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V T Cell Repertoire of CD8⁺ Splenocytes Selected on Nonpolymorphic MHC Class I Molecules¹

Dhafer Laouini^2,*, Armanda Casrouge^*, Sophie Dalle^*, François Lemonnier, Philippe Kourilsky^* and Jean Kanellopoulos^3,*

^* Laboratoire de Biologie Moléculaire du Gène, Institut National de la Santé et de la Recherche Médicale U277-Institut Pasteur, Paris, France; and Laboratoire d’Immunité Cellulaire Anti-Virale, Institut Pasteur, Paris, France

	Abstract

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In this work, we have studied the role of the MHC class Ib molecules in the selection and maintenance of CD8⁺ T splenocytes. We have compared the CD8⁺ T cell repertoires of wild-type, H-2K-deficient, H-2D-deficient, or double knockout C57BL/6 mice. We show that the different CD8⁺ repertoires, selected either by class Ia and class Ib or by class Ib molecules only, use the various V (AV) and V (BV) rearrangements in the same proportion and without biases in the CDR3 size distribution. Furthermore, we have estimated the size of the BV repertoire in the four different strains of mice. Interestingly, we have found that the BV repertoire size is proportional to the overall number of CD8⁺ splenocytes. This observation implies that BV diversity is positively correlated with the number of CD8⁺ cells, even when the number of CD8⁺ splenocytes is dramatically reduced (90% in the double knockout mice).

	Introduction

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Immune responses to intracellular pathogens involve the recognition of MHC class I molecules loaded with peptides derived from infectious agents. These peptides/MHC class I complexes activate CD8⁺ T lymphocytes bearing specific TCRs. In mice, the MHC class I-related molecules include the classical (class Ia) proteins encoded by highly polymorphic genes (H-2K, H-2D, and H-2L), nonclassical (class Ib) ones encoded in Q, T, and M regions, and, finally, the ₂-microglobulin-associated molecules such as CD1 encoded by genes not linked to the MHC region. The MHC class Ib molecules H2-M3, HLA-E, and the mouse CD1d have an overall structure comparable to class Ia molecules (1, 2, 3, 4). The major differences with the class Ia molecules are found in the peptide binding groove (1, 3, 4), which can accommodate various ligands such as peptides from class Ia hydrophobic leader sequences for Qa1 and HLA-E (5, 6), N-formylated peptides derived from the N termini of certain bacterial and mitochondrial encoded proteins for H2-M3 (7) and ceramides, as well as some protozoan glycosylphosphatidylinositol for CD1d (8, 9, 10, 11).

The three-dimensional structure of the TCR has shown that each V (AV)⁴ or V (BV) chain forms three loops that interact with the peptide/MHC class Ia molecule (12, 13). These loops correspond to the complementarity-determining regions (CDR). The CDR1 and 2 are encoded within the AV and BV genes. The third CDRs are generated by the somatic rearrangement of V and J segments for AV and V, D, and J segments for BV. The diversity of the TCR repertoire results from rearrangements of various gene segments, their imprecise joining, the addition of template-independent N nucleotides during this process, and the pairing of different - and -chains (14, 15).

During development, the TCR repertoire of thymocytes is shaped by two different mechanisms of selection. Positive selection rescues thymocytes from cell death and warrants that mature T lymphocytes are capable of recognizing peptides/MHC complexes. Negative selection ensures that high avidity self-reactive thymocytes are eliminated through clonal deletion (16, 17). Both selective processes involve specific interactions with self-MHC molecules (18, 19, 20, 21) but lead to two different cell fates. The role of naturally processed self-peptides, eluted from MHC class Ia molecules, has recently been addressed by several groups (22, 23). Some of these self-peptides, even though they had few homologies with the antigenic peptide, were able to positively select some but not all CD8⁺ thymocytes bearing transgenic TCRs (23). These findings imply that weakly specific interactions between TCRs and self-peptides/MHC class Ia complexes promote positive selection of CD8⁺ thymocytes. Recent results of Berg et al. (24) suggest that positive selection by self-peptides/MHC class Ib molecules may be more specific than for peptides/MHC class Ia. Thus, a transgenic TCR specific for a Listeria-derived peptide presented by the MHC class Ib molecule H2-M3 is selected by a single self-peptide, i.e., the formyl-methionine peptide from an NADH dehydrogenase (24).

Selection of TCRs by self-peptides/MHC class Ia or Ib complexes is rather similar. However, the size and diversity of the CD8⁺ T lymphocytes selected by these complexes has not been determined yet. To study the size and diversity of the repertoire of CD8⁺ cells selected on class Ib molecules only, we took advantage of C57BL/6 mice deficient for H-2K and H-2D genes (H-2K°^/°, H-2D°^/°) (25). In the double knockout (KO) mice, only class Ib molecules persist because the MHC class Ia H-2L^b is missing in the C57BL/6 strain.

Using a method recently described by Casrouge et al. (26), we have estimated the size of the TCR repertoire from naive T splenocytes of wild-type, H-2K°^/°, H-2D°^/°, or double KO C57BL/6 mice.

	Materials and Methods

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Animals

All mice used in this study were 6- to 8-wk-old C57BL/6, H-2K°^/°, H-2D°^/°, or double KO C57BL/6 mice raised at the Pasteur Institute animal facility.

Abs and isolation of T cell subpopulations

FITC-labeled anti-CD8 Abs were purchased from PharMingen (San Diego, CA). Splenocytes from 6- to 8-wk-old mice were depleted of B220⁺ and CD4⁺ cells using biotinylated mAbs and streptavidin beads (Dynals, Oslo, Norway). Depleted splenocytes were then incubated with rat anti-mouse CD8 mAb (PharMingen) and anti-rat IgG magnetic-activated cell sorting microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), passed through MS⁺/RS⁺ columns (Miltenyi Biotec), and separated into CD8⁺ and CD8^- lymphocytes. The CD8⁺ cell populations were counted and their purity was assessed by flow cytometry analyses and found to be 98%.

RNA extraction and cDNA synthesis

Total RNA was extracted using Trizol reagent (Life Technologies, Gaithersburg, MD) as recommended by the manufacturer, with the addition of 20 mg/ml of glycogen (Boehringer Mannheim, Mannheim, Germany) to the samples. Total RNAs were reverse transcribed into cDNA using oligo(dT)₁₇ primers, 1 mM dNTP, 40 U Rnasin (Promega, Madison, WI), and 200 U of reverse transcriptase from Moloney murine leukemia virus (Life Technologies) at 43°C for 1 h, followed by an incubation at 53°C for 10 min.

Semiquantitative CD3 PCR

The number of CD3 copies in each cDNA preparation was quantified as described by Apostolou et al. (27). A plasmid containing a four-nucleotides-deleted form of a mouse CD3 cDNA fragment was used as competitor. Copies (10⁶–10²) of the plasmid were mixed with 1 µl of cDNA preparation, and a 40-cycle competitive PCR was performed. The amplified products were subjected to run-off reaction with fluorescent anti-sense CD3-specific primer designed to hybridize equally well to wild-type and deleted CD3. These products were loaded on a sequencing gel and analyzed with Immunoscope software, then the number of CD3 copies was determined.

Immunoscope analyses of AV and BV repertoires

Semiquantitative analyses of the TCR repertoires were conducted in 25 µl containing 10⁵–10⁶ copies of CD3 transcript equivalents, with 2 U of Taq polymerase (Goldstar; Eurogentec, Brussels, Belgium) in the buffer provided by the supplier. cDNA was amplified (25–28 cycles) using TCR AV- or BV-specific sense primers and fluorescent antisense primer designed to hybridize in the AC or BC segments (28, 29).

The fluorescent products, corresponding to the elongation of individual AV or BV PCR products with various CDR3 sizes, were loaded on polyacrylamide gels and subjected to electrophoresis in an automated DNA sequencer. CDR3 size distribution and signal intensities were then analyzed with Immunoscope software (29, 30). The patterns observed from unprimed splenocytes usually contain six to eight size peaks spaced each by three nucleotides, corresponding to the lengths of in-frame transcripts. The area of each size peak is proportional to the quantity of the TCR transcripts of the corresponding CDR3 length in the sample. Each peak corresponding to a given CDR3 length usually contains multiple distinct sequences. An increase in the height and area of a size peak signals clonal expansion occurring against polyclonal background.

Each product was then diluted v/v in 25 mM EDTA-formamide solution. This mix was heat denatured for 10 min at 80°C, and a 2-µl aliquot was loaded on a 6% (or 4.25%) polyacrylamide 8 M urea gel. Gel electrophoresis was performed on a 373A or 377 DNA sequencer (Applied Biosystems, Foster City, CA). Peak size and fluorescence intensity were determined with Immunoscope software (30).

Cloning of BV-TCR rearrangements

PCRs were performed using specific BV-BJ primers in 50 µl containing 10⁵ copies of CD3 transcript equivalents of the cDNA with 2 U of PFU polymerase (Stratagene, La Jolla, CA) in the supplier’s buffer. The elongation starts with 1 min at 94°C, followed by 40 cycles each consisting of 45 s at 94°C, 45 s at 60°C, 45 s at 72°C, and ending with a step at 72°C for 10 min. The PCR product was ethanol precipitated and resuspended in 10 µl formamide containing 0.05% bromophenol blue and 0.05% xylene cyanol. This mixture was heat denatured for 10 min at 80°C and then loaded on an 8% polyacrylamide 7 M urea gel. After migration, PCR products were visualized by silver staining (31) (DNA Silver Staining System; Promega) following manufacturer’s instructions. We usually obtained six to eight bands spaced each by three nucleotides, corresponding to in-frame transcripts of the V-J combinations. Bands of interest, corresponding to a given CDR3 length, were cut out from the gel and disrupted in 40 µl water. A second PCR was conducted using the same primers on 2 µl of the isolated PCR product with 2 U of PFU polymerase (Stratagene) in the supplier buffer for 20 cycles. Further purification was performed on a 15% nondenaturing acrylamide gel in Tris-borate EDTA electrophoresis buffer one time. Staining of this gel was obtained with a 30-min bath in a 0.5 mg/ml ethidium bromide solution. PCR product was electroeluted in Tris-borate EDTA electrophoresis buffer one time, and purity of the sample was estimated by a runoff reaction with the fluorescent BJ primer. We usually obtained 90–95% purity of the final product with the expected size. PCR products were then cloned in pCR Blunt II-TOPO vector using the Zero Blunt TOPO PCR cloning kit (InVitrogen, Carlsbad, CA) as recommended by the manufacturer.

BV rearrangement sequencing

For sequencing purpose, PCR was conducted directly on TOP 10 Escherichia coli colonies (InVitrogen) in a final volume of 30 µl using universal primers RP and M13(-40) designed to hybridize on each side of the polylinker where the BV-BJ PCR product was cloned. Five microliters of this PCR product was treated for 40 min at 37°C with 0.3 U of shrimp alkaline phosphatase (Amersham, Little Chalfont, U.K.) and 3 U of exonuclease I (Amersham) in a final volume of 7 µl. Both enzymes were heat denatured at 80°C for 20 min. Sequencing reactions were conducted directly on these products using M13(-20) primer and with the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit or the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit following manufacturer’s instructions (Applied Biosystems). Reaction products were loaded on a 5% Long Ranger polyacrylamide (FMC Bioproducts, Rockland, ME) 8 M urea gel. Gel electrophoresis was performed on a 373 or 377 DNA sequencer (Applied Biosystems), and CDR3 region corresponding sequences were extracted and analyzed using a software designed for this purpose. The BV segment sequences were taken from Arden et al. (32) and from the sequence of BV locus submitted by Rowen et al. (DNA Data Base in Japan/European Molecular Biology Laboratory/GenBank databases under accession numbers AE000663 and AE000522).

Statistical calculations

The equation used by Barth et al. (33) and Behlke et al. (34) was used to estimate the maximum probable number of distinct CDR3 sequences found in the cDNA preparation. Namely, the maximum likelihood estimate (MLE) of the number of distinct sequences is a value that maximizes the equation:

in which L is the number of distinct sequences in the cDNA preparation and M is the number of observed distinct sequences among the N sequences performed. A 95% confidence interval was computed as the narrowest interval in which the cumulated values of P(L) is >0.95.

Calculation of the BV repertoire size:

Size of the BV repertoire = Number of distinct sequences found in a CDR3 peak (MLE value) divided by (frequency of BV x frequency of BJ segment x frequency of the CDR3 peak in the Immunoscope profile of the BV-BJ rearrangements under study).

Number of cells present in a CDR3 peak of given length = Total number of T cells in the analyzed sample x frequency of BV x frequency of BJ segment x frequency of the CDR3 peak in the Immunoscope profile of the BV-BJ rearrangements under study.

Number of cells bearing the same CDR3 sequence = Calculated number of cells present in a CDR3 peak of given length divided by the number of distinct nucleotide sequences (MLE value) found in the CDR3 peak.

	Results

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CDR3 size distribution of TCR repertoire from CD8⁺ T splenocytes purified from wild-type, H-2K°^/°, H-2D°^/°, or double KO C57BL/6 mice

CD8-positive splenocytes were purified from different animals, and their mRNA was extracted and reverse transcribed into cDNA. Twenty-three BV- and 22 AV-specific PCRs were then conducted in semiquantitative conditions as described in Materials and Methods. BC- or AC-specific fluorescent primers were used for the PCR. These fluorescent products were visualized on a 373A sequencer and analyzed by Immunoscope software. It was shown previously in our laboratory that the CDR3 size profiles in functional AV and BV rearrangements from naive splenocytes display six to eight peaks separated by three nucleotides, corresponding to in-frame transcripts. These peaks give a typical bell-shaped distribution of the CDR3 lengths. However, for BV17 and BV19, which are pseudogenes in C57BL/6 mice (35, 36), in- and out-of-frame transcripts are mixed (data not shown).

In Fig. 1 are shown four representative profiles of AV (A) and BV (B) rearrangements obtained from the CD8⁺ splenocytes of wild-type, H-2K°^/°, H-2D°^/°, or double KO C57BL/6 animals; they all show bell-shaped patterns characteristic of polyclonal T lymphocytes. All other rearrangements have been tested in the different strains of mice, and the results clearly show that 21 BV and 22 AV are used and that the CDR3 size profiles of all combinations have a Gaussian-like pattern.

View larger version (27K): [in this window] [in a new window]   FIGURE 1. Profiles of the fluorescent AV-AC (A) and BV-BC (B) semiquantitative PCR products obtained from splenocytes of wild-type, H-2K°/°, H-2D°/°, or double KO C57BL/6 mice. The CDR3 patterns shown are representative of the 21 BV and 22 AV combinations tested. The fluorescence intensity is represented in arbitrary units as a function of the CDR3 size in amino acids.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Profiles of the fluorescent AV14-AC PCR products from CD4+ (left), CD8+ (middle), or CD4CD8 double negative splenocytes from the four categories of animals. The fluorescence intensity is represented in arbitrary units as a function of the CDR3 size in amino acids.

BV and AV gene segments usage by TCR from CD8⁺ T splenocytes of wild-type or of different MHC class I molecule-deficient mice

The relative BV and AV usage in the CD8⁺ T cell population from wild type and the three mutant mice is shown in Fig. 3. We used a semiquantitative PCR to precisely compare the different AV and BV. The relative frequency of each BV or AV was calculated by adding the areas of the six or eight peaks corresponding to a single AV or BV combination and dividing this value by the sum of the areas of all peaks from all AV or BV rearrangements. This analysis revealed that the BV and AV usage is similar between the different mice except for the higher usage of the BV5.1 segment in the three mutant mice comparatively to wild-type animals (4–7% in the mutants vs 1% in the wild type).

View larger version (49K): [in this window] [in a new window]   FIGURE 3. AV (A) and BV (B) usage of CD8+ splenocytes from C57BL/6 , H-2K°/° , H-2D°/° , and double KO mice. In the abcissa are shown the AV and BV chains analyzed. In the ordinate are represented the relative frequency of AV and BV usage calculated as follows: % = area of the six or eight peaks corresponding to a single AV or BV combination/area of all peaks from all AV or BV rearrangements.

Analysis of the CDR3 diversity in CD8⁺ T splenocytes from different strains of mice

To test the effect of different class I molecules on the BV diversity of CD8⁺ T cells, we determined the size of the BV repertoire in the four categories of mice used in this study. For this purpose, we used a method that we have recently described (26). Briefly, this method involves the isolation of a CDR3 band of a given length from a defined BV-BJ rearrangement, the cloning of these PCR fragments into a plasmid vector, and an extensive sequencing of the plasmid present in individual bacterial clones. This allowed us to determine the number of distinct nucleotide sequences found in a single CDR3 peak and to evaluate the size of the BV repertoire of naive T splenocytes as indicated in Table I. The equation used by Barth et al. (33) and Behlke et al. (34) was used to estimate the maximum probable number of distinct CDR3 sequences (MLE of the number of distinct sequences) present in a cDNA preparation. Importantly, the values calculated with the MLE were very close to or identical with the number of different sequences obtained experimentally, indicating that we had reached a plateau by exhaustive sequencing (Table I).

View larger version (20K): [in this window] [in a new window]   FIGURE 4. Size estimates of the BV repertoire of CD8+ splenocytes. In the abcissa are plotted the number of CD8+ splenocytes found in wild-type, H-2D°/°, H-2K°/°, and double KO C57BL/6 animals. The size estimates of the BV repertoires are shown in the ordinate. The estimates are calculated from the BV10-BJ1.2 and from the BV7-BJ1.2 rearrangements. The linear regression is drawn from all values. The average number of T cells sharing the same BV chain was calculated from the equation of the linear regression curve and found to be equal to 29.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Perarnau et al. (25) have observed that the CD8 T cell number is reduced by 30% in H-2D°^/°, 60% in H-2K°^/°, and 90% in double KO animals when compared with C57BL/6 mice. Because class Ia-negative mice, like wild-type animals, develop potent Qa1-restricted CTL responses against Listeria monocytogenes, it is most likely that observations made in double KO mice also apply to wild-type animals. It is striking that, although the pattern and level of expression of class I molecules detected on thymus cryosections with anti-mouse ₂-microglobulin mAb (25) are similar in class Ia negative and wild-type mice, very few CD8 lymphocytes are found in class Ia-negative animals (25). Many nonexclusive possibilities may account for this paucity: 1) expression of class Ib molecules often is restricted to certain tissues, and may be missing or expressed at low levels on cells responsible for positive selection; 2) a weaker CD8 interaction than with class Ia molecules has been documented with Q7 and Q9 molecules; 3) class Ib molecules may present a limited set of self-peptides as shown for H-2M3 and Qa1; 4) class Ib molecules may not be expressed in the periphery in sufficient amounts and/or on the right cells, resulting in a reduced T cell survival; and 5) finally, the class Ib-mediated positive and negative selection processes may be unbalanced as compared with class Ia in favor of the latter. Considering the large number of class Ib genes and molecules and the limited number of specific reagents at hand, none of these possibilities should be excluded.

The limited number of CD8⁺ T cells educated and maintained by class Ib molecules only led us to analyze more precisely their AV and BV repertoire. Some class Ib molecules have been shown to promote the education of T cells with a limited TCR diversity. NK1 T lymphocytes, which are restricted by CD1, a class Ib-like molecule not encoded in the MHC, have a limited repertoire diversity. These NK1 T lymphocytes are CD4⁺ or CD4^-CD8^- and display an invariant -chain with a 10-amino-acid long CDR3 associated with a limited set of BV segments (42). More recently, another subset of T lymphocytes expressing an invariant -chain was described in human, mouse, and cattle (43). This invariant chain, AV19AJ33 in mouse, is expressed by a subpopulation of CD4^-CD8^- T lymphocytes selected on a yet unidentified MHC class Ib molecule different from CD1 (43). This AV19AJ33 chain pairs predominantly with BV8 and BV6 segments (43). The existence of MHC class Ib-restricted T cell subpopulations, which display a limited T cell repertoire, led us to evaluate the diversity of the CD8 T lymphocytes in mice lacking one or all classical MHC class I molecules.

We first analyzed RNA from purified CD8 splenocytes from H-2K°^/°, H-2D°^/°, double KO, and C57BL/6 animals by the Immunoscope method. In the three different strains of mice tested, CD8 profiles were bell-shaped and similar to those found in wild-type animals. No clonal or oligoclonal expansion was detected. Moreover, using a semiquantitative PCR, we observed that the AV and BV usage in the three deficient strains closely matched that seen in C57BL/6 (Fig. 3). The only significant difference between mutant mice and wild-type animals concerns the BV5.1 segment. The decrease of BV5.1 in wild-type mice is probably not due to a superantigenic effect because 1) no endogenous superantigen restricted by MHC class I molecules has been described yet; and 2) in single KO mice, the expression of only one of the class Ia molecules (H-2K^b or H-2D^b) does not result in size reduction of the BV5.1 CD8⁺ T cell subset.

These analyses showed clearly that the CD8 T cell repertoires of the three mutant mice match closely the wild-type one and use similarly the different AV and BV rearrangements without biases in the CDR3 size distribution. However, the methods used provided no information on the number of distinct CDR3 sequences present in a given BV rearrangement. To evaluate the diversity of the BV repertoires in the four strains of mice, we used a method recently described by Casrouge et al. (26). In the four strains of mice, the BV repertoire sizes were calculated from two different rearrangements, the BV7-BJ1.2 and the BV10-BJ1.2. When we plotted the BV repertoire sizes vs the number of CD8 lymphocytes, a linear regression curve was obtained. From it we calculated the average number of T cells sharing the same BV chain: 29 (Fig. 4). This value is in good agreement with those previously found for unfractionated T splenocytes from C57BL/6 and DBA/2 mice (26).

In summary, this work shows that the diversity (usage of AV and BV segments, CDR3 sizes) of the CD8⁺ T cell repertoire selected by class Ib molecules is similar to the diversity of the CD8⁺ repertoire in normal mice, taking into account the 90% reduction of CD8⁺ splenocytes. Furthermore, we have estimated the size of the BV repertoire in the four different strains of mice and we have found that the BV repertoire size is proportional to the overall number of CD8⁺ splenocytes. This observation implies that BV diversity is positively correlated with the number of CD8⁺ cells. Considering that certain class Ib molecules have evolved to present peculiar sets of peptides, i.e., N-formylated peptides for H-2 M3, endoplasmic reticulum signal peptides for Qa1 and complement functionally class Ia molecules, significant diversification of their corresponding CD8⁺ T cell repertoire is evidently advantageous.

	Acknowledgments

 
We thank Dr. Irina Apostolou, Dr. Iris Motta, and Dr. Jean-Pierre Cabaniols for discussion and critical review of the manuscript. The help of Annick Lim for technical advice with the semiquantitative PCR is greatly appreciated.

	Footnotes

 
¹ This work was supported by Association de Recherches contre le Cancer, Ligue de Recherches contre le Cancer, and by a grant from the European Economic Community (no. 96-0077). D.L. was supported by fellowships from Association des Universités Partiellement ou Entièrement de Langue Française-Université des Réseaux d’Expression, from the International Network of the Pasteur Institute, and from Collège de France.

² Present address: Harvard Medical School/Children’s Hospital, Enders Building, 8th Floor, 300 Longwood Avenue, Boston, MA 02115--5747.

³ Address correspondence and reprint requests to Dr. Jean Kanellopoulos, Unité de Biologie Moléculaire du Gène, Institut National de la Santé et de la Recherche Médicale U277-Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15.

⁴ Abbreviations used in this paper: AV, V; BV, V; KO, knockout; CDR, complementarity-determining regions; °^/°, deficient; MLE, maximum likelihood estimate.

Received for publication June 28, 2000. Accepted for publication September 6, 2000.

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