Transitional B cell cytokines risk stratify early borderline rejection after renal transplantation
2022; Elsevier BV; Volume: 103; Issue: 4 Linguagem: Inglês
10.1016/j.kint.2022.10.026
ISSN1523-1755
AutoresAravind Cherukuri, Khodor I. Abou‐Daya, Raad Chowdhury, Rajil Mehta, Sundaram Hariharan, Parmjeet Randhawa, David M. Rothstein,
Tópico(s)Cytomegalovirus and herpesvirus research
ResumoBorderline rejection (BL) in renal transplantation is associated with decreased allograft survival, yet many patients with BL maintain stable graft function. Identifying patients with early BL at risk for shortened allograft survival would allow for timely targeted therapeutic intervention aimed at improving outcomes. 851/1187 patients transplanted between 2013-18 underwent early biopsy (0-4 mos). 217/851 (25%) had BL and were compared to 387/851 without significant inflammation (NI). Serial surveillance and for-cause biopsies and seven-year follow-up were used to evaluate histological and clinical progression. To identify high-risk patients, we examined clinical/histological parameters using regression and non-linear dimensionality reduction (tSNE) and a biomarker based on peripheral blood transitional-1 B cell (T1B) IL-10/TNFα ratio. Compared to NI, early BL was associated with increased progression to late acute rejection (AR; 5-12 mos), premature interstitial fibrosis and tubular atrophy (IFTA) and decreased seven-year graft survival. However, decreased graft survival was limited to BL patients who progressed to late AR or IFTA, and was not influenced by treatment. Although tSNE clustered patients into groups based on clinical factors, the ability of these factors to risk stratify BL patients was modest. In contrast, a low T1B IL-10/TNFα ratio at 3 months identified BL patients at high risk for progression to AR (ROC AUC 0.87) and poor 7-yr graft survival (52% vs. 92%, p=0.003), while BL patients with a high ratio had similar graft survival to patients with NI (91%, p=NS). Thus, progressive early allograft inflammation manifested as BL that progresses to late AR in the first post-transplant year represents a high-risk clinical state for poor allograft outcomes. Such high-risk status can be predicted by the T1B IL-10/TNFα ratio before irreversible scarring sets in, thus allowing timely risk stratification. Borderline rejection (BL) in renal transplantation is associated with decreased allograft survival, yet many patients with BL maintain stable graft function. Identifying patients with early BL at risk for shortened allograft survival would allow for timely targeted therapeutic intervention aimed at improving outcomes. 851/1187 patients transplanted between 2013-18 underwent early biopsy (0-4 mos). 217/851 (25%) had BL and were compared to 387/851 without significant inflammation (NI). Serial surveillance and for-cause biopsies and seven-year follow-up were used to evaluate histological and clinical progression. To identify high-risk patients, we examined clinical/histological parameters using regression and non-linear dimensionality reduction (tSNE) and a biomarker based on peripheral blood transitional-1 B cell (T1B) IL-10/TNFα ratio. Compared to NI, early BL was associated with increased progression to late acute rejection (AR; 5-12 mos), premature interstitial fibrosis and tubular atrophy (IFTA) and decreased seven-year graft survival. However, decreased graft survival was limited to BL patients who progressed to late AR or IFTA, and was not influenced by treatment. Although tSNE clustered patients into groups based on clinical factors, the ability of these factors to risk stratify BL patients was modest. In contrast, a low T1B IL-10/TNFα ratio at 3 months identified BL patients at high risk for progression to AR (ROC AUC 0.87) and poor 7-yr graft survival (52% vs. 92%, p=0.003), while BL patients with a high ratio had similar graft survival to patients with NI (91%, p=NS). Thus, progressive early allograft inflammation manifested as BL that progresses to late AR in the first post-transplant year represents a high-risk clinical state for poor allograft outcomes. Such high-risk status can be predicted by the T1B IL-10/TNFα ratio before irreversible scarring sets in, thus allowing timely risk stratification. Lay SummaryBorderline rejection (BL), a diagnostic category that uses a lower threshold of inflammation within the kidney allograft than that used for a definitive diagnosis of acute rejection, is clinically heterogeneous. While some patients with BL remain stable, others develop progressive inflammation, premature scarring, and graft loss. Such patients at increased risk for premature graft loss can be accurately identified early by assessing the ratio of interleukin 10 to tumor necrosis factor alpha expressing transitional-1 B cells in their blood, thus allowing timely risk stratification. Borderline rejection (BL), a diagnostic category that uses a lower threshold of inflammation within the kidney allograft than that used for a definitive diagnosis of acute rejection, is clinically heterogeneous. While some patients with BL remain stable, others develop progressive inflammation, premature scarring, and graft loss. Such patients at increased risk for premature graft loss can be accurately identified early by assessing the ratio of interleukin 10 to tumor necrosis factor alpha expressing transitional-1 B cells in their blood, thus allowing timely risk stratification. Improving long-term graft survival is a major unmet need in kidney transplantation. Currently, ∼40% of kidney transplants are lost by 10 years.1Hariharan S. Israni A.K. Danovitch G. Long-term survival after kidney transplantation.N Engl J Med. 2021; 385: 729-743Crossref PubMed Scopus (106) Google Scholar Growing evidence indicates that late graft loss results primarily from cumulative immune-mediated damage that may be overt or clinically silent.2Rush D. Nickerson P. Gough J. et al.Beneficial effects of treatment of early subclinical rejection: a randomized study.J Am Soc Nephrol. 1998; 9: 2129-2134Crossref PubMed Google Scholar, 3Shishido S. Asanuma H. Nakai H. et al.The impact of repeated subclinical acute rejection on the progression of chronic allograft nephropathy.J Am Soc Nephrol. 2003; 14: 1046-1052Crossref PubMed Scopus (194) Google Scholar, 4Cosio F.G. Grande J.P. Wadei H. et al.Predicting subsequent decline in kidney allograft function from early surveillance biopsies.Am J Transplant. 2005; 5: 2464-2472Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 5Mehta R. Bhusal S. Randhawa P. et al.Short-term adverse effects of early subclinical allograft inflammation in kidney transplant recipients with a rapid steroid withdrawal protocol.Am J Transplant. 2018; 18: 1710-1717Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 6Seifert M.E. Yanik M.V. Feig D.I. et al.Subclinical inflammation phenotypes and long-term outcomes after pediatric kidney transplantation.Am J Transplant. 2018; 18: 2189-2199Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 7Cherukuri A. Salama A.D. Mehta R. et al.Transitional B cell cytokines predict renal allograft outcomes.Sci Transl Med. 2021; 13: eabe4929Crossref PubMed Scopus (19) Google Scholar Such damage may result from episodes of acute rejection (AR) or from milder allograft inflammation that does not meet the diagnostic threshold for AR, termed "borderline changes suspicious for acute rejection," commonly called "borderline rejection" (BL). Although patients with BL exhibit an increased frequency of premature allograft loss, a majority (>75%) maintain excellent renal function in the long term.8Nankivell B.J. Agrawal N. Sharma A. et al.The clinical and pathological significance of borderline T cell-mediated rejection.Am J Transplant. 2019; 19: 1452-1463Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar,9Wiebe C. Rush D.N. Gibson I.W. et al.Evidence for the alloimmune basis and prognostic significance of borderline T cell-mediated rejection.Am J Transplant. 2020; 20: 2499-2508Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Although mild grades of early allograft inflammation are associated with increased subsequent AR, the true incidence of BL diagnosed early after transplantation, and its clinical impact in terms of long-term allograft survival, is unclear.5Mehta R. Bhusal S. Randhawa P. et al.Short-term adverse effects of early subclinical allograft inflammation in kidney transplant recipients with a rapid steroid withdrawal protocol.Am J Transplant. 2018; 18: 1710-1717Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar,10Mehta R.B. Tandukar S. Jorgensen D. et al.Early subclinical tubulitis and interstitial inflammation in kidney transplantation have adverse clinical implications.Kidney Int. 2020; 98: 436-447Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Thus, a diagnosis of BL creates a both prognostic and therapeutic dilemma, as the clinician is left to try to decide, often on a case-by-case basis, whether BL does or does not represent early or mild AR that might progress without additional anti-rejection therapy. Unsurprisingly, no consensus has been reached on whether or how to treat these patients. Delineating the natural history of early BL and identifying early clinical or biological markers that could identify patients at risk for poor outcomes, would allow for selective treatment of high-risk patients before further maturation of the immune response and irreversible allograft damage occurs. Although several biomarkers have been shown to predict an increased incidence of BL, none allow risk stratification.9Wiebe C. Rush D.N. Gibson I.W. et al.Evidence for the alloimmune basis and prognostic significance of borderline T cell-mediated rejection.Am J Transplant. 2020; 20: 2499-2508Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar,11Friedewald J.J. Kurian S.M. Heilman R.L. et al.Development and clinical validity of a novel blood-based molecular biomarker for subclinical acute rejection following kidney transplant.Am J Transplant. 2019; 19: 98-109Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar,12Park S. Guo K. Heilman R.L. et al.Combining blood gene expression and cellfree DNA to diagnose subclinical rejection in kidney transplant recipients.Clin J Am Soc Nephrol. 2021; 16: 1539-1551Crossref PubMed Scopus (21) Google Scholar In this study, we compared paired early and late surveillance biopsies (sBxs) and for-cause biopsies (fcBxs) in patients diagnosed with BL within the first 4 months after kidney transplantation, to elucidate its natural history of histologic progression. Patients with early BL that progressed to late AR (5–12 months) developed more scarring and graft loss than did patients who had either no significant inflammation (NI) or BL that did not progress to AR. Using both supervised and unsupervised analytical methods, we identified clinical factors that could help risk-stratify outcomes in patients with BL, with modest predictive ability. In contrast, the ratio of interleukin 10 (IL-10)/tumor necrosis factor (TNF)α expression by the transitional-1 B cell (T1B) subset, a marker of regulatory B cells in the peripheral blood, could effectively risk-stratify patients with early BL by identifying those at risk for progressive inflammation/damage and graft loss who might benefit from an early increase in immunosuppression.7Cherukuri A. Salama A.D. Mehta R. et al.Transitional B cell cytokines predict renal allograft outcomes.Sci Transl Med. 2021; 13: eabe4929Crossref PubMed Scopus (19) Google Scholar This retrospective observational cohort study included all adult kidney transplant recipients who underwent transplantation between January 2013 and December 2018 at the University of Pittsburgh Medical Center and aims to risk-stratify kidney transplant recipients with early BL. The study protocol was approved by the University of Pittsburgh Institutional Review Board (#PRO13060220, REN13040245/PRO12030552). Unless medically contraindicated, patients at the University of Pittsburgh Medical Center underwent 2 sBxs at 3 and 12 months in addition to fcBxs. sBxs were not performed if patients had an fcBx within 1 month. On the basis of the timing, biopsies were classified as either early (0 to 4 months) or late (5 to 12 months; Supplementary Figure S1). The primary study group comprised patients whose early biopsy was diagnosed as BL, based on the Banff 2019 classification of renal allograft pathology: foci of tubulitis (t1, t2, or t3) with mild interstitial inflammation (i1), or mild tubulitis (t1) with moderate to severe interstitial inflammation (i2 or i3).13Loupy A. Haas M. Roufosse C. et al.The Banff 2019 Kidney Meeting report (I): updates on and clarification of criteria for T cell- and antibody-mediated rejection.Am J Transplant. 2020; 20: 2318-2331Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar While clinical BL (cBL) was diagnosed on an fcBx, subclinical BL (scBL) was diagnosed on an sBx. All BL patients (n = 217) were included irrespective of coexistent interstitial fibrosis, tubular atrophy, or donor-specific antibodies (DSAs). The comparator group comprised patients with NI on their early biopsy, defined as no inflammation or not reaching the threshold for BL (n = 387). This definition was chosen because this degree of minor inflammation does not affect graft survival.14Nankivell B.J. P'Ng C.H. Chapman J.R. Does tubulitis without interstitial inflammation represent borderline acute T cell mediated rejection?.Am J Transplant. 2019; 19: 132-144Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar Patients who had Banff ≥1A AR or other inflammatory conditions diagnosed on the early biopsy (n = 247) were excluded from this analysis (Figure 1). Blood was drawn at 0, 1, 3, 6, 9, and 12 months to monitor for DSAs. Presence of DSAs was assessed by One Lambda single antigen beads, per manufacturer instructions. An adjusted median fluorescence intensity of ≥1000 was used to detect DSA presence. Induction immunosuppression consisted of Thymoglobulin (6 mg/kg total) or basiliximab (in ∼5% of the cohort), and methylprednisolone (500 mg) followed by a 7-day prednisolone taper. Patients were then maintained on mycophenolate mofetil and tacrolimus (target trough concentrations: 8–12 ng/ml for 3 months, and 6–10 ng/ml after 3 months). Patients with calculated panel reactive antibodies (PRAs) of >90% were also maintained on oral prednisolone (5 mg/d). Banff 1A or 1B T cell–mediated rejection, whether clinical or subclinical, was treated with 3 doses of methylprednisolone (250 mg each). Banff ≥2A and steroid-resistant T cell–mediated rejection were treated with Thymoglobulin (maximum 6 mg/kg). Acute antibody- mediated rejection was treated with plasmapheresis and i.v. IgG. Maintenance prednisolone (5 mg/d) was added for all patients with rejection. Outcomes were as follows: late biopsy-proven AR between 5 and 12 months post-transplant, interstitial fibrosis and tubular atrophy (IFTA) at 1 year, patient survival, and death-censored graft survival at 7 years. Patients were followed for a maximum of 7 years. Mean follow-up was 59 ± 21 months. Biopsies were scored and confirmed by a single pathologist blinded to the results of the study using Banff 2019 criteria to define BL, Banff ≥1A T cell–mediated rejection, or antibody- mediated rejection.13Loupy A. Haas M. Roufosse C. et al.The Banff 2019 Kidney Meeting report (I): updates on and clarification of criteria for T cell- and antibody-mediated rejection.Am J Transplant. 2020; 20: 2318-2331Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar Of the 244 patients who consented to enroll in the University of Pittsburgh Medical Center biorepository between 2013 and 2015 and had peripheral blood mononuclear cells cryopreserved, A total of 158 patients had either NI (n = 105) or BL (n = 53) diagnosed on their early biopsy and were used in the biomarker portion of this study. The biomarker was assessed at the time of the index biopsy (0–4 months), utilizing flow cytometry to assess IL-10 and TNFα expression by T1B cells,7Cherukuri A. Salama A.D. Mehta R. et al.Transitional B cell cytokines predict renal allograft outcomes.Sci Transl Med. 2021; 13: eabe4929Crossref PubMed Scopus (19) Google Scholar as detailed in the Supplementary Methods. Continuous variables are presented as means ± SD or SEM, and categorical variables are presented as percentages. Continuous variables were compared using the independent-samples t-test or the Mann–Whitney U test. For multiple-group comparison, analysis of variance (ANOVA) was used with Dunnett's post hoc correction or the Kruskal–Wallis test. The χ2 test was used to compare categorical variables. For nonparametric paired comparisons, we used the Wilcoxon matched-pairs signed-rank test. Univariate and multivariable logistic regression models were used to examine risk factors for BL, and to assess risk factors for AR subsequent to the diagnosis of BL. Overall graft loss and death-censored graft loss were analyzed using the Kaplan–Meier method, and survival curves between various patient groups were compared using the Log rank test. A multivariable Cox proportional hazards model was used to assess independent risk factors for graft loss. Receiver operating characteristic curve analysis was used to obtain optimal sensitivity and specificity of the biomarker based on predicted probabilities derived from logistic regression. Unsupervised analyses were conducted using dimensionality reduction with T-distributed stochastic neighbor embedding (tSNE). For each diagnostic category (NI and BL), 41 independent features were included (Supplementary Table S1). Nonlinear dimensionality reduction using T-distributed stochastic neighbor embedding was done using Rtsne (version 0.15), using the following parameters: perplexity = 30; maximum iterations (max_iter) = 10,000; learning rate (eta) = 500; initial dimensions (initial_dims) = 8. A total of 1187 patients underwent kidney transplantation between 2013 and 2018 at the University of Pittsburgh Medical Center, for whom the intent was to perform sBx at approximately 3 and 12 months, in addition to fcBx. Of the 1187 patients, 851 (72%) underwent either sBx or fcBx between 0 and 4 months post-transplantation (Figure 1). Of these 851 patients, 217 (25%) had BL on their early biopsy (0–4 months), and these were compared to the 387 patients (45%) whose early biopsies showed NI. Unless otherwise noted, the terms BL and NI refer to the findings on this early "index" biopsy. Supplementary Table S2 summarizes key baseline demographic and clinical differences between the BL and NI patient groups. A significantly greater proportion of BL patients had a higher kidney donor profile index (KDPI > 50%) than that of patients with NI (32% vs. 21%, respectively; Supplementary Table S2). More BL patients than NI patients received basiliximab induction, although the numbers in both groups were small (7.7% vs. 3.6%). Further, BL patients received kidneys from slightly older donors (43 vs. 40 years), and they had a higher serum creatinine level at 3 months (1.68 vs. 1.5 mg/dl) than did NI patients (Supplementary Table S2). Of the 217 patients with BL, 171 (79%) had scBL diagnosed on the 3-month sBx, and the remaining 46 (21%) had cBL diagnosed on fcBx between 0 and 4 months post-transplantation (Figure 2a ). Both scBL and cBL were associated with similarly increased inflammation (tubulitis, interstitial inflammation, and peritubular capillaritis) and chronic damage (tubular atrophy, interstitial fibrosis, and vascular fibrosis/intimal thickening) compared to NI (Figure 2b–h). We next examined risk factors for development of BL in this patient cohort. By univariate logistic regression, cardiac death donors, donor age, KDPI >50%, basiliximab induction, and serum creatinine level at 1 and 3 months were all associated with increased incidence of BL (Supplementary Table S3). Further, female gender and class I PRAs >50% exhibited a trend toward increased BL. However, by multivariate logistic regression, only female gender (odd ratio [OR] 1.6, 95% confidence interval [CI] 1.1–2.3, P = 0.015) and basiliximab induction (OR 2.7, 95% CI 1.2–5.8, P = 0.01) were independent risk factors for BL (Supplementary Table S3). The availability of paired early (0–4months) and late (5–12 months) biopsies in ∼80% of patients (Figure 1) allowed us to delineate the natural history of histologic progression in individual patients with BL, compared to those with NI. Analysis of paired biopsies from patients with BL showed significant worsening of tubulointerstitial inflammation (increased Banff tubulitis [t] and interstitial inflammation [i] scores; Supplementary Figure S2A and B), macrovascular inflammation (increased intimal arteritis [v] score; Supplementary Figure S2C), and microvascular inflammation (increased glomerulitis [g] and peritubular capillaritis [ptc] scores; Supplementary Figure S2D and E) over time. However, there was no difference in the degree of C4d (a fragment of complement component C4) deposition between paired early and late biopsies (Supplementary Figure S2F). Compared to NI patients, those with BL developed worse late tubulointerstitial (Supplementary Figure S3A and B), macrovascular (Supplementary Figure S3C), and microvascular (Supplementary Figure S3D and E) inflammation. This pattern translated into a 2-fold increase in late Banff ≥1A AR (late AR, 40% vs. 20%) and a 58% decrease in the incidence of "clean" late biopsies without significant inflammation (33% vs. 52%) in BL versus NI patients (Figure 3a ). In a multivariable logistic regression model, BL was associated with increased odds for late AR (OR: 2.5, 95% CI 1.6–3.8, P < 0.0001), independent of potential confounders (Supplementary Table S4). Among patients with either BL or NI that did progress to late AR, there was no significant difference in the severity of rejection (defined by Banff grade). However, late AR with microvascular inflammation (MVI; Banff g + ptc score ≥2) was twice as frequent in patients with BL (P = 0.06; Figure 3a). Importantly, among BL patients, those with cBL and scBL had comparable incidence and severity of late AR (defined by Banff grades or MVI; Figure 3b). As shown in Figure 4a and b , BL was associated with significantly worse late IFTA (Banff ct + ci scores) than NI. To exclude the influence of worse initial IFTA on early biopsies (Figure 2f and g), we compared late IFTA in BL and NI patients lacking any significant early IFTA (IFTA score <1) and found that BL was still associated with significantly worse late IFTA (Figure 4c and d). Furthermore, BL patients who developed late AR had significantly worse late IFTA than did patients with BL without late AR (Figure 4e and f). In fact, BL patients who developed late AR also had significantly worse late IFTA than did NI patients who developed late AR (Supplementary Figure S4). Finally, a greater proportion of BL patients than of NI patients had a combination of both bad IFTA (score ≥2) and late AR (Figure 4g). Taken together, these results indicate that BL patients, compared to NI patients, exhibit progressively worsening inflammation compounded by premature chronic damage. Although mortality was comparable, BL patients had worse 7-year death-censored graft survival (80% vs. 91%, P = 0.001) than NI patients (Figure 5a and b ). In a multivariable Cox model, BL was associated with increased hazard for death-censored graft loss independent of potential confounders (hazard ratio: 2.1, 95% CI, 1.2–3.5, P = 0.005; Supplementary Table S5). Importantly, BL patients that progressed to late AR had significantly worse 7-year death-censored graft survival than did BL patients who did not progress to late AR (72% vs. 87%, P = 0.001; Figure 5c). In fact, BL patients without late AR had graft survival comparable to that of patients with NI who remained rejection-free and those with NI who developed late AR (88% vs. 87% vs. 91%, respectively, Figure 5c). The association between BL plus late AR and worse death-censored graft loss was independent of potential confounders (hazard ratio: 2.3, 95% CI 1.1-4.7, P = 0.02; Supplementary Table S5). Thus, early inflammation that resolves (i.e., BL without late AR) and late AR without early/prolonged inflammation have little effect on graft survival. In contrast, early inflammation manifested as BL that progresses to late AR represents a high-risk state for premature allograft loss. Given that BL patients had a higher incidence of late AR associated with MVI or IFTA (Figure 3a; Figure 4), we examined the influence of these variables on graft survival. BL patients who progressed to late AR with MVI had remarkably poor death-censored 7-year graft survival (41%; P < 0.001 vs. all other groups; Figure 5d). In contrast, death-censored graft survival in BL patients who developed late AR without MVI (87%) was comparable to that in patients who had BL without late AR (90%), and in those with NI (91%). Similarly, BL patients who developed late IFTA had significantly worse graft survival than did NI patients (64% vs. 91%, P = 0.0007) or BL patients who did not develop IFTA (84%; Figure 5e). Finally, in patients with BL, late MVI and development of DSAs after the early biopsy were both independent risk factors for death-censored graft loss in a multivariable Cox model (Supplementary Table S6). Thus, BL patients who develop late AR, and especially those smaller groups that later develop IFTA or AR with MVI, are at significantly increased risk for graft loss. A total of 38% of patients with BL were treated—29% received corticosteroids, and 9% had dose escalation of tacrolimus or mycophenolate. Two patients received plasmapheresis with i.v. Ig (Supplementary Figure S5A). Interestingly, neither the degree of inflammation on the index biopsy (not shown) nor the diagnosis of cBL versus scBL (Supplementary Figure S5B) impacted treatment choice. Moreover, these treatment measures did not reduce the incidence of either AR or chronic damage on late biopsies, nor did they improve long-term death-censored graft survival (Supplementary Figure S5C and D; Supplementary Figure S6). To determine whether baseline clinical or histologic features could provide early prognostic insight into BL, we evaluated 41 independent features (Supplementary Table S1) using T-distributed stochastic neighborhood embedding, an unsupervised nonlinear dimensionality reduction algorithm. Based on shared features, this approach grouped BL patients into 5 distinct neighborhoods (N1–N5; Figure 6a). It is important to note that baseline histologic characteristics were not the distinguishing features of these neighborhoods (Supplementary Table S7). Neighborhood N1 (n = 11) predominantly comprised presensitized female recipients of high-KDPI deceased-donor allografts with prolonged cold ischemia time (CIT; Supplementary Table S7; Figure 6b–d). N2 (n = 125) was similar to N1 (high-KDPI deceased-donor allografts with prolonged CIT) but lacked the female predominance. In contrast, N3 (n = 44) predominantly contained living-donor recipients with very short CIT, whereas N4 (15 patients) comprised predominantly Caucasian recipients of living-donor kidneys. Finally, N5 (22 patients) primarily contained older recipients (Supplementary Table S7; Figure 6b–d). Therefore, N1 and N2 were relatively comparable and distinct from N3–N5, and neighborhoods were grouped accordingly to allow meaningful statistical analysis (Figure 6e). Compared to N3–N5, the N1–N2 group exhibited a greater incidence of late AR and a lower incidence of late biopsies without significant inflammation (Figure 6f). Furthermore, the N1/N2 group had worse IFTA and significantly worse 7-year death-censored graft survival (78% vs. 86%, P = 0.03; Figure 6g and h). Of note, the very small N1 neighborhood had lowest graft survival (60%) of all neighborhoods (Supplementary Figure S7). Thus, T-distributed stochastic neighbor embedding analysis identified a high-risk BL subgroup defined by deceased-donor transplants, longer CIT, worse KDPI, and higher rates of presensitization—already well-established risk factors for poor outcomes. Although T-distributed stochastic neighbor embedding provides early risk stratification, its performance is inferior to late risk stratification using progression to late AR (which identifies patients with 72% vs. 87% 7-year graft survival). When T-distributed stochastic neighbor embedding analysis of the same features was performed in NI patients, 4 patient neighborhoods (N1–N4) were identified (Supplementary Figure S8A). N1 comprised predominantly living-donor recipients, and N2–N4 were characterized by deceased-donor recipients with long CIT and higher KDPI (Supplementary Figure S8B–D). Surprisingly, categorization based on CIT, KDPI, and donor type that risk-stratified BL patients did not differentiate the clinical course of NI patients in terms of late rejection, IFTA, or death-censored graft survival (Supplementary Figure S8E and F). Taken together, these findings highlight a key interaction of these clinical features, which are commonly associated with decreased graft survival. When present with early BL, they increase the risk for progressive inflammation, premature scarring, and graft loss. However, in patients without early inflammation, these same features have no discernable impact on clinical course. B-cell subsets or their cytokine expression can predict transplant outcomes.7Cherukuri A. Salama A.D. Mehta R. et al.Transitional B cell cytokines predict renal allograft outcomes.Sci Transl Med. 2021; 13: eabe4929Crossref PubMed Scopus (19) Google Scholar,15Cherukuri A. Mohib K. Rothstein D.M. 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