In utero fetal liver cell transplantation in the treatment of immunodeficient or thalassemic human fetuses

Transfus.

Sci. 1993; 14:299-304

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Invited Report In Utero Fetal Liver Cell Transplantation in the Treatment of Immunodeficient or Thalassemic Human Fetuses J.-L. Touraine

been proven (Y-chromosome in this female patient) and immunological reconstitution is in progress, allowing a normal life at home. The third patient also has evidence of donor cell take (Ychromosome in a female patient) and a partial effect on thalassemia has been documented (donor hemoglobin present in peripheral blood). In all three cases, no side-effect of any kind developed in the mother nor in the fetus. In the fourth patient, the cell infusion was followed by bradycardia and fetal loss. Several advantages appear to be associated with in utero FLT over postnatal FLT: increased probability of graft take, ideal isolation of patient (in the uterus) and optimal environment for fetal cell development (in the fetal host). n

n Following 18 years’ experience in postnatal fetal liver transplantation PLTI, we have developed a new therapeutic method, namely the in utero transplantation of stem cells from the human fetal liver. This early transplant takes advantage of the immunological tolerance that exists in young fetal recipients. The four fetuses that we treated were 28, 26, 17 and 12 weeks of gestation. The first two patients had immunodeficiencies, the two others had thalassemia major. Donor cells were obtained from 7- to 12-week-old fetuses, with conditions approved by the National Committee for Bioethics. Donors and recipients were not matched. The fetal cells were infused through the umbilical vein of three patients and injected intraperitoneally into the other one, under ultrasonic visualization. The first patient, born in 1988, has evidence of engraftment and reconstitution of cell-mediated immunity: initially 10% then 26% of lymphocytes of donor origin (with distinct phenotype), T-cell responses to tetanus toxoid, CMV and candida antigens. This child, who had bare lymphocyte syndrome, has no clinical manifestation of the disease and lives normally at home. The second child was born in 1989; donor cell engraftment has

INTRODUCTION

normal The transplantation of hemopoietic stem cells from the bone marrow or the fetal liver can cure a number of diseases, in experimental animals as well as in humans. Postnatal transplantation of fetal liver cells, alone or in association with syngenic fetal thymic (epithelial) cells, leads to full immunological reconstitution of patients with severe combined immunodeficiency disease (SCID) and to improvement in patients with a variety of other congenital or hematological disorders. Especially when no compatible

Se_+e de Transplantation et Immunologic Clinique, I-I-IwI,~ Edward Herriot, pav. P, 69437 Lyon Cedex 03,

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donor is available for a bone marrow transplant to patients with these diseases, fetal liver transplantation represents a very valuable therapeutic solution and it cures a significant proportion of the patients.’ Over the last 18 years, 230 fetal tissue transplants (especially FLTs associated with syngenic fetal thymus) have been performed in our institution to treat 62 patients with severe immunodeficiency diseases (IDD), severe aplastic anemia or inborn errors of metabolism (IEM). At present, 77% of all patients with IEM and 50% of all patients with IDD are alive and well, with a follow-up of 1-18 years. They are either completely cured or significantly improved. Despite HLA mismatch between donor and recipient, in these cases, there is no noticeable restriction of T-cell function in vivo, ex vivo and in vitro, provided that investigations are carried out several years after the transplant.24 FLT therefore represents an efficient postnatal therapy in severe hematological, immunological or metabolic disturbances, and does not require any HLA matching between donor and recipient. As described below, it can also be very effective when performed prenatally, even in cases in which postnatal FLT usually does not result in graft take.5 Between 1988 and 1991, we have treated four sick fetuses with prenatal FLT. Two of these in utero transplants were carried out for inherited immunodeficiency, the two others for thalassemia major. Recommendations from the French National Committee for Bioethics were carefully followed for tissue procurement as well as for transplant conditions, both in postnatal and in prenatal FLTs.~ The three first children are now born. They are the first three patients showing engraftment of donor cells following in utero transplantation. They confirm the basic knowledge on the possibility of inducing immunological tolerance by administration of allogenic cells before immune maturation of the recipient fetus.6,7

ETHICAL CONSIDERATIONS AND METHODS The four patients treated prenatally by in utero transplantation of fetal liver cells were fetuses with severe immunodeficiency diseases or with p” thalassemia. Informed consent from the parents was obtained prior to any initiation of the treatment. The procurement of fetal organs strictly followed the recommendations of the French National Committee for Bioethics.’ The ethical considerations are described in detail elsewhere.3 A few hours after fetal death, the liver and the thymus were removed aseptically and processed with an homogeneizer to prepare a single cell suspension. Only fresh, not previously frozen, tissues and cells were used for transplantation. Cell viability was checked by the trypan blue method and it exceeded 80% in the cell suspension injected in utero. Special attention was directed toward the prevention of microorganism transmission. Serological tests for several viruses (HIV, HBV, HCV, CMV) were routinely performed on the donor’s maternal serum. The cell phenotype was determined by cytofluorometry, using fluoresceinlabeled monoclonal antibodies. HLA typing was performed both by microcytotoxicity immunoand by fluorescence, with the appropriate antibodies. Gene amplification using the Polymerase Chain Reaction (PCR) followed the technique of Mullis and Faloona.’ RESULTS In Utero Transplantation of Stem Cells in Immunodeficiencies The case reports of the first two patients treated by in utero FLT are summarized below. Both of them were fetuses with immunodeficiency diseases severe diagnosed in mid-gestation. The first patient suffered from bare lymphocyte

In Utero Stem Cell Transplantation

syndrome (BLSJ, i.e. a genetically transmitted form of combined immunodeficiency disease due to lack of expression of HLA antigens.” Infecespecially with opportunistic tions, are responsible for microorganisms, death of these infants, unless they grow up isolated in a fully sterile atmosphere while they are successfully reconstituted with stem cell transplants (SCTs). When carried out postnatally, however, such an SCT-in the form of either a bone marrow transplant or an FLT-is usually associated with graft failure due to the presence of allogenic reactions in the host (persisting transplant immunity) and to a high susceptibility to infections (defective to infectious antigens]. immunity Prenatal diagnosis of BLS can be performed by HLA analysis of fetal blood lymphocytes. ’ ’ Mrs T’s first child died of BLS before reaching his second birthday, despite an attempted SCT which did not result in stable graft take and immunological reconstitution. When she became pregnant again, she asked for prenatal diagnosis which demonstrated the presence of type III BLS (virtually complete lack of expression of both class I and class II HLA antigens on the cell surface, in contrast with findings in immunologically normal fetuses). Three choices were offered to this family: (i) therapeutic abortion; [ii) no

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treatment before birth and stem cell transplant after birth and (iii] in utero stem cell transplant followed by postnatal SCT. The parents were informed that the latter treatment was unprecedented and that its results were therefore uncertain. The mother and the father decided for the earliest possible transplant. In June 1988, at the 28th week after fertilization, the transplant was carried out involving the infusion of 7mL of culture medium containing a suspension of 16~10~ fetal liver cells and fetal thymic epithelial cells into the umbilical vein.5 Liver and thymic cells were obtained from two dead fetuses whose ages were 7 and 7.5 weeks after fertilization (Table 1J. Over the months following the in zzterotransplant, no adverse effects were observed in the pregnant woman nor in her baby and the child was normally born in August 1988 (Table 1). At birth, the diagnosis of BLS was again confirmed but some cells with class I HLA antigens became progressively detectable: 10% of the lymphocytes had a normal expresson of class I HLA antigens at the age of 1 month,’ and these cells were of donor origin since their HLA specificities, of donor type, were not inherited from the child’s parents. In particular, these cells expressed the HLA A9 specificity of donor origin, which made transplanted cells readily detectable in the initial test, at

Table 1. Modalities and Results of In Utero Transplants into Human Fetuses Patients

D.T.

Diseases

BLS

Age of Fetal Patients*

Age of Fetal Donors’

Date of Transplant

Date of Birth

Outcome

28

7 h 7.5

30 June 88

17 August 88

Graft take (HLAJ & immunological reconstitution

M.H.

KID

26

7.5

08 June 89

07 August 89

Graft take (Y chrom.) & immunological reconstitution in progress

M.R.

Thalassemia major

12

9.5

10 October 89

25 March 90

Graft take (Y chrom.) & thalassemia partially improved

Thalassemia

17

11.5

24 April 91

-

Bradycardia & fetal death

R.M.

major ‘Fertlization weeks. n Ill3-G

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birth, and in the subsequent tests performed since. These results demonstrated the persisting engraftment of the fetal liver cells infused into the sick fetus. The expression of class II HLA antigens remained comparatively low on the surface of resting lymphocytes. This finding confirms that B-cell development does not occur as rapidly as T-cell differentiation from stem cell transplants in immunodeficient patients.’ As initially scheduled, the newborn was placed in a sterile bubble and to accelerate reconstitution he further received seven additional SCTs from nine fetal donors. This complementary treatment was carried out after the tests demonstrating engraftment of in utero transfused stem cells. No engraftment of the cells infused after birth could be demonstrated, confirming the “resistance” to transplantation in these patients. However, the number of cells deriving from the in utero transplant increased and was found to be 26% among PBLs at 1 year. In parallel, T-cell maturity and immunological reconstitution progressed. T-lymphocyte reconstitution can now be considered to be virtually complete, and the proliferative responses to antigens (candida antigens, CMV antigens and tetanus toxoid) have occurred and increased up to a normal degree. Immunoglobulin still levels are relatively low, a finding consistent with the previous observation of slow reconstitution of humoral immunity following FLT.’ Because of this T-cell reconstitution from in utero transplanted cells and in view of his good health, the patient was allowed to leave the isolator after 16 months. The child’s present condition is excellent. He has not experienced infections and he lives a normal life at home; he only receives immunoglobulins every month until he produces sufficient amounts of IgM and IgG. After this first in utero FLT, and in view of the most encouraging results observed, a second fetus with IDD was

treated prenatally, in 1989. This second patient was a younger fetus with a complete form of severe combined immunodeficiency disease. He was treated with FLT in June 1989, at the age of 26 weeks of fertilization, after the prenatal diagnosis of severe IDD (complete lack of CD2+, CD3+, CD4+ and CD8+ lymphocytes in the peripheral blood of this fetus while B-cells were present, findings superimposable with those obtained in the first child of the family who also had SCID). The transplant was carried out under the same conditions as in the first fetus treated in utero. It involved the intravenous infusion of 37X106 fetal liver cells into the umbilical vein of the fetal patient, under ultrasound control. The age of the donor was 7.5 weeks after fertilization (Table 1). There were no side-effects and the child was born normally in August 1989. At birth, the girl still had some immunological manifestations of SCID. She has therefore been maintained in sterile isolation and has received additional infusions of fetal liver and thymus cells, with the aim of accelerating development of the in utero transplanted stem cells. Now, cell-mediated immunity has progressed sufficiently for an adequate immunity against microorganisms, so that the child has been allowed to leave the isolator. She lives normally at home and only receives immunoglobulin infusions. By PCR gene amplification techniques, DNA fragments specific for the Y-chromosome have been demonstrated in the DNA material prepared from peripheral blood lymphocytes of this girl at the ages of 8 and 10 months. Engraftment has therefore been obtained and donor cells have differentiated into Tlymphocytes with adequate immune functions. In Utero Trans lantation of Stem Cells in B Thalassemia The results obtained in IDD prompted us to attempt in utero FLT in fetal

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with severe non-immune patients hematological disorders. In such conditions, however, graft take may not be facilitated by immune incompetence of the fetal host and we assumed that FLT had to be carried out during the first trimester of pregnancy, at a time when normal fetuses have not yet developed cell-mediated immunity. l2 A pregnant woman with a family history of thalassemia solicited early prenatal diagnosis. By molecular biology techniques, the fetus was demonstrated to have #Ic thalassemia major. The mother refused abortion for religious reasons, and she asked for in utero FLT. She was informed of the uncertainty of the results in such a disease. FLT was carried out in the fetal patient whose age was 12 weeks postfertilization. The transplant consisted of the intraperitoneal injection of 3 x lo* viable cells from a fetus of 9.5 weeks fertilization (Table 1). There were no adverse effects in the mother nor in the fetus. Birth occurred in March 1990. The site of puncture could hardly be seen on the abdominal skin of the newborn and ultrasound investigations demonstrated no modification of the abdominal wall. Studies performed after birth showed the presence of thalassemia. However, there were a few cells of donor origin: PCR gene amplification techniques revealed Y-chromosome-specific DNA fragments in the PBL of this girl. In addition, hemoglobin A was found to account for 0.9% of all hemoglobin at 6 months. No further transplant was done in this infant who is presently in very good general condition. More recent investigations have been carried out, 1 year after birth; this young girl has received a single blood transfusion; her total hemoglobin level is slightly below normal, and the HbA percentage is presently 30%, 3 months after the blood transfusion. These data suggest that the engraftment of a few donor cells has been followed by some cell proliferation resulting in some improvement and partial correction of this hematologic disorder.

The fourth fetal patient also had thalassemia major. Unfortunately, the intravenous infusion into the umbilical vein was followed by bradycardia and fetal death (Table 1J. DISCUSSION The results obtained in the three initial patients demonstrated the feasibility of in utero FLT (before the end of the first trimester in one case).13 They confirm experimental data in animals,’ and show that the procedure can be effective in humans: engraftment is obtained and IXIl result in cure of inherited immunodeficiency. It is not yet established whether the present conditions are sufficient to significantly improve other inborn errors of metabolism such as thalassemia. To further increase the effectiveness of this procedure in the cases that do not involve immunodeficiency, it may be useful to increase the number of donor cells and to carry out FLT in a fetal host of an even lower age. The main reasons for developing in utero FLT are the following: increased probability of graft take and chimerism, especially in diseases such as BLS In which a residual immunity can induce rejection, and even more so in diseases without immunodeficiency (provided that FLT is performed in very young fetuses); improved isolation at the time of transplant, since the uterus is even better than a sterile bubble; and more optimal environment for fetal cell development when transplanted in the fetus rather than in the infant. The earlier the transplant is performed, the greater will be the chance of full and rapid development of the transplanted cells. Whether or not the use of additional therapy, in fetuses with normal immunity, might be safe and beneficial has not been investigated and will obviously have to be studied carefully in

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animal models before any use in humans. Fetal cell transplants can completely cure half of the patients with severe congenital immunodeficiencies treated after birth.’ Immune immaturity of the fetal donor prevents acute graftversus-host disease. When these cells are transplanted in utero, before any immune maturation in the recipient (in conditions such as BLS, adenosine deaminase deficiency or even inherited diseases without immunodeficiency), rejection is prevented. In most cases, prenatal diagnosis of these various diseases resulted in the decision to terminate the pregnancy. This new opportunity of in utero transplantation may represent a more satisfying solution than abortion of the sick fetus. In inherited hematological diseases and in other inborn errors of metabolism, it also offers great promise and, in the future, it will share, with somatic gene therapy, the possibility of curing many of these severe diseases. REFERENCES Touraine J-L, Roncarolo MG, Royo C, Touraine F: Fetal tissue transplantation, bone marrow transplantation and prospective gene therapy in severe immunodeficiencies and enzyme deficiencies. Thymus 19873 10:75-87. Touraine J-L: Bone marrow and fetal liver transplantation in immunodeficiencies and inborn errors of metabolism: lack of significant restriction of Tcell function in long-term chimeras despite HLA-mismatch. Zmmzmol Rev 1983; 71:103-121. Touraine J-L: Transplantation of fetal haemopoietic and lymphopoietic cells in humans, with special reference to in utero transplantation, in Edwards RG (ed.): Fetal Tissue Transplants in Medicine, Chapter 6. U.K., Cambridge University Press, 1992, pp. 155-176.

4. Roncarolo MG, Touraine J-L, Banchereau

J: Cooperation between major histocompatibility complex mismatched mononuclear cells from a human chimera in the productionof antigen-specific antibody. j Cfin Invest 1986;

77:673-680. 5. Touraine J-L, Raudrant D, Royo C, Rebaud A, Roncarolo MG, Souillet G, Philippe N, Touraine F, Betuel H: In utero transplantation of stem cells in a patient with the bare lymphocyte syndrome. Lancet 1989j 1:1382. 6. Billingham RE, Brent L, Medawar PB: Actively acquired tolerance of foreign cells. Nature 1953; 172:603-606. 7. Harrison MR, Slotnick RN, Crombleholme TM, Globus MS, Tarantal AF, Zanjani ED: In utero transplantation of fetal liver haemopoietic stem cells in monkeys. Lancet 1989; 2:1425-1427. 8. Touraine J-L: Hors de la B&e, vol. 1. Paris, Flammarion, 1985, 242~~. 9. Mullis KB, Faloona FA: Specific synthesis of DNA in vitro via a polymerasecatalyzed chain reaction. Methods Enzymol 1987; 155:335-350. 10. Touraine J-L, Betuel H, Souillet G, Jeune M: Combined immunodeficiency disease associated with absence of cell-surface HLA A and B antigens. / Pediatr 1978j 93:47-51. 11. Durandy A, Cerf-Bensussan N, Dumez Y, Griscelli C: Prenatal diagnosis of combined immunodeficiency severe with defective synthesis of HLA molecules. Prenat Diagn 19873 7:27-31. 12. Royo C, Touraine J-L, de Bouteiller 0: Ontogeny of T lymphocyte differentiation in the human fetus: acquisition of phenotype and functions. Thymus 19871 10:57-73. 13. Touraine J-L, Raudrant D, Vu110 C, Frappaz D, Freycon F, Rebaud A, Brabier F, Roncarolo MG, Geburher L, Betuel H, Zabot MT: New developments in stem transplantation with special cell reference to the first in utero transplants in humans. Bone Marrow Transplant 1991; 792-97.