Autoantibody production significantly decreased with APRIL/BLyS blockade in murine chronic rejection kidney transplant model.

Chronic antibody mediated rejection (cAMR) remains a significant barrier to achieving long-term graft survival in kidney transplantation, which results from alloantibody production from B lymphocytes and plasma cells. APRIL (A proliferation-inducing ligand) and BLyS (B lymphocyte stimulator) are critical survival factors for B lymphocytes and plasma cells. Here we describe the results of APRIL/BLyS blockade in a murine cAMR kidney transplant model. c57/B6 mice underwent kidney transplantation with Bm12 kidneys (minor MHC mismatch), a well-described model for chronic rejection where animals cannot make donor specific antibody but rather make antinuclear antibody (ANA). Following transplantation, animals received TACI-Ig (to block APRIL and BLyS) or no treatment. Animals were continued on treatment until harvest 4 weeks following transplant. Serum was analyzed for circulating anti-nuclear autoantibodies using HEp-2 indirect immunofluorescence. Spleen and transplanted kidneys were analyzed via H&E. ANA production was significantly decreased in APRIL/BLyS blockade treated animals (p<0.0001). No significant difference in autoantibody production was found between syngeneic transplant control (B6 to B6) and APRIL/BLyS blockade treated animals (p = 0.90). Additionally, disruption of splenic germinal center architecture was noted in the APRIL/BLyS blockade treated animals. Despite the significant decrease in autoantibody production and germinal center disruption, no significant difference in lymphocyte infiltration was noted in the transplanted kidney. APRIL/BLyS blockade resulted in a significant decrease of autoantibody production and disrupted splenic germinal center formation in a chronic kidney transplant model, however in this model no difference in kidney transplant pathology was seen, which may have to do with the absence of any T cell centric immunosuppression. Regardless, these findings suggest that APRIL/BLyS blockade may play a role in decreasing antibody formation long-term in kidney transplantation. Future investigations will use APRIL/BLyS blockade in conjunction with T lymphocyte depleting agents to determine its efficacy in chronic rejection.

Introduction

Antibody mediated rejection (AMR) is widely recognized as a common cause for late kidney allograft failure.[1-3] Mature B lymphocytes and plasma cells (terminally differentiated B lymphocytes) play a critical role not only in the development of AMR through donor specific antibody (DSA) production, but they also function as effector cells in T lymphocyte activation, which may result in cellular rejection.[4, 5] Current strategies used to treat AMR include anti-CD20 antibodies (rituximab), antibody removal (plasmapheresis, intravenous immunoglobulin), and proteasome inhibitors to target plasma cells, which are the producers of alloantibody.[6, 7] Despite these efforts, chronic antibody mediated rejection (cAMR) remains a significant barrier to achieving long-term graft survival in kidney transplantation. As a result, novel and effective strategies to treat antibody mediated rejection remain an unmet need.[8]

Due to the multiple functions of B lymphocytes as both effector and alloantibody-producing cells, it is our hypothesis that B lymphocytes will need to be targeted at various stages of development in order to successfully prevent DSA production and rejection. APRIL (A proliferation-inducing ligand) (TNFSF 13a) and BLyS (B lymphocyte stimulator) (TNFSF 13b) are critical survival factors for plasma cells and B lymphocytes, respectively. APRIL plays an important role in plasmablast and plasma cell survival.[9, 10] BLyS acts as an important co-stimulator of B lymphocyte survival and proliferation.[4, 11, 12] APRIL and BLyS have been individually investigated as potential therapeutic targets in oncology and transplant clinical trials, respectively. A recent randomized-controlled trial investigated the efficacy of anti-BLyS antibody (belimumab, GlaxoSmithKline) in addition to standard-of-care immunosuppression to reduce naïve B cells. Although this endpoint was not met, belimumab reduced memory B cells, which suggests it may have a role in long-term desensitization strategies in patients with pre-existing DSA.[13] B cell maturation antigen (BCMA), which APRIL and BLyS bind to on the surface of memory B and plasma cells, has been targeted in multiple myeloma clinical trials using anti-BCMA antibody with promising results.[14, 15]

We have previously reported the significant B lymphocyte depletion and DSA reduction that occurs with APRIL/BLyS blockade in allosensitized mice.[16] However, APRIL/BLyS blockade did not result in any difference in acute rejection rates, which may be due to a need for extended treatment. Therefore, our goal of the current study was to determine the effect of targeting both APRIL and BLyS in a chronic rejection kidney transplant model. Previously, MHC class II-mismatched Bm12 (B6.H-2bm12) to B6 (c57/BL6) has been described as a murine model of chronic heart rejection but has not been applied to murine kidney transplant models.[17-19] Here we describe the results of APRIL/BLyS blockade in a novel murine chronic rejection kidney transplant model.

Materials and methods

Animals

C57BL/6 (H-2b) and B6.H-2bm12 (H-2bm12) mice were purchased from Jackson Laboratories (Bar Harbor, ME) and housed in the University of Wisconsin Laboratory Animal Facility. All procedures were performed in accordance with the Animal Care and Use Policies at University of Wisconsin. Animal health including animal deaths, room temperature, 12-hour light/dark cycles, and cage cleaning among other sanitation duties were performed daily by WIMR housing staff. Food and water were available ad libitum. This research was prospectively approved by School of Medicine and Public Health Institutional Animal Care and Use Committee at the University of Wisconsin (M005204). Animals that underwent transplantation were monitored daily post-transplant. Animal health was evaluated by activity level, weight gain or loss, hunched posture, and other signs of distress. C57BL/6 animals were transplanted with a Bm12 kidney with simultaneous nephrectomy as previously described.[20] Animals were then randomized to no treatment or APRIL/BLyS blockade (treated with TACI-Ig (Transmembrane activator and calcium modulator and cyclophilin ligand interactor-Immunoglobulin) (100 μg TACI-Ig in PBS, i.p. injection 3x/week for 28d)) post-transplantation. TACI-Ig blocked both APRIL and BLyS. Animals were anesthetized with isoflurane during surgery or injections and sacrificed via cardiac puncture. Buprenorphine (1mg/kg SQ) was used administered post-transplant and every 72 hours for the first week following surgery. C57BL/6 animals transplanted with a c57BL/6 kidney were used as syngeneic transplant control. The contralateral native kidney was removed on day 1 post-transplant, leaving the animal to rely fully on the transplanted kidney. Spleen, blood, urine, and kidney were collected at 28d post-transplant for immediate utilization, storage in 10% formalin for immunohistochemistry (IHC), or were processed to single cells and cryopreserved in liquid nitrogen.

Quantification of circulating autoantibodies and biochemical

Serum samples were collected from mice 1 month post-transplant at the time of harvest. Circulating autoantibody levels were determined using NOVA Lite HEp-2 indirect immunofluorescent assay. Briefly, the serum was diluted to 1:40 with PBS, incubated with antigen substrate slides and unreacted antibodies are washed off. The substrate was then incubated with anti-mouse IgG FITC and the unbound reagent was washed off. A fluorescent microscope was used to image autoantibody positive samples. Quantification was performed using a custom macro written for ImageJ software (NIH, imagej.nih.gov/ij/). Three to five non-overlapping pictures of representative images were taken from animals for ImageJ analysis.

Urine protein and creatinine was measured on an IdexxVetTest 8008 bioanalyzer (Idexx Laboratories, West Sacramento, CA) using compatible assay chips according to manufacturer’s instructions. Proteinuria severity was calculated using the urine protein to creatinine (UPC) ratio and graded as none (UPC <0.5), mild (UPC 0.5–1.0), moderate (UPC 1.0–2.0), and severe (UPC >2.0).

Flow cytometry

Single cell suspensions of splenocytes were prepared from fresh cells. Flow methods were similar to Allman and Gross.[21, 22] After Ficoll purification, splenocytes underwent ACK lysis of red blood cells. After counting and re-suspension in R10 (RPMI with 10% Fetal Calf Serum), 500,000 cells were added to cluster tubes and stained. Cells from each tissue were stained for B lymphocyte subsets, T lymphocyte subsets, and regulatory T cells (Tregs). Antibodies used for B lymphocyte subsets include: Alexa Fluor 488 anti-mouse IgD (11-26c.2a BioLegend), PerCP rat anti-mouse CD45R/B220 (RA3-6B2 BD Pharmingen), PE rat anti-mouse CD24 (M1/69 BD Pharmingen), PE/Cy7 rat anti-mouse IgM (R6-60.2 BD Pharmingen), BV421 rat anti-mouse CD3 (17A2 BioLegend), BV605 hamster anti-mouse CD27 (LG.3A10 BD Horizon), BV711 rat anti-mouse CD38 (90/CD38 BD OptiBuild), FITC rat anti-mouse CD21/CD35 (7G6 BD Pharmingen), PE rat anti-mouse CD5 (clone 53–7.3 BD Pharmingen), APC anti-mouse CD23 (B3B4 BioLegend), and viability dye Ghost Dye Red 780 (13-0865-T100 Tonbo Biosciences). Antibodies used for T lymphocyte subsets and Tregs include: PE rat anti-mouse CD25 (PC61 BD Pharmingen), PE/Cy5 rat anti-mouse CD4 (GK1.5 BioLegend), BV605 rat anti-mouse CD8α (53–6.7 BD Horizon), Alexa Fluor 647 anti- mouse FOXP3 (150 D BioLegend), and viability dye Ghost Dye Red 780 (13-0865-T100 Tonbo Biosciences). Flow cytometry was performed on a BD LSR II at the UWCCC Flow Cytometry Laboratory and data analyzed with FlowJo (TreeStar, Inc., Ashland, OR).

B lymphocyte subset gating

Cells were gated to remove non-singlets, then gated through a live/dead gate, and lastly through tight lymphocyte gate based on forward and side scatter. CD3- lymphocytes were gated in and then visualized as IgD versus CD45R. Memory B lymphocytes were defined as CD3-CD27+CD45R+ from the CD3- gate. From the CD3- lymphocyte gate, mature B (CD3-CD21+IgM+), transitional 1 (T1) (CD3-CD21-IgM++), and transitional 2 marginal zone (T2 MZ) B (CD3-CD21++IgM++) lymphocytes were gated. Additionally, CD45R versus CD5 were gated as CD45R+CD5- cells then visualized as CD23 versus CD21. Gates were drawn for CD21-CD23- (newly formed B lymphocytes) and CD21intCD23+ (follicular (Fo) B lymphocytes).

Plasma cell gating

Cells were gated to remove non-singlets, then through a large gate to ensure capture of the typically larger plasma cells, and were subsequently visualized in an IgD versus CD45R gate. IgD-CD45R- cells were then visualized as CD27 versus IgM. Plasma cells were defined as CD3-IgD-CD45R-CD27+IgM-CD38+. Normalized cell counts for plasma cells were calculated based on the large gate, rather than the lymphocyte gate as was used for other lymphocyte populations.

T lymphocyte subset gating

Cells were gated to remove non-singlets, then gated through a tight lymphocyte gate based on forward and side scatter. Cells were then visualized as CD4 versus CD3 and CD8 versus CD3. T lymphocytes were defined as CD4+CD3+ or CD8+CD3+. CD4+CD3+ cells were further gated as CD25 versus FOXP3. Tregs were defined as CD4+CD3+CD25+FOXP3+.

Histology

Kidneys were collected at time of euthanasia and 1/3 of the spleen was preserved in 10% formalin for at least 24 hours, processed, paraffin embedded, and cut into 5 μm sections. After deparaffinizing and rehydrating, spleen sections were stained with anti-PAX5 (Abcam, ab140341 polyclonal). The ImmPRESS HRP reagent and ImmPACT DAB substrate were used to detect anti-PAX5 primary antibody as a brown pigment. Kidney sections were washed in distilled water, counter-stained with hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS) stain and dehydrated through an ethanol series. Slides were imaged on a Nikon Eclipse E600 supplied with an Olympus DP70 camera. Automated quantification was performed using a custom macro written for ImageJ software (NIH, imagej.nih.gov/ij/). All H&E and PAS slides were reviewed by a transplant pathologist (WZ) blinded to study groups and scored for rejection according to Banff 2013 guidelines.[23]

Statistics

Statistics were performed using the statistical packages that are part of Prism 7 for Windows, v 7.0b. ANOVA, T-tests, and chi-square were primarily used. P values of 0.05 or less were considered significant. Statistical calculations to determine power were determined prior to implementation of this experiment.

Results

APRIL/BLyS blockade significantly decreased anti-nuclear autoantibody production

Animals were harvested 4 weeks following transplant and their tissues were collected to evaluate for changes in ANA production, chronic kidney rejection, and B and T lymphocyte populations. This model has previously been established in preclinical cardiac transplant models as a method to study chronic rejection without immunosuppression because all allografts will develop vasculopathy.[24] ANA was used as a marker for antibody production because the minor MHC mismatch between Bm12 and C57/Bl6 (3 amino acids) does not result in alloantibody production, but rather autoantibody.[25] Post-transplant treatment with APRIL/BLyS blockade resulted in a significant reduction of serum ANA (p<0.0001) (Fig 1). Serum ANA detected in the APRIL/BLyS blockade treated group was not significantly different from that seen in the syngeneic kidney transplant group.

Fig 1

APRIL/BLyS blockade resulted in significant reduction of anti-nuclear autoantibodies (ANA).

Representative images from indirect immunofluorescence assay detecting circulating anti-nuclear autoantibodies (ANA). (A) B6-B6 transplant group, (B) Bm12-B6 transplant group with no treatment. Arrowheads indicate high-intensity staining. (C) Bm12-B6 transplant group treated with APRIL/BLyS blockade. Arrows indicate low-intensity staining. (D) Densitometry using FITC anti-IgG to detect ANA formation in serum. Graph depicts percentage of total area that was fluorescent. APRIL/BLyS blockade resulted in a significant decrease of total fluorescence indicating a significant decrease in circulating ANA.

APRIL/BLyS blockade reduces mature B lymphocytes but does not alter newly formed B lymphocytes

Splenic B lymphocyte populations were evaluated using flow cytometry. Overall, mature B lymphocyte populations, which rely on BLyS for development, were significantly decreased in the APRIL/BLyS blockade treated group. Follicular B cells (CD3-CD21intCD23+) were reduced in APRIL/BLyS blockade treated animals compared to both untreated transplant (p<0.008) and syngeneic transplant (p<0.05) (Fig 2A). Newly formed B cells (CD3-CD21-CD23-) remained unchanged between groups (Fig 2B). Additionally, early stages of transitional 1 (T1) B cells were not significantly different between treated and untreated groups (Fig 3A). However, transitional zone B cells in later stages of development were depleted with APRIL/BLyS blockade as seen in the transitional 2 (T2) marginal zone B cell population (CD3-CD21++IgM++) (p<0.009) (Fig 3B). Mature B cells (CD3-CD21+IgM+) were also significantly reduced in the APRIL/BLyS blockade treated group compared to untreated transplant and syngeneic transplant (p<0.004) (Fig 3C).

Fig 2

Follicular B lymphocytes were significantly depleted with APRIL/BLyS blockade, but newly formed B cells remained unchanged.

Flow cytometry was used to assess immature and mature B lymphocyte populations in spleen. Each graph shows number of cells per 100,000 lymphocytes. (A) Follicular B lymphocytes were defined as CD3-CD21intCD23+. (B) Newly formed B cells were identified as CD3-CD21-CD23-. (C) Representative flow cytometry data of follicular and newly formed B cells by group. Number shown represents percentage of total cells in gate. *p<0.05, **p<0.008.

Fig 3

APRIL/BLyS blockade significantly depleted mature B and transitional 2 (T2) marginal zone B lymphocyte subsets, but preserved immature B lymphocytes.

Flow cytometry was used to assess immature and mature B lymphocyte populations in spleen. Each graph shows number of cells per 100,000 lymphocytes. (A) T1 B cells were defined as CD3-CD21-IgM++. (B) T2 marginal zone B cells were identified as CD3-CD21++IgM++. (C) Mature B cells were defined as CD3-CD21+IgM+. *p<0.03, **p<0.004.

Plasma cell, but not memory B cell, populations declined following treatment with APRIL/BLyS blockade

Memory B and plasma cells were assessed to determine if APRIL/BLyS blockade affected long-lived B lymphocyte populations. Memory B cells (CD3-CD27+CD45R+) were unchanged despite treatment with APRIL/BLyS blockade (Fig 4A). Plasma cells (CD3-IgD-CD45R-CD27+IgM-CD38+) were significantly decreased in APRIL/BLyS blockade treated group compared to untreated transplant groups (p<0.04) (Fig 4B).

Fig 4

Plasma cells, but not memory B cells, were significantly decreased with APRIL/BLyS blockade.

Flow cytometry was used to assess long-lived B cell populations in spleen. (A) Graph shows number of cells per 100,000 lymphocytes. Memory B cells were defined as CD3-CD27+CD45R+. (B) Graph shows number of plasma cells per 100,000 cells collected. Plasma cells were defined as CD3-IgD-CD45R-CD27+IgM-CD38+. (C) Representative flow cytometry data of memory B and plasma cells by group. Number shown represents percentage of total cells in gate. *p<0.05.

Germinal centers in spleen were significantly disrupted in groups treated with APRIL/BLyS blockade

Splenic germinal centers were evaluated via immunohistochemistry using PAX5 antibody in order to further characterize changes in B lymphocyte populations. PAX5 has previously been established as a marker for B lymphocytes.[26] Germinal centers demonstrated normal architecture in syngeneic and untreated groups but was completely disrupted in APRIL/BLyS blockade treated animals (Fig 5). The depletion of B lymphocytes in splenic germinal centers was also confirmed by an overall decrease in anti-PAX5 staining in APRIL/BLyS blockade treated groups compared to other groups (p<0.0001).

Fig 5

Spleen PAX5 significantly decreased in APRIL/BLyS blockade treated mice.

Preservation of splenic germinal center seen in (A) syngeneic transplant control and (B) untreated transplant group. B lymphocytes are shown in brown; T lymphocytes are shown in blue. (C) Destruction of normal germinal center seen in animals treated with APRIL/BLyS blockade as indicated black outline. (D) Densitometry using anti-PAX5 antibody to detect B lymphocytes in splenic germinal centers. Graph depicts percentage of total area of spleen staining for anti-PAX5.

APRIL/BLyS blockade results in significantly decreased regulatory T cells (Tregs) but increases effector T cell populations

After characterizing B lymphocytes with APRIL/BLyS blockade, T cell populations were assessed in order to determine if the changes seen in B lymphocyte populations resulted in T cell alterations. Interestingly, both CD3+CD4+ and CD3+CD8+ T cell populations were significantly increased in the APRIL/BLyS blockade treated group compared to both syngeneic transplant (p<0.009) and untreated transplant groups (p<0.005). Conversely, Tregs in untreated and treated transplant groups were significantly lower compared to syngeneic transplant (p<0.05) (Fig 6).

Fig 6

APRIL/BLyS blockade significantly reduces regulatory T cells (Tregs), but increases effector T cell populations.

Flow cytometry was used to assess T cell populations in spleen. Each graph shows number of cells per 100,000 lymphocytes. (A) CD3+CD4+ T cells. (B) CD3+CD8+ T cells. (C) Regulatory T cells (Tregs) were defined as CD3+CD4+CD25+FoxP3+. (D) Representative flow cytometry data of CD3+CD4+ and CD3+CD8+ from B6-B6 (Left), Bm12-B6 (Middle), and Bm12-B6 + APRIL/BLyS blockade treated group (Right). Number shown represents percentage of total cells in gate. *p<0.05. **p<0.009.

Despite changes in ANA production and B lymphocytes, APRIL/BLyS blockade did not prevent rejection or preserve kidney function

Finally, we evaluated the transplanted kidneys 4 weeks post-transplant. Histology was reviewed by a transplant pathologist (blinded) and scored for acute and chronic peritubular capillaritis (ptc), glomerulitis (g), tubulitis (t), vasculitis (v), interstitial inflammation (i), mi (microcirculation inflammation), and arteriolar hyalinosis (ah) according to Banff 2013. Overall, no difference in rates of cellular rejection were seen between untreated and treated transplant groups (Fig 7). The overwhelming level of cellular rejection noted in the treated group is consistent with the significant increase in T cells and decrease seen in Tregs. In the untreated group, 16.7% (N = 1) developed active AMR whereas no animals in the APRIL/BLyS blockade treated group developed AMR. Significant tubulitis, interstitial inflammation, and vasculitis was noted in both untreated and treated transplant groups. Syngeneic transplant group did not develop any ACR or AMR (Table 1).

Fig 7

APRIL/BLyS blockade did not prevent rejection in kidney transplant model.

Animals underwent kidney transplant and were randomized to receive no treatment or APRIL/BLyS blockade for 1 month post-transplant. (A) B6-B6 kidney transplant did not develop any evidence of rejection. Arrowhead indicates normal kidney tubule. (B) Bm12-B6 developed rejection in 100% of animals and 16.7% (N = 1) developed AMR. Arrow indicates damaged tubule. (C) Bm12-B6 animals treated with APRIL/BLyS blockade also overwhelmingly developed ACR. Arrow indicates damaged tubule. (D) Urine protein and creatinine (UPC) was measured to assess kidney function. UPC ratio was scored as none (UPC <0.5), mild (0.5–1.0), moderate (UPC 1.0–2.0), and severe (UPC >2.0) proteinuria. Proteinuria was not improved in animals who received APRIL/BLyS blockade.

Table 1

Banff scoring.

	Tx control(N = 3)		No treatment(N = 6)		APRIL/BLyS blockade (N = 6)
Banff score	Mean	SD	Mean	SD	Mean	SD	P
t	0.3	0.6	3.0	0.0	3.0	0.0	NS
i	0.0	0.0	3.0	0.0	3.0	0.0	NS
g	0.0	0.0	0.2	0.4	0.0	0.0	NS
ah	0.0	0.0	0.0	0.0	0.0	0.0	NS
v	0.0	0.0	1.7	0.5	1.5	1.0	NS
ptc	0.0	0.0	0.3	0.8	0.0	0.0	NS
cg	0.0	0.0	0.0	0.0	0.0	0.0	NS
ci	0.0	0.0	0.0	0.0	0.0	0.0	NS
ct	0.0	0.0	0.0	0.0	0.0	0.0	NS
cv	0.0	0.0	0.0	0.0	0.0	0.0	NS
mm	0.0	0.0	0.0	0.0	0.0	0.0	NS
mvi	0.0	0.0	0.5	1.2	0.0	0.0
AMR, % (N)Active	0% (0)	—	16.7% (1)	—	0% (0)	—	NS
ACR
Negative	100% (3)	—	0% (0)	—	0% (0)	—	NS
I	0% (0)	—	0% (0)	—	16.7% (1)	—	NS
IIA	0% (0)	—	33.3% (2)	—	33.3% (2)	—	NS
IIB	0% (0)	—	66.7% (4)	—	33.3% (2)	—	NS
III	0% (0)	—	0% (0)	—	16.7% (1)	—	NS

Renal function was measured in each group using a urine protein to creatinine (UPC) ratio. Consistent with the rejection seen on histology, untreated and treated transplant groups had worse renal function compared to syngeneic transplant, although not significantly different (Fig. 7D). In the syngeneic transplant group, 66.7% (N = 2) and 33.3% (N = 1) of animals had none (UPC <0.5) or mild proteinuria, respectively. APRIL/BLyS blockade treated group demonstrated slightly worse renal function than the untreated group. Seventy-five percent (N = 3) in the untreated group had no proteinuria compared to 33.3% (N = 1) in the treated group. No animals in the untreated group had moderate proteinuria (UPC 1.5–2.0), however 33.3% (N = 1) in the treated group developed moderate proteinuria (Fig 7D).

Discussion

Chronic rejection remains a significant cause of late kidney allograft loss; therefore, novel therapies are required to treat this ongoing problem. Bm12 (H-2bm12) to c57/B6 (H-2b) minor MHC mismatch has previously been described as a murine chronic heart rejection model with severe cardiac allograft vasculopathy and fibrosis evident within 4 weeks.[27-29] However, this chronic rejection model has yet to be described in the kidney transplant literature. Therefore, we investigated the effect of targeting the B lymphocyte survival factors APRIL and BLyS on chronic antibody mediated rejection in a murine kidney transplant model. APRIL/BLyS blockade via TACI-Ig resulted in significant changes in both B and T lymphocyte populations. Specifically, follicular, T2 marginal zone, mature B lymphocytes and plasma cells were depleted with APRIL/BLyS blockade. These changes in mature B lymphocyte populations were further confirmed by the disruption of splenic germinal center architecture in treated animals. Importantly, animals treated with APRIL/BLyS blockade demonstrated a significant decrease in anti-nuclear autoantibody that was reduced to syngeneic transplant levels. This autoantibody decrease is consistent with the significant depletion of plasma cells seen in APRIL/BLyS blockade treated animals, which are the primary source of antibody production. Immature B lymphocyte populations such as newly formed and T1 B lymphocytes, which are less dependent on APRIL and BLyS for survival, were not altered with APRIL/BLyS blockade. In addition to not altering immature B lymphocytes, long-lived memory B cells were not depleted.

Despite depleted mature B lymphocytes, these changes did not result in any difference in rejection seen in untreated versus treated groups. Significant cellular rejection was seen in APRIL/BLyS blockade treated animals, which corresponds to increased effector T lymphocyte and decreased regulatory T lymphocyte populations. No AMR developed in APRIL/BLyS blockade treated group whereas 16.7% (N = 1) of untreated animals demonstrated active antibody mediated rejection on biopsy. No differences in renal function, as measured by urine protein to creatinine ratio, were seen between untreated and treated groups.

Interestingly, CD3+CD4+ and CD3+CD8+ T lymphocytes significantly increased compared to both untreated animals and syngeneic transplant control. The increase in effector T lymphocytes demonstrated here may be due to the depletion of regulatory B lymphocytes, which would normally downregulate inflammation and autoimmunity through IL-10.[30]

APRIL and BLyS have been extensively studied as therapeutic targets to reduce autoantibody in several preclinical models of autoimmune diseases including systemic lupus erythematous (SLE), IgA nephropathy, Sjorgren’s syndrome and rheumatoid arthritis.[31-35] In a preclinical murine SLE model, dual APRIL and BLyS inhibition, but not BLyS blockade alone, reduced plasma cells and IgM levels.[36] Furthermore, APRIL/BLyS blockade with TACI-Fc completely prevented SLE disease progression including renal injury and arrested autoantibody production and onset of nephritis.[37] As indicated above, decreased B lymphocytes and autoantibody production through APRIL/BLyS blockade has been established in preclinical autoimmune disease models. However, the novelty of our current study is that we report decreased autoantibody production in a preclinical kidney transplant model using APRIL/BLyS blockade. Although changes in B lymphocyte populations and decreased autoantibody production did not translate to improved renal function or decreased rejection, these initial findings are important to note and may indicate modifications that need to be made to the chronic rejection model presented here. It is possible that if the post-transplant period was extended beyond 4 weeks, then significant differences in AMR would be seen between untreated and treated groups as was seen in circulating autoantibody levels. Additionally, future investigations will use APRIL/BLyS blockade in conjunction with T lymphocyte depleting agents to determine its efficacy in chronic rejection. By including a T lymphocyte depleting agent

Conclusions

APRIL/BLyS blockade successfully reduced autoantibody production in a novel chronic kidney transplant rejection model. Decreased autoantibody production is likely due to depleted mature B lymphocyte populations including plasma cells. The findings presented here are further supported by the autoimmune literature in which APRIL/BLyS inhibition has been extensively studied as a method to reduce autoantibody and disease severity. However, this study remains novel due to the finding of decreased autoantibody with APRIL/BLyS blockade in a kidney transplant model. Additionally, the commonly used Bm12 to B6 chronic heart transplant model has not previously been described in kidney transplant literature. Despite these changes, cellular rejection and kidney function were not improved, which may be due to the absence of any T cell centric immunosuppression. Regardless, these findings suggest that APRIL/BLyS blockade may play a role in decreasing antibody formation long-term in kidney transplantation.

22 Aug 2019

PONE-D-19-21251

Autoantibody Production Significantly Decreased with APRIL/BLyS Blockade in Murine Chronic Rejection Kidney Transplant Model

PLOS ONE

Dear Dr. Redfield III,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Please address our reviewers comments.

==============================

We would appreciate receiving your revised manuscript by Oct 06 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Maria Lourdes Gonzalez Suarez, MD, PhD

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Additional Editor Comments:

Decreased production of autoantibodies is an expected result of the therapy with APRIL/BLyS blockade. This study does help to direct efforts toward answering the question on whether therapy for AMR would have a direct effect on cellular mediated rejection in kidney transplantation. I agree with the authors that perhaps if follow up time was increased, it would be possible to see a difference in the treated versus untreated group. Study was well conducted.

Please address the comments of our reviewers.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Relevant manuscript, well written and provided testing the use of APRIL/BLyS blockade as a potential therapeutic target to prevent allograft rejection in an in vivo mouse model of kidney transplant, comparing pathological findings with clinical measures.

Comments

1. Line 316: "Seventy-five percent (n=3) in the untretaed group had no proteinuria compared to 33.33% (n=1) in the treated group". Please change 33.33% to 25%, if in deed your total N=4.

2. Figure 1, panel C. Arrow is supposed to indicated negative staining, but the arrow is actually pointing to a green area of less intensity when compared to panel B. This could be still interpreted as a positive staining, although difference in intensity is noticeable. Is this auto-fluorescence?

Reviewer #2: This study done by Dr. N. M. Bath et al is a well done study. They have described outcomes of blockade of April/BlyS in a murine cAMR kidney transplant model, showing decrease in autoantibody production and altered splenic germinal center however it also shows there were no differences in kidney transplant pathology. The results from this study suggest that APRIL/BLyS blockade may have a role in decreasing antibody formation in long-term in kidney transplantation. Over all the methods applied are satisfactory and the figures represent mentioned results. Also this study enhances our knowledge about chronic anti body medicated rejection animal model in kidney transplant.

Reviewer #3: Overall, I found the manuscript interesting as the management of chronic antibody mediated rejection of the transplanted kidney remains a major challenge for practicing nephrologists and has a significant impact on the allograft longevity. The problem is a relevant one and newer/better therapies are much needed.

One of my concerns is about the novel chronic kidney rejection model used. The previously described vasculopathic changes from the heart transplant literature could not be reproduced in any of the transplant kidney biopsies. Along the same lines, no other histological changes consistent with chronic AMR or chronic active AMR were seen in the sensitized animals irrespective of the treatment arm.

The experiments were well described and consistent with previous publications by your group. The small sample size and short follow-up time added to the concerns about the model actually representing the disease certainly limit the conclusions.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Aditya Singh Pawar

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

26 Sep 2019

Reviewer #1: Relevant manuscript, well written and provided testing the use of APRIL/BLyS blockade as a potential therapeutic target to prevent allograft rejection in an in vivo mouse model of kidney transplant, comparing pathological findings with clinical measures.

Comments

1. Line 316: "Seventy-five percent (n=3) in the untretaed group had no proteinuria compared to 33.33% (n=1) in the treated group". Please change 33.33% to 25%, if in deed your total N=4.

The numbers listed (lines 311-319) are correct. Unfortunately, we had very little urine collected on animals so we were not able to perform a urine protein:creatinine ratio (UPC) for every animal in this study. Number of urine specimens available for each group are as follows: syngeneic transplant (N=3); untreated transplant (N=4); treated transplant (N=3).

2. Figure 1, panel C. Arrow is supposed to indicated negative staining, but the arrow is actually pointing to a green area of less intensity when compared to panel B. This could be still interpreted as a positive staining, although difference in intensity is noticeable. Is this auto-fluorescence?

This is correct that the arrow in Figure 1C is pointing to an area that is green, although less intense than 1B. Figure 1A is truly negative (no green staining), whereas 1C is of lesser intensity (as also indicated in 1D with B6-B6 being near 0% and treated group being near 1%). We have changed the language to in the figure caption to refer to “lower intensity staining.” Changes made to lines 208-209.

Reviewer #2: This study done by Dr. N. M. Bath et al is a well done study. They have described outcomes of blockade of April/BlyS in a murine cAMR kidney transplant model, showing decrease in autoantibody production and altered splenic germinal center however it also shows there were no differences in kidney transplant pathology. The results from this study suggest that APRIL/BLyS blockade may have a role in decreasing antibody formation in long-term in kidney transplantation. Over all the methods applied are satisfactory and the figures represent mentioned results. Also this study enhances our knowledge about chronic anti body medicated rejection animal model in kidney transplant.

Reviewer #3: Overall, I found the manuscript interesting as the management of chronic antibody mediated rejection of the transplanted kidney remains a major challenge for practicing nephrologists and has a significant impact on the allograft longevity. The problem is a relevant one and newer/better therapies are much needed.

One of my concerns is about the novel chronic kidney rejection model used. The previously described vasculopathic changes from the heart transplant literature could not be reproduced in any of the transplant kidney biopsies. Along the same lines, no other histological changes consistent with chronic AMR or chronic active AMR were seen in the sensitized animals irrespective of the treatment arm.

The experiments were well described and consistent with previous publications by your group. The small sample size and short follow-up time added to the concerns about the model actually representing the disease certainly limit the conclusions.

We agree that the model presented here is not perfect due to the fact that chronic AMR was not noted in any sensitized animals. As stated (lines 377-379), if the post-transplant period had been extended beyond 4 weeks, the changes in ANA production and immune cell types may have also been seen in histology. In future models, the follow up time will likely need to be extended past 4 weeks. However, we believe the changes in ANA production and B cell populations seen from this model at 4 weeks are important findings.

2 Oct 2019

Autoantibody Production Significantly Decreased with APRIL/BLyS Blockade in Murine Chronic Rejection Kidney Transplant Model

PONE-D-19-21251R1

Dear Dr. Redfield III,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Maria Lourdes Gonzalez Suarez, MD, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for addressing reviewers comments. Manuscript is improved and ready for acceptance.

Reviewers' comments:

10 Oct 2019

PONE-D-19-21251R1

Autoantibody Production Significantly Decreased with APRIL/BLyS Blockade in Murine Chronic Rejection Kidney Transplant Model

Dear Dr. Redfield III:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Maria Lourdes Gonzalez Suarez

Academic Editor

PLOS ONE