Supplemental Information Belatacept-based Maintenance Immunosuppression Controls the Post-transplant Humoral Immune Response in Highly Sensitized Non-human Primates

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Supplemental Information

Belatacept-based Maintenance Immunosuppression Controls the Post-transplant Humoral Immune Response in Highly Sensitized Non-human Primates

Robin Schmitz¹, Zachary W. Fitch¹, Miriam Manook¹,Paul M. Schroder¹, Ashley Y. Choi¹, Danae Olaso¹, Janghoon Yoon¹, Yeeun Bae¹, Brian I. Shaw¹, Mingqing Song¹, Maragatha Kuchibhatla², Alton B. Farris³, Allan Kirk¹, Jean Kwun^1,*, and Stuart J. Knechtle^1,*

Affiliations:

1 Duke Transplant Center, Department of Surgery, Duke University School of Medicine, Durham, NC.

2 Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, NC 27710

3 Department of Pathology, Emory University School of Medicine, Atlanta, GA.

*To whom correspondence should be addressed:

Jean Kwun, PhD, 207 Research Drive, Jones 362, DUMC Box 2645, Durham, NC 27710, USA Phone:

919-668-6792; Fax: 919-684-8716; E-mail: [email protected]

and Stuart J Knechtle, MD, 330 Trent Drive, DUMC Box 3512, Durham, NC 27710, USA Phone: 919- 613-9687; Fax: 919-684-8716; E-mail: [email protected]

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Supplemental Tables

Supplemental Table 1. MHC class I (Mamu-A and -B) and II (Mamu-DRB, -DQB, and -DPB) haplotypes of all rhesus macaque pairs used in the study.

Supplemental Table 2.Early rejection phenotypes

Supplemental Table 3. Desensitization, induction and maintenance immunosuppression of individual NHP recipients in the multivariant analysis.

Supplemental Figures

Supplemental Figure 1. Pre-transplant DSA level, graft survival and post-transplant DSA level.

Supplemental Figure 2. T cell depletion with anti-CD4/CD8 monoclonal antibodies abolishes early acute rejection with belatacept and rapamycin maintenance immunosuppression.

Supplemental Figure 3. T Cell depletion and repopulation after kidney transplantation.

Supplemental Figure 4. Post-transplant lymphoproliferative disorder in animals under belatacept and tacrolimus maintenance immunosuppression.

Supplemental Figure 5. Progression of ACR and AMR under belatacept monotherapy.

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Supplemental Tables

Supplemental Table 1. MHC class I (Mamu-A and -B) and II (Mamu-DRB, -DQB, and -DPB) haplotypes of all rhesus macaque pairs used in the study.

Animal ID Mamu-A

Haplotype 1 Mamu-A

Haplotype 2 Mamu-B

Haplotype 1 Mamu-B

Haplotype 2 Mamu-DRB

Haplotype 1 Mamu-DRB

Haplotype 2 Mamu-DQA

Haplotype 1 Mamu-DQA

Haplotype 2 Mamu-DQB

Haplotype 1 Mamu-DQB

Haplotype 2 Mamu-DPA

Haplotype 1 Mamu-DPA

Haplotype 2 Mamu-DPB

Haplotype 1 Mamu-DPB Haplotype 2 Pair 1

H72F A019 A002a B015b/c B043b/c DR03a DR09b 26_01 26_01 18g3 06_12 02g3 06g 07_01 05_01

H49F A026 A004 B001a B012b DR16 DR04a 23_02 01g1 18g5 06_01 08g 02g1 04_01 15g

Pair 2

H65G A_unk* A004 B047a B001a DR03f DR04a 01_02 01g1 06g2 06_01 02g1 02g1 15g 08_01

H89T A019 A025 B015c B017a DR03a DR01a 26_01 26g2 18g3 18g1 02g3 06g 07_01 01g1

Pair 3

H87X A001 A008 B047a B069b DR04a DR04a 01g1 01g1 06_01 06_01 02g1 02g1 15g 15g

HADR A002a A012 B012a B001a DR03f DR06 01_02 23_01 06g2 18g4 07_01 02g1 19g1 08_01

Pair 4

H99H A002a A012 B012a B001a DR03f DR06 01_02 23_01 06g2 18g4 07_01 02g1 19g1 08_01

H48J A004 A023 B002 B043a DR06 DR06 23_01 23_01 18_02 18_02 02g1 02g1 15g1 15g1

Pair 5

H99F A002a A001 B001c B017a DR15ab DR03a 01g2 DQA_unk* 06g1 18g3 02g1 02g3 15g 07_01

HADN A004 A012 B048 B001a DR01a DR04a 26g2 01g1 18g1 06_01 02g1 06g 15g 01g1

Pair6

L196 A110-A111 A004 B043b B001a DR14b DR04a 05_02 01g1 16_01 06_01 07g2 02g1 DPB_unk* 15g

H97F A002a A008 B012a B028 DR03f DR09a 01_02 26g2 06g2 18g1 07_01 06g 19g1 01g1

Pair7

J978 A004 A012 B12b B055 DR04a DR02 01g1 24g1 06_01 18_10 02g1 02g1 15g 15g

J968 A002a A023 B12a B43b Dr16 DR10a 23_02 25g1 18g5 15g1 02g1 02g1 15g 05_01

Red indicates animals treated with Tacrolimus and belatacept and blue indicates rapamycin and belatacept for their maintenance immunosuppression.

Green indicates animals treated with anti-CD4/CD8 mAbs for induction and rapamycin and belatacept for their maintenance immunosuppression.

* A-unk, DQA-unk, and DPB_unk mean that there are an “unknown” Mamu-A1, DQA, and DPB allelic variant in the animal. This variant may only differ only by a single nucleotide compared to known sequences but it isn’t represented in the reference database.

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Supplemental Table 2. Early rejection phenotypes.

Name POD

Treatment*

t v i ti

i-IFTA

g ci ct cg mm cv ah ptc Diagnosis

[ACR = acute cellular rejection, AMR = antibody-mediated rejection, TMA = thrombotic microangiopathy, s/o = suggestive of]

H65G 93 T 1 0 0 0 0 3 1 0 3 3 1 0 0 No evidence of ACR; chronic transplant glomerulopathy & glomerultis, suggestive of CAMR &/or TMA

H99F 119 T 1 0 1 1 0 3 1 1 3 3 1 0 2 No evidence of ACR; chronic transplant glomerulopathy & glomerultis, suggestive of CAMR &/or TMA

H89T 9 R 2 3 0 0 0 3 0 0 3 3 1 0 3 ACR, type 3 (focal arterial fibrinoid necrosis with mural inflammation). Glomerular &

arteriolar fibrin s/o AMR &/or TMA.

HADR 35 R 0 0 0 0 0 2 0 0 0 0 0 0 0 Acute tubular injury. Glomerular fibrin s/o AMR &/or TMA.

HADN 51 R 2 0 2 2 3 3 1 1 0 0 0 0 2 ACR, type 1A. Fibrin & a cellular reaction is next to some arteries, suggestive of fibrinoid aneurysmal change; however, I don't see a clear connection to the arteries, so I can't definitively call it ACR, type 3, or TMA, although that's a consideration.

g, allograft glomerulitis; cg, allograft glomerulopathy; mm, mesangial matrix; ptc, peritubular capillary inflammation; i, interstitial inflammation; t, tubulitis; v, intimal arteritis; ci, cortical interstitial fibrosis; ct, cortical tubular atrophy; cv, vascular fibrous intimal thickening; ah, arteriolar hyalinosis; ti, total inflammation; i-IFTA, cortical inflammation; c4d, complement breakdown product that deposits in peritubular capillaries and medullary vasa recta.

*Treatment: T, Tacrolimus; R, Rapamycin

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Supplemental Table 3. Desensitization, induction and maintenance immunosuppression of individual NHP recipients in the multivariant analysis.

IDs Group Subgroup Desensitization Induction Maintenance POD reason for sacrifice (other than

rejection) rejection co- morbidity 1

1 a

no desensitization CD4/CD8 Tac/MMF/steroid 1 1 1

2 no desensitization CD4/CD8 Tac/MMF/steroid 4 1 1

5

b

no desensitization rhATG Tac/MMF/steroid 4 1 1

6 no desensitization rhATG Tac/MMF/steroid 4 1 1

10

c

no desensitization CD4/CD8 Tac/MMF/steroid 1 1 1

16

2 a

Belatacept+Cafilzomib CD4/CD8 Tac/MMF/Steroid 6 1 1

17 Belatacept+Cafilzomib CD4/CD8 Tac/MMF/Steroid 77 1 1

20 Belatacept+Cafilzomib CD4/CD8 Tac/MMF/Steroid 180 end of study 0 0

21 Belatacept+Cafilzomib CD4/CD8 Tac/MMF/Steroid 180 end of study 0 0

22

b

Lulizumab+Carfilzomib rhATG Tac/MMF/Steroid 12 1 1

23 Lulizumab+Carfilzomib rhATG Tac/MMF/Steroid 13 1 1

27

c

Belatacept+Carfilzomib+2C10 CD4/CD8 Tac/MMF/Steroid 1 unknown 0 1

28 Belatacept+Carfilzomib+2C10 CD4/CD8 Tac/MMF/Steroid 8 1 1

29 Belatacept+Carfilzomib+2C10 CD4/CD8 Tac/MMF/Steroid 16 infection 0 1

32 Belatacept+Carfilzomib+2C10 CD4/CD8 Tac/MMF/Steroid 120 end of study 0 0

33

3 a

Belatacept+Carfilzomib rhATG Belatacept/Tac/steroid 93 censored - PTLD 0 1

34 Belatacept+Carfilzomib rhATG Belatacept/Tac/steroid 119 1 1

35 Belatacept+Carfilzomib rhATG Belatacept/Tac/steroid 180 end of study 0 0

38

b

Belatacept+Carfilzomib rhATG Belatacept/Rapa/steroid 9 1 1

39 Belatacept+Carfilzomib rhATG Belatacept/Rapa/steroid 35 1 1

40 Belatacept+Carfilzomib rhATG Belatacept/Rapa/steroid 51 1 1

41 Belatacept+Carfilzomib rhATG Belatacept/Rapa/steroid 180 end of study 0 0

42 Belatacept+Carfilzomib rhATG Belatacept/Rapa/steroid 180 end of study 0 0

43

c

Belatacept+Carfilzomib CD4/CD8 Belatacept/Rapa/steroid 103 Stop Rapa 0 0

44 Belatacept+Carfilzomib CD4/CD8 Belatacept/Rapa/steroid 110 Stop Rapa 0 0

45 Belatacept+Carfilzomib CD4/CD8 Belatacept/Rapa/steroid 180 end of study 0 0

46 Belatacept+Carfilzomib CD4/CD8 Belatacept/Rapa/steroid 180 end of study 0 0

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Supplemental Figures

Supplemental Figure 1. Pre-transplant DSA level, graft survival and post-transplant DSA level. (A) T and B cell flow crossmatch of all animals immediately prior to kidney transplantation in the two treatment groups. (B) Kaplan-Meier curve and AMR scores of belatacept-based (belatacept/tacrolimus/steroid) immunosuppression (red) vs. tacrolimus-based (tacrolimus/MMF/steroid) immunosuppression (orange and green lines). AMR scores were calculated based on Banff grading glomerulitis (g) and peritubular capillaritis (ptc). CFZ, carfilzomib; DSA, donor-specific antibody; Rapa, rapamycin; rhATG, rhesus- specific anti-thymocyte globulin; Tac, tacrolimus.

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Supplemental Figure 2. T cell depletion with anti-CD4/CD8 monoclonal antibodies abolishes early acute rejection with belatacept and rapamycin maintenance immunosuppression. (A) Schema of the experimental design and immunosuppressive regimen. (B) Kaplan-Meier curve of 6-month graft survival and serum creatine level for animals with CD4/CD8 mAbs with belatacept and rapamycin maintenance immunosuppression. (C) Absolute count for lymphocytes, CD3, CD4 and CD8 T cells. (D) Post-transplant DSA kinetics measured by T and B cell flow crossmatch. (E) Pre-presentative renal pathology at 1- and 6- months post-transplantation from animals with CD4/CD8 depletion with belatacept and rapamycin maintenance immunosuppression. (F) The peak CMV viral titers in rapamycin and belatacept maintenance immunosuppression (rhATG vs. anti-CD4/CD8 mAbs) and hemoglobin level. *, p<0.05, **, p<0.01, ***, p<0.001; NS, not significant.

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Supplemental Figure 3. T cell depletion and repopulation after kidney transplantation. (A) Flow cytometric analysis of circulating CD4⁺ and CD8⁺ T cell numbers and frequencies after kidney transplantation. All available samples were analyzed from POD 0, 1, 4, 7, 14, 28, 42, 56, 84, 112, 140, 168 (n=5 - 10). P value was calculated based on comparison to POD 0 (paired or unpaired t-test). (B) Frequency of CD4⁺ and CD8⁺ T cell subsets defined by CD28 and CD95 (Tn = naïve T cells (CD28⁺CD95^-); Tcm = central memory T cells (CD28⁺CD95⁺); Tem = effector memory T cells (CD28^-CD95⁺). (C) Post-transplant circulating Tfh cell populations including ICOS⁺PD-1⁺, CXCR5⁺, and CXCR5⁺ICOS⁺PD-1⁺ CD4 T cells.

with proliferating Ki67⁺CD20 B cells in the peripheral blood. (D) ICOS⁺PD-1⁺cTfh cells from the day of transplantation and at the time of sacrifice in animals rejected without AMR (n=3) and with AMR (n=7).

(E) Representative H&E images of LN biopsies from pre-, post-desensitization with 1-and 3-months after kidney transplantation in lower magnification. The portion with higher magnification provided in figure 3 are indicated in as rectangles. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, not significant.

Rapa, rapamycin; Tac, tacrolimus.

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Supplemental Figure 4. Post-transplant lymphoproliferative disease in animals under belatacept and tacrolimus maintenance immunosuppression. (A) Macroscopic image taken during exploratory laparotomy for hydronephrosis. A mass in the right lower quadrant/retroperitoneum is causing obstruction of the transplanted ureter. This lesion was later identified as post-transplant lymphoproliferative disease (PTLD) on histopathology. (B) The mid-shaft femoral bone marrow is hypercellular (85%) and consists of solid sheets of large atypical pleomorphic lymphoid (neoplastic) cells with admixed cells having plasmacytoid appearance. The monomorphic population of these cells predominates with few normal myeloid and erythroid precursors noted. (C) Throughout sections of the hepatic parenchyma, there are numerous areas of large pleomorphic atypical (neoplastic) lymphoid cells. Infiltrates are present throughout all areas of the parenchyma and are evident within hepatic sinusoids. Throughout the areas of lymphoid infiltrate there is evidence of lymphoid necrosis.

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Supplemental Figure 5. Progression of ACR and AMR under belatacept monotherapy. ACR, Acute cellular rejection; AMR, antibody-mediated rejection.