold higher levels were detected in ⁺9D3^- cells than in ⁺9D3⁺ cells. In contrast, larger differences were observed in the levels of IL-4 produced by these two T cell subsets in the different organs. Thus, in thymus, spleen, and lymph nodes, ⁺9D3⁺ cells secreted 10- to 20-fold higher levels of IL-4 than ⁺9D3^- cells, while the difference was only 2-fold in the liver. It is likely that in the thymus, the low levels of IL-4 detected in the ⁺9D3^- cultures arise from the roughly 10% of Thy-1^dull thymocytes in D2B6F₁ animals, which express the V6.6 gene product and are not recognized by the 9D3 mAb. Similarly, the low levels of IL-4 detected in the ⁺9D3^- cultures from spleen and lymph nodes could arise from V6.6-bearing cells, although we do not have direct evidence for this interpretation. Taken together, these experiments suggest that the production of IL-4 by peripheral T cells is primarily accomplished by cells expressing the V6 chain and a restricted TCR repertoire.

View this table: [in this window] [in a new window]   Table III. Cytokine production by sorted +9D3+ (+) and +9D3- (-) cells in D2B6F1 V1Tg mice

Phenotypic analysis of ⁺9D3⁺ cells at different anatomical sites^.

We then investigated the surface phenotype of the ⁺9D3⁺ and ⁺9D3^- cells present at different anatomical sites, and their FACS profiles in thymus, spleen, and liver are illustrated in Fig. 5. Invariably, most ⁺9D3⁺ cells showed an HSA^low CD44^bright CD62L^low CD45RB^int CD69⁺ phenotype, similar to that of NK T cells and activated T cells. However, none of these markers could unambiguously define the ⁺9D3⁺ population. In fact, the same activated phenotype is expressed by the vast majority of T cells in the liver, and CD44^bright cells represent an important fraction of the ⁺9D3^- cells in thymus and spleen.

View larger version (24K): [in this window] [in a new window]   FIGURE 5. Phenotypic analysis of +9D3+ and +9D3- populations. Cells were prepared as described in Materials and Methods and three color stained as described in Fig. 2. Shown are the profiles of the indicated Ab in +9D3+ and +9D3- populations isolated from the indicated organs. Staining results of lymph node cells were similar to those presented for spleen cells.

View larger version (35K): [in this window] [in a new window]   FIGURE 6. CD4, CD8, and NK1.1 expression by +9D3+ and +9D3- populations. A, D2B6F1 Tg thymocytes treated with anti-HSA and anti-CD8 mAbs and complement were stained with FITC-labeled anti-CD4, PE-labeled anti-, and biotin-labeled 9D3 followed by streptavidin-Tricolor. The histogram shows CD4 expression in +9D3+ cells. B, DN thymocytes from D2B6F1 Tg mice were stained with FITC-labeled anti-NK1.1, PE-labeled anti-, and biotin-labeled 9D3 mAb followed by streptavidin-Tricolor. Histograms show NK1.1 expression in +9D3+ and +9D3- populations. C, Cells were prepared as described in Materials and Methods and three color stained as described above. Shown are the profiles of the indicated Ab in +9D3+ and +9D3- populations isolated from the indicated organs. All cells were analyzed with a FACScan. Staining results of lymph node cells were similar to those shown for spleen cells.

⁺9D3⁺ cells expressing a restricted TCR repertoire exist in the periphery of normal mice

Two types of experiments were performed to investigate the presence of this type of T cells in the periphery of normal mice and to estimate their numbers. First we performed RT-PCR reactions on total RNA isolated from different organs of DBA/2 mice with primers specific for the V6.4 and C gene segments. Because most ß T cells have deleted the TCR locus in both chromosomes, this PCR is expected to mainly, if not exclusively, amplify transcripts expressed by T cells. The PCR amplifications were submitted to run-off reactions with a FAM-conjugated J1 primer, and the labeled fragments were separated on a sequencing gel. The profiles obtained from one such experiment are shown in Fig. 7. As can be seen, a major peak protruding from the Gaussian-type curve and corresponding to a CDR3 length identical with that of the DTN40 hybridoma cell line is evident in thymus, spleen, and liver. The same peak is also evident, although to a lesser extent, in the lymph nodes and in the peritoneal cavity. These experiments suggested that cells expressing a restricted TCR repertoire are present in these organs in normal mice.

View larger version (25K): [in this window] [in a new window]   FIGURE 7. Cells with a restricted TCR repertoire exist in the periphery of normal mice. RNA isolated from the indicated organs of DBA/2 mice was amplified by RT-PCR with V6.4- and C-specific primers. An aliquot of the amplified products was submitted to a run-off reaction with a FAM-labeled J1 primer, and the final products were resolved with an automated sequencer. Plots show the profile of fluorescence intensity vs the length of the fragments. LN, lymph node cells; PC, peritoneal cavity cells.

View this table: [in this window] [in a new window]   Table IV. Representation of +9D3+ and +V1+ T cells in different organs from normal DBA/2 mice

The phenotype, TCR diversity level, and functional properties found in this T cell subset are also characteristic of the T NK population, and both T cell populations seem to home preferentially to liver and spleen. These common properties may reflect a similar differentiation program and/or a related function. However, the likely difference in their specificities implies separate aspects of function. Additional experiments will aim at characterizing the specific ligands recognized by this T cell population.

	Acknowledgments

 
We thank P. Minoprio for providing the anti-cytokine mAb. The Tg mice used here were produced in the laboratory of Prof. S. Tonegawa.

	Footnotes

 
¹ This work was supported by institutional grants and grants from the Association pour la Recherche sur le Cancer, Fondation pour la Recherche Medicale, and Association Nationale pour la Recherche contre le Sida.

² Address correspondence and reprint requests to Dr. Pablo Pereira, Unité du Développement des Lymphocytes, Centre National de la Recherche Scientifique, URA 1961, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France. E-mail address:

³ Abbreviations used in this paper: Tg, transgenic; CD62L, CD62 ligand; DN, double negative; FAM, 6-carboxy-fluorescein; HSA, heat-stable Ag.

Received for publication May 4, 1999. Accepted for publication June 30, 1999.

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