- Email: [email protected]
[22] Cloning and sequencing immunoglobulin and T-cell receptor variable regions involved in neuroimmune disorders
[221
Cloning and Sequencing Immunoglobulin and T-Cell Receptor Variable Regions Involved in Neuroimmune Disorders Curtis C. Maier and J. Edwin Blalock
Introduction The polymerase chain reaction (PCR) is a powerful tool which has greatly facilitated the rapidity and ease of cloning and sequencing variable (V) regions of immunoglobulins (Igs) and T-cell receptors (TCRs). However, the extraction and isolation of RNA for first-strand cDNA synthesis can still be a timeconsuming and costly venture, generally requiring at least 105 to 10 6 cells for a workable RNA yield. In this report we describe a rapid and inexpensive method for the generation of cDNA from very low numbers of lymphocytes involved in neuroimmune disorders. The method involves lysing as few as 10 cells in a 0.5% Nonidet P-40 (NP-40) detergent solution which should release cytoplasmic RNA yet not disrupt the nuclear membrane. Thus, most contaminating DNA and immature mRNA are easily removed with the cellular debris by centrifugation while the mature target mRNA remains in the supernatant. The cytoplasmic mRNA is then converted to cDNA and used as template in the amplification of the V regions of Igs and TCRs. This methodology was first worked out by amplifying V regions of Igs produced by hybridomas specific for encephalitogenic epitopes of myelin basic protein (MBP) and has now been applied to TCRs from encephalitogenic T-cell lines and clones as well as Ig V regions expressed by lymphocytes which have infiltrated the central nervous system (CNS) of multiple sclerosis (MS) patients.
Materials and Methods
Cells All cells used in this study have been donated by Drs. John N. Whitaker and Shan-Ren Zhou (UAB, Birmingham, AL). The hybridoma, denoted 845D3, secretes IgG1/K and is specific for human MBP peptide 80-89 (1). RT1 is a T-cell line established from Lewis rats immunized with guinea pig Methods in Neurosciences, Volume 24
Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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(gp) MBP and specifically responds to the encephalitogenic antigen gpMBP 68-88. Cerebral spinal fluid (CSF) cells are obtained by spinal taps and are processed as quickly as possible following the tap.
Oligonucleotide Primers Oligonucleotide primers are synthesized in this laboratory on a Dupont Coder 300 DNA synthesizer (Dupont, Wilmington, DE) and purified on Nensorb Prep columns (NEN, Boston, MA) or purchased from Oligos Etc. (Wilsonville, OR), unless otherwise noted. The random hexamers used to prime first-strand synthesis have a 3'-C and all four nucleotides at the remaining five positions. Sequences of primers used to amplify mouse and human Ig and rat TCR V regions are given in Table I.
Rapid Method for cDNA Synthesis Cultured and primary lymphocytes are processed in the same manner. Cells are washed two times with 1 ml cold phosphate-buffered saline (PBS) and are pelleted by centrifugation in an Eppendorf tabletop microcentrifuge at 5000 rpm for 1 min at room temperature. After the last wash the cells are counted in a hemacytometer and the percent viability is determined by trypan blue exclusion. We have found that first-strand cDNA synthesis will proceed successfully even at only 50% viability. The number of cells lysed for firststrand synthesis can vary from 10 to 106 with best yields generally in the 104 range. Transfer the desired number of cells to a fresh microfuge tube and pellet as above. Remove all residual PBS (recentrifuge, if necessary, to remove PBS on the sides of the tube). It is very important to do the following lysis and centrifugation at 4~ (i.e., move to the cold room) to impair any ribonuclease activity. Resuspend the pellet in ice-cold 20-/A lysis mix containing 1 x superscript reverse transcriptase first-strand buffer (GIBCO BRL, Gaithersburg, MD) [50 mM Tris-HC1 (pH 8.3), 75 mM KC1, 3 mM MgCI2], 10 mM dithiothreitol (DTT) (GIBCO BRL), 0.5% NP-40 (molecular biology grade, Sigma, St. Louis, MO; due to the high viscosity of NP-40, it is easier to manipulate if a large volume, such as 500/A, of a 10% solution is made as a stock), and 40 U recombinant RNasin (Promega, Madison, WI) as a ribonuclease inhibitor. Pipette the pellet several times to ensure it is resuspended and centrifuge 2 min, 12,000 rpm at 4~ Add 10/~1 of the supernatant to a fresh microfuge tube containing 11/A of first-strand mix. First-strand mix consists of lx superscript reverse transcriptase first-strand buffer (GIBCO BRL; see above), 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 1 mM
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dTTP (Ultrapure dNTPs, Pharmacia LKB Biotechnology, Piscataway, NJ), 50 pmol random hexamers, and 50 U superscript RNase H- reverse transcriptase (GIBCO BRL). Incubate this first-strand reaction at 42~ for 1 hr. Heat the reaction mix to 95-100~ for 5 min to destroy the superscript and dislocate it from the cDNA template. Reactions have been stored for a couple of months at -20~ avoiding multiple freeze-thaws, and can still be used as template for PCR; however, after several months the template will degrade unless otherwise purified. The same care taken to avoid template contamination in PCR (i.e., a set of pipettes for primers and buffers separate from those used for template and cells, using autoclaved doubly distilled H 2 0 , microfuge tubes, and pit tips) must also be followed when setting up the first-strand reaction. A sham control first-strand reaction is run simultaneously to determine if any contaminating template was introduced during the procedure. This simply consists of 10/xl of lysis mix combined with 11/xl of first-strand mix in the absence of any cells. This reaction is used later as template in the primer control PCR.
Polymerase Chain Reaction Amplification The V region cDNA generated in the first-strand reaction is specifically amplified by PCR using the primers from Table I with either Amplitaq (Perkin-Elmer Cetus, Norwalk, CT) or cloned Pfu DNA polymerase (Stratagene, La Jolla, CA). Amplifications performed with Amplitaq are done in 10 mM Tris-HC1, pH 8.3, 50 mM KC1, 2.0 mM MgCI2, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 1.25 U Amplitaq, 50 pmol of each of the appropriate primers, and 0.1 or 1.0/xl of the first-strand reaction. Cloned Pfu is supplied with a different buffer (10x reaction buffer No. 3); at the working concentration it contains 20 mM Tris-HCl, pH 8.75, 10 mM KC1, 10 mM (NH4)2SO 4, 20 mM MgCI2, 0.1% Triton X-100, and 0.1 mg/ml bovine serum albumin (BSA). Polymerase chain reaction amplifications done with Pfu DNA polymerase are carried out in this buffer and also include 0.:~ mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 2.5 U cloned Pfu, 50 pmol of each of the appropriate primers, and 1.0/xl of the firststrand reaction. The PCR conditions for Amplitaq and cloned Pfu are similar except higher annealing temperatures are generally more permissive with Amplitaq (we routinely amplify Ig V regions with Amplitaq at annealing temperatures of 62~ however, for the results presented in this paper, mouse Ig and rat TCR V regions are amplified with cloned Pfu and annealed at 58~ while human Ig V regions are amplified with Amplitaq and cloned Pfu and annealed at 54~ All V regions are subjected to 40 cycles of amplification in a thermal
[22] CLONING OF ANTIGEN RECEPTOR V-REGIONS
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cycler (Perkin-Elmer Cetus DNA thermal cycler) under the following conditions: denature 96~ for 1 min (the first 5 cycles to minimize primer dimers, 94~ for the remaining 35 cycles), anneal at the temperatures mentioned above for 1 min, and extend at 72~ for 2 min. Five to 10/zl of the reaction product is electrophoresed on a 1.2% (w/v) agarose gel, stained with ethidium bromide, visualized under UV light, and photographed. The expected size of the amplified V regions is 350-450 bp for these primer combinations. First-strand reactions can also be amplified with /3-actin (2) or GAPDH primers to ensure successful first-strand synthesis and correct PCR conditions.
Cloning and Sequencing o f V Regions The oligonucleotide primers used for PCR are designed to include SalI restriction sites to facilitate cloning of the PCR products. The V region PCR products are cloned into the SalI restriction site of the sequencing vector, M 13mp18, by the following protocol. Polymerase chain reaction products are extracted with phenol/chloroform, followed by a chloroform extraction and then precipitated with 0.5 volumes of 7.5 M ammonium acetate and 2.5 volumes of ethanol. When DNA loss is a concern, the organic phases are back extracted with an equal volume of doubly distilled H20 and the aqueous phases combined. The PCR products are recovered by centrifugation at 12,000 rpm for 30 min, washed with 70% ethanol, and digested with 8 U SalI (Promega), 37~ for 16 hr (overlay the reaction mix with mineral oil). The restriction digestion mix is loaded directly onto 5% Nusieve GTG agarose (FMC BioProducts, Rockland, ME) containing 0.5/xg/ml ethidium bromide and run in 1 x TAE buffer. The DNA band at the correct molecular weight is excised from the gel, keeping the exposure time to UV light and the amount of gel excised to the bearest minimum, and the agarose digestesd with GELase (Epicentre Technologies, Madison, WI), following the manufacturer's fast protocol. The recovered PCR products are quantitated on an ethidium bromide-stained gel. If one 100-/zl PCR is used as starting material, a yield of approximately 100-200 ng can be expected. Ten to 50 ng of V region PCR products is combined with 50 ng M13mpl8 and this mixture digested with 0.8 U SalI for at least 6 hr at 37~ The amount of SalI is reduced in this reaction to avoid cleavage at inappropriate sites. The reaction is extracted and organic phases are back extracted and precipitated as above. The digested V region PCR product and M13mpl8 is then ligated with 0.3 U T4 DNA ligase (Promega) in a 10-/zl volume for 4 hr at 16~ During this reaction, JM 109 strain Escherichia coli are made competent for transfection following the protocol outlined in Promega's "Protocols and Applications
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Guide" (3), except volumes are reduced 10-fold and the trituration buffer contains 50 mM CaCI2 rather than 100 mM CaC12. The ligation mix is diluted 10-fold and 2 and 8 ~1 are transfected into 200/xl of competent cells. This generally yields 200-800 plaques/plate, 1% of which are recombinant (clear rather than blue). Clear plaques are grown 5 hr in 4 ml 2XYT and assayed for carrying the correct size insert by PCR. Three microliters of selected plaque purified recombinants are added directly to PCR mix containing M13 forward and reverse primers (sequences can be obtained from New England Biolabs, Beverly, MA) and subjected to 30 cycles of amplification. Nonrecombinant clear plaques will yield a PCR product of 100 bp, while recombinant plaques will be 100 bp larger than the V region PCR product. Single-stranded recombinant M13 DNA is purified from 3 ml of selected recombinant M13 clones following the protocol provided by Sambrook et al. (4). Three to five isolates are then sequenced using a Sequenase 7-deaza-dGTP sequencing kit (United State Biochemical, Cleveland, OH) and the reactions electrophoresed on both 6 and 4% acrylamide wedge gels (0.4-1.2 mm, BRL, Gaithersburg, MD). This allows the entire sequence to be clearly read and any discrepancies in isolate sequences determined. We routinely perform multiple, identical PCRs starting with the same first-strand reaction as template and then sequence recombinant M13 isolates from each, to ensure nucleotides are not misincorporated during the amplifications. Following this protocol, starting with cells, we obtain complete sequence information on V regions in 1 week.
Sensitivity of Method Hybridoma cells, 845D3, secreting an antibody specific for the human MBP peptide 80-89 (1), are used to determine the minimum number of cells necessary to amplify Ig V regions. Figure 1 shows that both heavy-chain and lightchain V regions can be amplified from as few as 10 cells, but not 1 cell. This experiment was repeated three times with similar results. To verify that the PCR product amplified was the target sequence of interest, the PCR products were cloned into M13 and sequenced. Figure 2 shows the sequences of the 845D3-VH PCR products which indeed are heavy-chain V regions. Actually two populations of sequences exist in the PCR product, one is derived from the mRNA of the productively rearranged allele, while the other contains a frameshift at codon position 100d due to N-sequence additions, resulting in a nonproductive transcript encoded by the other allele. The sequence of the light-chain PCR product also comes from a nonproductively rearranged allele (data not shown); however, neither of the sterile transcripts are encoded by
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FIG. 1 Polymerase chain reaction products of 845D3 VL and VH amplified from low numbers of cells. Lane 1, molecular weight marker [1000, 700, 500, 400, 300, 200, 100, and 50 base pairs (bp) 50 ng of each; Biomarker Low, Bioventures, Inc., Murfreesboro,TN]. Bands at 1000, 500, and 300 bp are indicated on the left-hand side. Lanes 2-5 and 6-9 are PCR products of eDNA generated from 100, 10, 1 and 0 hybridoma 845D3 cells, respectively. The eDNA was amplified with primers mouse Ig VK and mouse and human Ig CK in lanes 2-5 to yield 360 bp products, while lanes 6-9 show the 400-bp PCR products from eDNA amplified with primers mouse Ig VH and mouse Ig C),.
the myeloma fusion partner, SP2/O, which commonly arises when cloning V regions from hybridomas. Avoiding cloning of the myeloma sterile transcript was accomplished by designing the VK and VH degenerate primers to have the most 3' nucleotide of the primer mismatched with the myeloma sequence, thereby preventing the amplification of myeloma V region cDNA.
Amplification of Rat T-Cell Receptor Regions The majority of T cells from Lewis rats specific for the encephalitogenic gp MBP peptide 68-88 express the V region gene V/38.2 (5, 6). Using a Vfl primer which is designed to specifically amplify Vfl8 family members we can amplify a PCR product of the expected size from 350 T cells of a T-cell line (as well as T-cell clones established from the line) specific for gp MBP 68-88 (Fig. 3). Analysis of the sequences of the PCR product verified that it is Vf18.2 (data not shown).
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845D3 VH Sterile VH
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40 G l n T h r Pro V a l H i s G l y L e u G l u T r p Ile G l y CAG ACA CCT GTG CAT GGC CTG GAA TGG ATT GGA --- T T T --A - G A A - C A - A . . . . . G . . . . . G --C
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70
T y r A s n G l n Lys Phe Lys G l y Lys A l a T h r L e u T h r A l a A s p Lys S e r S e r Ser T h r A l a TAC AAT CAG AAG TTC AAG GGC AAG GCC ACA CTG ACT GCA GAC AAA TCC TCC AGC ACA GCC . . . . . C - C A T C T C . . . . A A - T C G A A T - T - T A - C --- CG-C . . . . A A G -A- C A G TT-
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CDR 3 I00 a b ~ ~ ~ i01 , ii0 T y r G l y S e r Ser Ser Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala T A C G G T A G T A G T T C C *** *** C T T G C T T A C T G G G G C C A A G G G A C T C T G G T C A C T G T C T C T G C A G-- TAC TAT AAT TAC GAC GT CTT GCT TAC TGG GGC CAA GGG ACT CTG GTC ACT GTC TCT GCA ~-N-~-) J H 3
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A l a Lys T h r T h r Pro Pro Ser GCC AAA ACG ACA CCC CCA TCT GTC TAT CCA CTG GCC CCT GCG GCC AAA ACG ACA CCC CCA TCT GTC TAT CCA CTG GCC CCT ~G
Fic. 2 The nucleotide and deduced amino acid sequence of 845D3 VH and its sterile transcript. The primer sequences are underlined. Dashes indicate identical nucleotides and gaps are inserted (asterisks) to maximize homology. The amino acid numbering and placement of CDRs (overline) are according to E. A. Kabat, T. T. Wu, H. M. Pery, K. S. Gottesman, and C. Foeller, "Sequences of Proteins of Immunological Interest," Fifth Edition, U.S. Dept. Health and Human Services, National Institutes of Health, Bethesda, MD, 1991.
Va gene usage by rat T cells specific for gp MBP 68-88 has been previously shown by Southern blot analysis to be somewhat restricted as well (5, 6). Using the TCR Va degenerate primer described in Table I, we were able to amplify Va regions from the RT1 T-cell line (Fig. 3). Sequencing of gp MBP
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FI6.3 Va and V/3 PCR products amplified from cDNA of 350 cells ofthe encephalitogenic rat T-cell line, RT1. Lane 1 is a molecular weight marker with the 400-bp band indicated. Lane 2 shows the 430-bp Va PCR product amplified with the primers rat TCR Va and rat TCR Ca, while the 450-bp V/3 PCR amplified with primers rat TCR V/3 and rat TCR C/3 is seen in lane 4. No PCR product is produced when amplifying cDNA from 0 cells (Va and V/3 primers, lanes 3 and 5, respectively).
68-88 specific TCR Va elements by ourselves and others (7) reveal several other different Va families are utilized in addition to the predominant Va family (represented by RT1 Val in Fig.4). Sequences representing three different Va families expressed in the RT1 T-cell line are shown in Fig. 4. The TCR Va primer amplified at least one other family (data not shown) and possibly others; therefore, this primer may be useful in identifying several new families of the poorly studied rat TCR Va structures.
Amplification of Immunoglobulin V Regions of Human Cerebrospinal Fluid Lymphocytes The power of this technique is exemplified by the ability to amplify Ig V regions from lymphocytes which can only be obtained in small numbers, such as B cells infiltrating the CNS. From one patient 6 ml of CSF was obtained. This contained 2000 lymphocytes/ml, and approximately only 30% of these are expected to be B cells. B cells cannot be cultured, thus to
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AACTGCAGTT
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198 TGCACATCGA .........
248
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GG--TGCAGG
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298
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398
FIG. 4 The nucleotide sequences of three different Va elements occurring in the Va PCR product of the RT1 T-cell line. The primer sequences are underlined and nucleotides numbered starting at the first nucleotide of codon 1. Dashes indicate identical nucleotides, gaps inserted (asterisks) to maximize homology. The 5' end of the constant region is noted with the arrow.
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FIG. 5 V regions amplified from human CSF lymphocytes. Lanes 1 and 10 are the molecular weight marker with the 400-bp band indicated. Lanes 2-5 are cDNAs amplified with primers human Ig VK and mouse and human Ig CK. Lanes 6-9 are cDNAs amplified with primers human Ig VH and human Ig JHCy. Lanes 11-14 are cDNAs amplified with primers human Ig Vh and human Ig Ch. Lanes 15-18 are cDNAs amplified with/3-actin primers. The order of cDNAs for each primer combination is as follows: CSF of MS patient (12,000 cells; lanes 2, 6, 11, 15), human spleen cDNA (generous gift from D. R. Shaw, UAB, Birmingham, AL; lanes 3, 7, 12, 16), CSF of non-MS patient (6,000 cells; lanes 4, 8, 13, 17), and 0 cells (lanes 5, 9, 14, 18).
determine the repertoire of V regions utilized by B cells infiltrating the CNS, cDNA must be made from a very low number of cells. In the initial attempt to demonstrate that the procedure works on human CSF lymphocytes, degenerate primers listed in Table I were tested for their ability to amplify human Ig V regions. Shown in Fig. 5 are the PCR products generated by the VK, VH, and Vh primer combinations using MS and non-MS CSF lymphocytes (12,000 and 6,000 cells, respectively) and compared to spleen cDNA. /3Actin primers are included as a positive control. The VK and VH primers worked well while the degenerate Vh primer amplified poorly; however, a new degenerate primer has since been designed which should amplify more h family members. While these degenerate primers obviously do not amplify
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all VK, Vh, or VH elements, these results support the idea that with welldesigned primers, this can be an easy and rapid method for determining the repertoire of V elements utilized by lymphocytes in normal vs autoimmune neurological disorders (see Refs. 8-10 for extensive lists and rationale of V region PCR primer design).
Selection of Thermostable DNA Polymerase A major difference exists in commercially available thermostable DNA polymerases" The presence or absence of 3'-5' proofreading activity. The proofreading activity of cloned Pfu DNA polymerase results in a 12-fold increase in the fidelity of DNA synthesis when compared to a nonproofreading, thermostable DNA polymerase, such as Amplitaq. Thus, if maintaining the integrity of the template sequence is important, as in cloning projects, then a proofreading DNA polymerase should be used if possible. However, we have found that proofreading polymerases, such as cloned Pfu polymerase, will not amplify diverse templates with degenerate primers as efficiently as Taq polymerase. For example, in Fig. 6 the VK PCR products of CSF lymphocyte and spleen cDNA amplified with Amplitaq and cloned Pfu polymerase are compared. Less product is generated with Pfu polymerase. Furthermore, some Ig V region cDNAs from hybridomas involved in experimental autoimmune myasthenia gravis amplify nicely with Taq polymerase yet will not amplify at all with cloned Pfu polymerase when using the same degenerate primers (data not shown). Taken together, this suggests the Pfu polymerase is not as tolerant of primer-template mismatches as Taq polymerase. Thus repertoire analysis involving degenerate primers should be executed with Taq polymerase.
Summary Presented in this report is a method for the generation of cDNA from a small number of lymphocytes, as few as 10, that might be encountered in the study of human neuroimmune diseases. The method is inexpensive, does not require kits or extraordinary equipment, and is incredibly rapid, making it ideal for analysis of several samples at once. Nonidet P-40 lysis cDNA synthesis has been accomplished in every cell type and disrupted tissue thus far attempted, including hybridomas (11) and B and T lymphocytes, pituitary (2), hypothalamus (2), thymus (2), and liver tissue (unpublished, 1993), and is expected to work on other cell types as well. This technique is therefore
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FIG. 6 Comparison of Taq DNA polymerase to Pfu DNA polymerase when amplifying with VK primers. Lane 1, molecular weight marker with the 400-bp band indicated. Lanes 2 and 5 are human CSF lymphocyte cDNA amplified with Taq and Pfu, respectively. Lanes 3 and 6 are human spleen cDNA amplified with Taq and Pfu, respectively. Lanes 4 and 7 are cDNA of 0 cells amplified with Taq and Pfu, respectively.
a legitimate substitute to timely RNA extractions requiring voluminous cells and can be utilized for typical RT-PCR applications. A possible shortcoming in the method is the ability to convert low abundance or highly unstable mRNAs to cDNA. Because RNases are not immediately denatured with guanidinium and mRNA is not enriched, it is possible the sensitivity to reverse transcribe certain transcripts may be compromised. Parameters for detecting low abundance mRNAs by this methodology have not been analyzed. Furthermore, it is very important to design primers for PCR which flank a noncoding region or intron. Thus if DNA contaminates the first-strand reaction it can be distinguished from cDNA template based on the size of the PCR product.
Acknowledgments The authors greatly appreciate the helpful discussions with Drs. Robert D. LeBoeuf, John N. Whitaker, and Shan-Ren Zhou. This work was supported by PPG P01 NS29719, NIH DK38021, a Muscular Dystrophy Association grant, and a Multiple
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References 1. J. O. Price, J. N. Whitaker, R. I. Vasu, and D. W. Metzger, J. Immunol. 136, 2426 (1986). 2. C. C. Maier, B. Marchetti, R. D. LeBoeuf, and J. E. Blalock, Cell. Mol. Neurobiol. 12, 447 (1992). 3. "Promega Protocols and Applications Guide," (D. E. Titus, ed.), 2nd Ed., p. 52. Promega Corporation, U.S.A., 1991. 4. J. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual," 2nd Ed., p. 4.29. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. 5. E. Heber-Katz and H. Acha-Orbea, Immunol. Today 10, 164 (1989). 6. S. S. Zamvil and L. Steinman, Annu. Rev. Immunol. 8, 579 (1990). 7. A. E. Hinkkanen, J. M~/itt/i, Y.-F. Qin, C. Linington, A. Salmi, and H. Wekerle, Immunogenetics 37, 235 (1993). 8. R. D. LeBoeuf, F. S. Galin, S. K. Hollinger, S. C. Peiper, and J. E. Blalock, Gene 82, 373 (1989). 9. J. D. Marks, M. Tristem, A. Karpas, and G. Winter, Eur. J. Immunol. 21, 985 (1991). 10. M. A. Panzara, E. Gussoni, L. Steinman, and J. R. Oksenberg, BioTechniques 12, 728 (1992). l l. C. C. Maier, R. D. LeBoeuf, S.-R. Zhou, J. N. Whitaker, M. A. Jarpe, and J. E. Blalock, J. Neuroimmunol. 46, 235 (1993).
Cloning and Sequencing Immunoglobulin and T-Cell Receptor Variable Regions Involved in Neuroimmune Disorders Curtis C. Maier and J. Edwin Blalock
Introduction The polymerase chain reaction (PCR) is a powerful tool which has greatly facilitated the rapidity and ease of cloning and sequencing variable (V) regions of immunoglobulins (Igs) and T-cell receptors (TCRs). However, the extraction and isolation of RNA for first-strand cDNA synthesis can still be a timeconsuming and costly venture, generally requiring at least 105 to 10 6 cells for a workable RNA yield. In this report we describe a rapid and inexpensive method for the generation of cDNA from very low numbers of lymphocytes involved in neuroimmune disorders. The method involves lysing as few as 10 cells in a 0.5% Nonidet P-40 (NP-40) detergent solution which should release cytoplasmic RNA yet not disrupt the nuclear membrane. Thus, most contaminating DNA and immature mRNA are easily removed with the cellular debris by centrifugation while the mature target mRNA remains in the supernatant. The cytoplasmic mRNA is then converted to cDNA and used as template in the amplification of the V regions of Igs and TCRs. This methodology was first worked out by amplifying V regions of Igs produced by hybridomas specific for encephalitogenic epitopes of myelin basic protein (MBP) and has now been applied to TCRs from encephalitogenic T-cell lines and clones as well as Ig V regions expressed by lymphocytes which have infiltrated the central nervous system (CNS) of multiple sclerosis (MS) patients.
Materials and Methods
Cells All cells used in this study have been donated by Drs. John N. Whitaker and Shan-Ren Zhou (UAB, Birmingham, AL). The hybridoma, denoted 845D3, secretes IgG1/K and is specific for human MBP peptide 80-89 (1). RT1 is a T-cell line established from Lewis rats immunized with guinea pig Methods in Neurosciences, Volume 24
Copyright 9 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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(gp) MBP and specifically responds to the encephalitogenic antigen gpMBP 68-88. Cerebral spinal fluid (CSF) cells are obtained by spinal taps and are processed as quickly as possible following the tap.
Oligonucleotide Primers Oligonucleotide primers are synthesized in this laboratory on a Dupont Coder 300 DNA synthesizer (Dupont, Wilmington, DE) and purified on Nensorb Prep columns (NEN, Boston, MA) or purchased from Oligos Etc. (Wilsonville, OR), unless otherwise noted. The random hexamers used to prime first-strand synthesis have a 3'-C and all four nucleotides at the remaining five positions. Sequences of primers used to amplify mouse and human Ig and rat TCR V regions are given in Table I.
Rapid Method for cDNA Synthesis Cultured and primary lymphocytes are processed in the same manner. Cells are washed two times with 1 ml cold phosphate-buffered saline (PBS) and are pelleted by centrifugation in an Eppendorf tabletop microcentrifuge at 5000 rpm for 1 min at room temperature. After the last wash the cells are counted in a hemacytometer and the percent viability is determined by trypan blue exclusion. We have found that first-strand cDNA synthesis will proceed successfully even at only 50% viability. The number of cells lysed for firststrand synthesis can vary from 10 to 106 with best yields generally in the 104 range. Transfer the desired number of cells to a fresh microfuge tube and pellet as above. Remove all residual PBS (recentrifuge, if necessary, to remove PBS on the sides of the tube). It is very important to do the following lysis and centrifugation at 4~ (i.e., move to the cold room) to impair any ribonuclease activity. Resuspend the pellet in ice-cold 20-/A lysis mix containing 1 x superscript reverse transcriptase first-strand buffer (GIBCO BRL, Gaithersburg, MD) [50 mM Tris-HC1 (pH 8.3), 75 mM KC1, 3 mM MgCI2], 10 mM dithiothreitol (DTT) (GIBCO BRL), 0.5% NP-40 (molecular biology grade, Sigma, St. Louis, MO; due to the high viscosity of NP-40, it is easier to manipulate if a large volume, such as 500/A, of a 10% solution is made as a stock), and 40 U recombinant RNasin (Promega, Madison, WI) as a ribonuclease inhibitor. Pipette the pellet several times to ensure it is resuspended and centrifuge 2 min, 12,000 rpm at 4~ Add 10/~1 of the supernatant to a fresh microfuge tube containing 11/A of first-strand mix. First-strand mix consists of lx superscript reverse transcriptase first-strand buffer (GIBCO BRL; see above), 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 1 mM
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dTTP (Ultrapure dNTPs, Pharmacia LKB Biotechnology, Piscataway, NJ), 50 pmol random hexamers, and 50 U superscript RNase H- reverse transcriptase (GIBCO BRL). Incubate this first-strand reaction at 42~ for 1 hr. Heat the reaction mix to 95-100~ for 5 min to destroy the superscript and dislocate it from the cDNA template. Reactions have been stored for a couple of months at -20~ avoiding multiple freeze-thaws, and can still be used as template for PCR; however, after several months the template will degrade unless otherwise purified. The same care taken to avoid template contamination in PCR (i.e., a set of pipettes for primers and buffers separate from those used for template and cells, using autoclaved doubly distilled H 2 0 , microfuge tubes, and pit tips) must also be followed when setting up the first-strand reaction. A sham control first-strand reaction is run simultaneously to determine if any contaminating template was introduced during the procedure. This simply consists of 10/xl of lysis mix combined with 11/xl of first-strand mix in the absence of any cells. This reaction is used later as template in the primer control PCR.
Polymerase Chain Reaction Amplification The V region cDNA generated in the first-strand reaction is specifically amplified by PCR using the primers from Table I with either Amplitaq (Perkin-Elmer Cetus, Norwalk, CT) or cloned Pfu DNA polymerase (Stratagene, La Jolla, CA). Amplifications performed with Amplitaq are done in 10 mM Tris-HC1, pH 8.3, 50 mM KC1, 2.0 mM MgCI2, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 1.25 U Amplitaq, 50 pmol of each of the appropriate primers, and 0.1 or 1.0/xl of the first-strand reaction. Cloned Pfu is supplied with a different buffer (10x reaction buffer No. 3); at the working concentration it contains 20 mM Tris-HCl, pH 8.75, 10 mM KC1, 10 mM (NH4)2SO 4, 20 mM MgCI2, 0.1% Triton X-100, and 0.1 mg/ml bovine serum albumin (BSA). Polymerase chain reaction amplifications done with Pfu DNA polymerase are carried out in this buffer and also include 0.:~ mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 2.5 U cloned Pfu, 50 pmol of each of the appropriate primers, and 1.0/xl of the firststrand reaction. The PCR conditions for Amplitaq and cloned Pfu are similar except higher annealing temperatures are generally more permissive with Amplitaq (we routinely amplify Ig V regions with Amplitaq at annealing temperatures of 62~ however, for the results presented in this paper, mouse Ig and rat TCR V regions are amplified with cloned Pfu and annealed at 58~ while human Ig V regions are amplified with Amplitaq and cloned Pfu and annealed at 54~ All V regions are subjected to 40 cycles of amplification in a thermal
[22] CLONING OF ANTIGEN RECEPTOR V-REGIONS
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cycler (Perkin-Elmer Cetus DNA thermal cycler) under the following conditions: denature 96~ for 1 min (the first 5 cycles to minimize primer dimers, 94~ for the remaining 35 cycles), anneal at the temperatures mentioned above for 1 min, and extend at 72~ for 2 min. Five to 10/zl of the reaction product is electrophoresed on a 1.2% (w/v) agarose gel, stained with ethidium bromide, visualized under UV light, and photographed. The expected size of the amplified V regions is 350-450 bp for these primer combinations. First-strand reactions can also be amplified with /3-actin (2) or GAPDH primers to ensure successful first-strand synthesis and correct PCR conditions.
Cloning and Sequencing o f V Regions The oligonucleotide primers used for PCR are designed to include SalI restriction sites to facilitate cloning of the PCR products. The V region PCR products are cloned into the SalI restriction site of the sequencing vector, M 13mp18, by the following protocol. Polymerase chain reaction products are extracted with phenol/chloroform, followed by a chloroform extraction and then precipitated with 0.5 volumes of 7.5 M ammonium acetate and 2.5 volumes of ethanol. When DNA loss is a concern, the organic phases are back extracted with an equal volume of doubly distilled H20 and the aqueous phases combined. The PCR products are recovered by centrifugation at 12,000 rpm for 30 min, washed with 70% ethanol, and digested with 8 U SalI (Promega), 37~ for 16 hr (overlay the reaction mix with mineral oil). The restriction digestion mix is loaded directly onto 5% Nusieve GTG agarose (FMC BioProducts, Rockland, ME) containing 0.5/xg/ml ethidium bromide and run in 1 x TAE buffer. The DNA band at the correct molecular weight is excised from the gel, keeping the exposure time to UV light and the amount of gel excised to the bearest minimum, and the agarose digestesd with GELase (Epicentre Technologies, Madison, WI), following the manufacturer's fast protocol. The recovered PCR products are quantitated on an ethidium bromide-stained gel. If one 100-/zl PCR is used as starting material, a yield of approximately 100-200 ng can be expected. Ten to 50 ng of V region PCR products is combined with 50 ng M13mpl8 and this mixture digested with 0.8 U SalI for at least 6 hr at 37~ The amount of SalI is reduced in this reaction to avoid cleavage at inappropriate sites. The reaction is extracted and organic phases are back extracted and precipitated as above. The digested V region PCR product and M13mpl8 is then ligated with 0.3 U T4 DNA ligase (Promega) in a 10-/zl volume for 4 hr at 16~ During this reaction, JM 109 strain Escherichia coli are made competent for transfection following the protocol outlined in Promega's "Protocols and Applications
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Guide" (3), except volumes are reduced 10-fold and the trituration buffer contains 50 mM CaCI2 rather than 100 mM CaC12. The ligation mix is diluted 10-fold and 2 and 8 ~1 are transfected into 200/xl of competent cells. This generally yields 200-800 plaques/plate, 1% of which are recombinant (clear rather than blue). Clear plaques are grown 5 hr in 4 ml 2XYT and assayed for carrying the correct size insert by PCR. Three microliters of selected plaque purified recombinants are added directly to PCR mix containing M13 forward and reverse primers (sequences can be obtained from New England Biolabs, Beverly, MA) and subjected to 30 cycles of amplification. Nonrecombinant clear plaques will yield a PCR product of 100 bp, while recombinant plaques will be 100 bp larger than the V region PCR product. Single-stranded recombinant M13 DNA is purified from 3 ml of selected recombinant M13 clones following the protocol provided by Sambrook et al. (4). Three to five isolates are then sequenced using a Sequenase 7-deaza-dGTP sequencing kit (United State Biochemical, Cleveland, OH) and the reactions electrophoresed on both 6 and 4% acrylamide wedge gels (0.4-1.2 mm, BRL, Gaithersburg, MD). This allows the entire sequence to be clearly read and any discrepancies in isolate sequences determined. We routinely perform multiple, identical PCRs starting with the same first-strand reaction as template and then sequence recombinant M13 isolates from each, to ensure nucleotides are not misincorporated during the amplifications. Following this protocol, starting with cells, we obtain complete sequence information on V regions in 1 week.
Sensitivity of Method Hybridoma cells, 845D3, secreting an antibody specific for the human MBP peptide 80-89 (1), are used to determine the minimum number of cells necessary to amplify Ig V regions. Figure 1 shows that both heavy-chain and lightchain V regions can be amplified from as few as 10 cells, but not 1 cell. This experiment was repeated three times with similar results. To verify that the PCR product amplified was the target sequence of interest, the PCR products were cloned into M13 and sequenced. Figure 2 shows the sequences of the 845D3-VH PCR products which indeed are heavy-chain V regions. Actually two populations of sequences exist in the PCR product, one is derived from the mRNA of the productively rearranged allele, while the other contains a frameshift at codon position 100d due to N-sequence additions, resulting in a nonproductive transcript encoded by the other allele. The sequence of the light-chain PCR product also comes from a nonproductively rearranged allele (data not shown); however, neither of the sterile transcripts are encoded by
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FIG. 1 Polymerase chain reaction products of 845D3 VL and VH amplified from low numbers of cells. Lane 1, molecular weight marker [1000, 700, 500, 400, 300, 200, 100, and 50 base pairs (bp) 50 ng of each; Biomarker Low, Bioventures, Inc., Murfreesboro,TN]. Bands at 1000, 500, and 300 bp are indicated on the left-hand side. Lanes 2-5 and 6-9 are PCR products of eDNA generated from 100, 10, 1 and 0 hybridoma 845D3 cells, respectively. The eDNA was amplified with primers mouse Ig VK and mouse and human Ig CK in lanes 2-5 to yield 360 bp products, while lanes 6-9 show the 400-bp PCR products from eDNA amplified with primers mouse Ig VH and mouse Ig C),.
the myeloma fusion partner, SP2/O, which commonly arises when cloning V regions from hybridomas. Avoiding cloning of the myeloma sterile transcript was accomplished by designing the VK and VH degenerate primers to have the most 3' nucleotide of the primer mismatched with the myeloma sequence, thereby preventing the amplification of myeloma V region cDNA.
Amplification of Rat T-Cell Receptor Regions The majority of T cells from Lewis rats specific for the encephalitogenic gp MBP peptide 68-88 express the V region gene V/38.2 (5, 6). Using a Vfl primer which is designed to specifically amplify Vfl8 family members we can amplify a PCR product of the expected size from 350 T cells of a T-cell line (as well as T-cell clones established from the line) specific for gp MBP 68-88 (Fig. 3). Analysis of the sequences of the PCR product verified that it is Vf18.2 (data not shown).
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845D3 VH Sterile VH
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.
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70
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CDR 3 I00 a b ~ ~ ~ i01 , ii0 T y r G l y S e r Ser Ser Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala T A C G G T A G T A G T T C C *** *** C T T G C T T A C T G G G G C C A A G G G A C T C T G G T C A C T G T C T C T G C A G-- TAC TAT AAT TAC GAC GT CTT GCT TAC TGG GGC CAA GGG ACT CTG GTC ACT GTC TCT GCA ~-N-~-) J H 3
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A l a Lys T h r T h r Pro Pro Ser GCC AAA ACG ACA CCC CCA TCT GTC TAT CCA CTG GCC CCT GCG GCC AAA ACG ACA CCC CCA TCT GTC TAT CCA CTG GCC CCT ~G
Fic. 2 The nucleotide and deduced amino acid sequence of 845D3 VH and its sterile transcript. The primer sequences are underlined. Dashes indicate identical nucleotides and gaps are inserted (asterisks) to maximize homology. The amino acid numbering and placement of CDRs (overline) are according to E. A. Kabat, T. T. Wu, H. M. Pery, K. S. Gottesman, and C. Foeller, "Sequences of Proteins of Immunological Interest," Fifth Edition, U.S. Dept. Health and Human Services, National Institutes of Health, Bethesda, MD, 1991.
Va gene usage by rat T cells specific for gp MBP 68-88 has been previously shown by Southern blot analysis to be somewhat restricted as well (5, 6). Using the TCR Va degenerate primer described in Table I, we were able to amplify Va regions from the RT1 T-cell line (Fig. 3). Sequencing of gp MBP
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FI6.3 Va and V/3 PCR products amplified from cDNA of 350 cells ofthe encephalitogenic rat T-cell line, RT1. Lane 1 is a molecular weight marker with the 400-bp band indicated. Lane 2 shows the 430-bp Va PCR product amplified with the primers rat TCR Va and rat TCR Ca, while the 450-bp V/3 PCR amplified with primers rat TCR V/3 and rat TCR C/3 is seen in lane 4. No PCR product is produced when amplifying cDNA from 0 cells (Va and V/3 primers, lanes 3 and 5, respectively).
68-88 specific TCR Va elements by ourselves and others (7) reveal several other different Va families are utilized in addition to the predominant Va family (represented by RT1 Val in Fig.4). Sequences representing three different Va families expressed in the RT1 T-cell line are shown in Fig. 4. The TCR Va primer amplified at least one other family (data not shown) and possibly others; therefore, this primer may be useful in identifying several new families of the poorly studied rat TCR Va structures.
Amplification of Immunoglobulin V Regions of Human Cerebrospinal Fluid Lymphocytes The power of this technique is exemplified by the ability to amplify Ig V regions from lymphocytes which can only be obtained in small numbers, such as B cells infiltrating the CNS. From one patient 6 ml of CSF was obtained. This contained 2000 lymphocytes/ml, and approximately only 30% of these are expected to be B cells. B cells cannot be cultured, thus to
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AACTGCAGTT
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TACTTCCCAT
........ C--
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-C
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98
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198 TGCACATCGA .........
248
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TGAG-AGGC
GG--TGCAGG
CAGGAACCAG
ACTAACAGTC
AAACCAAATG
GTG ..... GC
AG--G--ATC
CT--T
ACG .......
GG ..... A--
GT-G--G
GTGTACCAGC
TGAAAGATCCC
....
C .........
.... G
298
.... AAG-TC
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TT--C
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ACATCACCCT
398
FIG. 4 The nucleotide sequences of three different Va elements occurring in the Va PCR product of the RT1 T-cell line. The primer sequences are underlined and nucleotides numbered starting at the first nucleotide of codon 1. Dashes indicate identical nucleotides, gaps inserted (asterisks) to maximize homology. The 5' end of the constant region is noted with the arrow.
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FIG. 5 V regions amplified from human CSF lymphocytes. Lanes 1 and 10 are the molecular weight marker with the 400-bp band indicated. Lanes 2-5 are cDNAs amplified with primers human Ig VK and mouse and human Ig CK. Lanes 6-9 are cDNAs amplified with primers human Ig VH and human Ig JHCy. Lanes 11-14 are cDNAs amplified with primers human Ig Vh and human Ig Ch. Lanes 15-18 are cDNAs amplified with/3-actin primers. The order of cDNAs for each primer combination is as follows: CSF of MS patient (12,000 cells; lanes 2, 6, 11, 15), human spleen cDNA (generous gift from D. R. Shaw, UAB, Birmingham, AL; lanes 3, 7, 12, 16), CSF of non-MS patient (6,000 cells; lanes 4, 8, 13, 17), and 0 cells (lanes 5, 9, 14, 18).
determine the repertoire of V regions utilized by B cells infiltrating the CNS, cDNA must be made from a very low number of cells. In the initial attempt to demonstrate that the procedure works on human CSF lymphocytes, degenerate primers listed in Table I were tested for their ability to amplify human Ig V regions. Shown in Fig. 5 are the PCR products generated by the VK, VH, and Vh primer combinations using MS and non-MS CSF lymphocytes (12,000 and 6,000 cells, respectively) and compared to spleen cDNA. /3Actin primers are included as a positive control. The VK and VH primers worked well while the degenerate Vh primer amplified poorly; however, a new degenerate primer has since been designed which should amplify more h family members. While these degenerate primers obviously do not amplify
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all VK, Vh, or VH elements, these results support the idea that with welldesigned primers, this can be an easy and rapid method for determining the repertoire of V elements utilized by lymphocytes in normal vs autoimmune neurological disorders (see Refs. 8-10 for extensive lists and rationale of V region PCR primer design).
Selection of Thermostable DNA Polymerase A major difference exists in commercially available thermostable DNA polymerases" The presence or absence of 3'-5' proofreading activity. The proofreading activity of cloned Pfu DNA polymerase results in a 12-fold increase in the fidelity of DNA synthesis when compared to a nonproofreading, thermostable DNA polymerase, such as Amplitaq. Thus, if maintaining the integrity of the template sequence is important, as in cloning projects, then a proofreading DNA polymerase should be used if possible. However, we have found that proofreading polymerases, such as cloned Pfu polymerase, will not amplify diverse templates with degenerate primers as efficiently as Taq polymerase. For example, in Fig. 6 the VK PCR products of CSF lymphocyte and spleen cDNA amplified with Amplitaq and cloned Pfu polymerase are compared. Less product is generated with Pfu polymerase. Furthermore, some Ig V region cDNAs from hybridomas involved in experimental autoimmune myasthenia gravis amplify nicely with Taq polymerase yet will not amplify at all with cloned Pfu polymerase when using the same degenerate primers (data not shown). Taken together, this suggests the Pfu polymerase is not as tolerant of primer-template mismatches as Taq polymerase. Thus repertoire analysis involving degenerate primers should be executed with Taq polymerase.
Summary Presented in this report is a method for the generation of cDNA from a small number of lymphocytes, as few as 10, that might be encountered in the study of human neuroimmune diseases. The method is inexpensive, does not require kits or extraordinary equipment, and is incredibly rapid, making it ideal for analysis of several samples at once. Nonidet P-40 lysis cDNA synthesis has been accomplished in every cell type and disrupted tissue thus far attempted, including hybridomas (11) and B and T lymphocytes, pituitary (2), hypothalamus (2), thymus (2), and liver tissue (unpublished, 1993), and is expected to work on other cell types as well. This technique is therefore
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FIG. 6 Comparison of Taq DNA polymerase to Pfu DNA polymerase when amplifying with VK primers. Lane 1, molecular weight marker with the 400-bp band indicated. Lanes 2 and 5 are human CSF lymphocyte cDNA amplified with Taq and Pfu, respectively. Lanes 3 and 6 are human spleen cDNA amplified with Taq and Pfu, respectively. Lanes 4 and 7 are cDNA of 0 cells amplified with Taq and Pfu, respectively.
a legitimate substitute to timely RNA extractions requiring voluminous cells and can be utilized for typical RT-PCR applications. A possible shortcoming in the method is the ability to convert low abundance or highly unstable mRNAs to cDNA. Because RNases are not immediately denatured with guanidinium and mRNA is not enriched, it is possible the sensitivity to reverse transcribe certain transcripts may be compromised. Parameters for detecting low abundance mRNAs by this methodology have not been analyzed. Furthermore, it is very important to design primers for PCR which flank a noncoding region or intron. Thus if DNA contaminates the first-strand reaction it can be distinguished from cDNA template based on the size of the PCR product.
Acknowledgments The authors greatly appreciate the helpful discussions with Drs. Robert D. LeBoeuf, John N. Whitaker, and Shan-Ren Zhou. This work was supported by PPG P01 NS29719, NIH DK38021, a Muscular Dystrophy Association grant, and a Multiple
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III NEUROIMMUNESYSTEM Sclerosis Society pilot grant (PP0317) to JEB. CCM was the recipient of a postdoctoral fellowship from NIH Training Grant T3207335.
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