Transfected human class I gene product adequately assembles minor Histocompatibility antigens.

Immunogenetics 34 270-272, 1991

Immunp-genetics

© Springer-Verlag 1991

Transfected human class I gene product adequately assembles

minor histocompatibility antigens

Eis Goulmy1, Jos Pool1, Eis Blokland1, and Dan Geraghty2

1 Department of Immunohaematology and Bloodbank, Umveisity Hospital, Leiden, The Netherlands 2 Fred Hutchinson Cancer Research Center, Seattle, USA

Received February 18, 1991, revised Version received March 27, 1991

The success of HLA genotypically identical bone marrow grafting is still hampered by graft vs host disease and re-jection of the graft. One of the causes of the latter com-plication could be attributed to minor histocompatibility (miH) antigen disparities between HLA genotypically identical siblings (Goulmy 1988). In previous in vitro studies, we demonstrated the presence of anti-host cytotoxic Τ cell (CTL) as well as proliferative (Th) Τ cell reactions in blood-samples taken after HLA identical bone marrow transplantation (van Eis 1990a, b). The latter ac-tivities are directed against miH antigens which are recognized in association with MHC class I (CTL) and class II (Th) products (Goulmy 1988; van Eis 1990c). We recently acquired CTL clones specific for five non-sex-linked miH antigens designated HA-1, -2, -3, -4, and -5, whereby HA-1, -2,-4, and -5 use the HLA-A2 gene pro-duct as restriction molecule. Using the latter CTL clones, immunogenetic studies were carried out to determine the miH antigen gene frequencies and to study the miH an-tigen segregation patterns in families. One complication in these studies is that it is necessary for the HLA-A2 molecule to be present in order to detect the miH antigens. As illustrated in Figure la, the HLA-A2 positive identical siblings 02 and 03 carry miH antigens HA-1 and -2. Since the father (01) of family Β oriy posseses HA-2, the miH antigen HA-1 must have been inherited from the mother. Therefore, the absence of the required MHC class IHLA-A2 restricting antigen hampers adequate genetic analysis in this and other families.

In order to solve this deficit, we used electroporation (Potter et al. 1984) to introduce cloned HLA genes into the Epstein-Barr transformed Β cell lines of selected members of this family. We transfected the HLA-A2 gene cloned in the pHEBO vector (Sugden et al. 1985; Shimizu et al. 1986) into the HLA-A2 negative cells of the mother Addrens correspondence and offpnnt requests to Ε Goulmy, Depart ment of Immunohaematology, Bldg I E3-Q, Umversity Hospital, PO Box 9600, 2300 RC Leiden, The Netherlands.

(00) and into three HLA identical, HLA-A2 negative family members 04, 05, and 06 (Fig. la). Fluorescence activated cell sorter (FACS) analyses using the class I and HLA-A2 specific monoclonal antibodies were carried out and demonstrated the surface expression of the HLA-A2 gene product on all transfected cells (Fig. 2). Subsequent-ly, the transfected cell lines were subjected to miH typing. Figure lb shows that we were indeed able to trace the miH antigen HA-1, which after HLA-A2 gene transfection ap-peared tobe inherited from the mother (00). Furthermore, the cells from three children (04,05,06) who did not carry the A2 allele and thus could not be tested for the presence of miH antigen HA-1 and -2, were now amenable to the CTL analysis. In addition to using the miH HA-1 and HA-2 specific CTL clones, we also tested HLA-A2 allo-specific and HLA-A2 restricted H-Y allo-specific CTL clones on the cells of all family members. As shown in Figure lb, A2 allo-specific, A2 restricted miH antigen H-Y, HA-1, and HA-2-specific CTLs lysed the transfected cells to the same extent as the naturally expressing HLA-A2 cells. To confirm that we were actually dealing with identical MHC/miH antigen peptide complexes, cold target Inhibition studies were carried out. Table 1 shows that HLA-A2 transfectants were equally potent as cold target inhibitors of HLA-A2 directed lysis as their natural-ly expressing HLA-A2 counterparts. Non-transfected target cells failed to show any Inhibition.

Ε Goulmy et al miH artigen recogmtion after MHC gene transfection ₂₇₁ IHA 1 » HA 2 _A29 B8 Cw7 DR3 A11 Bw62 Cw3 DRwe ab A3 A2 Bw62 Bw62 Cw3 Cw3 DRw6 DR7 A2 Bw62 Cw3 DR7 02 bo ) A29 B8 Cw7 DR3 A2 Bw62 Cw3 DR7 03 bc I A29 B8 Cw7 DR3

[

A3 Bw62 Cw3 DRw6 04 "ad~ A11 Bw62 Cw3 DRw6

[

A3 Bw62 Cw3 DRw6 _ 1 05 ad A11 Bw62 Cw3 DRw6 ad A3 Bw62 Cw3 DRw6 A11 Bw62 Cw3 DRw6 • HA 1 • HA 2 Ά electroporation bc A2 Bw62 Cw3 DR7 CTLS A2 HA 1 Κ A2HA2 # A 2 H Y A29 B8 Cw7 DR3 8238 92 61 n n 1 0 0 v— > • cd A29 B8 Cw7 DR3 CTLS A2 HA 1 • A2HA2 · A2 Η Υ A2 N— y bc A2 A29 Bw62 Ββ Cw3 Cw7 DR7 DR3 • 51 25 • 6455 0 0 ) A K Bw62 Cw3 DRw6 80 75 93 77 0 0 91 69

\L

4 • ad A3 Bw62 Cw3 DRwe • 1 A3 Bw62 Cw3 DRw6 CTLS A2 HA 1 A K Bw62 Cw3 DRw6 72 67 83 80 8371 A2 HA 2 A2 Η Υ A2 O l l 1 ab A2 Bw62 Cw3 DR7 0 0 • 62 39 69 45 46 16

ε

• ad A3 Bw62 Cw3 DRw6 • AK Bw62 Cw3 DRw6 94 90 92 85 i n on \_ y • ad A3 Bw62 Cw3 DRw6 • AK Bw62 Cw3 DRw6 87 70 S4 76 1 f)

Fig. 1. a, b. The presence of the miH

an-tigens HA-1 and HA-2 IS mdicated by their specific Symbols The % specific lysis ob-tained from the different CTL clones at two effector to target ratios (i e , 20 1 and 2 1) is shown *indicates that cells underwent HLA-A2 gene transfection, D males, Ο females

Antibody 9E8 Antibody P20 1

04

04+A2

Log Fluorescence Intensity

-Fig. 2. The EBV LCLs of family members 00, 04, 05, and 06 were

272 Ε Goulmy et al miH antigen recognition after MHC gene transfection Table 1. Companson of HLA-A2 transfectants and naturally HLA-A2 expressing cells as cold target Inhibitors of HLA-A2 directed lysis Target cells

hol cold

Cytotoxic Τ cell clones allo A2 97/541" 87/47 60/ 9 67/14 74/28 85/54 87/65 49/12 52/18 65/41 60/27 40/ 9 16/ 0 57/33 17/ 2 32/ 3 73/40 44/ 8 67/32 30/ 3 27/ 1 A2HY 1/ 4 82/70 82/64 65/17 84/74 64/53 62/54 41/19 67/50 71/47 34/ 2 72/57 0/ 0 A2HA 1 90/81 84/69 70/20 67/21 87/56 82/70 89/64 55/22 57/16 79/69 2/ 2 47/32 25/ 7 40/23 7/ 0 13/ 0 A2HA-2 70/25 78/28 35/ 3 33/ 3 44/ 6 70/25 66/25 26/ 3 36/ 7 33/10 49/15 31/ 1 21/ 3 56/15 18/ 0 23/ 4 53/19 31/ 4 61/24 30/ 7 27/ 3 00* 00* 00* 00* 00* 04* 04* 04* 04* 04* 01 01 01 01 01 01 03 03 03 03 03 none 00 00* 04* 03 none 04 04* 00* 0 1φ none 01 00* 04 04* 03 none 03 00 00* 04*

* Indicates the cells underwent tiansfection

f % Specific lysis at two effector/target ratio, ι e , 20 1 and 2 1

*Note that 01 is HA-1 negative but HA-2 positive

The generaüon of miH antigen specific CTLs has been descnbed in detail (Goulmy 1988) Specific cytotoxic Τ cell activity was measured by the cell-mediated lympholysis assay previously descnbed (Goulmy 1982) The hot cold target cell ratio used is 1 10

surface expressed MHC products resulting not only m high levels of specific lysis by both ailo- and MHC restncted miH antigen specific CTLs, but also in com-parable mhibiüng capaciües. Consequently, gene

trans-fection has proved to be a rehable technique for adequate miH antigen recognition and thus broadens lts lmmuno-genetic analyses

Acknowledgmcnts This work was supported by the J Α Cohen Insti tution for Radiopathology and Radiation Protection (IRS)

References

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