Controlling the Major Histocompatibility Antigens - Europe PMC

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Leonard Ellman,* Ira Green, William J. Martin, and Baruj Benacerraf .... gently ground in a glass hand homogenizer with twice their volume of saline. The.
proccedings of the National Academy of Sciences Vol. 66, No. 2, pp. 322-328, June 1970

Linkage between the Poly-L-Lysine Gene and the Locus Controlling the Major Histocompatibility Antigens in Strain 2 Guinea Pigs Leonard Ellman,* Ira Green, William J. Martin, and Baruj Benacerraf NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES, NATIONAL INSTITUTIKS OF HEALTH, BETHESDA, MARYLAND

Communicated by Henry G. Kunkel, March 6, 1970

Abstract. The data presented demonstrate linkage between the major histocompatibility locus of inbred strain 2 guinea pigs and a "specific immune response gene," the PLL gene, which controls responsiveness to poly-L-lysine and hapten conjugates of this polypeptide in these animals. This finding extends to another species and to a different immune system the linkage observed in mice between the H2 locus and specific immune response genes at the Ir-1 locus. The general significance of the linkage of specific immune response genes to histocompatibility loci is discussed. The immune response of guinea pigs to poly-L-lysine (PLL), to poly-L-arginine, to a copolymer of L-glutamic acid and L-lysine, and to hapten conjugates of these polypeptides is predicated upon the presence of an autosomal dominant gene which is referred to as the PLL gene.1' 2 The PLL gene is found in all inbred strain 2 guinea pigs and in a varying percentage of Hartley strain guinea pigs and is lacking in all inbred strain 13 guinea pigs. (2 X 13) F1 guinea pigs are all heterozygous for the gene. When immunized with 2,4-dinitrophenyl-polyL-lysine (DNP-PLL) in complete adjuvant, guinea pigs which possess the PLL gene (responder) produce high levels of specific antibodies and develop strong Arthus and delayed hypersensitivity reactions when challenged with the immunizing antigen intradermally. In recent years genetic control of specific immune responses has also been demonstrated in other immune systems, in mice,3-5 rabbits' and rats.7' 8 Thus, the immunogenicity in mice of branched synthetic polypeptides containing random sequences of L-tyrosine, or L-histidine, or L-phenylalanine, and Lglutamic acid on a backbone of D, L-alanine and L-lysine[(T,G), A-L, (H,G)A-L, (Phe,G)-A-L] has been shown to be largely determined by single dominant genes at a single locus referred to as Ir-i or by three closely linked genes.3' 4 The immune response of mice to a random copolymer of L-glutamic acid and L-lysine containing 5% L-alanine is also under the control of another dominant gene.5 In addition the ability to recognize porcine lactic dehydrogenase isoenzymes as antigens has been reported to be under single genetic control in rats7 and rabbits.6 The discovery within a short time of several specific systems under genetic control indicates that genes affecting immune responses are not rare.

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At the present time little is known of the process controlled by these specific immune response genes; however, a most interesting finding was made by MIcDevitt and Chinitz concerning Ir-1.9 The ability to respond to antigens controlled at this locus is linked to the H2 histocompatibility locus of mice. The ability to respond well to (T,G)-A-L is linked to the H2b allele, whereas the abilities to respond well to (H,G)-A-L and to (Phe,G)-A-L are linked to the H2k and H2q alleles, respectively. These observations led us to investigate whether the PLL gene is similarly linked to the major histocompatibility locus of guinea pigs. This study was performed by independently analyzing the inheritance of the PLL responder status and of genes determining strain 2 histocompatibility antigens in backcross progeny of the mating of (2 X 13)F, male with strain 13 female guinea pigs. Materials and Methods. Experimental animals: Adult inbred strain 2 and 13 guinea pigs were obtained from the NIH Animal Production Section. They were mated to produce (2 X 13)F1 animals. Male F, guinea pigs were mated with female strain 13 guinea pigs to produce (2 X 13)F1 X 13 back-cross offspring. Materials: A poly-L-lysine HBr (PLL) preparation with 110,000 average molecular weight was purchased from Pilot Chemicals, Watertown, Mass. Complete Freund's adjuvant was obtained from Difco Laboratories, Inc., Detroit, Mich., and contained 0.5 mg/ml M. butyricum. Thymidinemethyl-'H, 2 Ci/mM ('H-TdR) was purchased from New England Nuclear, Boston, Mass. '1Cr, 185 ACilg, was obtained from Amersham-Searle, Chicago, Ill. Lyophilized guinea pig serum, as a source of com-

plement, was purchased from BBL, Cockeysville, Md. PLL was reacted with 2,4-dinitrofluorobenze as described10 and DNP62PLL was prepared. (The subscript refers to the average number of DNP groups per molecule.) Identification of back-cross offspring with the PLL gene: Adult back-cross guinea pigs were immunized in the foot pads with 0.1 mg DNP-PLL emulsified in complete Freund's adjuvant. Two weeks after immunization the animals were tested intradermally with 0.01 mg DNP-PLL in 0.1 ml saline. Animals which possessed the PLL gene developed Arthus reactions followed by severe delayed hypersensitivity reactions. Guinea pigs lacking the PLL gene did not display evidence of hypersensitivity to DNP-PLL. In all cases the results were unequivocal. Of 18 back-cross guinea pigs, nine responded to DNP-PLL and nine failed to do so. The responder status was therefore inherited from the F, parent by 50% of the offspring, as would be expected for a process controlled by a dominant autosomal gene1 (Table 2). Identification of back-cross offspring possessing strain 2 histocompatibility antigens: Two techniques were selected to detect the presence of strain 2 histocompatibility antigens in back-cross progeny of (2 X 13)F1 X 13 matings-(1) mixed leucocyte interaction which is believed to detect histoincompatibility only at the major locus' and2 "1Cr release from lymph node cells exposed to dilutions of antistrain 2 isoantiserum in the presence of guinea pig complement.'3 Mixed leucocyte interaction: The experimental animals were bled by cardiac puncture into a heparinized syringe. The blood was mixed with one third its volume of 3 grams per cent sterile gelatin (Nutritional Biochemical Co.) in physiologic saline and was allowed to stand in a 370C water bath. Sedimentation of the red blood cells was complete by 1 hr and the white-blood-cell-rich supernatant was removed and washed in Eagle's medium twice. The proportion of mononuclear cells was determined by phase contrast microscopy and averaged 80-90%. The culture conditions were as follows: a total of 1 X 106 mononuclear cells were added to 10 X 75 mm plastic culture tubes (Falcon Plastics Co.) in a volume of 1 ml of Eagle's medium, with glutamine, antibiotics, and 10% strain 13 guinea pig serum. All cultures were established in quadruplicate. The cells were cultured at 370C in an atmosphere of 5% CO2 for 72 hr. Eighteen hr before harvesting, 1 ACi of 'H-TdR in 0.1 ml was added to each tube. The cells were

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harvested on Millipore filters and the radioactivity of the I)erchloric acid precipitates was measured according to the method of Robbins et al.'4 In preliminary experiments it was determined that the degree of blast transformation could be enhanced and made more reproducible by previously immunizing the strain 13 animals intradermally with strain 2 lymph node cells or intraperitoneally with strain 2 leukemic cells.'5 All experiments were therefore performed with previously immunized strain 13 animals. In each experiment, 5 X 105 leucocytes from either PLL positive or negative back-cross animals were mixed with 5 X 105 leucocytes from strain 13 animals. As a measure of maximum stimulation, 5 X 105 leucocytes from a strain 2 and strain 13 animal were mixed and run in parallel. Preparation of antiserum against histocompatibility antigens of strain 2 guinea pigs: Lung, liver, spleen, and lymph node tissues from strain 2 guinea pigs were gently ground in a glass hand homogenizer with twice their volume of saline. The tissue suspension was emulsified with complete Freund's adjuvant and strain 13 guinea pigs were immunized with 0.2 ml of the emulsion in each foot pad. The animals were then injected every week for 10 weeks with 1 ml of the strain 2 tissue mixture in saline, which was divided into five intradermal doses. The guinea pigs were bled weekly for a month and sera of individual animals were pooled separately and stored at -20°C. The antisera were tested for cytotoxicity against lymph node cells from strain 2, strain 13, and (2 X 13)F1 guinea pigs by the trypan blue exclusion test and by the "Cr release technique (Fig. 1).1" The antiserum with the highest cytotoxicity titer, in the presence of guinea pig complement, against strain 2 and (2 X 13)F1 cells was selected for the experiments. Detection of strain 2 histocompatibility antigens by "lCr cytotoxicity test: Inguinal and popliteal lymph nodes were removed from the guinea pigs and gently teased over a stainless steel sieve (80-gauge mesh) to yield single cell suspensions. The medium used throughout was Hanks' balanced salt solution supplemented with 10% fetal calf serum. The cells were washed once and resuspended to a concentration of 5 X 107 cells/ml. 50 liCi of 5"Cr/ml, were added to each of the cell suspensions and the mixtures were incubated at 370C for 30 min. The cells were then washed four times with chilled medium and finally'resuspended to a concentration of 107 cells/ml. 0.1 ml of cell suspension was added to 0.1 ml of twofold dilutions of antistrain 2 isoantiserum. The initial dilution was 1:5. After 15 min at room temperature, 0.1 ml of a 1:2 dilution of reconstituted lyophilized guinea pig serum pH 7.2 (complement source) was added to each serum dilution. The mixture was left at room temperature for a further 2 hr, and 2 ml of chilled medium was then added to each tube. The tubes were centrifuged, and a 0.2-ml sample of each supernatant fluid was removed. The radioactivity in each sample was measured and compared with the radioactivity present in an equal volume of the supernatants from tubes containing cells incubated in medium plus complement only, and with that present in tubes containing cells which were frozen and thawed four times. The percentage of maximum "Cr released specifically was calculated for each antiserum dilution as follows: Radioactivity released by antiserum dilution -Radioactivity released in absence of antiserum 1 Radioactivity released by frozen thawed cells X -Radioactivity released in absence of antiserum Each serum dilution was tested in duplicate and the controls were tested in quadruplicate. Results. Mixed leucocytes from strain 2 and strain 13 guinea pigs displayed reproducible stimulation of 'H-TdR incorporation into DNA. Strain 2 antigens on (2 X 13)F1 leucocytes also reproducibly induced stimulation of DNA synthesis by strain 13 leucocytes (one-way reaction). As expected, mixture of syngeneic leucocytes obtained from separate animals failed to induce blast trans-

formation (Table 1).

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TABLE 1. In vitro 'H-TdR incorporation in mixed leucocyte reactions of strain 2, strain 13, and (2 x 13)F1 guinea pigs. Leucocyte mixture E/C Mean* E/C Range 13 + strain 2 8.1 3.2-24 (6)t 13 + (2 X 13)F, 3.4 2.7-6.8 (3) 13 + strain 13 1.0 0.9-1.1 (3) 2 + strain 2 1.0 0.8-1.2 (2) * Experimental culture/control culture (E/C) represents the 3H-TdR incorporation into DNA in cultures in which 5 X 106 leucocytes from two animals are mixed, relative to half the sum of 3H-TdR incorporation observed when 106 leucocytes from each animal are incubated separately. A value greater than 1.0 represents stimulation due to histoincompatibility at the major locus. t The number in parentheses indicates the number of times the experiment was performed.

Strain Strain Strain Strain

The mixed leucocyte reaction between cells from strain 13 and back-cross guinea pigs was completely different depending upon whether or not the animals possessed the PLL gene (Table 2). Leucocytes from every "responder" backcross guinea pig stimulated significant increases of DNA synthesis by strain 13 leucocytes (one-way reaction), indicating the inheritance of the major histocompatibility genes of strain 2 guinea pigs by back-cross animals with the PLL gene. Leucocytes from back-cross animals lacking the PLL gene failed to stimulate significant blast transformation of strain 13 leucocytes, indicating histocompatibility with strain 13 cells. Identical results were obtained when lymph node cells from back-cross guinea pigs were exposed to antistrain 2 isoantiserum and complement. As a control, Figure 1 shows the sensitivity of (2 X 13)F1 cells to the cytotoxic effect of serial TABLE 2. Linkage between the PLL gene and the locus controlling the major strain 2 histocompatibility antigens in back-cross progeny of (2 X 13)F1 X 13 guinea pigs. Back-cross animal

1 2 3 4

5 6 7 8 9 10 11 12 13 14 15 16 17 18

PLL gene status -

Mixed leucocyte reaction of back-cross and strain 13 guinea pigs E/C*

0.9 1.2 1.4 0.9 1.1 1.0 1.0 1.1 1.2 Mean and standard error 1.08 4t .054 + 2.3 + 2.8 + 2.9 + 3.7 + 5.4 + 4.2 + 9.3 + 20.2 Not done + Mean and standard error 6.3 i 2.13

Percentage of 5"Cr release by 1:5 dilution of antistrain 2 serum

Not done Ot 0 0 0 0 0 0 0 62 66 47 39 60 65 62 48 53 56 ± 3. 1

* E/C incorporation of 311-TdR into DNA of experimental culture/control culture calculated as described in Table 1. E/C greater than 1 indicates stimulation due to histoincompatibility at the major locus. t 0 = No 5tCr release above values obtained without antiserum.

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FIG. 1.-Percentage of maximum 5Cr released from labeled lympho\cytes from strain (2 X 13)F1 and \strain 13 guinea pigs exposed to varying concentrations of antistrain 2 isoantiserum in the presence of complement. The number in paren\ theses represents the number of ex\periments performed and the verti20 _ represent the standard $\cal bars error.

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140 1-20 1:40 1:80 ANTISERUM DILUTION

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dilutions of the antistrain 2 antiserum used in these experiments. Table 2 and Figure 2 illustrate that lymph node cells from all nine responder back-cross guinea pigs showed similar degrees of sensitivity to antistrain 2 antiserum as cells from (2 X 13)F1 animals. In contrast, in no case could cells from back-cross guinea pigs lacking the PLL gene be distinguished from strain 13 cells by antistrain 2 antiserum. 80 70

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2.-Percentage of maxi~~~~~~~~~~~~~FIG. 6mCr released from labeled

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