A feasibility study on the prediction of acute graft-vs.-host disease before hematopoietic stem cell transplantation based on fetomaternal tolerance

Abstract

The contact between the immune systems of mother and child during pregnancy affects an immune response of the child against noninherited maternal antigens (NIMA) and the mother against inherited paternal antigens (IPA). However, the immunologic effects of developmental exposure to NIMA or IPA are heterogeneous, and can be either tolerogenic or immunogenic. Although we have reported that prediction of acute graft-vs.-host disease (GVHD) is feasible in a murine model, there has been no literature in human. We devised a novel method for predicting a tolerogenic effect by using mixed lymphocyte reaction combined with enzyme-linked immunospot (MLR-ELISPOT) assay. The assay can evaluate reactivity of interferon-γ spot-forming cells of donor against the recipient. Although we have shown only two examples of mother to child reactivity so far, our preliminary results suggest that this pre-screened assay may be used to predict acute GVHD. The clinical trial is in progress to evaluate MLR-ELISPOT assay as a predicting measure of acute GVHD in haploidentical transplantation from NIMA or IPA-mismatched family donor.

Human leukocyte antigen (HLA) broadly-sensitized patients commonly failed to produce antibodies against mismatched noninherited maternal antigens (NIMA), but were fully capable of producing anti-noninherited paternal antigens (NIPA).¹ This phenomenon is now referred to as fetomaternal tolerance, and suggests that perinatal exposure to NIMA may affect the developing immune system of neonates. Furthermore, pregnancy induces compromised immune response against inherited paternal antigens (IPA), i.e., the maternal anti-fetal responses.² These phenomena have been clinically utilized in organ transplantation and allogeneic hematopoietic stem cell transplantation (HSCT).^3,4 Ichinohe et al. have demonstrated the feasibility of HLA-haploidentical HSCT from NIMA-mismatched relatives without T-cell depletion.⁵ Maternal grafts were found to be associated with better outcome than paternal grafts.^4,6 These clinical studies have been performed based on the presence of fetomaternal microchimerism as a result of fetomaternal immunological tolerance. Nevertheless, some cases developed severe acute graft-vs.-host disease (GVHD) despite the existence of microchimeric cells.⁷

Several mechanisms of fetomaternal tolerance have so far been reported. One possible mechanism is the clonal deletion of NIMA-specific lymphocytes. Vernochet et al. described partial deletion of B cells having high affinity for the NIMA.⁸ However, B cells having low affinity for the NIMA were not clonally deleted. Another possible mechanism is the induction of regulatory T cells (Treg) for NIMA. Tsang et al. described the possibility of inducing NIMA-specific Treg in the direct and indirect presentation of maternal microchimerism.⁹ On the other hand, since oral tolerance is known to generate transforming growth factor-β-producing Treg,¹⁰ oral exposure to maternal major histocompatibility antigen complex (MHC) present in breast milk may generate NIMA-specific Treg, resulting in a high level of microchimerism.¹¹ Aoyama et al. reported that exposure to NIMA both in utero and by breastfeeding appears to generate higher levels of maternal microchimerism than in utero exposure alone, and that the degree of microchimerism.¹²

Predicting acute GVHD in vitro before transplantation has been tried in an HLA-mismatched setting, but satisfactory methods had not been established. The frequencies of cytotoxic T-lymphocyte precursor (CTLp) and helper T-lymphocyte precursor (HTLp) cells, as well as mixed lymphocyte reaction (MLR), were reported for the methods that had been evaluated to detect an individual’s reactivity against NIMA in vitro.^13-16 Moretta et al. described that the frequency of NIMA-specific CTLp in cord blood samples could be measured in order to better define the phenomenon of NIMA tolerance.¹⁴ NIMA-reactive cord blood cells were detectable, but the authors of that study could not show a difference in the CTLp frequency against NIMA and NIPA. Falkenburg et al. described that neither the CTLp nor HTLp frequencies against NIPA were not significantly different from those against NIMA.¹³ Indeed, Kircher et al. showed that the CTLp and HTLp frequencies were not predictive for the risk of acute GVHD in patients who received allogeneic HSCT.¹⁷ Collectively, established test systems are not available for predicting an alloreaction and the outcome after HSCT. CTLp reflects the alloreactivity of class I mismatch, and MLR and HTLp reflect alloreactivity of class II mismatch. Thus, all of the above-mentioned methods can detect MHC class I or class II separately, but it is difficult to detect them simultaneously.^18,19

We have recently reported a novel method that overcomes these disadvantages (Fig. 1).^18,19 We demonstrated that the number of interferon (IFN)-γ spot-forming cells was significantly lower in the NIMA-exposed low responder group than high responder group by using an MLR combined with enzyme-linked immunospot (MLR-ELISPOT) assay in a murine model.^19,20 Thus, the capacity for an individual to produce IFNγ against NIMA could differentiate low risk from high risk acute GVHD. This assay is easily applicable in humans, and is a versatile method to detect reactivity to MHC class I, as well as class II. Recently, we have started a clinical trial for prediction of acute GVHD before NIMA/IPA-mismatched HSCT, using MLR-ELISPOT assay (UMIN-CTR, UMIN000009674; www.umin.ac.jp/ctr/index/htm).

Figure 1. MLR-ELISPOT assay. ELISPOT assay combined with MLR (MLR-ELISPOT) is a sensitive functional assay to detect alloreactivity for both class I and class II histocompatibility antigens. Peripheral blood mononuclear cells from donor (responder cells) were incubated with irradiated peripheral blood mononuclear cells from recipient or control (stimulator cells). These stimulated, cytokine spot-forming responder cells were incubated in a 96-well membrane ELISPOT plate coated with purified anti-interferon-γ monoclonal antibody. After washing, the plate was incubated with secondary biotinylated anti-interferon-γ monoclonal antibody. The plate was then developed with streptavidin-alkaline phosphatase and a colorimetric substrate. Visualized spots were counted with an ImmunoSpot Analyzer.

Now, we have shown our preliminary results of MLR-ELISPOT assay in maternal haploidentical HSCT (Table 1). Case 1: A 14-y-old male with refractory malignant lymphoma received bone marrow transplantation from an HLA-mismatched mother. Engraftment was achieved on day 12. He did not develop acute GVHD after HSCT. Before transplantation, a possibility of acute GVHD was evaluated by MLR-ELISPOT assay. Donor reactivity against the patient was very low (3%). Case 2: A 6-y-old male in second complete remission of acute lymphoblastic leukemia received peripheral blood stem cell transplantation from an HLA-mismatched mother. Engraftment was evident on day 12. He developed grade III acute GVHD (stage 1 skin rash and stage 3 diarrhea). Before transplantation, MLR-ELISPOT assay for acute GVHD showed that donor reactivity against the patient was high (33%). Acute GVHD has been successfully treated with immunosuppressants. These preliminary results suggested that acute GVHD might be predictable before transplantation just by evaluating the ability of IFN-γ spot-forming cells against NIMA.

Table 1. Preliminary data for prediction of acute GVHD by MLR-ELISPOT assay.

Characteristic	Case 1	Case 2
Age, Sex	14y, Male	6y, Male
Diagnosis	NHL	ALL
Disease status	Refractory	2nd CR
Conditioning regimen	TBI+MEL	TBI+MEL
HLA histocompatibility	5/6 match	4/6 match
GVHD prophylaxis	TAC+sMTX	TAC+sMTX
Donor, source	Mother, BM	Mother, PBSC
MLR-ELISPOT data (spots/50,000 responder cells)
Anti-control response*	39 spots	58 spots
Anti-patient response	1 spots	19 spots
Relative alloreactivity	3%	33%
Acute GVHD	None	Grade III (skin 1, gut 3)

Abbreviations: NHL, non-Hodgkin lymphoma; ALL, acute lymphoblastic leukemia; CR, complete remission; TBI, total body irradiation; MEL, melphalan; TAC, tacrolimus; sMTX, short-term methotrexate; BM, bone marrow; PBSC, peripheral blood stem cell. *HLA histocompatibility against control was 0/6 match in case 1 and 1/6 match in case 2.

NIMA/IPA-mismatched haploidentical HSCT has been explored.^5,7,21 However, it is difficult to predict severe acute GVHD prior to transplantation. Our recent clinical trial has addressed this issue. Although NIMA-mismatched or maternal haploidentical transplantation without T cell-depletion has been performed, the individual reactivity of the donor is not evaluated at present. This MLR-ELISPOT assay might be useful to predict the immunological reaction of donor T cells against the recipient in NIMA-mismatched or maternal haploidentical HSCT.

Submitted

01/29/13

Revised

03/26/13

Accepted

04/16/13

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.