The Role of Human IgG/Donor Specific Antibodies, Complement Activation, and Neutrophils in Renal Graft Injury.

Original Article

Medical Student: Andres X. Crowley

Mentor: Kenneth A. Andreoni MD



Specific Aim: There is a growing observation in the renal transplant literature that the

appearance of anti-HLA (Human Leukocyte Antigen) antibodies, especially donor

specific anti-HLA antibodies (DSAs), after solid organ transplantation, correlates with

rapid loss of renal graft function. We and others hypothesize that upon kidney

transplantation, some patients generate donor-specific anti-HLA antibodies (DSAs).

These antibodies may bind to donor tissue and create organ dysfunction through

activation of complement. The complement system may either act alone to damage tissue

directly or recruit neutrophils that release proteolytic enzymes causing allograft injury.

To test this hypothesis, this study will 1) investigate the isotypes and subclasses of human

immunoglobulin (DSA) found in the serum of patients with clinically and pathologically

defined acute humoral rejections; 2) assess DSA activation of complement pathways

(both classical and alternative) and neutrophil activation (note: part 2 of the original

proposal was not completed due to time restraints when this draft of the results was

submitted)



Background and Significance: Both Acute and Chronic Humoral Rejection are now

accepted as major contributors to renal and extra-renal allograft loss. Graft injury

mediated by antibodies is clinically important during the early as well as late course post-

transplantation. The improving ability of immunosuppressive medications to decrease

the incidence and severity of acute cellular rejection has direct correlation to short-term

success in organ transplantation. Improvements in long term graft survival have been

more difficult to achieve1. "Chronic rejection" and "chronic renal allograft nephropathy"

are terms for renal graft long-term injury leading to fibrosis and graft dysfunction from a

combination of immunological and non-immunological causes. Acute and chronic,

cellular and humoral injuries are all well described in renal allografts. It has been

observed for many years that the combination of acute cellular and humoral rejection has

dismal renal graft outcome.



In a recent review, Dr. Terasaki cites over 33 publications that associate the presence of

HLA antibodies with acute and chronic rejection in kidney, heart, lung, pancreas, and

liver grafts. The majority of studies have been in kidney graft recipients with a

significant interest in highly sensitized cardiac recipients2. HLA antibodies of Class I and

Class II origin that can be defined against the specific allograft of interest are termed

"Donor Specific Antibodies". Detailed investigation of the actual DSA induced injury in

the renal tissue has not been thoroughly explored. Whether cellular injury results in a

byproduct of alloantibody production, or whether HLA antibody production causes direct

renal injury, such as by endothelial activation of other mechanisms, needs to be defined3-

7.

Since acute and chronic humoral rejections are associated with the presence of HLA

antibodies in the serum, we will seek to determine whether theses DSAs cause injury to

the allograft. We have obtained serum from renal transplant recipients after a clinically

and pathologically documented acute humoral rejection episode. We will define the

subclass and subtypes of these donor-specific HLA antibodies (DSAs), attempt to

demonstrate binding of these antibodies to donor tissue, and create an in-vitro model to

investigate whether these antibodies activate complement and lead to neutrophil

infiltration and subsequent tissue injury. Results obtained from the proposed studies

should advance our understanding of humoral rejection and may help us develop novel

treatments for renal graft recipients undergoing antibody-mediated graft injury.



Materials and Methods:

Sources of patient sera: Serial recipient serum samples and protocol renal graft biopsies

have been collected from patients with and without intravenous immunoglobulin (IVIG)

treatment at UNC under IRB approved patient consent. In this set of initial experiments,

the serum samples of 4 patients with clinically diagnosed acute humoral rejection were

examined. Positive and negative controls were provided by One Lambda (Canoga Park,

CA).



Analysis of donor specific antibody (DSA) levels and IgG subclasses in the recipient

sera: In order to determine the IgG subclass of the Donor Specific Antibodies present in

each patient's sera, the technique of flow cytometry was utilized. Sera samples from both

pre- and post- allograft transplantation were initially analyzed using pooled FlowPRA

HLA Class I and Class II Screening tests (One Lambda, Canoga Park, CA). Furthermore,

mouse FITC-conjugated antibody fragments specific for human IgG total and IgG

subclasses 1 through 4 (SouthernBiotech, Birmingham AL) were used instead of the

FITC-conjugated anti-IgG-total provided by One Lambda. This initial set of assays

allowed for a cost-effective approach to screen for the presence of IgG subclasses without

having to determine the HLA specificity of each antibody.



Once this set of screening assays was completed, it was possible to eliminate certain IgG

subclasses from further analysis, and thus conserve serum and other key reagents. The

post-transplant sera samples were then analyzed using FlowPRA Single Antigen Bead

HLA Antibody Detection Tests for Class I and Class II. The appropriate FITC

conjugated anti-IgG antibody fragments mentioned above were used again to determine

the subclass of the Donor Specific Antibodies and the HLA-specific (non-Donor)

antibodies in each serum sample.



(The following descriptions of additional methods, which were listed in the original

proposal, were not completed during this phase of the project due to time restraints but

are currently being pursued in the laboratory of Dr. Zhi Lui at UNC-Chapel Hill:

1) Detection and quantification of activated complement components in patients' sera, 2)

In vitro determination of complement and neutrophil activation using Donor Specific

Antibodies from patient sera, 3) Immunohistological staining of kidney graft tissue

sections.)



Results and Discussion:

Patient 1 Summary (M.H.): IgG-1 positive for anti-DR 1, 103, 4, 7, 9, 15, 51, 53 ( ? -

HLA class II) and IgG-3 positive for anti DR 1, 4, 7, 9, 53 ( ? -HLA class II) in post-op

serum sample dated 7/1/2004 (with thymoglobin removed). The presence of ? -HLA class

I specific antibodies of subclasses IgG-1 and IgG-3 were also detected during the initial

screening assays, but the specificities of these antibodies were not determined due to

limited sample aliquots. We focused only on defining the ? -HLA class II antibody

specificities and subclasses for this patient's sera.

Patient 2 Summary (J.W.): IgG-1 positive for anti-DQ 5,6,8,9 ( ? -HLA class II) in the

post-op serum sample dated 10/4/2004. No IgG-3 antibodies were detected. No ? -HLA

class I specific antibodies were detected in the initial screening assays.

Patient 3 Summary (L.R.): IgG-1 positive for anti-DR 1, 103, 4, 9, 7, 10, 51, 53 ( ? -

HLA class II) and IgG-3 positive for anti DR 1, 103, 7, 53 and DQ 5 ( ? -HLA class II) in

post-op serum sample dated 3/22/2005. In the initial screening assays, there was a

significant presence of ? -HLA class I specific antibody in both samples, but only of the

subclass IgG-1. Therefore, the specificities of these antibodies were not analyzed since

the subclass, which was the variable in question, was already determined.

Patient 4 Summary (J.K.): IgG-1 positive for anti-A 3, 8(BW6) ( ? -HLA class I) and

IgG-3 positive for anti A 3, 8(BW6) ( ? -HLA class I) in post-op serum sample dated

9/13/2002. In the initial screening assays, we detected ? -HLA class II specific antibody

in both pre-op and post-op serum samples, but only of subclass IgG-1. Therefore, the

specificities of these antibodies were not analyzed since the subclass, which was the

variable in question, was already determined.

The results obtained from this series of experiments confirm what has been reported in a

related study performed by Kushihata,et al.8. We determined that the predominant IgG

subclass that is produced during acute humoral rejection of a renal allograft is IgG-1.

The subclass IgG-3 is also present to a lesser degree in some of the samples, and there is

no detectable trace of IgG-2 or IgG-4 specific for any HLA type in the samples

examined. This study is unique compared to the experiments of Kushihata, et al. in that

we determined the IgG subclass of DSA detected in the sera of patients clinically

diagnosed with acute humoral rejection, as opposed to those of patients who had high

PRA values (ie high levels of HLA specific antibodies), but were not recipients of renal

allografts. This distinction is important because the presence of HLA specific antibodies

in the serum can be due to a variety of causes other than allograft transplantation, such as

pregnancy and blood transfusion. The common finding, however, is that regardless of the

source of sensitizing antigen, HLA antigen (both class I and class II) stimulates the

production of IgG-1 and IgG-3 antibody, but does not stimulate the production of IgG-2

and IgG-4.

The significance of this finding is related to the intrinsic properties of the different IgG

subclasses. IgG-1 and IgG-3 are very effective with regards to complement activation,

whereas IgG-2 is not as effective, and IgG-4 does not bind complement at all9.

Considering the activation of complement may lead to tissue injury by both lytic and

inflammatory pathways9, 10, it is not surprising that these patients experienced symptoms

of acute graft dysfunction.

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