- Email: [email protected]
HLA class II Eplet mismatch predicts De Novo DSA formation post lung transplant
Accepted Manuscript HLA class II Eplet mismatch predicts De Novo DSA formation post lung transplant
Duncan C. Walton, Linda Cantwell, Steven Hiho, Joseph Ta, Stephen Wright, Lucy C. Sullivan, Greg I. Snell, Glen P. Westall PII: DOI: Reference:
S0966-3274(18)30069-8 doi:10.1016/j.trim.2018.10.002 TRIM 1173
To appear in:
Transplant Immunology
Received date: Revised date: Accepted date:
30 May 2018 7 October 2018 12 October 2018
Please cite this article as: Duncan C. Walton, Linda Cantwell, Steven Hiho, Joseph Ta, Stephen Wright, Lucy C. Sullivan, Greg I. Snell, Glen P. Westall , HLA class II Eplet mismatch predicts De Novo DSA formation post lung transplant. Trim (2018), doi:10.1016/j.trim.2018.10.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Brief Communication
HLA Class II Eplet Mismatch Predicts De Novo DSA Formation Post Lung Transplant Duncan C. Walton 1, Linda Cantwell1, Steven Hiho 1, Joseph Ta 1, Stephen Wright1, Lucy C. Sullivan 2,3, Greg I. Snell2, Glen P. Westall2. Victorian Transplantation and Immunogenetics Service, Australian Red Cross Blood Service,
PT
1
Melbourne, Australia; 2Lung Transplant Service, Alfred Hospital, Melbourne, Australia; Department of Microbiology and Immunology, University of Melbourne, Peter Doherty
MA
NU
SC
Institute for Infection and Immunity, Melbourne, Australia.
RI
3
D
Corresponding Author
AC C
EP
[email protected]
TE
Glen Westall
ACCEPTED MANUSCRIPT Abstract
The use of algorithms such as HLAMatchmaker to redefine donor-recipient HLA matching is gaining increasing attention. Our research has previously demonstrated that higher HLA class II eplet mismatches correlated with the development of chronic lung allograft dysfunction (CLAD). In this study of lung transplant recipients we prospectively examined
PT
the association between donor-recipient HLA eplet mismatches as defined by HLAMatchmaker (version 2.1) and de-novo anti-HLA donor-specific antibody (DSA)
RI
formation, as assessed by single antigen-bead solid phase assay. HLA class II eplet mismatch,
SC
when split at the median for the cohort, predicted the development of de-novo HLA class II DSA at 3 months but not at 12 months. Having previously shown that high HLA class II eplet
NU
mismatches was associated with CLAD, we now show that the same factors are associated
AC C
EP
TE
D
MA
with de-novo HLA class II DSA post-transplant.
ACCEPTED MANUSCRIPT Abbreviations
AMR – antibody-mediated rejection CLAD - chronic lung allograft dysfunction CI – confidence interval
PT
DSA - donor-specific antibody
RI
HR – hazard ratio MFI – mean fluorescence intensity
SC
OR – Odds ratio
AC C
EP
TE
D
MA
NU
RAS - restrictive allograft syndrome
ACCEPTED MANUSCRIPT Introduction
Increasingly, HLA compatibility between a solid organ transplant recipient and their donor is being defined at the epitope level thereby providing greater structural discernment than has historically been provided at the allelic level [1]. This has led to the use of matching algorithms such as HLAMatchmaker [1] to better identify structurally and therefore
PT
immunologically compatible donor and recipient pairs, aiming to reduce subsequent alloreactivity and improve allograft survival. HLAMatchmaker epitopes are defined by
RI
eplets, which are short polymorphic amino acid sequences that are involved in cognate
SC
antibody binding. With increased precision we can now stratify donor and recipient HLA matching over a greater range of histocompatibility rather than that provided by the
NU
traditional HLA allelic mismatch values.
The link between HLA compatibility as defined by HLAMatchmaker and de-novo DSA has
MA
been demonstrated in renal transplantation. Weibe et al showed that epitope load analysis outperforms traditional antigen mismatch for risk stratification of de-novo DSA. This group
D
demonstrated that patients with HLA DQ eplets mismatch score of greater than 17 were
TE
significantly more likely to develop de-novo class II HLA DSA [2].
EP
De-novo DSA directed towards HLA-DQ has been shown to significantly impact graft survival. In heart transplantation, de-novo HLA-DQ DSA is associated with the primary end point of
AC C
death or graft dysfunction with no increase in risk seen in patients who develop non-DQ DSA [3]. In kidney transplantation, de-novo DQ DSA is a significant risk factor for late allograft failure, and was associated with a histological pattern suggestive of chronic AMR [4]. Tikkanen et al made similar observations in a single centre cohort study of 340 lung transplant recipients concluding that the increased risk of CLAD was driven by the development of de-novo DQ DSA [5].
We have previously demonstrated that HLA class II, HLA-DRB1/DQA1&DQB1 eplet mismatches predicted for the development of chronic lung allograft dysfunction (CLAD), and in particular the restrictive allograft syndrome (RAS) [6]. In this study, we have extended our
ACCEPTED MANUSCRIPT analysis of eplet mismatch scores using HLAMatchmaker to examine the influence on denovo anti-HLA donor specific antibody (DSA) formation.
Methods
From July 2014, 49 consecutive lung transplant recipients were included in this analysis. The
PT
study was approved by the Alfred Hospital’s ethics committee and all patients provided informed consent. All patients received triple immunosuppression (tacrolimus,
RI
azathioprine/mycophenolate mofetil and prednisolone) and patients at risk of renal
SC
dysfunction received Basiliximab as induction therapy. Recipients and donors were HLA typed (Labtype SSO, One Lambda Inc, Canoga Park, CA) for HLA-A, B, DRB1345 and DQA1/DQB1, and 4-digit, common and well documented alleles were assigned. HLA
NU
mismatch scores for each patient/donor pair were assessed using HLAMatchmaker (v2.1, [1]). HLA-A,-B, -DRB1345,-DQA/B were used to generate eplet scores. Recipient serum
MA
samples were screened pre-transplant, 3 months and 12 months post-transplant for antiHLA antibodies by single antigen beads (One Lambda Inc, Canoga Park, CA) as per standard
D
protocol and assessed for the presence of de-novo DSA with a mean fluorescence intensity
TE
(MFI) cut off >1000. Serum samples were pretreated using hypotonic dialysis and/or Absorb Out (One Lambda Inc, Canoga Park, CA) where required to remove non-specific binding
EP
and/or inhibitory factors.
AC C
The association between HLA eplet mismatch (Class I, Class II, Class I & II) and the development of de-novo anti-HLA DSA at 3 and 12 months after LTx was examined using Logistic Regression. Due to the small sample size, and in some instances the near separation of data, we used a Firth-type adjustment to bias correct the estimates and stabilise the estimated standard errors [7]. Models were adjusted for pre-transplant Class I and/or Class II HLA antibodies by including an indicator variable in the logistic regression models. The eplet mismatch variable was dichotomised based on the sample median. Additional confounding factors may impact this analysis but were not included due to the small sample size. All statistics were calculated using R and the logistf package (nominal =0.05).
ACCEPTED MANUSCRIPT Results
Two patients had DSA pre-transplant, of whom one developed additional de-novo DSA at 3 months post-transplant. Overall, 12 patients developed DSA at 3 months (Class I only, n=1; class II only, n=9; both Class I&II, n=2). Of the 11 patients with de-novo class II DSA at 3 months, only two continued to show reactivity at 12 months, conversely one patient that
PT
did not have DSA at three months went on to develop DSA at 12 months post-transplant (Table 1). The majority of DSA specificity at 3 months post-transplant were directed to
RI
either DQA1(10) or DQB1(9). Details including specificity and MFI of the de-novo DSA profile
SC
are shown in Table 2.
The median and ranges of eplet mismatches from the HLAMatchmaker analysis were 14,3-
NU
30 (HLA-I); 39,2-80 (HLA-II); 26,0-54 (DQB1/DQA1); and 59,19-94 (HLA class I&II). These
MA
values were used in all statistical regression analysis.
There was no significant increase in the adjusted odds ratio (OR) for the development of
D
HLA class I de-novo DSA when HLA class I eplet mismatch was analysed independently, at
TE
either 3 or 12 months post-transplant (Table 3). In contrast, class II and HLA-DQA1&DQB1 eplet mismatch was predictive of class II de-novo DSA formation at 3 post-transplant, with
EP
an OR of 5.5 (95% CI 1.3, 31.3; p=0.02) and OR 5.3 (95% CI 1.2,31.3; p=0.02), respectively (Table 3). However merging class I & II eplet mismatch in the analysis reduced the
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magnitude of odds ratio OR 4.8 (95% CI 1.3, 21.9; p=0.02). Predominately class II de-novo DSA were directed toward epitopes on HLA-DQA1 and DQB1 (Table 2). At 12 months posttransplant, eplet mismatch was not shown to be a significant predictor of de-novo DSA development although a trend was observed for HLA class II eplet mismatch.
Discussion
Having previously shown that high HLA class II eplet mismatches are associated with the RAS phenotype of CLAD, we now show that the same eplet mismatches are associated with denovo HLA class II DSA early post-Tx. Our novel finding that the HLAMatchmaker eplet
ACCEPTED MANUSCRIPT mismatch scores predicts the development of de-novo DSA following LTx is consistent with recent findings following renal transplantation [8]. Moreover, de-novo DSA to HLA class II antigens, and in particular to HLA-DQ, appear to be more alloreactive and have a greater influence on LTx outcomes compared to other anti-HLA DSA. In a recent study, the development of DSA largely directed to HLA-DQ was associated with a two-fold increased
PT
risk of CLAD [5].
This study demonstrates that the epitope matching provides not only greater information
RI
on HLA structural compatibility between lung transplant recipients and their donor, but that
SC
these differences are clinically important. Eplet mismatch scores allow the clinician to stratify lung transplant recipients into high and low risk groups not only related to the production of de novo DSA but also with regard to long-term graft survival. Data from this
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and our previous studies suggests a need to explore organ allocation protocols with a view to avoiding high HLA class II eplet transplants. Patients who are transplanted with a high
MA
eplet mismatch score may require increased monitoring for alloreactivity and augmented
D
immunosuppression.
TE
The antibodies detected post-transplant are predominantly directed to HLA-DQA1/DQB1 eplets and validates the current understanding that these antibodies appear more
EP
frequently in lung transplant recipients. Future studies on larger cohorts will aim to further
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define the most immunogenic HLA-DQ eplet mismatches.
Although our study was limited to a small sample size, we were still powered to show eplet mismatch correlated with de-novo DSA development. Using a Firth-type correction had a small impact on the odds ratio observed and did not change the overall significance when compared to the un-adjusted data. This adjustment was required due to the separation of data. Future studies are aimed at correlating the presence of such DSA with CLAD in larger cohort sizes.
Our data showing eplet mismatches correlated with the development of de-novo HLA class II DSA, coupled with our previous study linking HLA class II eplet mismatch to RAS, supports work by other groups suggesting that humoral immune pathways drive the development of
ACCEPTED MANUSCRIPT the RAS phenotype of CLAD [9]. Hence, we postulate that the restrictive phenotype of CLAD may represent chronic AMR.
Conclusion
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In conclusion, eplet mismatch scores represent a superior method of assessing compatible donor-recipient pairs and can be used to stratify risk of HLA de-novo DSA development and
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risk of CLAD post-transplant. This may lead to better allocation of resources post-transplant
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SC
and allow focus to be placed on high risk patients.
MA
ACKNOWLEDGEMENTS
DISCLOSURE
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One Lambda | A Thermo Fisher Scientific Brand. – for provision of Labscreen Single Antigen kits
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The authors of this manuscript have no conflicts of interest to disclose as described by the Transplant Immunology.
ACCEPTED MANUSCRIPT REFERENCES
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1. Duquesnoy RJ. HLA epitope based matching for transplantation. Transplant immunology 2014;31(1):1-6 doi: 10.1016/j.trim.2014.04.004[published Online First: Epub Date]|. 2. Wiebe C, Pochinco D, Blydt-Hansen TD, et al. Class II HLA epitope matching-A strategy to minimize de novo donor-specific antibody development and improve outcomes. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons 2013;13(12):3114-22 doi: 10.1111/ajt.12478[published Online First: Epub Date]|. 3. Cole RT, Gandhi J, Bray RA, et al. De novo DQ donor-specific antibodies are associated with worse outcomes compared to non-DQ de novo donor-specific antibodies following heart transplantation. Clinical transplantation 2017;31(4) doi: 10.1111/ctr.12924[published Online First: Epub Date]|. 4. Lee H, Min JW, Kim JI, et al. Clinical Significance of HLA-DQ Antibodies in the Development of Chronic Antibody-Mediated Rejection and Allograft Failure in Kidney Transplant Recipients. Medicine 2016;95(11):e3094 doi: 10.1097/md.0000000000003094[published Online First: Epub Date]|. 5. Tikkanen JM, Singer LG, Kim SJ, et al. De Novo DQ Donor-Specific Antibodies Are Associated with Chronic Lung Allograft Dysfunction after Lung Transplantation. Am J Respir Crit Care Med 2016;194(5):596-606 doi: 10.1164/rccm.201509-1857OC[published Online First: Epub Date]|. 6. Walton DC, Hiho SJ, Cantwell LS, et al. HLA Matching at the Eplet Level Protects Against Chronic Lung Allograft Dysfunction. American Journal of Transplantation 2016;16(9):2695-703 doi: 10.1111/ajt.13798[published Online First: Epub Date]|. 7. Heinze G. A comparative investigation of methods for logistic regression with separated or nearly separated data. Statistics in medicine 2006;25(24):4216-26 doi: 10.1002/sim.2687[published Online First: Epub Date]|. 8. Lachmann N, Niemann M, Reinke P, et al. Donor-Recipient Matching Based on Predicted Indirectly Recognizable HLA Epitopes Independently Predicts the Incidence of De Novo Donor-Specific HLA Antibodies Following Renal Transplantation. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons 2017;17(12):3076-86 doi: 10.1111/ajt.14393[published Online First: Epub Date]|. 9. Vandermeulen E, Lammertyn E, Verleden SE, et al. Immunological diversity in phenotypes of chronic lung allograft dysfunction: a comprehensive immunohistochemical analysis. Transplant international : official journal of the European Society for Organ Transplantation 2017;30(2):134-43 doi: 10.1111/tri.12882[published Online First: Epub Date]|.
ACCEPTED MANUSCRIPT Table 1 – HLA antibody status at time points pre- and post-transplantation. HLA Antibody
Pre-
HLA
De-novo DSA
HLA antibodies
De-novo
Transplant
antibodies
3 months
12 months
DSA 12
3 months
post-
post-
months
post-
transplant
transplant
post-
Status
transplant
8 (16%)
11 (22%)
1 (2%)
15 (31%)
1 (2%)
Class II only
3 (6%)
8 (16%)
9 (18%)
7 (14%)
2 (4%)
Class I & II
9 (18%)
19 (39%)
2 (4%)
9 (18%)
1 (2%)
SC
RI
Class I only
PT
transplant
AC C
EP
TE
D
MA
NU
*Note two patients had pre-transplant DSA at time of transplant.
ACCEPTED MANUSCRIPT Table 2 – Class II HLA antibodies at 3 and 12 months’ post-transplant, highest MFI values listed for each bead, highlighted specificities show the actual de-novo DSA.
15
HLA class II DSA3 months post-transplant
HLA class II DSA12 month post-tx Class II Abs
30
DQB1*06:03 (4732), DQA1*01:03 (4732) DQB1*06:01 (2295), DQB1*06:02 (1713), DR52 (1470), DP10 (1439), DR14 (1408), DP15 (1112), DQB1*06:09 (1096)
-
38
DR13 (4195), DQA1*06:01 (2833), DR17 (2369), DQA1*05:01 (2216), DR11 (1875), DR52 (1735), DR14 (1734), DQB1*04:02 (1100), DQA1*04:01 (1100)
29
DQB1*03:02 (3515), DQA1*03:02 (3515), DQB1*03:01 (2954), DQA1*05:03 (2954), DQB1*03:03 (1838), DQA1*06:01 (1277)
DQB1*03:03 (1852), DQA1*03:02 (1852), DQB1*03:01 (1764), DQA1*05:05 (1764), DQB1*03:02 (1509), DQA1*06:01 (1263), DRB1*04:04 (1180), DR16 (1093)
2
19
18
PT
12
DQA/B1 Eplet mismatch
-
RI
5
DRB1 Eplet mismatch
SC
Transplant ID
-
21
32
22
19
34
DQA1*03:03 (2494)
-
32
23
21
DQB1*03:01 (6687), DQA1*06:01 (6434), DR53 (6105), DQB1*03:03 (1394)
DR53 (9669), DQB1*03:01 (2791), DQA1*05:05 (2791), DQA1*05:03 (2461), DQA1*06:01 (2240)
40
5
42
41
18
30
44
5
19
47
51
14
11
MA
D
-
DQB1*04:01 (1764), DQA1*03:03 (1764)
-
DQB1*03:01 (2453), DQA1*06:01 (2453), DQA1*05:05 (1104)
-
53
DQB1*03:02 (6507), DQA1*03:02 (6507), DQB1*03:03 (4082), DQB1*03:01 (3318), DQA1*05:05 (3318), DQA1*06:01 (2967), DRB1*12:01 (1397)
-
27
DQB1*03:02 (8145), DQA1*03:02 (8145), DQB1*03:01 (7635), DQA1*06:01 (7635), DQB1*03:03 (7465), DP11 (2149)
-
-
DQB1*02:01 (14219), DQA1*04:01 (14219), DQA1*03:01 (14032), DQB1*03:03 (12198), DQA1*03:02 (12198), DQB1*02:02 (11754), DQB1*03:01 (11054), DQA1*06:01 (11054), DQA1*05:03 (10443), DQA1*05:05 (8734), DR4 (3058)
TE
DQB1*05:01 (2256)
EP
19
AC C
45
NU
19
DQB1*06:03 (2094), DQB1*03:02 (1805), DQA1*03:02 (1805), DQB1*03:01 (1573), DQB1*03:03 (1558)
32
AC C
EP
TE
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Table 3. Estimated Odds Ratio for development of de-novo DSA, when eplet mismatch
95% Cl
p-value
1.0 (ref)
-
-
(14,30)
0.6
[0.1,4.8]
0.63
Class II
1.0 (ref)
-
-
5.5
[1.3,31.3]
0.02
1.0 (ref)
RI
variable is split at the median. Eplet Mismatch
Adjusted OR
-
-
[1.2,31.3]
0.02
-
-
[1.3,21.9]
0.02
1.0 (ref)
-
-
1.0
[0.1,13.1]
0.99
1.0 (ref)
-
-
8.4
[0.8,1157.0]
0.09
1.0 (ref)
-
-
1.9
[0.2,24.6]
0.55
1.0 (ref)
-
-
1.3
[0.2,9.2]
0.76
(Split at median)
Class I (0,14]
(DRB1345,DQB1&DQA1)(0,39]
PT
3 months post-transplant
DQA1* & DQB1* (0,26] (26,54]
5.3 1.0 (ref)
(59,94]
4.8
NU
Class I & II (0,59]
SC
(39,80]
12 months post-transplant Class I (0,14]
MA
(14,30) Class II
(38,80]
TE
DQA1 & DQB1 (0,26]
D
(DRB1345,DQB1&DQA1)(0,39]
(26,54]
EP
Class I & II (0,59]
#
AC C
(59,94]
Model adjusted for the presence of HLA antibodies pre-transplant
ACCEPTED MANUSCRIPT Highlights
PT RI SC NU MA D TE EP
HLA class II eplet mismatch, when split at the median for the cohort, predicted the development of de-novo HLA class II DSA at 3 months but not at 12 months. The antibodies detected post-transplant are predominantly directed to HLA-DQA1/DQB1 eplets
AC C
Duncan C. Walton, Linda Cantwell, Steven Hiho, Joseph Ta, Stephen Wright, Lucy C. Sullivan, Greg I. Snell, Glen P. Westall PII: DOI: Reference:
S0966-3274(18)30069-8 doi:10.1016/j.trim.2018.10.002 TRIM 1173
To appear in:
Transplant Immunology
Received date: Revised date: Accepted date:
30 May 2018 7 October 2018 12 October 2018
Please cite this article as: Duncan C. Walton, Linda Cantwell, Steven Hiho, Joseph Ta, Stephen Wright, Lucy C. Sullivan, Greg I. Snell, Glen P. Westall , HLA class II Eplet mismatch predicts De Novo DSA formation post lung transplant. Trim (2018), doi:10.1016/j.trim.2018.10.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Brief Communication
HLA Class II Eplet Mismatch Predicts De Novo DSA Formation Post Lung Transplant Duncan C. Walton 1, Linda Cantwell1, Steven Hiho 1, Joseph Ta 1, Stephen Wright1, Lucy C. Sullivan 2,3, Greg I. Snell2, Glen P. Westall2. Victorian Transplantation and Immunogenetics Service, Australian Red Cross Blood Service,
PT
1
Melbourne, Australia; 2Lung Transplant Service, Alfred Hospital, Melbourne, Australia; Department of Microbiology and Immunology, University of Melbourne, Peter Doherty
MA
NU
SC
Institute for Infection and Immunity, Melbourne, Australia.
RI
3
D
Corresponding Author
AC C
EP
[email protected]
TE
Glen Westall
ACCEPTED MANUSCRIPT Abstract
The use of algorithms such as HLAMatchmaker to redefine donor-recipient HLA matching is gaining increasing attention. Our research has previously demonstrated that higher HLA class II eplet mismatches correlated with the development of chronic lung allograft dysfunction (CLAD). In this study of lung transplant recipients we prospectively examined
PT
the association between donor-recipient HLA eplet mismatches as defined by HLAMatchmaker (version 2.1) and de-novo anti-HLA donor-specific antibody (DSA)
RI
formation, as assessed by single antigen-bead solid phase assay. HLA class II eplet mismatch,
SC
when split at the median for the cohort, predicted the development of de-novo HLA class II DSA at 3 months but not at 12 months. Having previously shown that high HLA class II eplet
NU
mismatches was associated with CLAD, we now show that the same factors are associated
AC C
EP
TE
D
MA
with de-novo HLA class II DSA post-transplant.
ACCEPTED MANUSCRIPT Abbreviations
AMR – antibody-mediated rejection CLAD - chronic lung allograft dysfunction CI – confidence interval
PT
DSA - donor-specific antibody
RI
HR – hazard ratio MFI – mean fluorescence intensity
SC
OR – Odds ratio
AC C
EP
TE
D
MA
NU
RAS - restrictive allograft syndrome
ACCEPTED MANUSCRIPT Introduction
Increasingly, HLA compatibility between a solid organ transplant recipient and their donor is being defined at the epitope level thereby providing greater structural discernment than has historically been provided at the allelic level [1]. This has led to the use of matching algorithms such as HLAMatchmaker [1] to better identify structurally and therefore
PT
immunologically compatible donor and recipient pairs, aiming to reduce subsequent alloreactivity and improve allograft survival. HLAMatchmaker epitopes are defined by
RI
eplets, which are short polymorphic amino acid sequences that are involved in cognate
SC
antibody binding. With increased precision we can now stratify donor and recipient HLA matching over a greater range of histocompatibility rather than that provided by the
NU
traditional HLA allelic mismatch values.
The link between HLA compatibility as defined by HLAMatchmaker and de-novo DSA has
MA
been demonstrated in renal transplantation. Weibe et al showed that epitope load analysis outperforms traditional antigen mismatch for risk stratification of de-novo DSA. This group
D
demonstrated that patients with HLA DQ eplets mismatch score of greater than 17 were
TE
significantly more likely to develop de-novo class II HLA DSA [2].
EP
De-novo DSA directed towards HLA-DQ has been shown to significantly impact graft survival. In heart transplantation, de-novo HLA-DQ DSA is associated with the primary end point of
AC C
death or graft dysfunction with no increase in risk seen in patients who develop non-DQ DSA [3]. In kidney transplantation, de-novo DQ DSA is a significant risk factor for late allograft failure, and was associated with a histological pattern suggestive of chronic AMR [4]. Tikkanen et al made similar observations in a single centre cohort study of 340 lung transplant recipients concluding that the increased risk of CLAD was driven by the development of de-novo DQ DSA [5].
We have previously demonstrated that HLA class II, HLA-DRB1/DQA1&DQB1 eplet mismatches predicted for the development of chronic lung allograft dysfunction (CLAD), and in particular the restrictive allograft syndrome (RAS) [6]. In this study, we have extended our
ACCEPTED MANUSCRIPT analysis of eplet mismatch scores using HLAMatchmaker to examine the influence on denovo anti-HLA donor specific antibody (DSA) formation.
Methods
From July 2014, 49 consecutive lung transplant recipients were included in this analysis. The
PT
study was approved by the Alfred Hospital’s ethics committee and all patients provided informed consent. All patients received triple immunosuppression (tacrolimus,
RI
azathioprine/mycophenolate mofetil and prednisolone) and patients at risk of renal
SC
dysfunction received Basiliximab as induction therapy. Recipients and donors were HLA typed (Labtype SSO, One Lambda Inc, Canoga Park, CA) for HLA-A, B, DRB1345 and DQA1/DQB1, and 4-digit, common and well documented alleles were assigned. HLA
NU
mismatch scores for each patient/donor pair were assessed using HLAMatchmaker (v2.1, [1]). HLA-A,-B, -DRB1345,-DQA/B were used to generate eplet scores. Recipient serum
MA
samples were screened pre-transplant, 3 months and 12 months post-transplant for antiHLA antibodies by single antigen beads (One Lambda Inc, Canoga Park, CA) as per standard
D
protocol and assessed for the presence of de-novo DSA with a mean fluorescence intensity
TE
(MFI) cut off >1000. Serum samples were pretreated using hypotonic dialysis and/or Absorb Out (One Lambda Inc, Canoga Park, CA) where required to remove non-specific binding
EP
and/or inhibitory factors.
AC C
The association between HLA eplet mismatch (Class I, Class II, Class I & II) and the development of de-novo anti-HLA DSA at 3 and 12 months after LTx was examined using Logistic Regression. Due to the small sample size, and in some instances the near separation of data, we used a Firth-type adjustment to bias correct the estimates and stabilise the estimated standard errors [7]. Models were adjusted for pre-transplant Class I and/or Class II HLA antibodies by including an indicator variable in the logistic regression models. The eplet mismatch variable was dichotomised based on the sample median. Additional confounding factors may impact this analysis but were not included due to the small sample size. All statistics were calculated using R and the logistf package (nominal =0.05).
ACCEPTED MANUSCRIPT Results
Two patients had DSA pre-transplant, of whom one developed additional de-novo DSA at 3 months post-transplant. Overall, 12 patients developed DSA at 3 months (Class I only, n=1; class II only, n=9; both Class I&II, n=2). Of the 11 patients with de-novo class II DSA at 3 months, only two continued to show reactivity at 12 months, conversely one patient that
PT
did not have DSA at three months went on to develop DSA at 12 months post-transplant (Table 1). The majority of DSA specificity at 3 months post-transplant were directed to
RI
either DQA1(10) or DQB1(9). Details including specificity and MFI of the de-novo DSA profile
SC
are shown in Table 2.
The median and ranges of eplet mismatches from the HLAMatchmaker analysis were 14,3-
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30 (HLA-I); 39,2-80 (HLA-II); 26,0-54 (DQB1/DQA1); and 59,19-94 (HLA class I&II). These
MA
values were used in all statistical regression analysis.
There was no significant increase in the adjusted odds ratio (OR) for the development of
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HLA class I de-novo DSA when HLA class I eplet mismatch was analysed independently, at
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either 3 or 12 months post-transplant (Table 3). In contrast, class II and HLA-DQA1&DQB1 eplet mismatch was predictive of class II de-novo DSA formation at 3 post-transplant, with
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an OR of 5.5 (95% CI 1.3, 31.3; p=0.02) and OR 5.3 (95% CI 1.2,31.3; p=0.02), respectively (Table 3). However merging class I & II eplet mismatch in the analysis reduced the
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magnitude of odds ratio OR 4.8 (95% CI 1.3, 21.9; p=0.02). Predominately class II de-novo DSA were directed toward epitopes on HLA-DQA1 and DQB1 (Table 2). At 12 months posttransplant, eplet mismatch was not shown to be a significant predictor of de-novo DSA development although a trend was observed for HLA class II eplet mismatch.
Discussion
Having previously shown that high HLA class II eplet mismatches are associated with the RAS phenotype of CLAD, we now show that the same eplet mismatches are associated with denovo HLA class II DSA early post-Tx. Our novel finding that the HLAMatchmaker eplet
ACCEPTED MANUSCRIPT mismatch scores predicts the development of de-novo DSA following LTx is consistent with recent findings following renal transplantation [8]. Moreover, de-novo DSA to HLA class II antigens, and in particular to HLA-DQ, appear to be more alloreactive and have a greater influence on LTx outcomes compared to other anti-HLA DSA. In a recent study, the development of DSA largely directed to HLA-DQ was associated with a two-fold increased
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risk of CLAD [5].
This study demonstrates that the epitope matching provides not only greater information
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on HLA structural compatibility between lung transplant recipients and their donor, but that
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these differences are clinically important. Eplet mismatch scores allow the clinician to stratify lung transplant recipients into high and low risk groups not only related to the production of de novo DSA but also with regard to long-term graft survival. Data from this
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and our previous studies suggests a need to explore organ allocation protocols with a view to avoiding high HLA class II eplet transplants. Patients who are transplanted with a high
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eplet mismatch score may require increased monitoring for alloreactivity and augmented
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immunosuppression.
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The antibodies detected post-transplant are predominantly directed to HLA-DQA1/DQB1 eplets and validates the current understanding that these antibodies appear more
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frequently in lung transplant recipients. Future studies on larger cohorts will aim to further
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define the most immunogenic HLA-DQ eplet mismatches.
Although our study was limited to a small sample size, we were still powered to show eplet mismatch correlated with de-novo DSA development. Using a Firth-type correction had a small impact on the odds ratio observed and did not change the overall significance when compared to the un-adjusted data. This adjustment was required due to the separation of data. Future studies are aimed at correlating the presence of such DSA with CLAD in larger cohort sizes.
Our data showing eplet mismatches correlated with the development of de-novo HLA class II DSA, coupled with our previous study linking HLA class II eplet mismatch to RAS, supports work by other groups suggesting that humoral immune pathways drive the development of
ACCEPTED MANUSCRIPT the RAS phenotype of CLAD [9]. Hence, we postulate that the restrictive phenotype of CLAD may represent chronic AMR.
Conclusion
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In conclusion, eplet mismatch scores represent a superior method of assessing compatible donor-recipient pairs and can be used to stratify risk of HLA de-novo DSA development and
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risk of CLAD post-transplant. This may lead to better allocation of resources post-transplant
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SC
and allow focus to be placed on high risk patients.
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ACKNOWLEDGEMENTS
DISCLOSURE
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One Lambda | A Thermo Fisher Scientific Brand. – for provision of Labscreen Single Antigen kits
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The authors of this manuscript have no conflicts of interest to disclose as described by the Transplant Immunology.
ACCEPTED MANUSCRIPT REFERENCES
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1. Duquesnoy RJ. HLA epitope based matching for transplantation. Transplant immunology 2014;31(1):1-6 doi: 10.1016/j.trim.2014.04.004[published Online First: Epub Date]|. 2. Wiebe C, Pochinco D, Blydt-Hansen TD, et al. Class II HLA epitope matching-A strategy to minimize de novo donor-specific antibody development and improve outcomes. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons 2013;13(12):3114-22 doi: 10.1111/ajt.12478[published Online First: Epub Date]|. 3. Cole RT, Gandhi J, Bray RA, et al. De novo DQ donor-specific antibodies are associated with worse outcomes compared to non-DQ de novo donor-specific antibodies following heart transplantation. Clinical transplantation 2017;31(4) doi: 10.1111/ctr.12924[published Online First: Epub Date]|. 4. Lee H, Min JW, Kim JI, et al. Clinical Significance of HLA-DQ Antibodies in the Development of Chronic Antibody-Mediated Rejection and Allograft Failure in Kidney Transplant Recipients. Medicine 2016;95(11):e3094 doi: 10.1097/md.0000000000003094[published Online First: Epub Date]|. 5. Tikkanen JM, Singer LG, Kim SJ, et al. De Novo DQ Donor-Specific Antibodies Are Associated with Chronic Lung Allograft Dysfunction after Lung Transplantation. Am J Respir Crit Care Med 2016;194(5):596-606 doi: 10.1164/rccm.201509-1857OC[published Online First: Epub Date]|. 6. Walton DC, Hiho SJ, Cantwell LS, et al. HLA Matching at the Eplet Level Protects Against Chronic Lung Allograft Dysfunction. American Journal of Transplantation 2016;16(9):2695-703 doi: 10.1111/ajt.13798[published Online First: Epub Date]|. 7. Heinze G. A comparative investigation of methods for logistic regression with separated or nearly separated data. Statistics in medicine 2006;25(24):4216-26 doi: 10.1002/sim.2687[published Online First: Epub Date]|. 8. Lachmann N, Niemann M, Reinke P, et al. Donor-Recipient Matching Based on Predicted Indirectly Recognizable HLA Epitopes Independently Predicts the Incidence of De Novo Donor-Specific HLA Antibodies Following Renal Transplantation. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons 2017;17(12):3076-86 doi: 10.1111/ajt.14393[published Online First: Epub Date]|. 9. Vandermeulen E, Lammertyn E, Verleden SE, et al. Immunological diversity in phenotypes of chronic lung allograft dysfunction: a comprehensive immunohistochemical analysis. Transplant international : official journal of the European Society for Organ Transplantation 2017;30(2):134-43 doi: 10.1111/tri.12882[published Online First: Epub Date]|.
ACCEPTED MANUSCRIPT Table 1 – HLA antibody status at time points pre- and post-transplantation. HLA Antibody
Pre-
HLA
De-novo DSA
HLA antibodies
De-novo
Transplant
antibodies
3 months
12 months
DSA 12
3 months
post-
post-
months
post-
transplant
transplant
post-
Status
transplant
8 (16%)
11 (22%)
1 (2%)
15 (31%)
1 (2%)
Class II only
3 (6%)
8 (16%)
9 (18%)
7 (14%)
2 (4%)
Class I & II
9 (18%)
19 (39%)
2 (4%)
9 (18%)
1 (2%)
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RI
Class I only
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transplant
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EP
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D
MA
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*Note two patients had pre-transplant DSA at time of transplant.
ACCEPTED MANUSCRIPT Table 2 – Class II HLA antibodies at 3 and 12 months’ post-transplant, highest MFI values listed for each bead, highlighted specificities show the actual de-novo DSA.
15
HLA class II DSA3 months post-transplant
HLA class II DSA12 month post-tx Class II Abs
30
DQB1*06:03 (4732), DQA1*01:03 (4732) DQB1*06:01 (2295), DQB1*06:02 (1713), DR52 (1470), DP10 (1439), DR14 (1408), DP15 (1112), DQB1*06:09 (1096)
-
38
DR13 (4195), DQA1*06:01 (2833), DR17 (2369), DQA1*05:01 (2216), DR11 (1875), DR52 (1735), DR14 (1734), DQB1*04:02 (1100), DQA1*04:01 (1100)
29
DQB1*03:02 (3515), DQA1*03:02 (3515), DQB1*03:01 (2954), DQA1*05:03 (2954), DQB1*03:03 (1838), DQA1*06:01 (1277)
DQB1*03:03 (1852), DQA1*03:02 (1852), DQB1*03:01 (1764), DQA1*05:05 (1764), DQB1*03:02 (1509), DQA1*06:01 (1263), DRB1*04:04 (1180), DR16 (1093)
2
19
18
PT
12
DQA/B1 Eplet mismatch
-
RI
5
DRB1 Eplet mismatch
SC
Transplant ID
-
21
32
22
19
34
DQA1*03:03 (2494)
-
32
23
21
DQB1*03:01 (6687), DQA1*06:01 (6434), DR53 (6105), DQB1*03:03 (1394)
DR53 (9669), DQB1*03:01 (2791), DQA1*05:05 (2791), DQA1*05:03 (2461), DQA1*06:01 (2240)
40
5
42
41
18
30
44
5
19
47
51
14
11
MA
D
-
DQB1*04:01 (1764), DQA1*03:03 (1764)
-
DQB1*03:01 (2453), DQA1*06:01 (2453), DQA1*05:05 (1104)
-
53
DQB1*03:02 (6507), DQA1*03:02 (6507), DQB1*03:03 (4082), DQB1*03:01 (3318), DQA1*05:05 (3318), DQA1*06:01 (2967), DRB1*12:01 (1397)
-
27
DQB1*03:02 (8145), DQA1*03:02 (8145), DQB1*03:01 (7635), DQA1*06:01 (7635), DQB1*03:03 (7465), DP11 (2149)
-
-
DQB1*02:01 (14219), DQA1*04:01 (14219), DQA1*03:01 (14032), DQB1*03:03 (12198), DQA1*03:02 (12198), DQB1*02:02 (11754), DQB1*03:01 (11054), DQA1*06:01 (11054), DQA1*05:03 (10443), DQA1*05:05 (8734), DR4 (3058)
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DQB1*05:01 (2256)
EP
19
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45
NU
19
DQB1*06:03 (2094), DQB1*03:02 (1805), DQA1*03:02 (1805), DQB1*03:01 (1573), DQB1*03:03 (1558)
32
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EP
TE
D
MA
NU
SC
RI
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ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Table 3. Estimated Odds Ratio for development of de-novo DSA, when eplet mismatch
95% Cl
p-value
1.0 (ref)
-
-
(14,30)
0.6
[0.1,4.8]
0.63
Class II
1.0 (ref)
-
-
5.5
[1.3,31.3]
0.02
1.0 (ref)
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variable is split at the median. Eplet Mismatch
Adjusted OR
-
-
[1.2,31.3]
0.02
-
-
[1.3,21.9]
0.02
1.0 (ref)
-
-
1.0
[0.1,13.1]
0.99
1.0 (ref)
-
-
8.4
[0.8,1157.0]
0.09
1.0 (ref)
-
-
1.9
[0.2,24.6]
0.55
1.0 (ref)
-
-
1.3
[0.2,9.2]
0.76
(Split at median)
Class I (0,14]
(DRB1345,DQB1&DQA1)(0,39]
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3 months post-transplant
DQA1* & DQB1* (0,26] (26,54]
5.3 1.0 (ref)
(59,94]
4.8
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Class I & II (0,59]
SC
(39,80]
12 months post-transplant Class I (0,14]
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(14,30) Class II
(38,80]
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DQA1 & DQB1 (0,26]
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(DRB1345,DQB1&DQA1)(0,39]
(26,54]
EP
Class I & II (0,59]
#
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(59,94]
Model adjusted for the presence of HLA antibodies pre-transplant
ACCEPTED MANUSCRIPT Highlights
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HLA class II eplet mismatch, when split at the median for the cohort, predicted the development of de-novo HLA class II DSA at 3 months but not at 12 months. The antibodies detected post-transplant are predominantly directed to HLA-DQA1/DQB1 eplets
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