HLA-D typing with homozygous cells identified in an American indigenous isolate: II. Family studies...

Tissue Antigens (1982) 20, 86-99

HLA-D typing with homozygous cells identified in an American indigenous isolate 11. Family studies and D/DR relationship Z. LAYRISSE, H. D. HEINEN, N. SIMONNEY,

with the technical assistance of:

Z . STOIKOW, 0. BALBAS, I. LEETS and E. GARC~A

Instituto Venezolano De Investigaciones Cientificas, (I.V.I.C.) Caracas, Venezuela

Twelve American Indian nuclear families with 2-5 siblings have been HLA-D typed using mixed lymphocyte cultures and clusters of homozygous typing cells (HTC) of Caucasoid origin to detect DW1-DW7 and typing cells of American Indian origin to detect LDSA, LD15A and LDl5B antigens. Results obtained demonstrate complete absence of DWl-DW7 in these families and illustrate the inheritance and segregation of LDSA, LDl5A and LD15B. DR typing results obtained with the 8th International Histocompatibility Workshop genetic set of antisera indicate inheritance in coupling of DR2 with LD5A, of DR6.2 (DR3+6, MTl negative, MT2 positive) with LD15A, and of DRW8 with LDlSB. The existence of MLC activating antigen(s) different to DW4, yet associated to DRW4 in this population is postulated. The D/DR relationship present in this American Indian isolate demonstrate once more that DR2 can be inherited in combination with an HLA-D antigen different to DW2, and that LD15A HTC define a second sub-cluster of the broad DW6 specificity group, which is inherited with DRW6.2 and BW62 antigens in the Warao population.

Received for publication 7 October 1981, accepted 8 January I982

The Warao, an American Indigenous population inhabiting the Orinoco Delta in Eastern Venezuela, has been extensively studied for the HLA antigenic system both with serological and cellular methods (Layrisse et al. 1976). As expected from its isolation, a very

Supported in part by Grants S1-0663 and S1-1040 from the National Research Council (CONICIT) of Venezuela.

striking homogeneity in HLA specificities is found in this population compared to outbred Caucasoid human groups, and the incidence of individuals homozygous for the HLA-A & B antigens is 5 times that of Caucasoid or Af- rican populations. Mixed lymphocyte cultures between related and unrelated Warao individuals indicated also an extreme degree of homogeneity regarding the specificities con- trolled by the HLA-D locus and this was

0001-28151821070086-14 $02.5010 @ 1982 Munksgaard, Copenhagen

HLA-D/DR IN AMERICAN INDIAN FAMILIES 87

corroborated by D R W serology which has shown high incidence of only 4 D R W antigens (Layrisse et al. 1979, 1980). Six Warao homozygous typing cells (HTC) useful as reagents to identify 2 apparently new HLA-D determinants in population studies have been also described (Layrisse et al. 1978).

This paper shows results of HLA-D typing performed in 12 Warao nuclear families, using HlCs of Caucasian and American Indian origin, and demonstrates the inheritance and segregation of 3 HLA-D antigens, all of which can be identified solely with the use of HTC obtained within the same human population.

Material and methods

Families tested

Warao family groups comprising both parents and 2-5 siblings, living at Winikina and Nabasanuka villages of the Orinoco Delta, volunteered to donate blood during 13 field trips performed in the course of 2 years. The characteristics of the Warao population have been described previously (Layrisse & Wil- bert 1966). Peripheral blood was obtained in vacutainers containing heparin and trans- ported immediately without refrigeration a distance of 800 km to the laboratory in Caracas. Whenever possible, whole families were bled during the same field trip. Family relationship was ascertained through genealogies previously collected and cross- checked by the anthropologist of the team (H.D.H.).

Lymphocyte isolation (PBL) was performed 10-24 h after bleeding by Ficoll-Hypaque gradient centrifugation (Boyum et al. 1968). Cells were used immediately or kept frozen in liquid nitrogen for periods varying from one to 15 months.

Serological tests

HLA-A, B, C typing was done on lymphocytes isolated 12-24 h after bleeding using the standard lymphocytotoxicity dye exclusion test (Terasaki & McClelland et al. 1964) and 7th and 8th Workshop sets of typing sera. D R typing was done on B-lymphocytes enriched suspensions isolated from PBL by the throm- bin-nylon wool method (Danilovs et al. 1980) using anti-DR antibodies of the 8th Work- shop genetic set.

Mixed-lymphocyte cultures ( M L C )

Were set up following the technique used for the 8th Int. Histoc. Workshop (Dupont et al. 1980). Briefly, 5 x lo4 responding mononuclear cells in 0.1 ml of RPMI 1640 medium supplemented with antibiotics and 15% pooled human serum were co-cultured in triplicate with 5 X lo4 irradiated (4000 R from a ‘OCo source of 6000 Ci) stimulator cells in an equal volume of media in round-bottom microtiter plates; cultures were maintained in a humidified 5% C02 in air at- mosphere, for 5 days at which time 1 pCi of 3H-thymidine (New England Nuclear, 6.7 Ci/ mmole) was added to each well. Eighteen hours later, cultures were harvested into filter paper with an automatic machine (MASH 11, Microbiological Associates, Rockville, MD) and counted in a liquid scintillation counter. Each experiment of a total of 22 was performed with a variable number of responders and stimulators (between 14 and 32). Seven of the experiments were set up using as responders freshly isolated lymphocytes; all other MLC (15 out of 22) used as responder lymphocytes which had been kept frozen.

Homozygous typing cells (HTC)

The HTC of American Indian origin were obtained from selected Warao individuals

88 LAYRISSE ET AL

LDl5A CAI

R * 3 3 4

n i

LD15B LD15B SPA

R = Z l i

I - - do eil I20 160 ax 240

RELATIVE RESPONSE (DNV) RELATIVE RESPONSE (DNV) RELATIVE RESPONSE ( D N V I

Figure 1 . Histograms obtained with double normalyzed responses (DNV) of a variable number of ethnically different individuals and Warao homozygous typing cells (HTC) LDlSA (CRI) and LDI 5B (OLG & SPA) as stimulators, in mixed lymphocyte cultures.

who volunteered to travel to Caracas. Lym- phocytes were isolated from the buffy-coat of peripheral blood collected from 4-6 cycles on the Haemonetics blood separating machine, followed by Ficoll-Hypaque gradient centrifugation. Mononuclear cells were control- led-frozen in 7% DMSO-50% autologous plasma-RPMI 1640 mixture and kept in aliquots of 6 X 10'ivial in liquid nitrogen until used.

The families presented in this study were tested with 7 Warao HTC: REM & BAR defining the LD5A cluster; LI, AMA & CRI defining the LD15A cluster and SPA & OLG of the LD15B cluster (Layrisse et al. 1978). Histograms obtained with these HTC in population studies have been partially pub- lished (Layrisse et al. 1978). Those obtained with LD15A HTC CRI and with the mutually non-stimulating LD 15B HTC obtained from the unrelated donors SPA y OLG are shown in Fig. 1.

HTC of non-American Indian origin were obtained through exchanges with different laboratories and are listed in Table 1.

Data reduction and statistical analysis

The method of Ryder et al. (1975) was used to obtain double normalized values (DNV) from the median of triplicate counts per minute of each culture, in each experiment. Each individual was tested with 2-5 HTC of each cluster defining a different D W specificity and repeated tests (2-4) were performed whenever possible. Based on histograms drawn with results obtained with each HTC and population studies performed in our laboratory, DNV values equal to or below 35 were taken to indicate presence of the corresponding DW antigen in the responder. The scoring method used in the Central analysis of the HLA-D section of the 8th Workshop was also applied to the data obtained with all families (Dupont et al. 1980). In all cases, with only one exception, the HLA-D assignment was concordant with both methods. The exception was one responder tested only once giving an inter- mediate response (DNV = 45) with a single HTC, that is a negative assignment by the former method and a positive assignment by the scoring method.

HLA-DIDR IN AMERICAN INDIAN FAMILIES 89

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Results

Serological results

HLA-A, B, C & DR typing of the families here presented was performed during the 8th International Histocompatibility Workshop and the reaction patterns observed have been already reported (Layrisse et al. 1980). DR2, DR4 and DRW8 were assigned without diffi- culty as the cytotoxic reaction patterns observed did not differ considerably from patterns observed with cells from Caucasoid donors described during the 8th Workshop. DRW6 could be assigned based o n the strong cytotoxic reactions observed with DR3+6 sera, since DR3 is completely absent in this population. Post-Workshop analysis has demonstrated that the patterns obtained with DR3+6 positive Warao cells correspond exactly to reactions described for DRW6.2 (Bodmer et al. 1980), that is DR3+6 positive, MT1 negative, MT2 positive.

Warao HTC and DR specificities detected in them

DRW antigens present among 11 Warao HTC previously identified by family MLC and population studies were determined on B-lymphocytes using the 8th Workshop genetic set of antisera and the results are shown in Table 2. HTC defining the LDSA cluster were found to be DR2. According to their DR antigens, 6 HTC identifying D antigens associated to the B1S antigen can be divided into 2 different clusters: thus, 4 LD15A HTC are DRW6.2 by serology while the LDlSB HTC are DRW8.

HLA-D Typing of 12 Warao families

The results presented here are part of a total of 22 MLC set up with approximately 450 responders and an equal number of stimulators. Difficulties inherent to testing

Table 2. Homozygous typing cells (HTC) identified among the Warao population by inrra- family mixed lymphocyte cultures (MLC). HTC forming each cluster are mutually non-stimulatory in MLC.

HLA-BIDR antigens showing linkage dis-

specificity specificity in Warao Identification HLA antigens in HTC HLA-D equilibrium with

A C B DR population

- w5 1 2 6w5006* REM 7w53Y

LDSA BAR 8 ~ 3 0 6 2 8 w5 1 2 AME 2 - w5 1 2 Bw5 l,DR2

2 - w5 1 2 ROS 2 - w5 1 2

LIG 1 6 ~ 5 0 0 4 2 3 w62 w6.2

7w504 I** xw201 3 w62 w6.2 LDl5A Bw62,DRw6.2

2 1 ~ 3 1 31- ~ 6 2 1 ~ 5 1 ~ 6 . 2 ALE 2 1 ~ 3 1 31- w621w51 w6.2

SPA OLG LDlSB w31 1 w62 w8

w31 113 w62 wx Bw62,DRw8

* HTC submitted to 6th, 7th or 8th International Workshop. ** Siblings.

Table 3. Double normalized relative responses obtained in several M L C experiments with D WI-D W8, LDSA, L D I S A ai LDISB clusters as stiniulators and members of Warao family no. 7 as responders.

DW1 DW2 DW3 DW4 Exp.

1 * 2 3 4 1 2 3 4 1 2 4 5 1 2 3 4 5 7

Father 9 86 74 104 78 94 9s 51 I02 143.2 19 97 95 97 111 57 88 93 76

21 82 66 80 81 70 93 59 30 23

Mother 9 88 99 X Y 89 105 112 84 97 332.1 20 88 62 64 84 84 95 75 70

SIB.l 9 95 93 109 79 104 93 67 450.1 20 87 57 76 112 105 114 75 81

23 ~

SIB.2 9 98 87 101 91 89 77 77 450.2 20 75 68 71 88 105 100 109 56

23

SIB.3 9 95 90 93 98 82 84 90 88 450.3 19 99 88 106 73 87 100 98 67

22 98 68 23

*HTC belonging to a cluster are identified with numbers as referred to in Table 1. ** 75th percentile of responder expressed as counts per minute.


geographically distant, isolated populations, plus technical failures proper of the MLC technique, did not permit to gather results of complete families with the same HTCs. We have succeeded though, to test both parents of each family with 2-5 HTC of each cluster (mean of 3 HTC for DW1-DW7; mean of 2 for LDSA, LD15A and LD15B) and the en- tire families with 2-4 HTC for each specificity (mean of 3 HTC for DW1-DW4; mean of 2 HTC for the other specificities) with very few exceptions. Moreover, 20% of the total cultures (N = 1392) were repeated at least twice, with 38% of the HTC used. Two families with 5 siblings, 3 with 4, 4 with 3 and 3 families with 2 siblings have thus been tested. The complete data obtained by testing one family with several HTC for each HLA-D specificity in different experiments is shown in Table 3, as representative of the Warao families here reported. Due to the large amount of data gathered, the results observed with each cluster of HTC identifying each of the 10 HLA-D

specificities tested were used to calculate mean DNVs. Table 4 shows these mean DNV values given by each family member and complete HLA-A, B, C, D haplotype inheritance. Results obtained with DW8 HTC 8W203 and 8W204 were not used to calculate mean DNV due to the limited number of tests performed with these cells.

The results indicate complete absence of DW1-DW7 in these families and presence of LDSA, LD15A and LD15B traveling always with an HLA haplotype. Three families (nos. 3, 10 and 11) demonstrate segregation of LD15B from LD5A and LD15A. Three families (nos. 2, 5 and 10) illustrate indepen- dent inheritance of LD5A and LD15A. No triplets were observed.

DIDR Relationship

In all family cases here reported, with only 2 exceptions, LD5A was found in combination with DR2, LD15A with DRW6.2 and LD15B

DW5 DW6 DW7 DW8 LD5A LD15A LD15B 75p** CPM ~~

1 2 3 1 2 4 1 2 3 5 1 2 1 2 1 2 3 1 2

147 87 59 93 67 36 84 75 103 123 11842 75 129 81 87 100 87 100 67 111 87 54094 00 121 83 100 105 97 100 100 74 61 138 137 122 17025

46 36 100 109 78 100 4539

104 76 88 84 100 107 70 88 10 12 17463 74 100 82 71 79 100 49 58 10 9 44535

84 75 62 33 150 121 11 16 12234 89 105 96 88 98 98 74 97 26 17 37978

122 123 9 19 100 123 13073

100 69 79 93 100 100 21 26 14371 77 104 106 92 100 116 41 47 19 30 32797

104 59 59 51 240 173 8941

58 82 99 116 128 51 37 1004 1 00 111 92 93 78 73 178 376 47 21 29667

78 37348 93 167 16 15 132 147 2851

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2,4 up.M

& A A A b & 6 A A ~ $ 6 6 A 2,8 2,2 nt 6,6 nt 6,s 4,6 4,6 4,6 6,6 6,6 2,6 2.6

Figure 2. Inheritance in coupling of LDSA with DR2, LDISA with DRW6 (DRW6.2) and of LDISB with DRW8 in Warao nuclear families inter-related by marriage. The families shown in this figure include 7 of the families described in the text and 3 others studied less exhaustively. DR genotypes are represented by numbers shown under each individual. HLA-D antigens detected by mixed lymphocyte cultures and homozygous typing cells are indicated as follows: 4 or 1 = LDSA, = LDlSB; D or [I = undetectable HLA-D antigen, different to DWI-DWX, LDSA, LDISA or LDISB.

or & = LDISA; B or

with DRW8 (Fig. 2). One of the exceptions was given by the father in family no. 1 who is DR2 by serology but LD5A negative. l h e second exception observed concerns Sib 2 in family no. 9 (Table 4) which apparently car- ries DRW8 in the absence of LDlSB, as shown by results with 2 HTC in, unfortu- nately, only one experiment performed with this responder. DRW4 was found present in the parents of 8 of the I2 family groups tested, but always in the absence of DW4. Although 2 homozygous DRW4 Warao individuals have been identified by family testing (unpublished observations), population studies are necessary to prove that their lymphocytes are good typing cells. Thus, it has not been possible yet to identify the DW antigen(s) associated to the very frequent DRW4 specificity in this American indigenous isolate (Layrisse 1980).

A striking feature of the haplotypes more commonly observed in the families here reported is the constant inheritance of LD15B with the combination AW3 1BW62 (with CW1 or CW3); the other D W antigens showed preference for one particular combi-

nation: LD5A with A28BW51 and LD15A with A2BW62, but were also found in combination with BW60, BW35 and BW62 in the case of the former, and with BW5 1 and BW3.5 in the case of LD15A (Table 4).

The results also indicate that Sib 1 of Family no. 12 is a probable BID recombinant as he inherited DRW4DW blank instead of the expected DRW6.2 LD15A with the A28BW62 haplotype.

LD1.73-D W8 relationship

The very strong association between DRW8 and L D 15B observed among unrelated Warao and non-Warao individuals in our laboratory, suggests the possibility that LDlSB could be related to DW8. MLC testing of the families here reported with Caucasian HTC defining the DW8 specificity have been done only partially due to limited availability of the corresponding HTCs. Thus, out of 24 parents, 11 were tested with 8W204 (MAD) while 6 of the rest were tested with 8W203 (BtB). Nevertheless, the assignment


of LD15B and DRW8 in 7 parental haplotypes, was corroborated with typing responses with either 8W203 or 8W204 HTCs in 5 cases. Repeat testing of the 2 discrepant cases could not be performed. MLC between HTCs typing for LD15B and HTC typing for DW8 have been done again only partially in Caracas, but in repeated trials, both Warao cells OLG & SPA have shown typing responses (DNV below 30) to 8W203.

Discussion

The HLA-A, B, C profiles of American indigenous populations have been progressively unraveled in the last decade and several tribes living in North, Central and South America are today known to be strikingly homogenous in their HLA antigenic structure (as reviewed by Kostyu & Amos 1981). However, the specificities controled by the HLA-D/DR loci have been tested in only a limited number of tribes (Troup et al. 1978, Statsny et al. 1978, Layrisse et al. 1979), and this is not surprising considering the technical problems of the methods available to detect them and the dif- ficult access to truly pure American indigenous populations which remain unmixed with other ethnic groups only by geographic isolation.

The results here reported are important, not only because family studies are not fre- quently reported in the literature even, from more accessible human groups, but also because they show the inheritance of DW antigens detected until now only with HTC of American Indian origin.

The use of HTC and MLC to detect the presence of HLA-D determinants has been considered partly inadequate due to experi- mental variation and the limited access to typing cells. Modifications such as data editing and statistical treatment, have been incorpo- rated in an attempt to improve the method.

Nevertheless, alternative methods have not been found and during the 8th International Histocompatibility Workshop, the number of recognizable HLA-D antigens identified by this method was increased to 12, although family studies were performed only for DW1-7, as too few HTCs were available for the other specificities (Dupont et al. 1980). By studies performed in our laboratory, a re- latively large number of HTC defined by MLC performed within selected Warao families living at the Orinoco Delta in the villages of Winikina and Nabasanuka, have been identified. Two clusters of HTC were evident from early studies and they were described as LD5A and LD15A according to the HLA-B specificity they showed preferent association with (Layrisse et al. 1978). However, the existence of a second MLC activating deter- minant also associated to the B15 antigen in the Warao, was postulated already 6 years ago, to explain MLC reactions between the mother and one sibling of the original VD family (Layrisse et al. 1975). Clear definition of these two B15 associated D antigens was not possible until DR antigens could be defined in the corresponding HTCs. This was accomplished with reagents distributed during the 8th Workshop, which used in serologic tests permitted to separate the Warao HTCs into 2 clusters: LD15A-DRW6.2 and LD15B-DRW8. DR serology has allowed also to identify as DR2 the antigen present in LD5A HTCs.

We have now presented results of HLA-DIDR typing of 12 nuclear Warao families tested simultaneously with DW1-DW7 HTC clusters of Caucasian origin and with HTC clusters of American Indian origin. Complete absence of DW1-DW7 has been demonstrated in these families, while the inheritance and segregation of LDSA, LD15A and LD15B has been shown. The 3 HLA-D antigens present in these families have been identified in MLC with the use of

7 Tissue Antigens 20,Z

98 LAYRISSE ET AL

HTCs found within the same human isolate and have shown to be inherited in all families with DRW2, DRW6.2 and DRW8 respec- tively, as defined by B-cell serology with 8th workshop set of antisera. The existence of at least another MLC activating-DRW4 associated antigen for which no HTC(s) have been found is also postulated.

The relationship of several of the Warao HTC to HTCs of Caucasian origin has been tested in several International Workshops. N o correlation has been observed between LD5A (HTC 6W5006,7W539,8W306) and HTC of Caucasian or Japanese origin submitted to the 6th, 7th or 8th Workshops. LD15A has no relationship to antigens detected by HTC of Caucasoid origin described so far, as the early observation of a correlation (r = 0.55) with one of the HTC originally used to define the DW8 antigen (6W7003) has not been con- firmed with other DW8 HTC submitted to the 7th or 8th Workshop. The LD15B (DRW8) HTCs of Warao origin, on the other hand, are related to the DW8 cluster, as shown by typing responses obtained in our laboratory with these cells and 8W203. Population studies are necessary though to stablish the relationship of LD15B with other HTC defining DW8.

The DiDR relationship described in this study, is particularly interesting for several reasons. In the first place, the DRW8iLD15B association among unrelated Warao is stronger (r = 0.73), than the corresponding DRW8IDW8 in Caucasians (r = 0.588) (Dupont et al. 1980). Secondly, the very tight association of LD5A with DR2 in these families illustrates once more that, as opposed to results reported at the 1977 Oxford Workshop, one DR antigen can be found combined with different D antigens. Thus, DR2 has been found highly associated to DW2 (Bodmer 1978), but also with tb 24 (Richiardi et al. 1978) and LD14 (Suciu-Foca et al. 1981) in Caucasians and to LDHO/DB4 in Japanese (Sasasuki et al. 1977). Thirdly,

the finding of Warao HTCs typing for an HLA-D antigen different to DW6 (as defined by 8W149, 147, 148) which is inherited with a subgroup of the original DRW6 antigen, adds new insight into the broadly defined specificity group DW6. Thus, Warao HTC LD15A seem to define a second DW6 sub- cluster associated to DRW6.2 (DRW3+6, MT2 positive) and BW62 antigens. The relationship of LD 15A to the newly described DW6.1 antigen (Mickelson et al. 1980) re- mains to be determined, although they are clearly different by serology, the HTCs defining the latter being DRW3+6 negative, MT1 IMT2 positive.

Acknowledgments

The authors wish to thank the Corporacion Venezolana de Guayana (CVG) for facilities provided during the field trips and to the Radiophysical Department of I.V.I.C. for the irradiation of the blood samples.

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Address: Zulay Layrisse, Ph.Sc. Instituto Venezolano de Investigaciones Cientificas

Apartado 1827, Caracas 1010A, Venezuela

(I.V.I.C.)

HLA-D typing with homozygous cells identified in an American indigenous isolate: II. Family studies...

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