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
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-
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,
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
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
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.
1. Meier-Kriesche HU SJ, Srinivas TR, Kaplan B. Lack of improvement in renal
allograft survival despite a marked decrease in acute rejection rates over the most
recent era. Am J Transplant. 2004;4:378-383.
2. Teraski P. Humoral theory of transplantation. Am J Transplant. 2003(3):665-673.
3. Bian H HP, Mulder A, Reed EF. Anti-HLA antibody ligation to HLA class I molecules
expressed by endothelial cells stimulates tyrosine phosphorylation, inositol phosphate
generation, and proliferation. Hum Immunol. 1997(53):90-97.
4. Harris PE BH, Reed EF. Induction of high affinity fibroblast growth factor receptor
expression and proliferation in human endothelial cells by anti-HLA antibodies: a
possible mechanism for transplant atherosclerosis. J Immunol. 1997(159):5697-5704.
5. Liu C WK, Shi C, Heyner S, Komm B, Haddad JG. Post-transcriptional stimulation of
transforming growth factor beta 1 mRNA by TGF-beta 1 treatment of transformed human
osteoblasts. J Bone Miner Res. 1996(11):211-217.
6. McKenna RM TS, Terasaki PI. Anti-HLA antibodies after solid organ transplantation.
7. Russell PS CC, Winn HJ, Colvin RB. Coronary atherosclerosis in transplanted mouse
hearts. II. Importance of humoral immunity. J Immunol. 1994(152):5135-5141.
8. F. Kushihata JW, A. Mulder, F. Claas, J.C. Scornik. Human Leukocyte Antigen
Antibodies and Human Complement Activation: Role of IgG Subclass,
Specificity, and Cytotoxic Potential. Transplantation. October 15, 2004
9. Baldwin WM 3rd PS, Brauer RB, Daha MR, Sanfilippo F. Complement in organ
transplantation. Contributions to inflammation, injury, and rejection.
Transplantation. March 27, 1995 1995;59(6):797-808.
10. Saadi S PJ. Endothelial cell responses to complement activation In: Volanakis JE,
Frank MM, eds. The human complement system in health and disease. New York,
Marcel Dekker. 998 1998:335.
Medical Student: Andres X. Crowley