Intravenous human immunoglobulin treatment of serum from HLA-sensitized patients in kidney transplantation

Abstract

Objective: Intravenous immunoglobulin (IVIG) products are known to have beneficial immunomodulatory effects on several inflammatory and autoimmune disorders. These effects could be attributed to a different inhibitory action on complement factors, but other mechanisms could be implicated, e.g., immunocomplexes development and/or anti-idiotypic antibodies. Positive results on the reduction of anti-Human Leukocyte Antigens (HLA) antibodies in highly sensitized patients have also been found. The present study focuses on the effect of IVIG on the reduction of Panel Reactive Antibody level and crossmatch positivity in sensitized patients awaiting kidney transplantation. Methods: The study was performed adapting an in vitro assay on sensitized patients’ sera in waiting list for kidney transplantation. Sera of twelve highly sensitized patients were evaluated for the cytotoxicity inhibition after 10% IVIG treatment. Results: A reduction of anti- HLA antibody levels was observed in 75% (9/12) of treated patients in vitro, while 25% (3/12) resulted unresponsiveness. Particularly, our data showed a significantly higher Panel Reactive Antibody reduction for T lymphocytes (p < 0.010) than B lymphocytes (p < 0.032). Conclusions: In this study, we have used an in vitro assay to investigate susceptibility to desensitization with IVIG treatment of sensitized patient sera. These findings reveal that the variable effect of IVIG on reducing Panel Reactive Antibody in our immunized patients could be attributed to a different inhibitory action on complement, likely due to the type and the titre of anti-HLA antibodies.

• Desensitization of patient sera
• intravenous immunoglobulin
• kidney transplantation
• panel reactive antibody
• sensitized patients

Introduction

Renal transplant remains a highly significant challenge worldwide since it represents the best treatment for patients affected by the end-stage renal disease and it is a better choice than dialysis for life quality and long-term mortality risk.1 Preformed anti-Human Leukocyte Antigens (HLA) antibodies due to transfusions, pregnancies and prior transplants, are a major risk factor for the increased incidence of hyperacute/acute graft rejection and graft dysfunction. Moreover, they constitute the major barriers to the successful of transplantation.2,3 HLA-sensitized patients have waiting time longer than negative patients and their number in waiting list is constantly increasing.3 Furthermore, prolonged dialysis is an additional risk factor for these patients and for graft survival.4

Different therapeutic approaches and several desensitization protocols have been developed to increase the opportunity of transplantation in these patients including treatment with intravenous immunoglobulin (IVIG).5–7

IVIG is a preparation of pooled human plasma prepared from more than thousands of blood donors and can be used intravenously in the treatment of numerous pathological conditions. IVIG is increasingly used as treatment for autoimmune and systemic inflammatory diseases, as well as in the field of solid organ and bone marrow transplantation.8–10 Specifically, in organ transplantation IVIG is used, among other treatments, to reduce plasma anti-HLA antibody levels prior to transplantation and in the attempt to reverse acute humoral rejection.11,12

The immunomodulatory properties of IVIG are not fully understood. Proposed mechanisms of action include modulation of Fc-receptors, interference with complement and cytokine network, provision of anti-idiotypic antibodies, and modulation of effector lymphocytes function.13 IVIG is thought to affect humoral immunity, perhaps through binding the inhibitory FcγRIIb via the Fc portion of IgG, which leads to proliferation reduction and apoptosis induction.14 More recent reports suggest that IVIG has no direct effects on B-cell proliferation in vitro, nor it is able to alter mRNA levels of IgG and IgM in activated B cells.15 A limited number of in vivo studies have indicated T-cell-specific effects for IVIG.16 In vitro studies have shown conflicting results regarding the ability of IVIG to modulate mitogen- and anti-CD3-induced T-cell activation.17 Moreover, IVIG may also specifically alter certain cytokines and modify the Th1/Th2 ratio, although this finding was not consistently established.18

In the transplantation field, several evidences describe the anti-idiotypic activity of IVIG against anti-HLA antibodies and inhibition of complement as possible desensitization mechanisms. It is also unknown to what degree the IVIG-induced complement inhibition contributes to its clinical effect. In order to better investigate the activity of IVIG, we have evaluated the effect of IVIG on complement activation, induced by anti-HLA antibodies; in an in vitro cytotoxicity assay using highly HLA sensitized sera of patients awaiting kidney transplantation.

Patients and methods

Patients

The sera of patients from our waiting Regional list for deceased donor kidney were analyzed for anti-HLA antibodies by complement-dependent cytotoxicity (CDC), Enzyme-Linked ImmunoSorbent (ELISA) and Luminexx beads based assays.19 All transplant candidates with alloantibodies against class I and/or class II HLA antigens are defined HLA-sensitized patients. In this study, we have selected 12 sera of HLA-sensitized patients showing a PRA value in the range of 40–100% and a high cytotoxicity in vitro. In addition, all patients, except one, had received at least one previous transplant, 10/12 patients had experienced blood transfusions and 4 of 6 women had histories of pregnancies and/or abortions. Thus, 11/12 had already been transplanted or transfused or had undergone pregnancies.

CDC preparation assays and IVIG treatment

Individual sera from these patients were tested in a standard T- and B-cell CDC assay on an HLA typed lymphocyte panel of 20 individuals. Sera were used as undiluted, diluted 1:2 with glycine (0.2 M, Sigma-Aldrich, Milan, Italy) or diluted 1:2 with 10% IVIG (Endobulin 50 mg/mL, Baxter, Rome, Italy).

A CDC protocol was performed through dispensing 1 µL of sera (including negative and positive controls) in triplicate into 72-well oiled Terasaki trays. For doubling sera dilutions, equal volumes of glycine or IVIG were used. T and B cells were isolated by Dynabeads (Invitrogen Dynal AS), incubated with glycine- or IVIG-treated sera for 1 hr at 21 °C, followed by complement (Bio-Rad Medical Diagnostics GmbH) incubation at 21 °C for 2 hr. Inhibition was determined by comparing the IVIG-treated serum with the same serum diluted in glycine. Inhibition occurred when serum treated with IVIG gave a reading score of 1 or 2 compared to the same glycine-treated serum with a reading score of 4 or greater. The PRA score was expressed in percentage (range 0–100%).

Statistics

Statistical analysis was performed using t-test Student. Mean and standard deviation (SD) were calculated for PRA titer pre- and post-IVIG treatment in both T and B lymphocytes. The inhibition was considered significant with p < 0.05.

Results

The demographic characteristics for each patient such as waiting time in list, immunization events and maximum PRA value previously IVIG treatment are described in Table 1. The average of the pre-IVIG treatment CDC PRA values for each Class I and II was in the range of 40–100%; the average time in waiting list was found to be 56 ± 41 months.

Table 1. Demographic characteristics of analyzed patients.

							Pre-IVIG PRA
Patient	Sex	Age	Waiting time on list	Transplants	Pregnancies and/or abortions	Transfusions	Class I	Class II
		Years	Months	n.	n.	n.	(%)	(%)
1	M	41	15	1	—	1	90	90
2	F	55	15	1	1	1	50	60
3	M	65	31	1	—	2	80	80
4	F	34	60	2	0	2	45	90
5	M	49	17	1	—	0	40	85
6	M	36	38	1	—	0	90	95
7	F	50	111	0	2	5	100	100
8	F	62	30	1	3	6	60	85
9	F	54	60	2	1	1	80	95
10	M	44	122	1	—	10	100	85
11	F	46	47	2	0	2	100	85
12	M	28	125	1	—	1	60	70

We tested sera with and without IVIG by CDC crossmatch assay using a panel of 20 HLA-typing donor cells T and B separately. Patient sera showed a different PRA reduction after IVIG treatment. Particularly, reduction of anti-HLA antibody levels was observed in 75% (9/12) of treated patients in vitro, while 25% (3/12 patients) of them resulted unresponsiveness.

However, our data showed a significant PRA reduction of 46 ± 36% for T lymphocytes (p < 0.010) (Table 2) and of 13 ± 10% for B lymphocytes (p < 0.032) (Table 3).

Table 2. T cell panel reactive antibody (PRA) before and after IVIG sera treatment.

Patient	PRA before IVIG (%)	PRA after IVIG (%)	% PRA inhibition
1	90	55	39
2	50	5	90
3	80	50	38
4	45	5	89
5	40	0	100
6	90	25	72
7	100	100	0
8	60	60	0
9	80	60	25
10	100	100	0
11	100	50	50
12	60	30	50
Mean ± SD	75 ± 23	45 ± 34	46 ± 36
p Value	0.010

Note: Patient sera 7, 8 and 10 (italic values) did not respond to treatment.

Table 3. B cell panel reactive antibody (PRA) before and after IVIG sera treatment.

Patient	PRA before IVIG (%)	PRA after IVIG (%)	% PRA inhibition
1	90	80	11
2	60	40	33
3	80	70	13
4	90	80	11
5	85	70	18
6	95	75	21
7	100	100	0
8	85	85	0
9	95	80	16
10	85	85	0
11	85	70	18
12	70	60	14
Mean ± SD	85 ± 11	75 ± 15	13 ± 10
p Value	0.032

Note: Patient sera 7, 8 and 10 (italic values) did not respond to the treatment.

Discussion

The high impact of HLA antibodies on transplantation outcome and the ability to define and quantify antibody reactivity have encouraged the transplant community to develop novel strategies for the desensitization of patients awaiting organ transplantation.20

Several solutions have been proposed for timely and successful transplantation of sensitized patients. The goal of these therapies is to lower antibody levels enough to permit organ transplantation and minimize the risk of antibody-mediated rejection.21

IVIG are known to have powerful immunomodulatory effects on inflammatory and autoimmune disorders.21,22 The use of IVIG in the transplantation setting and desensitization protocols in vivo was proposed for successful transplantation of highly sensitized patients in order to reduce anti-HLA antibodies levels at a threshold considered to be safe at the time of transplantation.10,11,23

Glotz et al.24 and Tyan et al.25 first introduced IVIG treatment for desensitization of immunized recipients in kidney transplantation.

Despite IVIG has been widespread used, little is known about its effect on the repertoire of alloantibodies.

Several reports suggest that IVIG is able to lower PRA but is not successful in highly sensitized kidney transplant recipients with PRA levels higher than 90%; furthermore, the effect on single antibodies is not well characterized.26

In this study, we have used an in vitro assay to investigate susceptibility of sensitized patients to desensitization with IVIG. Our results showed a significant PRA reduction both for T (46 ± 36%; p < 0.01) and B lymphocytes (13 ± 10%; p < 0.032). Nevertheless, IVIG treatment was ineffective for three patient sera (25% of total analyzed cases) where no PRA cytotoxicity reduction was observed.

Some studies have shown that one of the mechanisms for allosensitization reduction by IVIG is the induction of an anti-idiotypic activity, but many other factors may be involved.9 Indeed, previous studies showed that inhibition observed in CDC or ELISA assays can be attributed to effect on complement rather than on anti-HLA antibodies.27,28 Furthermore, the inhibition by IVIG and the degree of C3 activation are related since, in order to achieve inhibition, a greater number of complement deposits requires a greater amount of IVIG concentration.28 Our results are in agreement with other studies reporting that absolute removal of alloantibodies by IVIG is not achievable in all sensitized patients.25,29 Despite our findings were obtained in vitro, we can speculate that, in non-responder patients, anti-HLA antibodies could activate the complement so efficiently to require higher doses of IVIG to achieve an inhibitory effect; however, also in this case the effect might be incomplete. This hypothesis is in according with the findings of Watanabe et al. that observed a relationship between the magnitude of complement activation and the complement inhibitory effect of IVIG.28

In addition, the unresponsiveness to IVIG treatment could be also explained, in accordance with some evidences, by a higher sensitization due to an increase of received blood units or to more pregnancies.30–32

In conclusion, our data reveal that the variable effect of IVIG on reducing PRA in the HLA-sensitized patients could be attributed to a different inhibitory effect on complement, likely due to the type and the titre of anti-HLA antibodies. Similarly, Wassmuth et al. showed that the immunomodulatory capacity is largely caused by the IgM/IgA fraction of IVIG when analyzed by lymphocytotoxicity.27 Several reports suggest that IVIG could reduce PRA value without affecting the HLA antibodies amount; but the effect on single anti-HLA antibodies is unclear, with some HLA specificities that may not be inhibited by IVIG.9,20 Furthermore, anti-HLA antibodies monitoring by solid-phase assay may help to predict the desensitization efficacy.21 Further studies are needed to clarify the in vitro IVIG mechanisms of action and for a more effective in vivo application of IVIG in the field of transplantation.

Declaration of interest

The authors report no conflict of interest. The authors alone are responsible for the content and writing of the article.