Immunodeficiency diseases and expression of HLA antigens

Immunodeficiency Diseases and Expression of HLA Antigens J.L. Touraine and H. B6tuel

ABSTRACT: The role of HLA antigens in lymphocyte differentiation is strongly suggested by the existence of a recently identified immunodeficiency associated with, and probably resulting from, the lack of expression ~[ HLA-A, B, and C antigens as well as [tz microglobulin on varies cells of hematopoietic origin. This "bare lymphocyte syndrome" has been described in a family where the transmission appeared to be autosomal recessive, and the responsible gone was n~ borne by the sixth chromosome. Another infant with a severe combined immunodeficiency disease has been treated u'ith fetal liter and thymus transplants (FLTTL A p, :ffsting chimerism has been documented: T cells derived from the donor and B cells from r ~ host. Despite complete HLA-A, B, and DR mismatch, T and B celL~did cooperate resultin/ /n significant antibody production, and defense against viral infection has been normal. Such an observation may suggest that "allogeneic restriction" of T-cell effectorfunctions con be circumvented.

INTRODUCTION Although severe combined immunodeficiency diseases (SCID) have common clinical manifestations, they represent a relatively heterogeneous group of diseases with varied etiologies and pathophysiologies [I, 2]. Some of the cases with an autosomal recessive transmission are associated with a deficiency of the adenosine deaminase activity [3]; most of the others result from still unknown metabolic disorders. We recendy have identified a combined immunodeficiency disease associated with an absence of expression of HLA-A and B antigens on some cells of hematopoietic origin in an infant of an Algerh~ family [4]. A second patient with the same disease in this family has been shown to lack expression of #~ microglobulin at th~ cell surface, and he has provided genetic information on the transmission of the disease. A treatment by bone marrow transplantation from an HLA-D-DR identical brother has been attempted in this patient. Besides providing a clue to the understanding o f an additional cause of immunodeficiency, this bare lymphocyte syndrome provides significant knowledge on the role of major histocompatibility complex (MHC) determinants in lymphocyte differentiation. Other information on the role of HLA antigens in T-lymphocyte functions have been obtained from the immunological analyses of patients with SCID reconstituted by an HLA mismatched fetal liver transplant [5].

From the IN$ERM Unit 80 and Histocompatibility Laboratory, Pay. P., HipitM E. H~eri~, , 69374 Lyon Codex2, France. Address requests for reprints to Dr. J.L. Touraine, Pay. P., H;pital E. Herriot, 69374 Lycu CtdGx2, Prance. RJ~imt 1980.

H~ ~ 2,147-153(1981) ~ i ~ r NorthHolland,lnc., 1981 52 V ~ Ave.,N~'wYork,NY 10017

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148

J. L Touraine and H. B6mel

MATERIALS A N D M E T H O D S HLA typing for A, B, and C locus antigens was performed on lymphocytes using the two-stage cytotoxicity method [6] with minor modifications [7]. HLA typing of platelets was carried out by complement fixation and HLA typing of skin-derived fibroblasts by an absorption technique. The presence of fie microglobulin at the cell surface was checked by cytotoxicity and by immunofluorescence with a specific anti-fie m serum. Since the HLA genotypes of the other family members were established, the patients' sera were used to absorb the anti-HLA ,era that reacted positively with the lymphocyte-o of the family [8]. Briefly, serial dilutions of the anti-HLA sera were made in veronal buffer; 1 /el of each dilution was mixed with an equal amount of neat patient's serum. After a 3-hr incubation at 20°C, the appropriate lymphocytes were added to each mixture and to the antisera diluted only in veronal buffer; the reaction was then allowed to proceed according to the usual two-stage technique. Absorption was considered positive when there was a fall of two dilutions in the reactivity of antisera incubated with the patient's serum as compared with the control. HLA-DR typing was carried out by cytotoxicity of B lymphocytes separated by adherence to nylon wool and u~ing local antisera, the specificities of which had been controlled during the Sevemh Histocompatibil/t~/Workshop. Mixed lymphocyte cultures were performed a~ previously described [9], with technical modifications to adapt them to micr~titer plates. Enumeration of T and B lymphocytes, cultures with mitogens, determination of marrow prothymocytes, and other immunological assays were carried out using already described methods [10~ RESULTS A N D DISCUSSION Bare Lymphocyte Syndrome Two siblings whose parents were fi~st cousins have been found to have a combined imraunodeficiency disease ~sociated with a lack of expression of HLA-A, B, and C antigens, and we have coined this new type of immunodeficiency, the Bare Lymphocyte Syndrome [4]. In both infants immunologkid dma were comparable. There was a moderate lymphopenia. Numbers and functions of T lymphocytes were extremely reduced. Plasma cells were not found. Plate~t counts were normal. Karyotype analysis did not evidence any gross ~dteration o f chromosomes. Delayed type hypersensitivity skin reactions to antigens ~ d DNFB were either negative or mildly positive. Serum levels of ]gM and IgA were decreased. IgG, of maternal TABLE 1 Detection by absorption of HIM. antigens in serum of Akim D Ami-HLASe~~ Absorption with

A9

AW30

BW35

Veronal buffer Serum of A k i m Presence o f

1/128 1/128 ---

1/64 1/16 AW30

1/32 1/16 --

B5

A2

A! !

BW21

1/32 !/8 B5

1/128 1/128 --

1/16 1/4 A 11

1/64 1/8 BW21

trim ther$ refer to ~he dgludon of ~nd-HLA serg~m~MMg ~0~ ,~to~*kity it~ the F ~ ¢ of ~¢l~roOrkite lymphocytes. A f~dlin t~o d/lutMn$ L*corn/tiered ~ p o ~ t / ~ ~forp6on ~t~ ~ t ~ - , , ,he pfe~en~¢ of HLA g ~ n in the serum retted.

Expression of HLA Antigens in Immunodeficiency

149

FATHER

MOTHER

a A24 CW4 BW35 DR5

C A2 CW4 BW35 OR6

b AW30 85 DR3

d A11 BW21 DR4

. ~ ~O, '' previously died in ac

infancy but had

I ac

ad

bc

© ~

bd - -

bd

not been investigated

FIGURE 1 HLA genotypes of familyD. origin, was close to normal. Alloheinaggl~ltinins were virtually absent. No antibody production was demonstrable. C2 and C4 complement levels were normal. Zinc levels were normal. Serum thymic factor activity (Drs. M. Dardel'me and J.F. Bach) was always below t (normal values in infants above ~L Marrow prothymocytes able to acquire the HTLA + phenotype were very few. The thymus and lymphoid organs were severely hypoplastic. Both lymphocytes and platelets were shown to lack HLA-A, B, and C antigens by cytotoxicity test and complement fixation. These antigens were detected however by absorption techniques in the serum (Table 1) [8] and on the surface of fibroblasts cultured from the skin. Normal expression of D ,and DR determinants was demonstrated by mixed lymphocyte culture and B-cel~ cytotoxicity. HLA genotypes of the family members are represented in Fig~are 1. Investigation of patient's T and B lymphocytes as well as EBV-tranfformed B-lymphoblastoid cells derived from this patient demonstrated an extremely low expressivity of cellular fl~ microglobulin, much lower than in normal ceLLs and onlt slightly higher than in Daudi cells [11]. After several weeks of cultare and several passages, however, expression of 82 microglobulin and of HLA antigens developed in cells of the bare lymphocyte syndrome line. In the serum the level of g2 microglobulin was normal. Very severe infections were present in both patients. The first infant died before bone marrow reconstitution could be attempted. The last infant born with this disease has been treated with two bone marrow transplants (2.6 and 5 x 108 nucleated celis/kg) from an HLA-A, B, and D-DR identical brother [11]. The first BMT was performed at 9 mo of age only, because of relative|y late referral of the patient, after four gastroenterids episodes. No graft-versushost reaction (GvHR) developed. T lymphocytes proliferated and matured, sequentially acquiring the HTLA + phenotype, the capacity to form E rosettes, and then to respond to mitogens and allogeneic ¢timuli. The donor origin of these cells was supported by their normal expression of HLA-A and B antigens, while host lymphocytes remained devoid of such surface antigens. Despite this progressive immunological reconstitution, the previously extreme malabsocpti~ and poor general condition led to a sudden death associated with a drastic and unexpected hypoglycemia, at age 13½ too, i.e., 4½ mo after the first BMT. 'rite analysis of this family, in which the two probands had different HLA haplotypes, normal HLA identical siblings, and a sister previously dead of immunodeficiency, suggests an autosomal recessive disease associated with genes different from those of the MHC region.

150

J.L. Touraine and H. B6mel After identification of the first family with the bare lymphocyte syndrome, two o~her patients from a Turkish family have been found to have a comparable syndrome. In the latter two children, however, both the immunodeficiency and the lack of expression of ~ microglobulin and HLA antigens were relatively less complete [12, 13]. In all of these four patients, the existence of the bare lymphocyte syndrome has confirmed the biological significance of HLA antigens in T-lymphocyte differentiation or function, or both. Transplantation of normal bone marrow cells appears to correct the main immunological abnormalities and perhaps would permit survival if performed earlier.

Chimera after Fetal Liver Transplantation The i~fant S. had a family history of SCID. From birth, this boy was brought up in strict isolation. Laboratory investigations revealed a typical SCID without ADA deficiency. In peripheral blood, T lymphocytes were virtually absent, and there was no proliferative response to mimgens. No marrow prothymocyte able to acquire the HTLA+ phenotype was found. Antibodies were not produced following antigen administration. No HLA identical donor was available in the family (Figure 2) and a FLTT (13-wk-old male fetus) was performed at age 23 days. A second FLTI', from a 10-wk-old male ferns, was done 5 ~.~o later because of the extremely limited increase o f t lymphocytes in peripheral blood. Over the following year, a total immunologic reconstitution developed: T lymphocyte numbers slowly increased, reaching normal values after 1 yr, and then proliferative response to mitogens augmented towards normal levels. Serum immunoglobulins progressively increased over the following months, and the distribution became normal afte~ a transient phase when an I.~G K peak of restricted heterogeneity was observed. No GvHR developed. After a complete isolation of 534 days, ~he child went ~ome. Chimerism was established by HLA typing: T lymphocytes derived from the transplanted cells (HLA-A2, A 11, B 15, B27) while B lymphocytes were of host ori#t~ {HLA-A3, AW33, CW4, CW6, BW47, B14, DRS, DR4). The fetal donor was not typed for HLA-DR determinants, but T lymphocytes, deriving from the feral donor, e~pressed DRI after in vitro culture with TCGF (Dr. J. BertoF~io). ~ p i t e complete HLA-A, B, and DR incompatibility between T and B lymphocyte, ~s well as between T lymphocytes and the various other host cells, andbody production--even against thymus-dependent antigens--became po, sible and all tested in vitro and in vivo functions of T lymphocytes appeared to be .vimially normal. Two infectious episo~les (pneumonia then meningitis owing to Hemophilus influenzae) had developed soon after ehe infant w~ taken off isolation, when immunoglobulin levels were still low. The general condition of the child has then been

FATHER

MOTHER

a A1 CW4 B17 DR7

¢ A3 GW4 BW47 DR5

b AW33CW6B14DR4

d AW'~GW4B12DH1

FIGURE 2 HLA geno~/pesof f~ily S.

Expression of HLA Antigens in Immunodeficiency

|5 |

completely normal: weight, height, and psychomotor development were ~ r m ~ . Anti-polio, diphtheria, and tetanus toxoid antibodies were produced in normal amounts after vaccinations. Some injections of y globulins were, however, performed. The child is presently in perfect health and lives normally, as shown by a follow-up of more than 3 yr later. H e resists efficiently to viruses and organisms. The addition of a thymus to the liver cell transplant seems to i n c r e ~ t,~probability of a successful and uneventful transplantation [14~. When both tissues are from the same donor, it not only enhances the maturation of T lymphocytes from the immature fetal liver cells, but it may also provide better conditions for this differentiation and lower the risk of the GvHR. In this child with a 3-yr follow-up, HLA typing of separated T and B lymphocytes demonstrated a persisting chimerism among lymphocyte populations: all T lymphocytes derive from the donor's precursor cells and all B lymphocytes from the recipient's (Table 2). Cooperation between such T and B cells of complexlydifferent HLA phenotypes has seemed to be possible in vivo and it results in the appearance of antibody production. In vivo defense mechanisms ~ viruses have been normal and preliminary data suggest that T-cell functions may not be severely restricted by the incompatibility at the major histocompadb~t~ complex (MHC). It is difficult ~o ascertain whether most of the T lymphocytes that derived from the donor cells differentiated in the environment of the grafted thymic epithelial cells (with identical HLA phenotype) or of the host thymus (with different HLA phenotype). Whatever the case, the T lymphocytes, expressing HLA antigens different from HLA antigens of B cells and of other host cells, were capable of at least some degree of activity on those cells. The restriction by the MHC of I-cell responses may not be absolute in huma~ transplantation as thought from mouse experiments [15]. Alternatively, this restriction can be circumvented in certain conditions. It may indeed be postulated that, in addition to the general mechanism of "self ÷ X" recognition, there are "allo ÷ X" recognition structures on some T cells, the number of which would expand under the appropriate solicitations in vivo. In conclusion, the study of HLA expression on cells from some immuaodeficient patients suggests that MHC determinants are of crucial importance both i~ T-cell differentiation and in interactions between T cells and other cells. The most important effector functions of T l,ymphocytes may, however, develop in long-term human chimeras, despite HLA-A, B, and DR incompatibility. Adaptative differentiation may thus be postulated to progressively circumvent the allogeneic restriction.

TABLE 2

Chi~erism following fetal liver ÷ thymus transplants

HLA v/pc of the proband A3-AW33-BW47-B14-DR 5-DR4 Appem'anceof A2-AI1-B15-B27-(DR 1) Progressive: 3 mo after 2nd graft (8 mo of age) Definitive: 10 mo after 2nd graft (15 mo of age) ~pression of l-ILAby complementdependent cytotoxicity On total peripheral blood lymphocytes:A2 (88%), B15, (65%). B27 (7(~%).A3 (30%:). BI4 (25%) After ~eparation:T cells = donor lymphocytes B cells -- host lymphocytes

152

J. L Touraine and H. l~mel ACKNOWLEDGMEHT We are grateful to Dr. G. Lenoir for the development of the EBV-transformed cell line, to Dr. F. Touralne for the investigations of cell-surface ~ microglobulin, to Dr. J. Berto#io for the culture with TCGF, and to Drs. H. Philippe, G. Sonillet, B. l~tend, and Prof. R. Frangois for help in the care of the Imtients. This work has been supported by the Centre de Transfusion and by grants from the DGRST, the INSERM, and the Fondation de France.

REFERENCES 1. Immunodeficiency, Geneva, WHO. Technical Report Series 630, 1978. 2. Touralne JL: Heterogeneity and pathophysiology of severe combined immunodeficiency. In: JL Touralne Ed. Bone Marrow Transplantation in Europe. Amsterdam, Excerpta Mediea, 1979, p 111. 3. Meuwissen HJ, Pickeriog RJ, Pollara B, Patter IH: Combined immunodeficiency disease and adenosine deamioase deficiency. A molecular defect. New York, Academic Press, 1975. 4. Touraine JL, B~mel H, Souillet G, Jeune M: Combined immunodeficiency disease associated with absence of cell surface HLA A and B antigens. J Pediatr 93:47, 1978. 5. Touraine JL: Transplan~ion of both feud liver and thymus in severe combined immunodeficiencies: imeracfion between donor's and recipient's cells. In: ThM Fiiedner, RP Gale, G Lucareili, Eds. Feud Liver Transplantation. Amsterdam, Excerpta Medica, 1980, p 276. 6. Mitud KK, Mickey MR, Singal DP, Ter',~ki Pl: Serotyping for homogransplantation. XVIII refinement of microdro#et lymphocyte cyunoxicity test. Tr~sphnt~ion 6:913, 1968. 7. B6tuel H, Canmun M, C o b m b ~ i J, Day HE, Ellouz R, de TI~ G: The retu~mship between nasopharynge~ c~cinoma and the HLA system ~,long Tunisian$. lnt J Cancer 16:249, 1975. 8. l~tuel H, Touralne JL, Souillet G, Jeune M: Abse~e of ce.|bmembrane HLA antigens in an immunodefident child. T~ssue A n ~ s i 1:68, 1918. 9. Touraine JL, Tougaine F, H ~ d e n JW, Hadden EM, Good RA: 5bromodeoxyuridine-liSht inacfiw~ion of h ~ |ymphocyte~ sfimulmed by miu~gens and aliogeneic cel~: evidence for disth~ct T lymphocyte subvert, lnr Arc!h AIMrgy Appi lmmuno152:105, 1976. 10. Touralne JL: Human T lymphocym dff~eren~u~on in inunu~def~iency ~ and after reconsfimtion by bone marrow or feud thymus ~ p L m t a t i o n . Clin lmmuaol Immunopathol 12:228, 1979. 11. Touraine JI., B6tuel H, Philippe M: The Imm lymphocyte syndrome. L lmmunoiog/cal studies before and after bone marrow ~ . Btut 41:198, 1980. 12. Schuurman RKB, van Rood JJ, Vo, sen JM, ScheUekem 7rhA, Feikamp.Vroom ThM, Doyer E, Gmelig-Meyling F, Visser HKA: Failure of lympho~te membrane HLA A and B expression in two siblings with c ~ immtmode~iency. Clin Immunol lmmuaopadml 14:418, 1979. 13. Schuurman RKB, Touraine JL, ~ d EW, van Rood J$: Lympho~te metabr~ne abnormalities associated with prinutry i ~ ¢ f g i c ~ y di~. In: M S e l i ~ , , WH Hitzig Eds. Primary l m m u n o d e f ~ Anu~rdm~ Elf~evief/No~ H ~ I , 1980, p 79.

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14. Pahwa R, Pahwa S, Good RA, Incefy GS, O'Reilly RJ: Rationale for combitg~d ¢~. of fetal liver and thymus for immunological reconstitudon in patients with vat/am, of severe combined immunodeficiency. Proc Nad Acad Sci USA 74:3002,1977, 15. Zinkernagel RM: The thymus: its influence on recognition of "'self major hhto~ompatibility antigens" by T cells and consequences for reconstitution of immunode~o ciency. Springer Semin lmmunopath 1:405, 1978.