British Journal of Haematology, 1986. 63, 231-239

Female marrow donors increase the risk of acute graft-versus-host disease: effect of donor age and parity and analysis of cell subpopulations in the donor marrow inoculum

KERRY ATKINSON, CAROLINE FARRELL, G R A E M E CHAPMAN, KATHRYN DOWNS, RONALD PENNY A N D JAMES BIGGS Departments of Huematology and Immunology, St Vincent's Hospital, Sydney, Australia

Received 12 March 1985; accepted for publication 17 September 1985

SUMMARY. We evaluated 2 7 factors for their influence on acute graft-versus- host disease (GVHD) in 40 recipients of HLA-identical sibling marrow transplants. These factors included the doses of mononuclear cell subpopula- tions present in the donor marrow inoculum quantitated using a panel of monoclonal antibodies. Female donors were associated with increased severity of acute GVHD, and the older the female donor the greater this effect. Increasing donor parity was also associated with an increased risk of acute GVHD. The number of T cells, T cells subsets, natural killer cells and monocytes infused did not influence the incidence or severity of acute GVHD in this study, and we could not explain the influence of female donors and of female donor age on acute GVHD by the cellular content of their marrow inocula. We postulate that non-HLA histocompatibility antigen disparity is a more important determinant for acute GVHD than the number of infused donor T cells, especially when female donors are used. The association between acute GVHD and increasing parity suggests that some female marrow donors have been pre-sensitized to their respective recipients by preceding pregnancies.

Acute graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality after human allogeneic marrow transplantation. From rodent experiments in which T cells are depleted from the donor marrow inoculum prior to its infusion into the recipient, it appears clear that T cells are the trigger mechanism for this disease (Korngold & Sprent, 1978). Indeed, for a given degree of histocompatibility antigen disparity between murine donor-recipient pairs, the severity of GVHD can be directly related to the dose of T cells infused (Okunewick et al, 1980).

Correspondence: Dr Kerry Atkinson, Department of Haematology, St Vincent's Hospital, Sydney, Australia.

231

232 Kerry Atkinson et al

A number of previous studies have identified a variety of factors predisposing to GVHD in recipients ofHLA-identical marrow transplants. Female donor sex (Bortin et al, 198 1). female recipient sex (Gluckman et al, 1981). increasing recipient age (Bortin et al, 1981; Gluckman et al, 1981: Bross et al, 1984) and inclusion of total body irradiation in the pretransplant conditioning regimen (Bortin et al, 1981; Gluckman et al, 1981) have each been shown to increase the severity of acute GVHD. Similarly, sex mismatch between donor and recipient has been reported as predisposing to GVHD in two studies (Storb et al, 1977; Bross et al, 1984), although Storb and colleagues in a subsequent analysis found it to be less important than in their first study (Storb et al, 1983); other studies have found it not to be important.

In this study we have analysed a number of pretransplant characteristics for their possible effect on incidence and severity of acute GVHD in 40 recipients of HLA-identical sibling marrow transplants in whom we enumerated, using monoclonal antibodies and flow cytometry, the numbers of T cells, helper-inducer T cells, cytotoxic-suppressor T cells, natural killer cells and monocytes in the donor marrow inoculum. We have included these as factors in the analysis.

MATERIALS A N D METHODS

Patients. After conditioning with cyclophosphamide alone (seven patients), cyclophos- phamide and fractionated total body irradiation (30 patients), or cyclophosphamide and meiphalan (three patients), 40 patients received HLA-identical sibling marrow grafts as treatment for haematological malignancy or severe aplastic anaemia. Each patient was given a unique patient number (UPN). The protocols for marrow transplantation have been previously reported (Biggs et al, 198 3), as have the clinical and histological features of acute GVHD (Glucksberg et al, 19 74; Lerner et al, 19 74). The grade of severity of acute GVHD was scored on the 0 4 scale described by Thomas et a l ( l975) . Additionally, however, a further grade (grade 5 ) was allocated to patients in whom acute GVHD was the primary cause of death. The day of marrow transplantation was designated day 0. Sustained engraftment occurred in all patients and this was shown to be of donor origin in 2 2 / 2 2 evaluable patients.

Bone marrow inoculum. Donor bone marrow was harvested from the anterior-posterior iliac crest under general anaesthesia. The monoclonal antibodies utilized in this study were as follows: anti-Leu 4 (Becton Dickinson, Mountain View, Calif.) (all peripheral blood T cells); anti-Leu 3a (Becton Dickinson) and OKT4 (Ortho Pharmaceutical Corp.. Raritan, N.J.) (helper-inducer T cells); anti-Leu 2a (Becton Dickinson) and OKT8 (Ortho) (cytotoxic- suppressor T cells); anti-Leu 7 (Becton Dickinson) (natural killer cells and a subset of cytotoxic-suppressor T cells); anti-HLA-DR (Becton Dickinson) (B cells, monocytes, activated T cells); anti-Leu M 1 (Becton Dickinson) (monocytes and granulocytes); OKTl 1 (Ortho) (E- rosette receptor bearing cells); OKTlO (Ortho) (haemopoietic stem cells, activated T and B cells, some null and NK cells); FMC 17,32,33 (Flinders Medical Centre, Bedford Park, South Australia) (monocyte subpopulations). The Becton Dickinson antibodies were generously donated by Dr Noel Warner, the Ortho antibodies by Dr Gideon Goldstein and Dr Leon Harris, and the FMC antibodies by Dr Hedy Zola.

An aliquot of marrow (in reality an aliquot of marrow and blood cells) was incubated for

Female Marrow Donors Influence Acute GVHD 233

30 min in an ice water bath with each of the monoclonal antibodies. All antibodies except OKTlO, anti-Leu M1 and the three FMC antibodies were directly conjugated with fluorescein. When unconjugated antibody was used, the cells were washed twice in phosphate-buffered saline (PBS) and incubated for a further 3 0 min in ice water with goat anti-mouse fluorescein-conjugated immunoglobulin (Coulter Electronics, Sydney). Ammonium chloride lysing reagent was then added and the cell suspension incubated at room temperature until the red cells had lysed (approximately 5 min). Cells were washed three times in PBS at 4OC, resuspended in PBS and kept on ice until analysed using an Epics V flow cytometer (Coulter Electronics).

From the proportion of cells positive with each monoclonal antibody, the number of cells

Table I. Univariate analysis of factors predisposing to acute graft-versus-host disease

Factor

Patient age Donor age Patient sex Donor sex Sex match M+M

Sex mismatch M+F F+M

Diagnosis (SAA = 1 /HM = 0) GVHD prophylaxis (CSP = 0, MTX = 2 1 ) Conditioning regimen (no TBI= 0. TBI= 1) RRI (donor v recipient) Total nucleated cell dose Total nucleated marrow cell dose OKTl1+ cell dose Leu-4 + cell dose OKT4/Leu-3 + cell dose OKT8/Leu-2 + cell dose Leu-7 + cell dose Leu-M1 + cell dose FMC 17+ cell dose FMC 32 + cell dose FMC 33 + cell dose OKTlO + cell dose HLA-DR +cell dose OKT6 + cell dose

F-+F

Correlation coefficient

Significance level (P)

0.3 1 0.44

-0.02 0.4 1 0.2 1

-0.26 0.23

-0.21 -0.14 -0.15

0.2 3 -0.17 -0.05 -0.03 -0.13 - 0.04

0.01 -0.05

0.2 -0.24 -0.18 -0.09 -0.19 -0.22

0.23 0.09

0.03 0.002 0.43 0.004 0.09 7 0.056 0.075 0.095 0.193 0.1 75 0.075 0.15 0.3 7 0.42 0.2 1 0.4 0.46 0.3 7 0.12 0.08 0.14 0.29 0.14 0.09 0.08 0.29

Abbreviations: M. male: F, female: SAA. severe aplastic anaemia: HM. haematolo- gical malignancy: CSP. cyclosporin: MTX, methotrexate: TBI, total body irradiation: RRI. relative response index.

2 34

infused x 107/kg recipient weight was calculated. The number of nucleated cells was counted using a Coulter S + counter (Coulter Electronics).

The number of nucleated marrow cells was calculated by subtracting the total blood white cell count from the total marrow nucleated cell count.

Statistical analysis. The relationship between leukaemia status at time of transplant and severity of acute GVHD was analysed using a chi-squared test. A univariate analysis was then carried out on the 40 patients using the factors listed in Table I, and the correlation of each factor with the severity of acute GVHD was assessed individually. All factors were then analysed together using a step-wise multivariate linear regression with acute GVHD as the response. As an extension of this, the patients were split by donor sex, and the regression rerun in order to examine the significance of these factors for male and female donors separately. The Student’s t test was used to compare the number of mononuclear cell subpopulations between patient groups with different grades of acute GVHD.

Kerry Atkinson et a1

RESULTS Clinical characteristics

The median age (range) of the patients was 2 5 (1 3-53) years: there were 2 3 males and 1 7

Table 11. Cell subpopulations present in the donor marrow inoculum analysed by incidence of acute graft-versus-host disease*

Patients with Patients with Patients without acute GVHD of acute GVHD Patients with

acute GVHD skin only gut and/or fatal acute Cell population (n=6) (n=22) liver (n=12) GVHD ( n = 5 )

Total nucleated cells Total nucleated marrow cells OKTll + cells Leu-4+ cells OKT4/Leu-3 + cells OKT8/Leu-2 + cells Leu-7 + cells Leu-M1 + cells FMC 17+ cells FMC 32+ cells FMC 33 + cells OKTlO+ cells HLA-DR+ cells OKT6+ cells

32.6f6.2 2 1.2 f 4 . 4

5.1 f 1.9 4.7f 1.0 2.7f 1.0 2.3 f 0 . 5 1.4f1.2

17.5 f 4 . 8 1.4 f 0 . 5 1.2 f 0 . 6 1.0 f0.4 4 . 4 f l . 1 3 .9f1.6 0.1 f 0 . 2

33.0f 6.9 25.0 f 6.8 4.8f2.1 4.2 f 1.3 2.4f0.9 2.2f0.7 0.5f0.4

20.9 f 6.1 1 . 7 f l . 0 1.7f 1.0 1.5f 1.3 4.7 f 2.8 4.1f1.2 0.2 f 0 . 4

31.3 f 8 . 6 22.6f6.0

4.4 f 3.4 4.2 f 1.8 2.5 f 1.0 2 . 2 f l . 0 1.2 f 1.6

16.5 f 6.9 l . l f 0 . 6 1 .150 .5 0.8f0.4 2.9 f 1.3 4.8f4.5 0.2 f 0 . 4

31.8 f 9.9 22.2 f 8 . 0

3.8 f 1.2 4.3 f 1.2 2.4 f 0 . 7 2.3 f 0 . 8 1.7f2.2

14.2f 10.2 1.2 f0.6 1 .4f0.4 0.8 f 0 . 2 3.4f0.4 6.3 f6 .9 0.2 f 0 . 2

Values for patients with acute GVHD of any grade of severity were not significantly higher than values for patients without acute GVHD. Values for patients with acute GVHD of skin only were not different from values for patients with more severe acute GVHD (two-tailed Student’s test).

* Results represent mean (f 1 SD) x 107/kg recipient weight.

Female Marrow Donors Influence Acute G V H D 2 3 5

females; seven had severe aplastic anaemia and 3 3 had haematological malignancy; 3 5 received cyclosporin and five methotrexate (MTX) as prophylaxis for GVHD; six had no evidence of acute GVHD, 22 had acute GVHD involving the skin only, and 12 had acute GVHD of the gut or liver. In five patients acute GVHD was the primary cause of death.

The median age (range) of the donors was 2 5 (7-54 years). There were 2 1 males and 19 female donors. Nine of the female donors had had a total of 3 7 pregnancies.

Analysis of cell subpopulations present in the donor marrow inoculum

The number of nucleated marrow cells and specific subsets of marrow mononuclear cells are shown according to the degree of organ involvement by acute GVHD in Table 11. There was no significant difference in the number of cells in any individual subpopulation between the different patient groups. The number of cells in each subpopulation was then entered as a variable in the univariate analysis of factors possibly predisposing to acute GVHD.

Univariate analysis of factors predisposing to acute GVHD

Of the factors shown in Table I, only three predisposed to acute GVHD. These were (increasing) patient age, (increasing) donor age and (female) donor sex. Specifically, the number of T cells, helper-inducer T cells, cytoxic-suppressor T cells, natural killer cells or monocytes infused did not influence the incidence or severity of subsequent acute GVHD.

Multivariate analysis of factors predisposing to acute GVHD

The three factors found to be significant in the univariate analysis were then re-examined in a multiple regression analysis. From this it was determined that only donor age and donor sex were significant factors in their own right (Table 111). When donor age and donor sex were taken into account patient age no longer remained significant.

Table 111. Multivariate analysis of factors predisposing to acute graft-versus-host disease

Regression Significance Factor coefficient level (P)

Donor sex 0.9441 0.001 Donor age 0.049 1 0.005

The regression model is as follows: Acute GVHD = - 0.1 1 52 + 0.049 1 donor age +

0.9441 donor sex where -0.1 152 is the y intercept. The significance levels in the Table measure the

influence of the factor on acute GVHD, having allowed for the influence of the previous factors.

236 Kerry Atkinson e t al

Table IV. Donor age and severity of acute graft-versus-host disease*

Patients with Patients with Patients with no acute acute GVHD acute GVHD of

Factor GVHD skin gut and/or liver

Age of male donor 2 5 f 1 6 2 4 f 8 3 0 f 1 7 (n=4) (n= 14) (n=4)

(n=2) ( n = 8 ) ( n = 8 ) Age of female donor 1 6 f 3 [24 f10 3 7 4 1 1 1

Boxed values significantly different from each other, P < 0.05, two-tailed

* Results represent mean age ( f 1 SD) in years. Student’s t test. All other values not significantly different from each other.

These conclusions were further examined by re-running the multiple regression analysis with the data divided on the basis of donor sex. For recipients with male donors, increasing (male) donor age was not a significant predisposing factor. In contrast, for recipients with female donors, increasing (female) donor age was a significant factor (P=0.03). These data were also examined by comparing the mean donor age for increasing grade of acute GVHD (Table IV). There was no significant difference in male donor age with increasing severity of acute GVHD. There was a significant increase in female donor age for recipients with gut and/ or liver acute GVHD compared to recipients with acute GVHD of the skin only.

We thus found that not only did a donor of female sex predispose to acute GVHD, but the older the female donor the greater the risk of acute GVHD.

Mechanism of influence of female donor sex and increasing female donor age on severity of acute graft-versus-host disease

We then explored possible mechanisms for the influence of the factors female donor sex and increasing female donor age on risk of acute GVHD.

(a) Increasing donor age. We determined whether there was any correlation between any marrow cell subpopulation and increasing donor age. Only OKT4 + and Leu M 1 + cells tended to increase in number in the marrow inoculum with increasing donor age. OKT4 t cells were associated with both older male and older female donors, and the association was stronger for male donors ( r = 0 . 3 8 , P=0.04).

Increasing numbers of Leu M 1 + cells were noted only in association with the increasing age of female donors ( r = 0.46, P = 0.04). The Leu-M1 antigen is present on granulocytes and monocytes (Hanjan et al, 1982). Since the monocyte numbers enumerated by antibodies FMC 17, 32 and 33 did not increase with increasing age, we can infer that the increase in number of Leu-M1 + cells represented an increase in granulocyte number. Since granulo- cytes have not been implicated in the pathogenesis of GVHD, this observation did not appear to explain the influence of female donor sex on severity of subsequent GVHD.

Female Marrow Donors Influence Acute GVHD 237

(b) Female donor sex. We could find no differences in male and female donor marrow for any of the cell subpopulations quantitated (data not shown). Likewise, we could not incriminate Y-chromosome-linked histocompatibility antigen disparities, because the sever- ity of acute GVHD was not different between male recipients of female marrow and female recipients of female marrow. We then examined female donor parity. We found a significant relationship between the severity of acute GVHD and the number of pregnancies (including abortions and miscarriages) of female donors ( r = 0.43. P= 0.03). Thus the greater the number of pregnancies the marrow donor had had pre-transplant, the greater the severity of subsequent acute GVHD in the recipient. In a multiple regression analysis with parity entered as a factor, however, donor age (and not parity) was again the only significant factor. This was accounted for by a strong correlation between increasing donor age and parity ( r = 0.69, P=O.O01). There was no relationship between degree of parity and any specific mono- nuclear cell subset in the donor marrow inoculum.

DISCUSSION

It is clear from rodent experiments that T cells trigger GVHD (Korngold & Sprent, 1978). In man the incidence of chronic GVHD is increased if irradiated donor buffy coat cells are infused in addition to donor bone marrow (Storb et al, 1982). Early attempts at depleting human bone marrow of T cells prior to its infusion into the recipient appear to be associated with a low incidence of acute GVHD (Prentice et al, 1984).

We therefore hypothesized that the number of T cells or T cell subsets present in the donor marrow inocula in this series of 40 recipients of HLA-identical sibling marrow allografts would correlate with the severity of subsequent acute GVHD. We were, however, unable to confirm this. Similar findings were made by Kay et ul (1 982) when the total lymphocyte number in the infused marrow was quantitated, and by Jansen et aZ(1983) when the number ofE rosette forming cells was quantitated. Furthermore, we were unable to demonstrate any correlation between the incidence and severity of acute GVHD with any other specific subpopulation of mononuclear cells, including natural killer cells and monocytes. There are several possible reasons to explain this. Firstly, the dose range of 'I' cells in unmanipulated human marrow inocula may be too narrow to allow a dose effect to be demonstrable: this is a factor beyond our control. Secondly, the T cell dose may be unimportant: in view of the data presented above, this would seem unlikely. Thirdly, the T cell (or T cell subset) dose may be important, but any effect of that dose may be obscured by the variation between recipients in their degree of non-HLA histocompatibility antigen disparity. Until more precise tests are generally available for typing such antigens, this possibility cannot be tested.

Three factors appeared important in predisposing to acute GVHD in the preliminary univariate analysis. These were patient age, donor age and donor sex. Patient age was found to predispose to acute GVHD in the analyses of Bortin et aZ (1981), Gluckman (1981) and Bross et aZ(1984). Each found that increasing patient age was associated with an increasing risk of acute GVHD. In contrast, however, in our study patient age was excluded as a significant factor by multivariate analysis: its apparent importance was due to its correlation with (increasing) donor age. As well as donor age, we found, in agreement with Bortin et al

238 Kerry Atkinson et a1

(1981). that female donor sex adversely affected the risk of acute GVHD. Indeed, the effect of donor age was restricted to female donors: female donors were associated with an increased risk of acute GVHD, and the older the donor the greater that risk. Sex mismatch between patient and recipient has been suggested by both Storb et a l ( l977) and Bross et al (1984) to predispose to acute GVHD; however, a subsequent report from the Seattle group has shown this to be less important in their enlarged series (Storb et al, 1983). The inclusion of irradiation in the conditioning regimes increased the risk of acute GVHD both in the series by Bortin and by Gluckman. In our univariate analysis, total body irradiation was not significant at the 5% level, although it was at the 10% level.

We were unable to explain the effect of female donor sex and increasing (female) donor age on acute GVHD in terms of the cellular content of the donor marrow inocula. There was no significant difference in the quantity of any specific mononuclear cell subpopulation between marrow inocula from male or female donors. However, when we examined the parity of female donors we found a significant relationship between increasing parity and the severity of acute GVHD. This suggests that preceding pregnancies can sensitize female donors to subsequent recipients of their marrow cells. In this regard experimental animals given lymphoid cells after either in vitro (Epstein et al, 1976) or in vivo (Weiden et al, 1976) sensitization to recipient alloantigens have demonstrated more severe GVHD than when non- sensitized cells were used. This effect of parity was not mediated by an increase in any particular mononuclear cell subpopulation in the marrow inoculum.

Since we could not detect an effect of T cell dose on the severity of acute GVHD, we postulate that non-HLA histocompatibility antigen disparity between donor and recipient is a more important determinant for risk of acute GVHD, especially when the marrow donor is female. The older the female donor the more likely she is to have been pregnant, and to have run the risk of being presensitized to the marrow recipient thereby.

This study suggests that male donors should be chosen before female donors if a choice of an HLA-identical sibling donor is available, that non-parous female donors be chosen before parous ones, and that controls for donor sex should be included in randomized trials of GVHD prophylaxis.

ACKNOWLEDGMENT

This work was supported by grants from the National Health and Medical Research Council of Australia and the New South Wales State Cancer Council.

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