High numbers of differentiated CD28null CD8+ T cells are associated with a lowered risk for late rejection and graft loss after kidney transplantation.

BACKGROUND: The hypothesis was tested that parameters of an aged T-cell compartment associate with the risk for late rejection after kidney transplantation.
METHODS: Recipients of a kidney transplant in the period 2007-2013 were (N = 365) were included. T cells were characterized prior to transplantation by flow cytometry as naive (CD45RO-CCR7+), central-memory (CD45RO+CCR7+), effector-memory (CD45RO-CCR7-) or terminally differentiated CD8+ Temra (CD45RO-/CCR7-/CD28-) cells. T cell telomere length and thymic output were assessed prior to transplantation in 202 recipients. Follow-up was until December 2018. The date of the first time of biopsy-proven late rejection (>6 months after transplantation) was used to calculate the rejection-free survival time.
RESULTS: Fifty cases of biopsy-proven rejection were recorded. Thymic output and T cell telomere length did not associate with late rejection-free survival. However, the percentage and absolute numbers of CD8+Temra and CD28null CD8+ T cells were significantly lower in patients with late rejection. Specifically, in the highest tertile of percentages of CD28null CD8+ T cells, the cumulative incidence of late rejection at 5 and 10 years was only 5% and 8% compared to 16% and 20% in the middle to lowest tertile (p = 0.002). Multivariate proportional hazard analysis showed that percentage and absolute number of CD28null CD8+ T cells remained significantly associated with late rejection and rejection-related graft loss.
CONCLUSION: High numbers of differentiated CD28null CD8+ T cells decrease the risk for late rejection and rejection-related graft loss after kidney transplantation.

Introduction

Progressive loss of renal function leading to end-stage renal disease (ESRD) is associated with premature ageing of the T-cell system. The pro-inflammatory environment resulting from loss of renal function results in a lower thymic output, increased T-cell differentiation, telomere shortening and skewing of the T cell receptor (TCR) repertoire[1-7].

The changes in the peripheral T-cell compartment of ESRD patients resemble the physiological changes in the immune system of healthy elderly individuals with the immunological T-cell age of ESRD patients on average being increased by 15–20 years compared to their chronological age[1].

Increasing age of the recipient decreases the risk for acute rejection after kidney transplantation, which is believed to be a result of an age-related less alloreactive immune system[8, 9]. We have investigated the relation between several immunological parameters of ageing (thymic output, T cell telomere length and T cell differentiation status) and early rejection of the kidney allograft. Only T cell differentiation status appeared to be associated with acute rejection after kidney transplantation[10-12]. Unexpectedly, in particular the presence of large numbers (both in percentage as absolute number) of T cells without surface expression of CD28 was associated with a decreased risk for allograft rejection[10, 12]. The CD28 molecule is a pivotal part of an important co-stimulatory pathway involving interaction with CD80/CD86 on antigen-presenting cells[13, 14].

The CD28null T cells are mainly present in the circulating memory T cell compartment and relatively few are found in the lymph nodes[15]. Their presence within the classical T cell subsets increases with progressive T cell differentiation. For example, all naïve T cell are CD28 positive while the vast majority of highly differentiated Temra cells has lost CD28 expression on their cell surface. As a consequence, CD28null T cells need exogenous cytokine signals like IL-15 and IL-21 to become activated and start proliferation after T cell receptor-allogeneic HLA interaction[16-18].

Currently, it is recognized that late rejection is a major cause of allograft loss in the long-term and the adequate treatment of this condition is considered an unmet need in the field of kidney transplantation[19-21]. Whether the status of the immune system of the recipient is a significant risk factor for late allograft rejection is currently unknown. To address this question we analyzed the status of the T cell system prior to transplantation, in particular the presence of CD28null T cells, with the incidence of late rejection.

Patients and methods

Patients

For this study we combined the datasets of 2 previous studies with at least 6 months follow-up after transplantation, including recipients (n = 365) of a kidney transplant within the period 2007–2013 at our transplantation center. The first dataset included consecutive recipients (n = 158) of a kidney transplant in the period 1 January 2007 to 1 December 2009 of whom the peripheral T cells were immunophenotyped before transplantation[10]. This study was approved by the Medical Ethical Committee of the Erasmus MC (MEC-2007-228). The second dataset included recipients (n = 207) that participated in a randomized-controlled clinical trial with the primary aim to study the efficacy of a genotype-based approach to tacrolimus dosing[22]. All patients undergoing a living-donor kidney transplantation (KT) in the period from 1 November 2010 to 1 October 2013 were considered for participation in this study. Donor kidneys were procured by Eurotransplant in case of deceased donors kidneys and the Erasmus MC for living donor kidney transplantations.

The trial and sub study involving the immunophenotyping of peripheral blood T cells was approved by the Medical Ethical Committee of the Erasmus MC (MEC-2010–080). All patients gave written informed consent to participate in the studies and it was conducted in accordance with the Declaration of Helsinki and the Declaration of Istanbul. Patients were excluded if they were younger than 18 years and if they received immunosuppressive medication (except for glucocorticoids) within 28 days prior to transplantation. None of the transplant donors was from a vulnerable population and all donors or next of kin provided written informed consent that was freely given. Consent for donation and donor registration in case of postmortem donation was obtained and regulate as required by national legislation within the Eurotransplant region (www.eurotransplant.org under legislation/eurotransplant). In our cohort, sixty percent of deceased donor kidneys were from deceased by cardiac death donors and 40% from deceased by brain death donors. The informed consent form for living kidney donors (translated in English) is shown in S1 Fig.

All medical costs were covered by the medical insurance.

Initial maintenance immunosuppression with tacrolimus, mycophenolate mofetil and glucocorticoids was given in >90% of patients (Table 1 As per protocol the following dosages and through levels were used: tacrolimus aiming for predose concentrations of 10–15 ng/mL in weeks 1–2, 8–12 ng/mL in weeks 3–4, and 5–10 ng/mL, thereafter, mycophenolate mofetil starting dose of 1 g b.i.d., aiming for predose concentrations of 1.5–3.0 mg/L. All patients received 50 mg prednisolone b.i.d. intravenously on days 0–3. Thereafter, 20 mg oral prednisolone was started and subsequently tapered to 5 mg at month 3.

Table 1

Clinical and demographical characteristics of kidney transplant recipients prior to transplantation.

	No late rejection (n = 315)	Late rejection (n = 50)	p-value
Age in years, median (range)	55 (18–79)	51 (19–78)	0.004
Male/female	205/110	31/18	0.9
Follow-up time in months, median (IQR)	82 (7–137)	71 (8–137)	0.03
Time to diagnosis of late rejection, months median (range)	-	44 (7–112)
Living kidney donor (% within group)	88.6%	91.8%	0.5
Previous kidney transplant	9.9%	24.5%	0.02
Pre-emptive transplantation	48.7%	20.4%	0.007
Early rejection (within first 6 months after KT)	17.5%	32.7%	0.02
PRA at time of transplantation (average with SD)	2.9% (10.3)	5.4% (17.8)	0.3
PRA peak serum (average with SD)	8.8% (19.4)	16.4% (17.7)	0.07
Total number of HLA mismatches (mean)	3.6	3.4	0.4
Induction therapy:			0.2
• Basiliximab	59.4%	44.9%
• ATG	2.9%	4.1%
Maintanance immune suppression			0.2
• tacrolimus/mycophenolate/prednisone	95.5%	91.8
• other	4.5%	8.2%
Distribution of underlying kidney disease			0.8
• Nephrosclerosis/hypertension	27.0%	26.5%
• Primary glomerulopathies	19.4%	18.74%
• Diabetes mellitus	17.1%	10.2%
• Urinary tract infections/ stones	1.6%	2.0%
• Reflux nephropathy	5.4%	8.2%
• Polycystic kidney disease	15.6%	14.3%
• Other	8.6%	14.3%
• Unknown	5.4%	6.1%
CMV seropositive*	65.3%	61.2%	0.6
Type of late rejection
• ABMR		39
• TCMR		5
• Mixed rejection		6

*CMV seropositive: detectable serum antibodies against cytomegalovirus at time of transplantation

The HLA-typing was assessed according to the international standards (American Society for Histocompatibility and Immunogenetics/the European Federation for Immunogenetics) using serologic and DNA-based techniques. The panel reactive antibodies (PRA) were determined at the laboratory of the blood bank in Leiden, the Netherlands.

All transplantations were ABO-compatible with a negative complement dependent cross match. Flowcytometry based cross matches were not performed and the presence of donor-specific anti-HLA antibodies by Luminex was not regularly assessed.

Late rejection was defined as biopsy-proven allograft rejection diagnosed at least 6 months after KT using the Banff criteria 2015. Graft loss was defined as the need for dialysis or retransplantation. The last day of follow-up was December 1, 2018.

PBMC isolation

By using Ficoll-Paque Plus (GE healthcare, Uppsala, Sweden), peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood samples. Blood was drawn from KT-recipients the day before KT. Isolated PBMCs were stored at -150°C with a minimum amount of 10×106 cells per vial until further analysis.

T cell differentiation status and absolute numbers of T cell subsets

To determine the T-cell differentiation status, staining for CD4 and CD8 was combined with the differential expression of CCR7 and CD45RA to identify naïve and memory T cells as has been described before in detail. Memory T cells were further subdivided into central-memory T cells, effector-memory T cells and the most differentiated Temra cells[23, 24]. In addition, CD28 expression was measured on T cells, which allows for a clear distinction between CD28 positive and negative T cells[25, 26].

Relative telomere length (RTL) and recent thymic emigrants

Only for the recipients of a kidney transplant after 2010 (n = 202), the RTL and number of recent thymic emigrants (RTE) were assessed. To determine the RTL of CD4+ and CD8+ T cells, flow fluorescent in situ hybridization was performed as described in detail previously[1]. RTE were defined as CD31-expressing naïve T cells[27].

Statistics

The primary outcome of this study was the incidence of late rejection after kidney transplantation.

The difference between continuous variables was assessed with the Mann–Whitney U test. The difference between categorical variables was analyzed either with the Pearson’s chi-squared test or with the Fisher’s exact test depending on the expected values in any of the cells of a contingency table. Univariate and multivariate Cox proportional hazard analysis was used to assess the association between immunological and clinical parameters, and the outcome late biopsy proven rejection. Clinical and immunological parameters that had a p-value of <0.1 in the univariate analysis were tested in the multivariate Cox regression analysis using forward and backward modeling. Kaplan-Meier survival curves were made for late rejection and graft loss, dividing the patients into groups according to the tertile of either percentage or absolute number of CD8CD28null T cells before transplantation.

The significance level (p-value) was two-tailed and an α of 0.05 was used for all analyses. Statistical analyses were performed using SPSS® version 21.0 for Windows® (SPSS Inc., IL, USA) and GraphPad Prism 5 (CA, USA). Figures were created with GraphPad Prism 5 (CA, USA).

Results

Recipients characteristics and late allograft rejection

Recipients characteristics are shown in Table 1. Fifty recipients (median age 51 year, range 19–78) were diagnosed with a late rejection at a median of 44 months after transplantation (range 7–112 months). The group of recipients with late rejection differed from the no late rejection group with respect to age at transplantation (younger), a previous kidney allograft (more frequently), an early acute rejection (more frequently) and the frequency of renal replacement therapy before kidney transplantation (more often). Twenty recipients lost their allograft because of late rejection.

Pre-transplant T cell ageing parameters and late allograft rejection

The average distribution of different T cell subsets given as percentages of the total of circulating CD4 or CD8 T cells, is shown in Table 2. The recipient group with late rejection had a significantly higher percentages of naïve CD4 and CD8 T cells, and lower percentages of the CD8 effector-memory and terminally differentiated Temra cells. The latter finding corresponded with a significant lower percentage of CD28null CD8 T cells. Of note, the relative telomere length and number of CD31 positive naïve T cells (reflecting thymus output of T cells) was not significantly different between both groups (Table 2).

Table 2

Characteristics of circulating T cells with subsets given in percentages prior to transplantation in relation to biopsy-proven late kidney rejection.

	no late rejection (n = 315)	late rejection (n = 50)	p-value
Naive CD4 T cells %	30.6 ± 0.9	36.0 ± 2.4	0.03
CD31pos naïve CD4 T cells* %	64.5 ± 1.1	67.6 ± 3.8	0.39
Central memory CD4 T cells %	37.7 ± 0.8	33.7 ± 1.9	0.07
Effector memory CD4 T cells %	25.1 ± 0.8	21.6 ± 2.0	0.10
CD28null CD4 T cells %	6.4 ± 0.6	4.6 ± 1.0	0.23
Naïve CD8 T cells%	22.7 ± 1.1	30.9 ± 2.8	0.005
CD31pos naïve CD8 T cells* %	95.4 ± 0.5	95.0 ± 1.2	0.78
Central memory CD8 T cells %	7.2 ± 0.3	8.1 ± 1.0	0.31
Effector memory CD8 T cells	29.3 ± 0.9	24.3 ± 2.3	0.04
Temra CD8%	30.8 ± 1.1	24.5 ± 2.3	0.02
CD28null CD8 T cells%	41.6 ± 1.3	32.5 ± 2.7	0.003
Relative telomere length CD4 T cells*	12.7 ± 0.4	12.5 ± 0.4	0.93
Relative telomere length CD8 T cells*	12.5 ± 0.9	11.7 ± 1.0	0.44

The absolute numbers of different T cells subsets (Table 3) revealed that the significant shifts in CD8 T cell subsets in the late rejection group could be attributed to an expansion of highly differentiated T cells as reflected by higher numbers of Temra cells and CD28null CD8 T cells. Average absolute numbers of naïve T cells were similar between the no late rejection and late rejection groups, underlying the importance of absolute cell counts for correct interpretation of relative shifts in T cell subsets.

Table 3

Circulating numbers of T cell subsets in cells/μl prior to transplantation in relation to late kidney rejection.

	no late rejection (n = 315)	late rejection (n = 50)	p-value
Naive CD4 T cells	142 ± 11	102 ± 23	0.16
Central memory CD4 T cells	240 ± 12	206 ± 20	0.15
Effector memory CD4 T cells	149 ± 8	120 ± 11	0.17
CD28null CD4 T cells	40 ± 4	31 ± 7	0.34
Naïve CD8 T cells	51 ± 5	50 ± 12	0.92
Central memory CD8 T cells	83 ± 7	116 ± 9	0.92
Effector memory CD8 T cells	25 ± 2	25 ± 3	0.13
Temra CD8	145 ± 10	85 ± 11	0.0001
CD28null CD8 T cells	179 ± 13	104 ± 16	0.0003

Multivariate analysis of CD28null T cells and late allograft rejection

Clinical and immunological parameters, which were significant different between the late rejection and no late rejection group were used in a multivariate Cox regression analysis. Of the immunological parameters only the percentage and the absolute number of CD28null CD8 T cells remained significantly independent associated with late rejection (Table 4).

Table 4

Multivariate cox regression analysis for outcome late rejection after transplantation.

Multivariate model with clinical parameters and percentages of T cell subsets
	Hazard ratio	95% confidence interval	p-value
Previous transplantation	2.19	1.12–4.34	0.024
Dialysis before transplantation	2.23	1.09–4.58	0.028
Early acute rejection	2.01	1.07–3.75	0.029
% CD28null CD8 T cells	0.98	0.96–0.99	0.007
Multivariate model with clinical parameters and absolute numbers of T cells subsets
Previous transplantation	2.21	1.12–4.35	0.022
Dialysis before transplantation	2.16	1.05–4.36	0.035
Early acute rejection	2.12	1.13–3.95	0.019
CD28null CD8 T cells/ul	0.99	0.94–0.99	0.036

Kaplan-Meier curves for late rejection-free survival were made according to the tertile of percentage (Fig 1A) or absolute number (Fig 1B) of CD28null CD8 T cells.

Fig 1

CD28 null CD8 T cells prior to transplantation and risk for late rejection.

Tertiles of CD28null CD8 T cells in percentage (A) and absolute number of cells (B) in relation to the cumulative hazard for late rejection after transplantation. P-values for difference between different strata were calculated by log-rank statistical analysis.

In particular the recipients falling in the highest tertile of CD28null CD8 T cells had the lowest incidence of late allograft rejection.

In the highest tertile of percentages of CD28null CD8+ T cells, the cumulative hazard of late rejection at 5 and 10 years was only 4% and 8% compared to 16% and 22% in the lowest tertile (p = 0.002).

This finding correlated with the risk for allograft loss because of late rejection, which was very low in recipients with a high percentage of CD28null CD8 T cells prior to transplantation (Fig 2, lowest versus highest tertile p-value = 0.001).

Fig 2

CD28null CD8 T cells prior to transplantation and late rejection-related graft loss.

Tertiles of percentage of CD28null CD8 T cells and late rejection-related graft loss after transplantation. Differences between the Kaplan-Meier survival curves for the different strata were calculated by log-rank statistical analysis.

Multivariate regression analysis showed that both the tertile (HR 0.64 (95% CI 0.41–0.99), p = 0.04) and the % of CD28null CD8 T cells (HR 0.98 (95% CI 0.96–0.99), p = 0.02) were significantly associated with allograft loss because of late rejection.

Discussion

In this study the hypothesis was tested that increased immunological ageing at the time of renal transplantation carries a lower risk for late allograft rejection. The results show that specifically increased numbers of CD28null CD8 T cells are associated with a lower frequency of late rejection and graft loss because of late rejection.

At present we have no obvious explanation for the inverse relation between an expanded pool of CD28null CD8 T cells and less late rejection. This relation appears to be independent of other markers of T cell ageing such as telomere length and thymus function, indicating a specific role of CD28null CD8 T cells in rejection. The data are in line with previous studies showing a similar relation of highly differentiated T cells with early acute rejection[10, 12]. Also, a lower frequency of CD28null CD8 T cells after alemtuzumab as induction therapy was associated with late rejection[28].

Alloreactive T cells can be identified in the CD28null T cell population and these cells are able to proliferate, but only in the presence of exogenous IL-15 or IL-21[16]. The CD28null T cells are predominantly present in the peripheral blood and only at low frequencies in the lymph nodes, consistent with the absence of the lymph node homing receptor CCR7 on these cells[15]. Given these findings it is most likely that CD28null T cells directly migrate into tissues like the renal allograft. However, as more of these cells appear to protect against rejection their modus of operandi may actually be of a CD8 suppressor/regulatory T cell as identified by different studies[13]. Direct suppressor function of CD28null CD8 T cells on alloreactive T cell proliferation in vitro could not be shown by our group[10], but an indication of suppressor function for alloreactive CD4 T cells was shown by Trzonkowski et al[28]. Most in vitro studies on CD8 T cells suppressor function use some time of stimulation rather than a direct use of cells. Reasoning along the same line, some time period of stimulation in the tissues may be needed for full development of suppressor function.

Of note, this cell population may expand after CMV infection providing a potential confounder[29]. However, in this study the prevalence of a positive CMV serostatus was similar for both late rejection and non-rejecting groups.

The diagnosis of late rejection was made after a median time period of 44 months post-transplantation. Therefore, the dynamics of CD28null T cells after transplantation is of interest but there are only few studies that have studied this. Our group found very stable levels of these cells within the first year after kidney transplantation[30] and a similar stability of highly differentiated CD8 T cells was found at a prolonged time after transplantation[31]. In the latter study, a higher percentage of CD28null CD8 T cells was associated with increased risk for squamous cell cancer again indicative of a decrease in T cell immunity.

Of interest is the observation that after T cell depleting therapy the highly differentiated memory T cells are among the first to repopulate within the circulation[28]. This further underlines that CD8 CD28null T cells are not per se dangerous to the graft and may actually suppress T cell alloreactivity. Most likely the CD28null CD8 T cells are a heterogeneous cell population containing both effector cells and suppressor cells[13].

The strength of the current study is the number of recipients forming a relatively homogenous group largely transplanted with a kidney from a living donor and receiving the same immune suppressive drugs without initial depleting T cell therapy. In addition, the duration of follow-up and number of events is adequate for meaningful analysis. However, the potential weakness is that the present data do not allow for extrapolation of the findings to e.g. other immune suppressive drugs regimens and deceased donor kidney recipients. Also, there is no additional information on markers of senescence on CD28null T cells, which could have given more insight into the type of cell subset involved. Validation of the findings in a different cohort is of course essential but this will require a similar large number of recipients with immunophenotyping before transplantation and a follow-up of at least 5–10 years.

In conclusion, recipients with high numbers of CD28null CD8 T cells prior to transplantation are at a lower risk for late allograft rejection. Late allograft rejection contributes significantly to graft loss in the long term and prevention and treatment is considered an unmet need in transplantation. This is the first study showing that recipients may actually have an immunological profile protecting them from this threatening long-term complication. This knowledge may be used to guide tapering of immune suppressive medication and warrants further studies to elucidate the underlying mechanisms involved.

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Informed consent kidney donation by living donor.

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14 Oct 2019

PONE-D-19-20193

High numbers of differentiated CD28null CD8+ T cells are associated with a lowered risk for late rejection and graft loss after kidney transplantation

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Reviewer #1: In this study the authors characterized the T cell population prior to kidney transplantation in a cohort of 365 patients who received a kidney transplant form a living donor in the period 2007-2013 with the hypothesis that parameters indicative of an aged T-cell population associate with a lower risk for late rejection.

They found that the number and percentage of differentiated T cells were lower in patients with biopsy-proven late rejection (n. 50, diagnosed at a median of 44 months) and were independently and inveresely associated with rejection occurrence.

The study is well-conducted and the main strength is that a relatively large and homogeneous cohort of patients was analysed, with a relatevely long follow-up (median 70-80 months).

In my opinion, there are some points to be elucidated:

- were all the biopsies performed on clinical indication?

- was there a correlation between the T cell population before the transplantation and the type of rejection?

- The data about DSA pre-transplantation is missing; this would l be an important information in the evaluation of the immunological status of the recipients. Most of the rejection (39/50) were categorized as ABMR: were DSA searched at the time of the kidney biopsy? If so, it would be appropriate to evaluate the correlation beetwen DSA and T cell population before the transplantation, and the association of DSA with graft loss.

- in the Kaplan-Meier curves (Fig 1A, 1B and 2) the number of patients at risk should be reported

- In the Kaplan-Meier analysis for late rejection-related graft loss (Fig 2) the time considered should be the time after the rejection diagnosis, not the time after transplantation

- A multivariate analysis for graft loss would be needed

- page 11, line 176 should be "....had a significantly HIGHER percentage s of naive CD4 and CD8 T cells and LOWER percentages of the CD8 effector..."

- page 11, line 178 should be: "...corresponded with a significant LOWER percentage of CD28null CD8 T cells.."

Reviewer #2: This is a retrospective study in kidney transplant recipients to determine if there is an association between T cell telomere length and T cell subset analysis (CD28 and CD8) correlated with late (>6 months) acute rejection. Several questions arise:

1. The overall population is of relatively immunologic risk - unsensitized, living donors. Despite this, the "late rejection" group had a very high early acute rejection rate -33%. Additionally, nearly 20% of the entire population had a late acute rejection. Can you explain the very high rate of early and late acute rejection in this low risk group

2. No data is supplied regarding immunosuppressive levels at any time point post-tx. Given the very high rejection rates, this is an important variable that must be accounted for in your multi-variant analysis.

3. There is a very high rate of anti-body mediated rejection compared to cell mediated rejection. Please explain

4. Do you have data on development of donor specific antibody formation.

5. Nearly 40% did not receive any induction therapy. How was induction therapy chosen?

6. Can you expound on why telomere length had no influence on long-term outcomes?

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Reviewer #1: No

Reviewer #2: No

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12 Nov 2019

PLOS ONE

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2. We note that your study involved tissue/organ transplantation. Please provide the following information regarding tissue/organ donors for transplantation cases analyzed in your study.

1. Please provide the source(s) of the transplanted tissue/organs used in the study, including the institution name and a non-identifying description of the donor(s).

Eurotransplant in case of deceased donors kidneys and Erasmus MC for living donor kidney transplantations. This information is now provided in the revised manuscript.

2. Please state in your response letter and ethics statement whether the transplant cases for this study involved any vulnerable populations; for example, tissue/organs from prisoners, subjects with reduced mental capacity due to illness or age, or minors.

- If a vulnerable population was used, please describe the population, justify the decision to use tissue/organ donations from this group, and clearly describe what measures were taken in the informed consent procedure to assure protection of the vulnerable group and avoid coercion.

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A vulnerable population was not used and the required ethics statement is provided in the revised manuscript.

3. In the Methods, please provide detailed information about the procedure by which informed consent was obtained from organ/tissue donors or their next of kin. In addition, please provide a blank example of the form used to obtain consent from donors, and an English translation if the original is in a different language.

This procedure is now given in the Methods by referring to the legislation within the Eurotransplant region (Eurotransplant.org under legislation/eurotransplant) and the informed consent form for donors (translated in English) is included.

4. Please indicate whether the donors were previously registered as organ donors. If tissues/organs were obtained from deceased donors or cadavers, please provide details as to the donors’ cause(s) of death.

All deceased donor kidneys were allocated via Eurotransplant and donor registration was regulated per country according to national legislation. In our cohort, sixty percent of deceased donor kidneys were from deceased by cardiac death donors and 40% from deceased by brain death donors. This information is now given in the Methods section.

5. Please provide the participant recruitment dates and the period during which transplant procedures were done (as month and year)

The period during which the transplant procedures were performed is now provided in the revised manuscript.

6. Please discuss whether medical costs were covered or other cash payments were provided to the family of the donor. If so, please specify the value of this support (in local currency and equivalent to U.S. dollars).

All medical costs were covered by the medical insurance. This information is now provided in the revised manuscript.

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Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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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: In this study the authors characterized the T cell population prior to kidney transplantation in a cohort of 365 patients who received a kidney transplant form a living donor in the period 2007-2013 with the hypothesis that parameters indicative of an aged T-cell population associate with a lower risk for late rejection.

They found that the number and percentage of differentiated T cells were lower in patients with biopsy-proven late rejection (n. 50, diagnosed at a median of 44 months) and were independently and inveresely associated with rejection occurrence.

The study is well-conducted and the main strength is that a relatively large and homogeneous cohort of patients was analysed, with a relatevely long follow-up (median 70-80 months).

In my opinion, there are some points to be elucidated:

- were all the biopsies performed on clinical indication? Yes

- was there a correlation between the T cell population before the transplantation and the type of rejection? No

- The data about DSA pre-transplantation is missing; this would l be an important information in the evaluation of the immunological status of the recipients. Most of the rejection (39/50) were categorized as ABMR: were DSA searched at the time of the kidney biopsy? If so, it would be appropriate to evaluate the correlation between DSA and T cell population before the transplantation, and the association of DSA with graft loss.

We agree with this comment and data about DSA pre-transplantation and of most patients at time of biopsy would be of value. Unfortunately, this information is largely absent as DSA were not structurally assessed before and at time of rejection within the time period of this study. This is stated in the manuscript.

- in the Kaplan-Meier curves (Fig 1A, 1B and 2) the number of patients at risk should be reported

This is now provided in the KM curves in the revised manuscript.

- In the Kaplan-Meier analysis for late rejection-related graft loss (Fig 2) the time considered should be the time after the rejection diagnosis, not the time after transplantation

The research question is whether pre-transplant numbers of CD28null T cells are related to late rejection and rejection-related graft loss. Therefore, we considered time after transplantation to graft loss. If we would take time after rejection to graft loss then we would study the question whether the numbers of CD28null T cells are associated with progression to graft loss after rejection is diagnosed. This is obviously a different approach and would test the hypothesis that CD28null T cells can mitigate or worsen the clinical course of rejection. We did this analysis but could not find such an effect.

- A multivariate analysis for graft loss would be needed

We now have included the results of the multivariate analysis for rejection-related graft loss in the results section of the revised manuscript.

- page 11, line 176 should be "....had a significantly HIGHER percentage s of naive CD4 and CD8 T cells and LOWER percentages of the CD8 effector..."

- page 11, line 178 should be: "...corresponded with a significant LOWER percentage of CD28null CD8 T cells.."

These errors have now been corrected.

Reviewer #2: This is a retrospective study in kidney transplant recipients to determine if there is an association between T cell telomere length and T cell subset analysis (CD28 and CD8) correlated with late (>6 months) acute rejection. Several questions arise:

1. The overall population is of relatively immunologic risk - unsensitized, living donors. Despite this, the "late rejection" group had a very high early acute rejection rate -33%. Additionally, nearly 20% of the entire population had a late acute rejection. Can you explain the very high rate of early and late acute rejection in this low risk group

The group of recipients has a mixed profile for their immunological risk as panel reactive antibodies were present in some recipients (either at time of transplantation or historically) and re-transplantations were performed. The % of early rejection diagnosed within the whole population is not that different from data obtained from registries which is always higher than most clinical trials with study drugs including low-immunological risk patients only.

For instance, in a recent paper from the ANZDATA registry the % early AR was 21% in a large cohort of first kidney transplantations and a significant risk factor for late rejection (Clayton et al, J Am Soc Nephrol. 2019 Sep;30(9):1697-1707).

The percentage of late rejections (which is not synonymous with acute rejection) is a cumulative percentage obtained at a rather long follow-up. Patients transplanted at our center have their regular visits at their referral medical center but are once-yearly seen by a transplantation-nephrologist in our center. When a steady decline in renal allograft function is observed we perform a diagnostic kidney biopsy to rule out late (chronic) rejection as a cause. In this way we try to diagnose all cases of late rejection which yields a much higher number of late rejections than most studies with a low % of diagnostic biopsies late after transplantation (e.g. Naesens et al, Transplantation. 2014 Aug 27;98(4):427-35)

2. No data is supplied regarding immunosuppressive levels at any time point post-tx. Given the very high rejection rates, this is an important variable that must be accounted for in your multi-variant analysis.

This is a very difficult question to address. The large majority of patients were given tacrolimus in combination with MMF and prednisone within the first 6 months after transplantation with through levels for tacrolimus by protocol. This protocol is now mentioned within the revised manuscript. Thereafter the data on through levels are incomplete as most patients were seen in their referral nephrology center and combinations of immune suppressive drugs could be changed. In addition, emerging data strongly suggest that the degree of intra-individual variability is a much stronger predictor of graft survival than through levels only. In a recent paper we did analyze the through levels and IPV in patients with late humoral rejection in great detail (Sablik et al, Transplant Int 31: 900-908, 2018 ) and found that the average tacrolimus through level was 6 which is above the general recommended through level of 5 ug/ml.

3. There is a very high rate of anti-body mediated rejection compared to cell mediated rejection. Please explain

Late rejection is frequently caused by anti-body mediated rejection and in the years after transplantation the incidence of cell mediated rejection becomes (very) low.

4. Do you have data on development of donor specific antibody formation.

Unfortunately, we have only data on DSA in the minority of patients before and after kidney transplantation as DSA assessment was not routinely performed. This is stated in the manuscript.

5. Nearly 40% did not receive any induction therapy. How was induction therapy chosen?

Within the study period, the use of anti-CD25 antibody was not yet the standard induction therapy. Induction with anti-CD25 antibody or a T cell depleting agent was done when the risk for acute rejection was thought to be high (e.g. high %PRA, high number of MM, frequent rejections in the past or with a repeated mismatch with a previous transplant).

6. Can you expound on why telomere length had no influence on long-term outcomes?

Telomere length is assessed on the whole populations of CD4 and CD8 T cells. It has a significant but variable relation with the recipient age and therefore is a “bulk” marker of immunological ageing. The association of an expanded CD28null T cell population and less rejection seems to be more cell specific and, although speculative, may indicate a role of e.g. suppressor CD8 T cells. This has been stated in the discussion of the manuscript.

________________________________________

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This has been performed.

Submitted filename: response to the reviewers.docx

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27 Nov 2019

PONE-D-19-20193R1

High numbers of differentiated CD28null CD8+ T cells are associated with a lowered risk for late rejection and graft loss after kidney transplantation

PLOS ONE

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Reviewer #1: (No Response)

Reviewer #2: This is a revised manuscript of a retrospective study in kidney transplant recipients to determine if there is an association between T cell telomere length and T cell subset analysis (CD 28 and CD 8) correlated with late (>6 months) acute rejection.

I appreciate your response to both reviewers. Several questions still exist:

1. It is still not clear why there is a high rate of acute rejection is a very low immunological risk population. The overwhelming majority of recipients were unsensitized, living donor recipients. Acute rejection rates of 19.5% at 6 months in this group is still far higher than data from the Europenan EKiTE registry (Lorent, M., Foucher, Y., Kerleau, K. et al. The EKiTE network (epidemiology in kidney transplantation - a European validated database): an initiative epidemiological and translational European collaborative research. BMC Nephrol 20, 365 (2019) doi:10.1186/s12882-019-1522-8) showing a 79% 2 year rejection free in a population consisting of 81% deceased donors with a large portion that were sensitized or repeat transplants.

2. How was the diagnosis of "late" acute rejection made? You mention that late rejections are not synonymous with acute rejection

3. You note that late rejections are most often antibody mediated but you have limited DSA data. Was the diagnosis of AMR made exclusively on the basis of C4D?

4. The lack of immunosuppressive drug levels is a significant shortcoming

5. The selection of induction agents remains unanswered. The choice of anti-DC25 or ATG is a very different class of agents vs IL2-RA. The immunologic effect can be very different on your T cell subsets. Have you analyzed these groups separately to determine if there is any difference in outcomes.

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3 Jan 2020

Dear editor,

The comments of reviewer #2 are adressed in a point-by-point fashion as shown below.

We hope the manuscript is now suitable for publication.

Sincerely,

Dr Michiel Betjes

I appreciate your response to both reviewers. Several questions still exist:

1. It is still not clear why there is a high rate of acute rejection is a very low immunological risk population. The overwhelming majority of recipients were unsensitized, living donor recipients. Acute rejection rates of 19.5% at 6 months in this group is still far higher than data from the Europenan EKiTE registry (Lorent, M., Foucher, Y., Kerleau, K. et al. The EKiTE network (epidemiology in kidney transplantation - a European validated database): an initiative epidemiological and translational European collaborative research. BMC Nephrol 20, 365 (2019) doi:10.1186/s12882-019-1522-8) showing a 79% 2 year rejection free in a population consisting of 81% deceased donors with a large portion that were sensitized or repeat transplants.

We agree with the reviewer that in our patient population a relatively high rate of acute rejection is observed. The reasons are difficult to identify as we use a triple immune suppressive regimen including prednison/MMF/tacrolimus with adequate through levels defined by protocol (see M&M). Comparing patient populations is always difficult with potential unrecognized bias. For instance, the EKiTE data show T cell depletion as induction therapy in 37% of recipients while we rarely give this kind of induction therapy in the Netherlands. In other countries like the USA this percentage can be as high as 70% in some centres. On the other hand, publications from Japan and Korea show a very low AR percentage without T cell depletion and a standard IS regimen.

Virtually all our episodes of rejection are biopsy proven. In other studies, underreporting of rejection may occur as no biopsy was done before anti-rejection therapy. Our kidney biopsies are scored by a board-certified renal pathologist but we cannot rule out the possibility that our pathologists are leaning more to a diagnosis of rejection (for instance in cases of borderline acute rejection) than their colleagues in other centres.

In summary, we cannot satisfactorily address this comment but can only speculate on possible explanations. Of note, despite a relatively high AR rate the one-year graft and patient survival of our living donor kidney transplant program is excellent (>98%).

2. How was the diagnosis of "late" acute rejection made? You mention that late rejections are not synonymous with acute rejection

Late rejections are by definition occurring 6 months (admittedly our definition but a commonly used definition) after kidney transplantation. A rapid decline in renal function with a biopsy showing little chronicity but predominantly an influx of immune cells occurring many months after transplantation is compatible with an acute rejection and can be observed when e.g. patients are significantly under immune supressed. However, in most cases there is a progressive decrease in eGFR loss over a period of weeks to months with a biopsy showing rejection with signs of chronicity.

3. You note that late rejections are most often antibody mediated but you have limited DSA data. Was the diagnosis of AMR made exclusively on the basis of C4D?

All our renal biopsies were evaluated by an experienced renal pathologist who scores >150 renal transplant biopsies each year in our centre. The diagnosis of AMR was based on using the Banff criteria but C4D staining can be absent. However, a renal biopsy with all histological hallmarks of AMR was diagnosed as AMR after discussing the case with the nephrologists at our weekly renal pathology meeting. The Banff 2015 allows for this by having a category of suspicious ABMR when DSA and/or C4d are negative while all other criteria are met.

4. The lack of immunosuppressive drug levels is a significant shortcoming

We have a protocol in place for through levels of MMF and tacrolimus and at our centre we adhere quite strictly to this protocol as outlined in the M&M section. However, patients are eventually referred to their own nephrologist for follow-up and we cannot account for this period. Single through levels are not that reliable and there is a growing body of evidence that high intrapatient variability of tacrolimus through levels is actually more predictive of future rejection and graft loss. Unfortunately, we do not have these data but also cannot think of a reason why IPV could give a structural bias in our data (that is consistently more IPV in a subgroup of patients with high or low numbers of CD28null T cells).

5. The selection of induction agents remains unanswered. The choice of anti-DC25 or ATG is a very different class of agents vs IL2-RA. The immunologic effect can be very different on your T cell subsets. Have you analyzed these groups separately to determine if there is any difference in outcomes.

We are not sure what to answer. A very low number of patients received ATG induction and this is to small a group for meaningful statistical analysis. Anti-CD25 is synonymous with IL-2-receptor antagonist.

Within a multivariate analysis, the use of basiliximab (anti-CD25) was not a significant variable associated with late rejection. All these date are shown in table 1.

Within the discussion we recognize that the results obtained are possibly not generalizable when other IS drug regimes are used, notably induction with T cell depleting agents.

Submitted filename: rebuttalPlOs.docx

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8 Jan 2020

High numbers of differentiated CD28null CD8+ T cells are associated with a lowered risk for late rejection and graft loss after kidney transplantation

PONE-D-19-20193R2

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15 Jan 2020

PONE-D-19-20193R2

High numbers of differentiated CD28null CD8+ T cells are associated with a lowered risk for late rejection and graft loss after kidney transplantation

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