Heterospecific Immunity Help to Sustain an Effective BK-virus Immune Response and Prevent BK-virus-associated Nephropathy

doi:10.21203/rs.3.rs-1650939/v1

PDF

Article

Heterospecific Immunity Help to Sustain an Effective BK-virus Immune Response and Prevent BK-virus-associated Nephropathy

https://doi.org/10.21203/rs.3.rs-1650939/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted 26 May, 2022

You are reading this latest preprint version

BK-virus-associated nephropathy (BKvAN) is a major complication in kidney transplant recipients, associated with a higher level of BK-virus (BKv) replication and leading to poor graft survival. In a large cohort of kidney transplant recipients with various BKv reactivation levels, we found that BKvAN was associated with a low BKv-specific memory CD8 T-cell functionality and an exhausted-like phenotype. This severe lymphocyte impairment was associated with a low level of class II HLA mismatches. In vitro experiments showed that allogeneic CD4 T cells partly restored BKv-specific memory CD8 T-cell responses. Our results suggest that in kidney transplant recipient, allogeneic CD4 T cells may provide a heterospecific help that maintain an effective BKv-specific memory CD8 T-cell response, and allows a better control of BKv replication in the kidney graft.

BK-virus-associated nephropathy

kidney transplantation

BK virus-specific T cells

HLA mismatches

With the development of potent immunosuppressive therapies, kidney transplantation has become the standard treatment for patients with end-stage renal disease, improving patient survival over dialysis¹. Over the last decade, BK-virus (BKv) has emerged as a major cause of opportunistic infections in kidney transplant recipients (KTRs).

BKv reactivation occurs in 30 to 40% of KTRs, and may lead to BK-virus-associated nephropathy (BKvAN)², which affects almost 10% of KTRs, and may result in premature kidney allograft failure and irreversible graft loss³. In the context of kidney transplantation, the initial event is BKv replication in urine, and then in blood, which may be associated with BKvAN. There is currently no specific antiviral treatment against BKv. Decreasing the intensity of therapeutic immunosuppression to restore BKv-specific immune responses remains the only therapeutic option, but can lead to allogeneic graft rejection². The management of BKv replication and BKv-specific immune restoration therefore remains a major challenge in kidney transplantation.

BKv is a widespread double-stranded DNA polyomavirus, with seroprevalence rates of up to 80% in adults worldwide^4,5. Primary infection frequently occurs during childhood, after which, BKv establishes viral latency in the reno-urinary tract, in which the virus is controlled by the BKv-specific T-cell response⁴. The early-gene region encodes proteins involved in viral replication, such as the “large tumor antigen” (LT-Ag). These regulatory proteins facilitate replication of the viral genome. By contrast, the late-gene region encodes capsid proteins, such as viral protein-1 (VP1)⁶. LT-Ag and VP1 are immunodominant proteins widely recognized by BKv-specific T cells, which contribute to the immune response and the control of viral quiescence in immunocompetent patients⁷.

The mechanisms of BKv reactivation and BKvAN are poorly understood. Immunosuppressive therapy is known to impair BKv-specific T-cell responses, which may result in a failure to control BKv replication⁸. Viral replication is thought to result from viral reactivation and a defective virus-specific immune response^9,10. Monitoring of the BKv-specific T-cell response and BKv viral load may therefore be useful for assessing the risk of BKv reactivation and the occurrence of BKvAN.

We prospectively studied BKv-specific T-cell responses and the impact of human leukocyte antigen (HLA) matching in a cohort of kidney transplant recipients with various levels of BKv reactivation (no reactivation, viruria, viremia, or BKvAN, 25 patients per group).

We compared BKv-specific T-cell responses in 100 kidney transplant recipients with various degrees of BKv reactivation (no BKv reactivation, BKv-viruria, BKv-viremia and BKvAN, 25 patients per group). The patients were followed prospectively for a median of 36 [26–38] months, to identify risk factors for BKvAN.

Clinical characteristics of KTRs according to level of BKv reactivation

We found no significant differences between the KTR groups in terms of characteristics before or at the time of transplantation (Table 1). There were no significant differences in graft function during the first month after transplantation, the occurrence of graft rejection during follow-up or in CD4 and CD8 T-cell counts (Table 1). Due to the study design, the time from kidney transplantation to inclusion was longer for patients without BKv reactivation and for the BKv-viruria group than for the BKv-viremia and BKvAN groups (Table 1, p = 0.0014).

Table 1

Demographics of the study population, comparison of immunosuppressive treatments and outcome after kidney transplantation
Demographic comparison	Without	Viruria	Viremia	BKvAN	pⁱ
n	25	25	25	25
Age of patients at inclusion, years	49 [39–63]	59 [47–67]	62 [49–70]	60 [54–64]	0.129
Male, n (%)	16 (64)	18 (72)	18 (72)	17 (68)	0.916
DIALYSIS, n (%)	20 (80)	23 (92)	23 (92)	23 (92)	0.431
Time on dialysis, months	32 [16.8–46]	28 [11–43]	40 [20.8–61.8]	47 [28–74]	0.137
Hemodialysis, n (%)	18 (90)	19 (82.6)	21 (91.3)	22 (95.7)	0.526
Peritoneal dialysis, n (%)	2 (10)	4 (17.4)	2 (8.7)	1 (4.3)	0.526
CAUSE OF END-STAGE RENAL DISEASE
Glomerulonephritis, n (%)	6 (24)	10 (40)	8 (32)	6 (24)	0.611
Hypertensive or diabetic nephrosclerosis, n (%)	5 (20)	7 (28)	10 (40)	9 (36)
Polycystic kidneys, n (%)	6 (24)	3 (12)	3 (12)	2 (8)
Tubular or interstitial diseases, n (%)	2 (8)	1 (4)	1 (4)	4 (16)
Unknown and other causes, n (%)	6 (24)	4 (16)	3 (12)	4 (16)
TRANSPLANTATION CHARACTERISTICS
Donation after brain death, n (%)	16 (64)	21 (84)	17 (68)	20 (80)	0.319
Donation after cardiac death, n (%)	4 (16)	0 (0)	5 (20)	2 (8)
Living donation, n (%)	5 (20)	4 (16)	3 (12)	3 (12)
Highly sensitized patientsⁱⁱ, n (%)	7 (28)	6 (24)	6 (24)	5 (20)	0.932
First transplant, n (%)	19 (76)	22 (88)	24 (96)	19 (76)	0.147
Pre-existing DSA, n (%)	10 (40)	8 (32)	10 (40)	10 (40)	0.917
De novo DSA status, n (%)	9 (36)	7 (28)	7 (28)	5 (20)	0.662
CMV R + status, n (%)	17 (68)	22 (88)	22 (88)	19 (76)	0.212
CMV D+/R- status, n (%)	4 (16)	1 (4)	2 (8)	3 (12)	0.528
BKV REACTIVATION
Plasma BKv viral load (copies/mL)	NA	0	2.2 x 10³ [8.9 x 10² − 1.2 x 10⁴]	3.5 x 10⁵ [5.0 x 10⁴ − 7.8 x 10⁵]	< 0.0001
Time from kidney transplantation to plasma BKv reactivation, months	NA	NA	5.6 [3.2–9.1]	3.4 [2.9–13.4]	0.859
Urinary BKv viral load (copies/mL)	NA	2.4 x 10⁴ [3.9 x 10³ − 2.9 x 10⁵]	8.5 x 10⁷ [2.2 x 10⁶ -2.9 x 10⁸]	1.0 x 10⁹ [1.9 x 10⁸ -1.6 x 10⁹]	< 0.0001
Time from kidney transplantation to urinary BKv reactivation, months	NA	14.8 [2.6–51]	3.9 [1.7–11]	3.6 [1.7–13.4]	0.047
BKvAN diagnosis, months	NA	NA	NA	11 [5–24]
IMMUNOSUPPRESSIVE TREATMENT COMPARISON at BKvAN diagnosis or 12 months after transplantationⁱⁱⁱ
INDUCTION and MAINTENANCE TREATMENTS
Thymoglobulin, n (%)	NA	6 (24)	13 (52)	15 (60)	0.027
Tacrolimus, n (%)	NA	19 (76)	16 (64)	21 (84)	0.516
Levels of tacrolimus, ng/mL	NA	9.1 [8.7–9.7]	9.1 [7.7–11.1]	8.6 [6.7–10.2]	0.670
Mycophenolate mofetil exposure, AUC h.mg/L	NA	35.8 [22.8–48.8]	49.3 [33.9–64.7]	60.4 [46.5–74.3]	< 0.001
CURATIVE TREATMENT FOR REJECTION
Intravenous steroid boluses, n (%)	NA	5 (20)	9 (36)	10 (40)	0.130
Thymoglobulin, n (%)	NA	1 (4)	4 (16)	1 (4)	> 0.9999
Plasma exchanges, n (%)	NA	4 (16)	8 (32)	6 (24)	0.508
Rituximab, n (%)	NA	4 (16)	8 (32)	4 (16)	> 0.9999
Bortezomib, n (%)	NA	5 (20)	3 (12)	4 (16)	0.743
OUTCOME AFTER TRANSPLANTATION
Delayed graft function, n (%)	11 (44)	6 (24)	7 (28)	8 (32)	0.462
eGFR at month 1 after transplantation, mL/min/1.73 m²	40 [32.3–68.8]	51 [31–81]	45.5 [31.3–63.5]	41 [26.8–56.8]	0.740
eGFR at BKvAN diagnosis or 12 months after transplantation, mL/min/1.73m²	51 [42.5–69.5]	43 [35-69.5]	48 [38–56]	29 [21-42.5]	< 0.0001
eGFR at the end of the follow-up, mL/min/1.73 m²	49 [37-67.5]	47 [31.5–58.5]	45 [28.5–60]	13 [9.5–29]	< 0.0001
Decrease in eGFR at the end of follow-up, mL/min/1.73 m²	0 [0–7]	3 [0-6.5]	4 [0–13]	12 [3.5–17]	0.0079
CMV reactivation, n (%)	10 (40)	8 (32)	7 (28)	9 (36)	0.828
EBV reactivation, n (%)	13 (52)	18 (72)	16 (64)	15 (60)	0.531
Graft rejection, n (%)	6 (24)	5 (20)	9 (36)	4 (16)	0.381
Graft loss, n (%)	0 (0)	0 (0)	1 (4)	9 (36)	< 0.0001
Patient death after transplantation, n (%)	1 (11.1)	3 (33.3)	2 (22.2)	3 (33.3)	0.720
Time from transplantation to inclusion, months	26 [16–34]	27 [9–79]	9 [7–18]	8 [4–28]	0.0014
CD4 T-cell count, absolute number/µL	247 [137–514]	342 [260–495]	253 [156–443]	210 [76–376]	0.472
CD8 T-cell count, absolute number/µL	210 [161–271]	311 [162–448]	172 [92–278]	159 [122–353]	0.197
Without: patients without plasma or urinary BKv reactivation; Viruria: patients with urinary BKv reactivation; Viremia: patients with plasma BKv reactivation; BKvAN: patients with BKv-associated nephropathy; BKv: BK-virus; CMV: Cytomegalovirus; CMV R + status: recipient seropositive for CMV; CMV D+/R- status: recipient seronegative for CMV transplanted with a graft from a CMV-seropositive donor; DSA: Donor-specific alloantibodies; EBV: Epstein Barr Virus; eGFR: estimated glomerular filtration rate (MDRD4-formula); n: number of patients; NA: not applicable; p: p-values. Continuous data are expressed in medians and interquartile ranges. (i) p-values indicate the significance of differences between groups in Kruskal-Wallis or χ2 tests. (ii) defined as > 85% panel reactive antibodies. (iii) The comparison of immunosuppressive treatments between KTR groups was performed at BKvAN diagnosis or 12 months after transplantation.

BKv reactivation in KTRs

We compared the plasma and urinary BKv viral loads between the KTR groups (Table 1).

At inclusion, the BKvAN group had higher plasma BKv viral loads than the BKv-viremia group (Fig. 1a, p = 0.0019). Similarly, patients in the BKvAN group also had a higher urinary BKv viral load than those with BKv-viruria or viremia (Fig. 1b, p < 0.0001 and p = 0.022 respectively).

Impact of BKv reactivation on allograft outcome and graft survival

We then analyzed the impact of BKv reactivation on the outcome of kidney transplantation (Table 1). The median time from transplantation to BKvAN diagnosis was 11 [5–24] months. Despite having a similar renal function and proportion of delayed graft function one month after renal transplantation, patients diagnosed with BKvAN had poorer renal function (eGFR of 29 [21-42.5] mL/min/1.73m²) than those of the other groups at month 12 (p < 0.0001) (Fig. 1c and Table 1). Immunosuppressive treatment was modified for patients with BKvAN, with a decrease in tacrolimus dose and decreases in antimetabolite exposure or antimetabolite withdrawal (Supplementary data: Table S1). Treatment with mTor inhibitor was initiated before BKvAN diagnosis in patients displaying sustained BKv replication in the plasma. Despite the decrease in tacrolimus level (p < 0.0001) (Table S1), graft function was significantly altered in the BKvAN group at the end of follow-up (eGFR: 13 [9.5–29] mL/min/1.73m² versus eGFR at BKvAN diagnosis: 29 [21-42.5] mL/min/1.73m², p < 0.0001). Patients with BKvAN displayed severe graft dysfunction, with a larger decrease in eGFR at the end of follow-up than was observed in the other groups (decrease in eGFR: 12 [3.5–17] mL/min/1.73m² versus 0 [0–7], 3 [0-6.5] and 4 [0–13] mL/min/1.73m^2, respectively, p = 0.0079) (Fig. 1c and Table 1). Graft survival was poorer in patients with BKvAN, for whom the rate of graft loss was 36%. The rate of graft loss in the other groups was < 5% (p < 0.0001) (Fig. 1d and Table 1). By contrast, allograft outcome was similar between the other groups (Figs. 1c and 1d, Table 1).

Risk factors for BKvAN

We analyzed factors potentially associated with the occurrence of BKvAN in cases of BKv reactivation.

Identification of risk factors associated with BKv reactivation

At BKvAN diagnosis or 12 months post-transplantation, patients with BKvAN had significantly higher levels of exposure to thymoglobulin (p = 0.027) and mycophenolate mofetil (p < 0.001) (Table 1).

We analyzed the relationship between donor/recipient [HLA-A,-B,-DR,-DQ] matching and BKv replication. BKv-viremia was associated with a larger number of total [HLA-A,-B,-DR,-DQ] and [HLA-DR] mismatches (p = 0.011 and p = 0.0036, respectively) (Figs. 2a, 2d and Table 2a). By contrast, KTRs with BKvAN had a significantly smaller number of HLA-DR mismatches than patients with BKv-viremia (p = 0.039) (Fig. 2d). We assessed the impact of the degree of HLA-matching as a risk factor for BKvAN, by stratifying the absolute number of HLA mismatches into subgroups. We found that cutoff values of at least five [HLA-A,-B,-DR,-DQ] mismatches and of two [HLA-DR] mismatches were associated with BKv-viremia without the occurrence of BKvAN (Figs. 2f, 2i and Table 2b). In the presence of at least five [HLA-A,-B,-DR,-DQ] mismatches, 23.8% of patients had viruria, 50% had BKv-viremia without BKvAN and 26.2% had BKvAN. In the presence of fewer than five mismatches, 45.5% of patients had viruria, 12.1% had BKv-viremia without BKvAN and 42.4% had BKvAN (p = 0.0025 - Fig. 2f and Table 2b). In terms of HLA-DR compatibility, in the presence of two [HLA-DR] mismatches, 20.8% of patients had viruria, 54.2% had BKv-viremia without BKvAN and 25% had BKvAN. In the presence of one [HLA-DR] mismatch, 28.6% of patients had viruria, 35.7% had BKv-viremia without BKvAN and 35.7% had BKvAN. In the absence of HLA-DR mismatches, 52.2% of patients had viruria, 8.7% had viremia without nephropathy and 39.1% had BKvAN (p = 0.018 - Fig. 2i and Table 2b). Compared to BKv-viremia without BKvAN, we identify HLA-DR compatibility as a risk factor associated with BKvAN (p = 0.030), while there are no statistical differences in terms of HLA-A,-B and DQ-compatibility (Figs. 2g, 2h and 2j).

Table 2. Comparison of HLA-A,-B,-DR,-DQ mismatches between KTR groups defined on the basis of BKv reactivation

Absolute number of HLA mismatches

Comparison of HLA-A,-B,-DR,-DQ mismatches	Viruria	Viremia	BKvAN	pⁱ
n	25	25	25
Total [HLA-A,-B,-DR,-DQ] mismatch number	4 [3-6]	6 [5-7]	4 [3-6]	*0.011*
HLA-A mismatch number	1 [1-1.5]	1 [1-2]	1 [0-2]	0.710
HLA-B mismatch number	2 [1-2]	2 [2-2]	2 [1-2]	0.052
HLA-DR mismatch number	1 [0-1]	2 [1-2]	1 [0-1.5]	*0.0036*
HLA-DQ mismatch number	1 [0-2]	1 [1-2]	1 [0-1]	0.310

Stratification of the HLA mismatches into subgroups

Comparison of HLA-A,-B,-DR,-DQ mismatches	Total [HLA-A,-B,-DR,-DQ] < 5 mismatches	Total [HLA-A,-B,-DR,-DQ] ≥ 5 mismatches	HLA-DR no mismatches	HLA-DR one mismatch	HLA-DR two mismatches
Viruria, n (%)	15 (45.5)	10 (23.8)	12 (52.2)	8 (28.6)	5 (20.8)
Viremia, n (%)	4 (12.1)	21 (50)	2 (8.7)	10 (35.7)	13 (54.2)
BKvAN, n (%)	14 (42.4)	11 (26.2)	9 (39.1)	10 (35.7)	6 (25)
pⁱ	*0.0025*		*0.018*

Viruria: patients with urinary BKv reactivation; Viremia: patients with plasma BKv reactivation; BKvAN: patients with BKv-associated nephropathy; BKv: BK-virus; n: number of patients; p: p-values. Continuous data are expressed in medians and interquartile ranges. (i) p-values indicate the significance of difference between groups in Kruskal-Wallis or χ² tests.

We then analyzed the BKv-specific T-cell functionality. We first evaluated the antiviral T-cell response by studying T-cell phenotype and functionality after stimulation with BKv or CEF (cytomegalovirus, Epstein Barr virus and influenza virus) in KTRs without BKv reactivation. A polyfunctional T-cell response was observed, and the antiviral T cells were able to proliferate, produce cytokines, and develop CD8 cytotoxicity in the presence of BKv or CEF peptides (Figs. 3a, 3b, 4a, 4b, Supplementary data: Figures S1, S2a, S2b and Table S2).

For the specific response to BKv in the context of BKv reactivation, we observed a gradual or total loss of BKv-specific T-cell polyfunctionality according to BKv reactivation level (Table 3). Based on the distribution of data for the BKv-specific T-cell response, we defined a cutoff value of 2.4 proliferative cells and 0.3 cytokine-producing cells for distinguishing between responder and non-responder patients. The proliferative capacity of BKv-specific CD4 and CD8 T cells was significantly lower in KTRs with BKvAN than in those with BKv-viruria or BKv-viremia (for CD4 T cells: p = 0.0002; for CD8 T cells: p < 0.0001 and p = 0.0026, respectively) (Figs. 3a, 3b). Using a cutoff value of 2.4 proliferative CD4 or CD8 T cells to distinguish between responders and non-responders, we observed a gradual decrease in the number of patients able to respond to BKv peptides with increasing BKv reactivation level (p = 0.032 and p < 0.001, respectively) (Figs. 3c, 3d and Table 3). The TNFα secretion capacity of BKv-specific CD8 T cells was significantly lower in KTRs with BKvAN than in those with BKv-viremia (p = 0.0012) (Fig. 4a). Similarly, the co-secretion of TNFα and IFNγ (TNFα/IFNγ) was less frequently observed in patients with BKvAN than those with BKv-viruria or viremia (p = 0.0004 and p = 0.025, respectively) (Fig. 4b). Using a cutoff value of 0.3 cytokine-producing CD8 T cells to distinguish between responders and non-responders, we observed a gradual decrease in the number of patients able to respond to BKv peptides with increasing BKv reactivation level, in terms of IFNγ/TNFα secretion capacity (p = 0.013) (Figs. 4c, 4d and Table 3). We observed no significant differences in IFN_γ secretion or cytokine-secreting CD4 T-cell levels after BKv-specific stimulation (data not shown). We then assessed the specific cytotoxicity of CD8 T cells against BKv. Specific cytotoxicity tended to be lower in the BKvAN group than in the other groups, with a mortality of BKv-target cells of 0.32 [0.32–3.61] dead cells in the BKvAN group, versus 3.63 [0.32–8.6] in the BKv-viremia group and 6.04 [0.32–10.22] in the BKv-viruria group. Nevertheless, this difference was not significant (p = 0.267) (Table 3 and Supplementary data: Figures S3).

Table 3

Comparison of BKv-specific T-cell functionality between KTR groups defined on the basis of BKv reactivation
BKv-specific T-cell functionality	Viruria	Viremia	BKvAN	pⁱ
PROLIFERATION CAPACITIES (nⁱⁱ)	16	13	15
CFSE^low BKv (LT-Ag and VP1) CD4 T cells, cell nb	4.36 [3.15–5.37]	3.39 [0.10–5.09]	2.01 [0.10–3.31]	0.0003
Response for LT-Ag, cell nb	3.90 [3.0-4.61]	1.80 [0.10–4.59]	2.12 [0.10–3.26]	0.023
Response for VP1, cell nb	4.92 [3.44–5.42]	3.60 [1.57–5.30]	1.93 [0.10–3.46]	0.008
No BKv-specific peptide response, n (%)	2 (12.5)	2 (16.6)	6 (40.0)	0.032
BKv-specific response against LT-Ag or VP1 peptides, n (%)	2 (12.5)	5 (41.7)	6 (40.0)
BKv-specific response against LT-Ag and VP1 peptides, n (%)	12 (75.0)	5 (41.7)	3 (20.0)
Correlation between LT-Ag CD4 T cells and BKv viral load	NA	-0.02 [-0.42;0.39]		0.94
Correlation between VP1 CD4 T cells and BKv viral load	NA	-0.53 [-0.77;-0.16]		0.006
CFSE^low BKv (LT-Ag and VP1) CD8 T cells, cell nb	4.88 [4.10–5.95]	2.33 [0.09–4.22]	0.09 [0.09–2.51]	< 0.0001
Response for LT-Ag, cell nb	4.52 [3.72–5.01]	2.29 [0.10–3.68]	0.10 [0.10–1.65]	< 0.0001
Response for VP1, cell nb	5.51 [4.75–6.05]	2.58 [0.67–4.84]	1.60 [0.10–2.57]	< 0.0001
No BKv-specific peptide response, n (%)	1 (6.25)	5 (38.5)	8 (53.3)	< 0.001
BKv-specific response against LT-Ag or VP1 peptides, n (%)	1 (6.25)	2 (15.4)	6 (40.0)
BKv-specific response against LT-Ag and VP1 peptides, n (%)	14 (87.5)	6 (46.1)	1 (6.7)
Correlation between LT-Ag CD8 T cells and BKv viral load	NA	-0.15 [-0.52;0.27]		0.48
Correlation between VP1 CD8 T cells and BKv viral load	NA	-0.45 [-0.72;-0.05]		0.025
CYTOKINE SECRETION CAPACITIES (nⁱⁱ)	16	14	14
TNF + BKv (LT-Ag and VP1) CD8 T cells, cell nb	0.03 [0.03–1.33]	1.23 [0.03–2.11]	0.03 [0.03–0.03]	0.0018
Response for LT-Ag, cell nb	0.03 [0.03–1.31]	1.43 [0.03–2.23]	0.03 [0.03–0.03]	0.037
Response for VP1, cell nb	0.03 [0.03–1.30]	1.10 [0.03–1.74]	0.03 [0.03–0.37]	0.153
No BKv-specific peptide response, n (%)	7 (43.7)	3 (21.4)	9 (64.3)	0.116
BKv-specific response against LT-Ag or VP1 peptides, n (%)	4 (25)	4 (28.6)	4 (28.6)
BKv-specific response against LT-Ag and VP1 peptides, n (%)	5 (31.3)	7 (50)	1 (7.1)
Correlation between LT-Ag CD8 T cells and BKv viral load	NA	-0.56 [-0.78;-0.23]		0.002
Correlation between VP1 CD8 T cells and BKv viral load	NA	-0.46 [-0.72;-0.10]		0.012
TNF⁺/IFN⁺ BKv (LT-Ag and VP1) CD8 T cells, cell nb	0.2 [0.03–0.54]	0.03 [0.03–0.593]	0.03 [0.03–0.03]	0.0005
Response for LT-Ag, cell nb	0.12 [0.03–0.52]	0.03 [0.03–0.58]	0.03 [0.03–0.03]	0.197
Response for VP1, cell nb	0.31 [0.03–0.73]	0.03 [0.03–0.61]	0.03 [0.03–0.03]	0.021
No BKv-specific peptide response, n (%)	5 (31.3)	7 (50)	13 (92.9)	0.013
BKv-specific response against LT-Ag or VP1 peptides, n (%)	8 (50)	4 (28.6)	1 (7.1)
BKv-specific response against LT-Ag and VP1 peptides, n (%)	3 (18.7)	3 (21.4)	0 (0)
Correlation between LT-Ag CD8 T cells and BKv viral load	NA	-0.33 [-0.63;0.06]		0.09
Correlation between VP1 CD8 T cells and BKv viral load	NA	-0.33 [-0.63;0.07]		0.09
CYTOTOXIC CAPACITIES (nⁱⁱ)	8	9	9
7AAD⁺ BKv (LT-Ag and VP1) target cells (cell nb)	6.04 [0.32–10.22]	3.63 [0.32–8.6]	0.32 [0.32–3.61]	0.267
Response for LT-Ag (cell nb)	6.04 [0.32–8.60]	6.09 [1.03–11.17]	0.85 [0.32–3.54]	0.522
Response for VP1 (cell nb)	5.16 [0.32–11.57]	0.32 [0.32–7.05]	0.32 [0.32–3.01]	0.342
No BKv-specific peptide response, n (%)	5 (62.5)	6 (66.7)	7 (77.8)	0.622
BKv-specific response against LT-Ag or VP1 peptides, n (%)	2 (25)	3 (33.3)	2 (22.2)
BKv-specific response against LT-Ag and VP1 peptides, n (%)	1 (12.5)	0 (0)	0 (0)
PD1 EXPRESSION ON BKv CD4 T cells, % of cells	NA	1.82 [0.46–5.30]	8.11 [2.00-14.88]	0.028
PD1 EXPRESSION ON BKv CD8 T cells, % of cells	NA	1.16 [0.39–4.95]	2.70 [1.23–9.86]	0.041
Viruria: patients with urinary BKv reactivation; Viremia: patients with plasma BKv reactivation; BKvAN: patients with BKv-associated nephropathy; BKv: BK-virus; n: number of patients; cell nb: normalized BKv-specific T-cell frequencies; NA: not applicable; p: p-values. Continuous data are expressed in medians and interquartile ranges. (i) p-values indicate the significance of differences between groups in Kruskal-Wallis or χ² tests. Correlations were evaluated with the nonparametric Spearman’s rank correlation test. (ii) For each patient, two BKv-specific responses were analyzed, after stimulation with LT-Ag and VP1 peptides.

We ruled out a global inhibition of antiviral T cells in patients with BKvAN, by assessing the ability of CD8 T cells to respond to viral peptides other than BKv peptides. In the presence of a CEF peptide pool, we observed a similar polyfunctional CD8 T-cell response for T-cell proliferation, cytokine secretion and CD8 cytotoxicity in all KTR groups, regardless of the level of BKv reactivation (Supplementary data: Figure S2 and Table S3).

These results indicate that a global antiviral response was maintained regardless of BKv status, and suggest a specific impairment of the BKv-specific T-cell response in patients with BKvAN.

Multivariate analysis to identify risk factors associated with BKv reactivation

For the identification of factors associated with BKv viremia and BKvAN, we performed a multivariate analysis with the factors identified as relevant in the univariate analysis (Table 4a and 4b). Three independent risk factors were found to be associated with BKv viremia and/or BKvAN: the thymoglobulin use, the total number of [HLA-A,-B,-DR,-DQ] mismatches and the loss of the proliferative BKv-specific CD8 T-cell response (Table 4b).

Table 4. Risk factors associated with BKv viremia and BKvAN in the context of BKv reactivation

Univariate analysis

Risk factors associated with BKv viremia and BKvAN in the context of BKv reactivation	Viremia (versus viruria)		BKvAN (versus viruria)		BKvAN (versus viremia)
	OR [95% CI]	pⁱ	OR [95% CI]	pⁱ	OR [95% CI]	pⁱ
Immunosuppressive treatment
Thymoglobulin	3.43 [1.03-11.48]	*0.068*	4.75 [1.41-16.05]	*0.036*	1.38 [0.45-4.25]	0.569
Antimetabolite exposure	1.12 [1.02-1.23]	*0.030*	1.17 [1.06-1.30]	*0.006*	1.05 [1.00-1.10]	0.058
HLA mismatches
Total [HLA-A,-B,-DR,-DQ] mismatch number	1.57 [1.13-2.19]	*0.024*	1.08 [0.81-1.45]	0.605	0.69 [0.50-0.95]	*0.036*
No [HLA-DR] mismatch number	1 (ref)	*0.036*	1 (ref)	0.692	1 (ref)	0.070
One [HLA-DR] mismatch number	7.50 [1.29-43.69]		1.67 [0.47-5.93]		0.22 [0.04-1.30]
Two [HLA-DR] mismatch number	15.60 [2.53-96.08]		1.60 [0.37-6.95]		0.10 [0.02-0.63]
Proliferative BKv (LT-Ag and VP1) CD4 T-cell response
Response for LT-Ag (cell nb)	0.72 [0.48-1.07]	0.159	0.62 [0.42-0.92]	0.054	0.86 [0.59-1.27]	0.453
Response for VP1 (cell nb)	0.82 [0.56-1.20]	0.298	0.57 [0.38-0.84]	*0.015*	0.69 [0.47-1.02]	0.093
No BKv-specific peptide response	2.40 [0.26-22.10]	0.278	12.00 [1.56-92.29]	*0.048*	5.00 [0.58-42.80]	0.34
BKv-specific response against LT-Ag or VP1 peptides	6.00 [0.86-41.90]		12.00 [1.56-92.29]		2.00 [0.31-12.84]
BKv-specific response against LT-Ag and VP1 peptides	1 (ref)		1 (ref)		1 (ref)
Proliferative BKv (LT-Ag and VP1) CD8 T-cell response
Response for LT-Ag (cell nb)	0.58 [0.36-0.92]	*0.030*	0.39 [0.23-0.66]	*0.003*	0.67 [0.43-1.05]	0.084
Response for VP1 (cell nb)	0.54 [0.33-0.87]	*0.017*	0.37 [0.21-0.64]	*<0.001*	0.68 [0.45-1.04]	0.075
No BKv-specific peptide response	11.67 [1.11-122.38]	0.090	112.00 [6.13-2,045.17]	*0.006*	9.60 [0.88-105.17]	0.09
BKv-specific response against LT-Ag or VP1 peptides	4.67 [0.35-61.83]		84.00 [4.48-1,576.52]		18.00 [1.27-255.74]
BKv-specific response against LT-Ag and VP1 peptides	1 (ref)		1 (ref)		1 (ref)

Multivariate analysis

Risk factors associated with BKv viremia and BKvAN in the context of BKv reactivation	Viremia (versus viruria)		BKvAN (versus viruria)		BKvAN (versus viremia)
	OR [95% CI]	pⁱⁱ	OR [95% CI]	pⁱⁱ	OR [95% CI]	pⁱⁱ
Thymoglobulin	34.15 [2.44-477.71]	*0.027*	15.85 [0.88-284.45]	0.092	0.46 [0.06-3.44]	0.452
Total [HLA-A,-B,-DR,-DQ] mismatches	1.90 [1.06-3.38]	*0.045*	1.02 [0.56-1.87]	0.94	0.54 [0.31-0.94]	*0.045*
Proliferative BKv CD8 T-cell response
No BKv-specific peptide response	14.34 [0.65-314.56]	0.17	214.88 [7.11-6,498.61]	*0.009*	14.99 [0.92-243.80]	0.156
BKv-specific response against LT-Ag or VP1 peptides	9.39 [0.38-229.70]		239.98 [6.99-8,237.53]		25.56 [1.11-586.42]
BKv-specific response against LT-Ag and VP1 peptides	1 (ref)		1 (ref)		1 (ref)

Viruria: patients with urinary BKv reactivation; Viremia: patients with plasma BKv reactivation; BKvAN: patients with BKv-associated nephropathy; BKv: BK-virus; cell nb: normalized BKv-specific T-cell frequencies, OR [95% CI]: odds ratio with 95% confidence interval; p: p-values. p-values indicate the significance of differences between groups in (i) multinomial univariate logistic regression analyses with correction for false discovery rate or (ii) Wald tests (multivariate multinomial logistic regression) with correction for false discovery rate.

Thymoglobulin, which was used as an induction treatment, was significantly associated with BKv-viremia compared to BKv-viruria (p = 0.027 - OR [95% CI] = 34.15 [2.44-477.71]), but not with BKvAN (p = ns) when compared to BKv viruria or BKv Viremia (Table 4b).

The total number of [HLA-A,-B,-DR,-DQ] mismatches was significantly associated with BKv-viremia compared to BKv-viruria (p = 0.045 - OR [95% CI] = 1.90 [1.06–3.38]). Interestingly, [HLA-A,-B,-DR,-DQ] mismatches were significantly associated with BKvAN compared to BKv-viremia (p = 0.045), but with an OR [95% CI] of 0.54 [0.31–0.94] highlighting that the presence of HLA mismatches in greater numbers reduces the risk of BKvAN (Table 4b).

A decrease in the proportion of patients displaying a BKv-proliferative CD8 T-cell response was associated with BKvAN (p = 0.009). In this group, when a BKv-specific CD8 T-cell response against LT-Ag and VP1 peptides was used as the reference, the OR [95% CI] was to 214.88 [7.11-6,498.61] for no BKv-specific peptide response and to 239.98 [6.99-8,237.53] for a BKv-specific CD8 T-cell response against LT-Ag or VP1 peptides. This result highlights that the decrease of the proliferative BKv-specific CD8 T-cell response increases the risk of BKvAN. Following introduction of the specific CD4 response into the model in place of the CD8 response, the occurrence of BKvAN was also found to be associated with a defect of the proliferative CD4 T-cell response to the VP1 peptide (p = 0.024).

Exhaustion of BKv-specific CD4 and CD8 T cells

BKv-specific T cells were not able to elaborate a functional response against BKv-peptides, but exhibited a preserved response against CEF-peptides, suggesting an exhaustion of BKv-specific T cells in patients with BKvAN.

We have evaluated the memory T-cell subpopulations of CD4 and CD8 T cells (Supplementary data: Figure S4). We detected an expansion of the TEMRA CD8 T-cell population in KTRs with BKv-viruria compared to the BKv-viremia and BKvAN groups (p = 0.0007) (Supplementary data: Figure S5h and Table S4).

To confirm the exhaustion of BKv-specific T cells in context of BKvAN, we assessed the relationship between BKv-specific lymphocyte functionality and the plasma BKv viral load. We observed a significant inverse correlation between BKv-specific CD4 and CD8 T-cell proliferation capacity and plasma BKv viral load (for VP1 peptides: p = 0.006 and p = 0.025, respectively) (Figs. 5a, 5b and Table 3). There was also a significant inverse correlation between the TNFα secretion capacities of BKv-specific CD8 T cells and plasma BKv viral load (for LT-Ag peptides: p = 0.002 and for VP1 peptides: p = 0.012) (Table 3).

We then assessed the expression of the inhibitory receptor PD1 and CTLA4 on BKv-specific CD4 and CD8 T cells (Supplementary data: Figure S6). The level of PD1 expression on BKv-specific CD4 and CD8 T cells was significantly higher in KTRs with BKvAN than in those with BKv-viremia (p = 0.028 and p = 0.041, respectively) (Figs. 5a, 5b and Table 3). We observed no significant differences in CTLA4 expression. Combined with a severe impairment of BKv T-cell functions and a highest plasma BKv loads in patients with BKvAN, these results are consistent with a BKv-specific CD4 and CD8 T-cell exhaustion in context of BKvAN.

Allogeneic CD4 T cells restore BKv-specific CD8 T-cell functionality

We then investigated the link between the severe impairment of BKv-specific T-cell functions, the exhausted-like phenotype of BKv-specific CD4 and CD8 T-cells and the smaller number of class II HLA mismatches in patients with BKvAN.

As we previously showed, heterospecific CD4 help provided by CD4 T cells generated from a distinct antigenic source could rescue memory CD8 T cell responses¹¹. We hypothesize that heterospecific immunity, provided by allogeneic CD4 T cells, may contribute to sustain the effective BKv CD8 T-cell response. Therefore, PBMCs from 11 KTRs with plasma BKv reactivation were stained with a fluorescent dye, depleted from autologous CD4 T cells, and incubated for five days with BKv-specific peptide pools in the presence of autologous CD4 T cells or allogeneic third-party CD4 T cells (heterospecific allogeneic CD4-help). Unstimulated CD4-depleted PBMCs incubated in the presence of autologous CD4 T cells or allogeneic third-party CD4 T cells were used as controls. At day 5, we evaluated the proliferation capacity of BKv-specific CD8 T cells by fluorescent dye dilution (Fig. 5c). We observed a significant increase in the proliferative capacities of BKv-specific CD8 T cells in the presence of third-party allogeneic CD4 T cells (with autologous CD4 T cells = 1.03 [0.06;2.32]; with allogeneic CD4 T cells = 2.2 [1.77;4.82] – p = 0.0137 – Fig. 5d). Interestingly, the proportion of proliferative BKv-specific CD8 T cells in conditions of allogeneic CD4 T cells was restored near from the response range of the BKv-viruria group (p = ns – Fig. 5d). There was no statistical difference in unstimulated CD4-depleted PBMCs incubated with autologous or allogeneic CD4 T cells (p = ns – Fig. 5d).

Our results highlight the loss of BKv-specific T-cell responses during the course of BKv replication, with an exhausted-like phenotype of BKv-specific CD4 and CD8 T-cells in KTRs with BKvAN.

BKv infection remains a major challenge in kidney transplantation, because it can lead to BKvAN, which causes graft loss in 50% of cases in this context^12–14. There is currently no specific antiviral treatment for BKvAN, and decreasing the intensity of immunosuppression is the only treatment shown to date to be effective^15,16. We show here that decreasing immunosuppression does not prevent high rates of graft loss in cases of established BKvAN (histologically proven), probably because of the delayed restoration of BKv T-cell responses and irreversible tissue lesions. Immunosuppressive regimens should therefore be adapted sooner after the detection of BKv in plasma, but achieving the balance between the control of BKv replication and the risk of acute allogeneic graft rejection when lowering immunosuppressive treatments remains a major challenge¹⁷. Indeed, after immunosuppression reduction for BKv replication, more than 20% of patients experience acute rejection, with lower graft function and survival¹⁸.

The use of intensive immunosuppressive strategies has been identified as a key factor in BKv reactivation^19,20. We found that thymoglobulin, a polyclonal antibody induction therapy that causes profound T-cell depletion, was a risk factor for BKv-viremia, consistent with current knowledge²¹, but not for BKvAN. This result implies that thymoglobulin could be a trigger for the blood replication of BKv, but not sufficient for the occurrence of BKvAN. Other authors have also reported a higher risk of BKvAN with certain immunosuppressive regimens, including high levels of exposure to steroids or tacrolimus/mycophenolate mofetil combinations^22–24. The choice of immunosuppressive drugs plays a role in the risk of BKvAN, as the use of tacrolimus was associated with a higher risk of developing BKvAN than the use of cyclosporine A (CsA)²⁵. The effect of tacrolimus may be explained by an activation of BKv replication, possibly in relation to its interaction with FKBP-12, whereas CsA reduces BKv replication in vitro^25–27. We also found that mycophenolate mofetil exposure was associated with a higher rate of BKvAN, but this factor was not identified as an independent risk factor in our model. Nevertheless, precise AUC determinations may be useful for monitoring the inhibition of T-cell activation and proliferation in kidney transplant patients^28,29.

Despite stepwise immunosuppression reduction, some patients clear BKv, whereas others develop persistent BKv replication, highlighting the role of individual mechanisms in viral control. In solid organ transplantation, the individual risk of opportunistic infection is largely determined by the “net state of immunosuppression”. Many factors contribute to this net state, including the immunosuppressive regimen and other individual predisposing factors, such as immune-aging, concomitant viral infection, and antiviral T-cell efficacy³⁰.

The BKv-specific T-cell response plays a crucial role in controlling BKv replication, although the precise pathophysiological mechanisms involved remain unknown^8,31,32. To date, there is several evidence to underline that the presence of BKv-specific CD8 T cells is predictive of the successful control of BKv reactivation after transplantation^33–35. We provide here an exhaustive characterization of BKv-specific T cells, in terms of their phenotype and functional assessment. We describe the changes in the BKv-specific T-cell response occurring during the course of BKv reactivation, with the progressive loss of BKv-specific T-cell responses. We observed changes in T-cell functionality that appeared to be limited to BKv-specific T cells, as none of the other antiviral responses tested were affected in patients with BKvAN.

Viruses during chronic infection can make use of the negative regulatory signal mediated by the immune checkpoint pathway to escape immune system control³⁶. In our work, the inverse correlation between BKv viral load and BKv-specific T-cell responses, and the higher levels of PD1 inhibitory receptor expression suggest an exhaustion of BKv-specific T cells in patients with BKvAN, as observed in other chronic viral infections^37–39.

HLA matching is a major challenge for successful transplantation, as HLA mismatches have a significant negative impact on allograft survival in kidney transplant patients^40–42. However, the impact of HLA matching on the development and progression of BKvAN remains a matter of debate⁴³. HLA is important for viral peptide presentation to both CD4 and CD8 T cells. In this point of view, BKv-specific CD8 T cells directed against the LPLMRKAYL 9-mer epitope are associated with the clearance of BKv^44,45, which binds to several HLA-B molecules^44,46,47.

In transplant recipients, antiviral T cells must operate under suboptimal (immunosuppressed) conditions, with differential individual effects. In addition, allogeneic kidney transplantation constitutes a complex immunological situation in which viral peptides may be presented by the donor or recipient HLA. In mismatch situations, the recognition, by recipient BKv-specific memory T cells, of the BKv peptides presented by donor HLA molecules on kidney epithelial cells may be altered, leading to BKv replication. Conversely, allogeneic responses may provide a stimulatory environment to BKv-specific T cells and induce additional help to restore or elaborate a functional BKv-specific CD8 T-cell response. We showed that BKvAN was mostly observed in cases of HLA-DR compatibility and was associated with an impairment of BKv-specific T-cell functions. This is consistent with previous reports showing that BKvAN occurs even in the context of good HLA matching^13,48, with a negative impact of HLA matching in the outcome of BKvAN⁴⁹. Conversely, in some cases, HLA mismatching may promote the development of BKvAN^21,50,51 because of the use of heavier immunosuppression⁵². In such cases, a similar inhibition of viral responses, not restricted to BKv, would be attempted.

Interestingly, we found that class II HLA-DR mismatches played an important role in BKv control, suggesting the contribution of CD4 T cells. CD4 T cells play a crucial role for the generation, maintenance and reactivation of memory CD8 T-cell responses^53–55. We hypothesized that mismatched CD4 T cells receiving allogeneic stimuli would be able to provide the necessary help to rescue BKv-memory CD8 T-cell responses (Fig. 6). This hypothesis was confirmed by replacing in vitro autologous CD4 T cells with allogeneic CD4 T cells and evaluating the improvement in the BKv-specific CD8 T-cell response. We previously reported that functional CD4 T cells generated in response to a different antigen (heterospecific helpers) can provide effective help to memory CD8 T cells in experimental mouse models¹¹. A higher degree of HLA mismatching can provide heterospecific CD4 help to BKv-specific memory CD8 T cells. This heterospecific help may increase the ability of the host to maintain an effective BKv-specific memory CD8 T-cell response that can protect against BKvAN. Along similar lines, Zeng et al. showed that kidney biopsy specimens from patients with BKv viremia contained more abundant alloreactive T-cell clones than virus-reactive clones, with a cell ratio of up to 7.7⁵⁶. In addition, Sawinski et al. showed that persistent BKv-viremia does not increase graft loss but was associated with the development of class II de novo donor-specific antibodies⁵⁷. Furthermore, we cannot rule out the possibility that, in the context of allogeneic transplantation, heterospecific immunity involves a potential cross-reactivity of virus-specific T cells with multiple HLA complexes⁵⁸.

In summary, we show that BKvAN is associated with a progressive severe loss of BKv-specific T-cell responses and an exhausted-like phenotype. These BKv-specific T-cell responses are crucial for the control of viral replication in the kidney. Our results suggest that a higher degree of HLA mismatching provides more effective control of BKv replication, possibly due to the involvement of heterospecific CD4 help processes. Thus, although a high degree of HLA matching may protect against graft rejection, it may, paradoxically, also be a significant risk factor for BKvAN. This finding should be taken into account in strategies aiming to mitigate the risk of BKvAN.

Patients and group classification

We performed a longitudinal observational study in the Kidney Transplant Department, University Hospitals, AP-HP. We assigned KTRs with BKv reactivation to three groups on the basis of the level of BKv reactivation, and we compared these groups. KTRs without BKv reactivation were used as the control group. The groups were defined as follows:

Patients with BKv viruria (urine BKv viral load > 200 copies/mL and plasma BKv viral load < 200 copies/mL in the last 12 months),
Patients with BKv viremia (stable or increasing plasma BKv viral load > 200 copies/mL in the last 6 months, without BKvAN diagnosis on kidney biopsy)
Patients with BKvAN (histologically proven BKvAN with a plasma BKv viral load > 200 copies/mL),
Patients without BKv reactivation (plasma and urine BKv viral loads < 200 copies/mL in the last 12 months).

We prospectively included 100 KTRs over the age of 18 years in the four groups (25 patients per group). The patients were followed prospectively between April 2014 and April 2018.

Patients who had undergone combined multiple-organ transplantation were excluded, as were those with chronic viral infections, such as viral hepatitis or human immunodeficiency virus infection. KTRs with BKv reactivation not meeting the study criteria were not included.

Written informed consent was obtained from all patients. The study was performed in accordance with the rules of the local ethics committee.

We evaluated clinical characteristics before kidney transplantation, kidney graft outcome, immunosuppressive regimen, plasma and urine BKv viral load, HLA mismatches between donor and recipient, and BKv-specific T-cell functionality.

Collection of clinical and biological data

Demographic data, for age and sex, the cause of end-stage renal disease, and dialysis and transplantation characteristics, were recorded for all patients (Table 1).

We recorded median plasma and urine BKv viral loads and the duration of BKv reactivation for each patient. Plasma and urine samples were obtained during routine visits to our center, and during acute allograft failure. BKv screening was performed in accordance with international guidelines, by assessing plasma BKv load monthly in the first six months after transplantation, and then every three months until two years after transplantation, and annually thereafter, with additional determinations in the event of an unexplained increase in serum creatinine concentration or after treatment for acute rejection^2,20. Urinary BKv load was also determined during plasma screening for BKv. We quantified BKv DNA by BKv-specific real-time PCR (BKV R-GENE, BioMerieux®).

Glomerular filtration rate was estimated with the MDRD-4 formula (eGFR), in the first month after transplantation, 12 months post-transplantation or at BKvAN diagnosis, and at the end of follow-up for each patient. The end of follow-up was defined as the occurrence of graft loss or the time at which the last follow-up visit took place. We determined levels of tacrolimus, everolimus, and mycophenolate mofetil exposure (area under the curve – AUC in h.mg/L) before and after BKvAN diagnosis or 12 months after transplantation.

Histological assessment

Kidney biopsy was performed, in accordance with the transplantation protocol, three and 12 months after transplantation or in cases of acute kidney injury (increase in basal serum creatinine concentration of more than 25%). BKvAN was diagnosed histologically by the Histopathology Department of Bicêtre Hospital, in accordance with international guidelines. The lesions observed included enlarged nuclei with smudgy chromatin changes, and intranuclear viral inclusions associated with a mononuclear inflammatory infiltrate and/or fibrosis. The presence of the virus was confirmed by immunohistochemistry with an antibody against large-SV40 tumor antigen^59,60.

Assessment of HLA matching

We collected donor and recipient HLA typing data from the French organ allocation organization. For each donor–recipient combination, the degree of HLA matching was determined by counting the number of HLA-A, HLA-B, HLA-DR and HLA-DQ mismatches between donor and recipient. We then assessed the effect of HLA matching on BKv reactivation and the occurrence of BKvAN.

HLA typing was performed on recipients and living donors, with DNA molecular biology methods, using reverse sequence-specific oligonucleotides (SSOs) for the A/B/DRB1/DQB1 loci from the standard kit, and from a high-definition kit from November 2016 onwards (One Lambda, Canoga Park, CA). For deceased donors, HLA typing was performed with low-resolution sequence-specific primers (SSPs) for the A/B/DRB1/DQB1 loci (Olerup Reagents, Bionobis, France). These techniques provide at least split antigen-level resolution (most probable allele, depending on the technique, locus and antigen).

Assessment of BKv-specific T-cell functionality

BKv-specific T-cell functionality was evaluated for all patients at inclusion. Absolute values of BKv-specific CD4 or CD8 T-cells were normalized to CD4 or CD8 T-cell frequencies within PBMCs and expressed per 10,000 CD4 or CD8 T cells.

We isolated peripheral blood mononuclear cells (PBMCs) from a whole-blood sample by centrifugation on a Ficoll gradient. PBMCs were frozen at a final concentration of 1 × 10⁷ cells/mL. For all patients, one or two tubes of cells were cryopreserved for a mean of less than 12 months, in a room strictly maintained at the same temperature so as to preserve T-cell viability and function^61,62.

For BKv stimulation, PBMCs were incubated with two different BKv-specific antigens (LT-Ag and VP1). These overlapping BKv-specific peptides activate both CD4 and CD8 T cells (PepTivator BKV LT-Ag or VP1 used at a final concentration of 1 µg/mL/peptide, Miltenyi Biotec®). We also used CEF peptides (pool of peptides from class I-restricted T-cell epitopes from Cytomegalovirus, Epstein-Barr and Influenza viruses) as a global antiviral immune control. All stimulations were performed relative to a negative control (unstimulated cells) and a positive control (staphylococcal enterotoxin B – SEB). We assessed lymphocyte functionality⁹ by measuring proliferation, cytokine production and cytotoxic capacities⁶³ by flow cytometry with a BD LSRFortessa™ (Supplementary data: Figure S1). Due to the lymphopenia presented by the KTRs, the low frequencies of BKv-specific T cells as previously described^7,64 and cell mortality during the freezing and thawing processes, lymphocyte function was assessed on a limited number of KTRs in each group.

BKv-specific T-cell proliferation in response to BKv antigens

We assessed lymphocyte proliferation in response to BKv peptides. PBMCs were stained with 0.5 µM CFSE and incubated for five days with BKv-specific peptide pools. The stimulated cells were then stained with antibodies against surface markers (antibodies against CD3, CD4, and CD8, BD Bioscience®) and their proliferation capacity was evaluated by CFSE dilution (Supplementary data: Figure S1).

BKv-specific T-cell cytokine secretion in response to BKv antigens

We assessed cytokine secretion in response to BKv peptides. PBMCs were incubated overnight, in the presence of brefeldin A (at a final concentration of 1 µl/mL, BD®), and BKv-specific peptide pools. After overnight culture, the stimulated cells were stained with antibodies against surface markers, then fixed and permeabilized before incubation with anti-interferon-γ (IFNγ) and anti-tumor necrosis factor-α (TNFα) antibodies (BD Bioscience®). Lymphocyte polyfunctionality was evaluated by determining the capacity of BKv-specific T cells for single (IFNγ⁺ or TNFα⁺) or double (IFNγ⁺ and TNFα⁺) cytokine secretion (Supplementary data: Figure S1).

BKv-specific T-cell cytotoxicity in response to BKv antigens

We measured BKv-specific CD8 T-cell cytotoxicity by incubating PBMCs overnight with BKv peptide-coated autologous target cells (ratio 3/1). Target cells were distinguished from BKv-specific CD8 T cells by CFSE staining. After stimulation, the mortality rate of target cells was evaluated by 7-amino-actinomycin D staining (Supplementary data: Figure S1).

BKv-specific T-cell proliferation in response to heterospecific immunity provided by allogeneic CD4 T-cell help

We assessed lymphocyte proliferation in response to BKv peptides in the presence or absence of allogeneic CD4 T-cells. PBMCs from 11 KTRs with plasma BKv reactivation were stained with a fluorescent dye, depleted of autologous CD4 T cells by magnetic depletion (CD4 MicroBeads human, Miltenyi®), and incubated for five days with BKv-specific peptide pools in the presence of autologous or allogeneic third-party CD4 T cells, at a ratio of 1 CD4 to 5 non-CD4 T cells. We distinguished PBMCs from allogeneic third-party cells by using two fluorescent dyes (CFSE or VPD). Unstimulated CD4-depleted PBMCs incubated in the presence of autologous CD4 T cells or allogeneic third-party CD4 T cells were used as controls. After five days of culture, the stimulated cells were stained for CD3, CD4, and CD8 and their proliferation capacity was evaluated by fluorescent dye dilution (CFSE or VPD - Fig. 5c).

Assessment of T-cell phenotype and inhibitory receptor expression

We evaluated the ex vivo phenotype of CD4 and CD8 T cells. CD4 and CD8 T cells were stained for memory T-cell subpopulation identification^65–67 (stem-cell memory [TSCM], central memory [TCM], effector memory [TEM], and terminally differentiated effector T cells [TEMRA]) (Supplementary data: Figures S4).

We also evaluated the expression of lymphocyte inhibitory receptors (programmed cell death 1 - PD1, and cytotoxic T-lymphocyte-associated protein 4 - CTLA4). Cytokine-secreting BKv-specific CD4 and CD8 T cells (boolean gate from IFNγ and/or TNFα secreting cells) were assessed for the expression of PD1 and/or CTLA4 and compared to non-responding T cells (without cytokine secretion) (Supplementary data: Figures S6).

• Statistical analysis

Percentages and bar charts were used for categorical data. Medians and interquartile ranges were calculated for the analysis of continuous data. For the normalization of data distributions, the frequencies of BKv-specific T cells were subjected to natural logarithm or square root transformation. For categorical data, groups were compared in chi-squared tests. Kaplan-Meier survival curves were plotted and compared in log-rank tests. For continuous data, comparisons between two groups were performed with nonparametric Mann-Whitney tests, whereas comparisons of more than two groups were performed with nonparametric Kruskal-Wallis tests, followed by Dunn’s test for multiple comparisons. All the tests used were two-sided. Correlations were evaluated with the nonparametric Spearman’s rank correlation test. Odds ratios with 95% confidence intervals (OR [95% CI]) were estimated and compared by univariate multinomial logistic regression with correction for the false discovery rate. Multivariate analysis was performed with Wald tests (multivariate multinomial logistic regression). Statistical analyses were performed with GraphPad Prism or STATA v15.0 (StataCorp, College Station, TX, US) software, and differences were considered significant if p < 0.05.

BKv

BK-virus

BKvAN

BK-virus-associated nephropathy

CEF

pool of cytomegalovirus, Epstein Barr virus and influenza virus peptides

CTLA4

cytotoxic T-lymphocyte-associated protein 4

DNA

deoxyribonucleic acid

eGFR

estimated glomerular filtration rate

HLA

human leukocyte antigen

IFNγ

interferon-γ

KTR

kidney transplant recipients

LT-Ag

large tumor antigen

mTor

mammalian target of rapamycin

OR [95% CI]

odd ratio with 95% confidence interval

PD1

programmed cell death 1

PBMC

peripheral blood mononuclear cell

PCR

polymerase chain reaction

TNFα

tumor necrosis factor-α

VP1

viral protein-1

ACKNOWLEDGMENTS

M.D. received a grant from La Société Francophone de Néphrologie, Dialyse et Transplantation (SFNDT).

The study was financed by a research found from Agence de la Biomédecine and Sandoz.

The authors wish to thank the patients, the medical and non-medical crews, and notably Dr Séverine Beaudreuil, Dr Michelle Elias and Dr Anne Grunenwald, for their involvement in the recruitment of patients at the Department of Nephrology and Transplantation, Le Kremlin-Bicêtre and Henri Mondor Hospitals from Assistance Publique - Hôpitaux de Paris (AP-HP).

DISCLOSURE

The authors have no conflicts of interest to disclose.

DATA AVAILABILITY STATEMENT

The data supporting the findings of this study are available from the corresponding

author upon reasonable request.

Tonelli, M. et al. Systematic review: kidney transplantation compared with dialysis in clinically relevant outcomes. Am. J. Transplant. 11, 2093–2109 (2011).
Hirsch, HH. et al. European perspective on human polyomavirus infection, replication and disease in solid organ transplantation. Clin. Microbiol. Infect. 20, 74–88 (2014).
Hirsch, HH. et al. Prospective study of polyomavirus type BK replication and nephropathy in renal-transplant recipients. N. Engl. J. Med. 347, 488–496 (2002).
Hirsch, HH. & Steiger, J. Polyomavirus BK. Lancet Infect. Dis. 3, 611–623 (2003).
Egli, A. et al. Prevalence of polyomavirus BK and JC infection and replication in 400 healthy blood donors. J. Infect. Dis. 199, 837–846 (2009).
Seemayer, C. A. et al. BK virus large T and VP-1 expression in infected human renal allografts. Nephrol. Dial. Transplant. 23, 3752–3761 (2008).
Schmidt, T. et al. BK polyomavirus-specific cellular immune responses are age-dependent and strongly correlate with phases of virus replication. Am. J. Transplant. 14, 1334–1345 (2014).
Dekeyser, M., François, H., Beaudreuil, S. & Durrbach, A. Polyomavirus-Specific Cellular Immunity: From BK-Virus-Specific Cellular Immunity to BK-Virus-Associated Nephropathy? Front. Immunol. 6, 307 (2015).
Sester, M., Leboeuf, C., Schmidt, T. & Hirsch, H. H. The ‘ABC’ of Virus-Specific T Cell Immunity in Solid Organ Transplantation. Am. J. Transplant. 16, 1697–1706 (2016).
Dekeyser, M., Ladrière, M., Audonnet, S., Frimat, L. & Bittencourt, M. D. C. An Early Immediate Early Protein IE-1–Specific T-Cell Polyfunctionality Is Associated With a Better Control of Cytomegalovirus Reactivation in Kidney Transplantation. Kidney Int. Rep. 2, 486–492 (2017).
de Goër de Herve, M. G., Cariou, A., Simonetta, F. & Taoufik, Y. Heterospecific CD4 help to rescue CD8 T cell killers. J. Immunol. 181, 5974–5980 (2008).
Ramos, E. et al. BK virus nephropathy diagnosis and treatment: experience at the University of Maryland Renal Transplant Program. Clin. Transpl. 143–153 (2002).
Ramos, E. et al. Clinical course of polyoma virus nephropathy in 67 renal transplant patients. J. Am. Soc. Nephrol. 13, 2145–2151 (2002).
Vasudev, B. et al. BK virus nephritis: risk factors, timing, and outcome in renal transplant recipients. Kidney Int. 68, 1834–1839 (2005).
Hirsch, H. H., Randhawa, P. S., & AST Infectious Diseases Community of Practice. BK polyomavirus in solid organ transplantation-Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice. Clin. Transplant. 33, e13528 (2019).
Halim, M. A. et al. Long-Term Follow-Up of Active Treatment Versus Minimization of Immunosuppressive Agents in Patients With BK Virus-Associated Nephropathy After Kidney Transplant. Exp. Clin. Transplant. 14, 58–65 (2016).
Shen, C.-L., Wu, B.-S., Lien, T.-J., Yang, A.-H. & Yang, C.-Y. BK Polyomavirus Nephropathy in Kidney Transplantation: Balancing Rejection and Infection. Viruses 13, 487 (2021).
Baek, C. H. et al. Risk Factors of Acute Rejection in Patients with BK Nephropathy After Reduction of Immunosuppression. Ann. Transplant. 23, 704–712 (2018).
Hirsch, HH. et al. Polyomavirus-associated nephropathy in renal transplantation: interdisciplinary analyses and recommendations. Transplantation 79, 1277–1286 (2005).
Kidney Disease: Improving Global Outcomes (KDIGO) Transplant Work Group. KDIGO clinical practice guideline for the care of kidney transplant recipients. Am. J. Transplant. 9, S1-155 (2009).
Thangaraju, S. et al. Risk Factors for BK Polyoma Virus Treatment and Association of Treatment With Kidney Transplant Failure: Insights From a Paired Kidney Analysis. Transplantation 100, 854–861 (2016).
Demey, B. et al. Risk factors for BK virus viremia and nephropathy after kidney transplantation: A systematic review. J. Clin. Virol. 109, 6–12 (2018).
Hirsch, HH. et al. Polyomavirus BK replication in de novo kidney transplant patients receiving tacrolimus or cyclosporine: a prospective, randomized, multicenter study. Am. J. Transplant. 13, 136–145 (2013).
Dharnidharka, V. R., Cherikh, W. S. & Abbott, K. C. An OPTN analysis of national registry data on treatment of BK virus allograft nephropathy in the United States. Transplantation 87, 1019–1026 (2009).
Gard, L. et al. A delicate balance between rejection and BK polyomavirus associated nephropathy; A retrospective cohort study in renal transplant recipients. PloS One 12, e0178801 (2017).
Hirsch, HH., Yakhontova, K., Lu, M. & Manzetti, J. BK Polyomavirus Replication in Renal Tubular Epithelial Cells Is Inhibited by Sirolimus, but Activated by Tacrolimus Through a Pathway Involving FKBP-12. Am. J. Transplant. 16, 821–832 (2016).
Acott, P. D., O’Regan, P. A., Lee, S. H. & Crocker, J. F. S. In vitro effect of cyclosporin A on primary and chronic BK polyoma virus infection in Vero E6 cells. Transpl. Infect. Dis. Off. J. Transplant. Soc. 10, 385–390 (2008).
Staatz, C. E. & Tett, S. E. Pharmacology and toxicology of mycophenolate in organ transplant recipients: an update. Arch. Toxicol. 88, 1351–1389 (2014).
Prémaud, A. et al. Inhibition of T-cell activation and proliferation by mycophenolic acid in patients awaiting liver transplantation: PK/PD relationships. Pharmacol. Res. 63, 432–438 (2011).
Roberts, M. B. & Fishman, J. A. Immunosuppressive Agents and Infectious Risk in Transplantation: Managing the ‘Net State of Immunosuppression’. Clin. Infect. Dis. Off. Publ. Infect. Dis. Soc. Am. 73, e1302–e1317 (2021).
Kaur, A., Wilhelm, M., Wilk, S. & Hirsch, H. H. BK polyomavirus-specific antibody and T-cell responses in kidney transplantation: update. Curr. Opin. Infect. Dis. 32, 575–583 (2019).
Comoli, P., Hirsch, H. H. & Ginevri, F. Cellular immune responses to BK virus. Curr. Opin. Organ Transplant. 13, 569–574 (2008).
Schachtner, T., Stein, M., Babel, N. & Reinke, P. The Loss of BKV-specific Immunity From Pretransplantation to Posttransplantation Identifies Kidney Transplant Recipients at Increased Risk of BKV Replication. Am. J. Transplant. 15, 2159–2169 (2015).
Schaenman, J. M. et al. Increased Frequency of BK Virus-Specific Polyfunctional CD8 + T Cells Predict Successful Control of BK Viremia After Kidney Transplantation. Transplantation 101, 1479–1487 (2017).
Schachtner, T. et al. BK virus-specific immunity kinetics: a predictor of recovery from polyomavirus BK-associated nephropathy. Am. J. Transplant. 11, 2443–2452 (2011).
Cai, H. et al. Immune Checkpoints in Viral Infections. Viruses 12, E1051 (2020).
Fenwick, C. et al. T-cell exhaustion in HIV infection. Immunol. Rev. 292, 149–163 (2019).
McLane, L. M., Abdel-Hakeem, M. S. & Wherry, E. J. CD8 T Cell Exhaustion During Chronic Viral Infection and Cancer. Annu. Rev. Immunol. 37, 457–495 (2019).
Blank, C. U. et al. Defining ‘T cell exhaustion’. Nat. Rev. Immunol. 19, 665–674 (2019).
Williams, R. C., Opelz, G., McGarvey, C. J., Weil, E. J. & Chakkera, H. A. The Risk of Transplant Failure With HLA Mismatch in First Adult Kidney Allografts From Deceased Donors. Transplantation 100, 1094–1102 (2016).
Williams, R. C., Opelz, G., Weil, E. J., McGarvey, C. J. & Chakkera, H. A. The Risk of Transplant Failure With HLA Mismatch in First Adult Kidney Allografts 2: Living Donors, Summary, Guide. Transplant. Direct 3, e152 (2017).
Shi, X. et al. What is the impact of human leukocyte antigen mismatching on graft survival and mortality in renal transplantation? A meta-analysis of 23 cohort studies involving 486,608 recipients. BMC Nephrol. 19, 116 (2018).
Scadden, J. R., Sharif, A., Skordilis, K. & Borrows, R. Polyoma virus nephropathy in kidney transplantation. World J. Transplant. 7, 329–338 (2017).
Leboeuf, C. et al. BK Polyomavirus-Specific 9mer CD8 T Cell Responses Correlate With Clearance of BK Viremia in Kidney Transplant Recipients: First Report From the Swiss Transplant Cohort Study. Am. J. Transplant. (2017) doi:10.1111/ajt.14282.
Cioni, M., Leboeuf, C., Comoli, P., Ginevri, F. & Hirsch, H. H. Characterization of Immunodominant BK Polyomavirus 9mer Epitope T Cell Responses. Am. J. Transplant. 16, 1193–1206 (2016).
Wunderink, H. F. et al. Reduced Risk of BK Polyomavirus Infection in HLA-B51-positive Kidney Transplant Recipients. Transplantation 103, 604–612 (2019).
Willhelm, M., Wilk, S., Kaur, A., Hirsch, H. H., & Swiss Transplant Cohort Study. Can HLA-B51 Protect Against BKPyV-DNAemia? Transplantation 103, e384–e385 (2019).
Helanterä, I. et al. BK virus viremia in a well-HLA-matched kidney transplant population mainly on low-dose cyclosporine-based immunosuppression. Clin. Transplant. 26, E596-601 (2012).
Drachenberg, C. B. et al. Negative impact of human leukocyte antigen matching in the outcome of polyomavirus nephropathy. Transplantation 80, 276–278 (2005).
Hässig, A. et al. Association of BK viremia with human leukocyte antigen mismatches and acute rejection, but not with type of calcineurin inhibitor. Transpl. Infect. Dis. 16, 44–54 (2014).
Wunderink, H. F. et al. Source and Relevance of the BK Polyomavirus Genotype for Infection After Kidney Transplantation. Open Forum Infect. Dis. 6, ofz078 (2019).
Awadalla, Y., Randhawa, P., Ruppert, K., Zeevi, A. & Duquesnoy, R. J. HLA mismatching increases the risk of BK virus nephropathy in renal transplant recipients. Am. J. Transplant. 4, 1691–1696 (2004).
Laidlaw, B. J., Craft, J. E. & Kaech, S. M. The multifaceted role of CD4(+) T cells in CD8(+) T cell memory. Nat. Rev. Immunol. 16, 102–111 (2016).
de Goër de Herve, M. G., Jaafoura, S., Vallée, M. & Taoufik, Y. FoxP3⁺ regulatory CD4 T cells control the generation of functional CD8 memory. Nat. Commun. 3, 986 (2012).
de Goër de Herve, M. G. et al. Direct CD4 help provision following interaction of memory CD4 and CD8 T cells with distinct antigen-presenting dendritic cells. J. Immunol. 185, (2010).
Zeng, G. et al. Antigen-specificity of T-cell Infiltrates in Biopsies with T-cell Mediated Rejection and BK Polyomavirus Viremia: Analysis by Next Generation Sequencing. Am. J. Transplant. (2016) doi:10.1111/ajt.13911.
Sawinski, D. et al. Persistent BK viremia does not increase intermediate-term graft loss but is associated with de novo donor-specific antibodies. J. Am. Soc. Nephrol. JASN 26, 966–975 (2015).
Karahan, G. E., Claas, F. H. J. & Heidt, S. Heterologous Immunity of Virus-Specific T Cells Leading to Alloreactivity: Possible Implications for Solid Organ Transplantation. Viruses 13, 2359 (2021).
Nickeleit, V. et al. The Banff Working Group Classification of Definitive Polyomavirus Nephropathy: Morphologic Definitions and Clinical Correlations. J. Am. Soc. Nephrol. JASN 29, 680–693 (2018).
Drachenberg, C. B., Hirsch, H. H., Ramos, E. & Papadimitriou, J. C. Polyomavirus disease in renal transplantation: review of pathological findings and diagnostic methods. Hum. Pathol. 36, 1245–1255 (2005).
Angel, S. et al. Toward Optimal Cryopreservation and Storage for Achievement of High Cell Recovery and Maintenance of Cell Viability and T Cell Functionality. Biopreservation Biobanking 14, 539–547 (2016).
Ford, T. et al. Cryopreservation-related loss of antigen-specific IFNγ producing CD4 + T-cells can skew immunogenicity data in vaccine trials: Lessons from a malaria vaccine trial substudy. Vaccine 35, 1898–1906 (2017).
Dekeyser, M. et al. Refractory T-Cell Anergy and Rapidly Fatal Progressive Multifocal Leukoencephalopathy After Prolonged CTLA4 Therapy. Open Forum Infect. Dis. 4, ofx100 (2017).
Schulze Lammers, F. C. et al. Antiviral T-Cell Frequencies in a Healthy Population: Reference Values for Evaluating Antiviral Immune Cell Profiles in Immunocompromised Patients. J. Clin. Immunol. (2022) doi:10.1007/s10875-021-01205-1.
Farber, D. L., Yudanin, N. A. & Restifo, N. P. Human memory T cells: generation, compartmentalization and homeostasis. Nat. Rev. Immunol. 14, 24–35 (2014).
Gattinoni, L. et al. A human memory T cell subset with stem cell-like properties. Nat. Med. 17, 1290–1297 (2011).
Lugli, E. et al. Superior T memory stem cell persistence supports long-lived T cell memory. J. Clin. Invest. 123, 594–599 (2013).

Thawed PBMCs were incubated at 37°C, under an atmosphere containing 5% CO₂, in Roswell Park Memorial Institute (RPMI) medium alone (unstimulated cells), with BKv-specific antigen stimulation (LT-Ag or VP1 at a final concentration of 1 µg/mL/peptide), or with CEF peptides (cytomegalovirus, Epstein-Barr virus, and influenza virus peptide pools at a final concentration of 1 µg/mL/peptide). The positive control consisted of incubation with staphylococcal enterotoxin B (SEB) at a final concentration of 0.2 µg/mL.

For T-cell proliferation, we stained 2 x 10⁶ PBMCs with 0.5 μM CFSE (THERMO FISHER SCIENTIFIC^®) and incubated them for five days. The stimulated cells were stained with antibodies directed against surface markers (CD3-BV605, CD4-V500, and CD8-APC H7 antibodies, BD Bioscience^®) and proliferative capacity was evaluated by CFSE dilution (Figure S1).

For T-cell cytokine secretion, we incubated 4 x 10⁶ PBMCs overnight in the presence of brefeldin A (at a final concentration of 1 µl for every 1 mL of cell culture, BD Bioscience^®). The cells were then stained with antibodies against surface markers (CD3-BV605, CD4-VioGreen, CD8-APC Vio770 antibodies, BD Bioscience^®), fixed and permeabilized for intracellular cytokine staining (IFN_γ-PE and TNF_α-Alexa 700 antibodies, BD Bioscience^®). Lymphocyte polyfunctionality was evaluated by determining the capacity of BKv-specific T cells to secrete one (IFN_γ⁺ or TNF_α⁺) or two (IFN_γ⁺ and TNF_α⁺) cytokines (Figure S1).

For T-cell cytotoxicity, we incubated 4 x 10⁶ PBMCs overnight with BKv peptide-coated autologous target cells (ratio of 3 CD8 T cells to 1 target cell). Target cells were distinguished from CD8 T cells by CFSE staining (THERMO FISHER SCIENTIFIC^®). Target cell mortality after stimulation was evaluated by 7-amino-actinomycin D staining (BD Bioscience^® - Figure S1).

We performed multicolor flow cytometry analysis with a BD LSRFortessa™ flow cytometer and FlowJo^®software. We ensured that the analysis of BKv-specific T-cell responses was robust, by analyzing a median of 40,492 [19,962-80,339] total T cells for CD4 T cells and 24,560 [11,443-65,902] for CD8 T cells, with a median of BKv-specific responding T cells of 590.5 [142.3-2,070].

There is NO Competing Interest.

SupplementarydataTableS1.docx
Supplementary data: Table S1. Therapeutic management before and after BKvAN diagnosis
SupplementarydataTableS2.docx
Supplementary data: Table S2. Assessment of antiviral T-cell responses in the context of kidney transplantation (KTRs without BKv reactivation)
SupplementarydataTableS3.docx
Supplementary data: Table S3. CEF-specific memory T-cell responses in KTR groups defined on the basis of BKv reactivation
SupplementarydataTableS4.docx
Supplementary data: Table S4. Comparison of memory T-cell subpopulations of CD4 and CD8 T cells between KTR groups defined on the basis of BKv reactivation
SupplementarydataFigureS1.pdf
Figure S1. Assessment of cellular immune response polyfunctionality in terms of cytokine secretion, proliferative, and cytotoxic capacities We assessed lymphocyte functionality by measuring cytokine secretion, proliferative and cytotoxic capacities by multicolor flow cytometry (BD LSRFortessa™). Peripheral blood mononuclear cells were incubated alone (unstimulated cells) or with overlapping BKv (VP1 or LT-Ag) peptide pools, CEF peptide pools, or with staphylococcal enterotoxin B (SEB). The frequency of responsive T cells was calculated by subtracting the frequency of cells detected in the absence of stimulation to correct for background staining. T-cell functionality was evaluated in healthy donors and in the groups of KTRs defined on the basis of BKv reactivation. Due to the cell mortality induced by the freezing and thawing processes, lymphocyte polyfunctionality was assessed on a limited number of KTRs from each group.Proliferative capacity. Lymphocyte proliferation was measured by carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution. Numbers indicate the percentage of CFSE^low cells among CD4 and CD8 T cells. Cytokine production capacity. The production of interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα) was evaluated by intracellular staining. Numbers indicate the percentages of CD4 or CD8 T cells secreting IFNγ, TNFα, or IFNγ/TNFα.Cytotoxic capacity. PBMCs were incubated overnight with autologous target cells coated with various antiviral stimulation (BKv or CEF) peptide pools, at a ratio of 3 CD8 T cells to one target cell. Target cells were distinguished by CFSE staining. CD8 cytotoxicity was measured as the number of dead target cells, identified by 7-amino-actinomycin D (7AAD) staining. Numbers indicate the percentage of 7AAD⁺ target cells.7AAD: 7-amino-actinomycin D; BKv: BK-virus; CEF: cytomegalovirus, Epstein-Barr virus, and influenza virus peptide pools; CFSE: carboxyfluorescein diacetate succinimidyl ester; IFNγ: interferon-γ; TNFα: tumor necrosis factor-α.
SupplementarydataFigureS2.pdf
Figure S2. Polyfunctionality of antiviral CD8 T cells (after CEF stimulation) in groups of KTRs defined on the basis of BKv reactivationCD8 T-cell functionality (expressed as the normalized responding T-cell number) after CEF stimulation in the groups of KTRs defined on the basis of BKv reactivation, with assessment of (a) proliferation, (b) IFNγ/TNFα secretion and (c) cytotoxic activity. Without BKv: patients without plasma or urinary BKv reactivation; Viruria: patients with urinary BKv reactivation; Viremia: patients with plasma BKv reactivation; BKvAN: patients with BK-virus-associated nephropathy; 7AAD: 7-amino-actinomycin D; CEF: cytomegalovirus, Epstein-Barr virus, and influenza virus peptide pools; CFSE: carboxyfluorescein diacetate succinimidyl ester; IFNγ: interferon-γ; n: number of patients; normalized cell nb: normalized CEF-specific T-cell frequencies; p: p-values; TNFα: tumor necrosis factor-α. p-values indicate the significance of differences between groups in Kruskal-Wallis tests. Scatter dot plots are shown, with the median and interquartile range. ns = non-significant.
SupplementarydataFigureS3.pdf
Figure S3. Assessment of BKv-specific CD8 cytotoxicity in KTRs in the context of BKv reactivationCytotoxic capacity of BKv-specific CD8 T cells for KTR groups defined according to BKv reactivation levels.The cytotoxic response is expressed as (a) the number of normalized dead BKv-coated target cells and (b) the proportion of patients responding to BKv peptides.PBMCs were incubated overnight with autologous target cells coated with BKv-peptide pools (ratio: 3 CD8 T cells to one target cell). CD8 cytotoxicity was measured as the normalized number of dead target cells identified by 7-amino-actinomycin D (7AAD) staining. A cutoff of 3.16 normalized dead target cells was used to distinguish between responders and non-responders. (n) represents the number of patients. For each patient, two BKv-specific responses were analyzed, after LT-Ag and VP1-peptide stimulations.Viruria: patients with urinary BKv reactivation; Viremia: patients with plasma BKv reactivation; BKvAN: patients with BK-virus-associated nephropathy; 7AAD: 7-amino-actinomycin D; BKv: BK-virus; LT-Ag: large tumor antigen; n: number of patients; normalized cell nb: normalized number of dead target cells; VP1: viral protein-1.p-values indicate the significance of differences between groups in Kruskal-Wallis tests (left side) and χ2 for trend (right side) tests. Scatter dot plots are shown, with the median and interquartile range. *p < 0.05, ns = non-significant.
SupplementarydataFigureS4.pdf
Figure S4. Gating strategy of memory CD4 and CD8 T-cell subpopulationsWe evaluated the ex vivo phenotype of CD4 and CD8 T cells. For the identification of memory T-cell subpopulations (stem-cell memory [TSCM], central memory [TCM], effector memory [TEM], and terminally differentiated effector T cells [TEMRA]), CD4 and CD8 T cells were stained with antibodies directed against surface markers (CD3-BV605, CD4-Viogreen, CCR7-PE-CF594, CD27-BV786, BD Bioscience^® and CD8-APC-Vio770, CD62L-V450, CD45RA-PC5, CD45RO-PC7, CD95-APC Miltenyi Biotec^®).
SupplementarydataFigureS5.pdf
Figure S5. Assessment of memory subpopulations of CD4 and CD8 T cells in KTRs in the context of BKv reactivationLymphocyte memory subpopulations were evaluated in CD4 (left side) and CD8 (right side) T cells, as the frequency of (a) and (b) stem-cell memory T cells [TSCM], (c) and (d) central memory T cells [TCM], (e) and (f) effector memory T cells [TEM], and (g) and (h) terminally differentiated effector T cells [TEMRA], in KTRs in the context of BKv reactivation. Without BKv: patients without plasma or urinary BKv reactivation; Viruria: patients with urinary BKv reactivation; Viremia: patients with plasma BKv reactivation; BKvAN: patients with BK-virus-associated nephropathy; BKv: BK-virus; n: number of patients. p-values indicate the significance of differences between groups in Kruskal-Wallis tests followed by Dunn’s tests for multiple comparisons. Box and whiskers plots showing the median and [5th-95th percentile]. **p < 0.005, ns = non-significant.
SupplementarydataFigureS6.pdf
Figure S6. Gating strategy of the expression of inhibitory receptors on BKv-specific CD4 and CD8 T cellsWe evaluated the expression of lymphocyte inhibitory receptors (programmed cell death 1 - PD1, and cytotoxic T-lymphocyte-associated protein 4 - CTLA4). Cytokine-secreting BKv-specific CD4 and CD8 T cells (boolean gate from IFNγ and/or TNFα secreting cells) were assessed for the expression of PD1 and/or CTLA4 and compared to non-responding T cells (without cytokine secretion). For evaluation of the expression of lymphocyte inhibitory receptors (PD1 and CTLA4), PBMCs were stained with the following antibodies (PD1-BV650, CTLA4-BV711, BD Bioscience®).

PDF

Version 1

posted 26 May, 2022

You are reading this latest preprint version

Heterospecific Immunity Help to Sustain an Effective BK-virus Immune Response and Prevent BK-virus-associated Nephropathy

Status:

Version 1

Abstract

Figures

Introduction

Results

Clinical characteristics of KTRs according to level of BKv reactivation

BKv reactivation in KTRs

Impact of BKv reactivation on allograft outcome and graft survival

Risk factors for BKvAN

Identification of risk factors associated with BKv reactivation

Multivariate analysis to identify risk factors associated with BKv reactivation

Exhaustion of BKv-specific CD4 and CD8 T cells

Allogeneic CD4 T cells restore BKv-specific CD8 T-cell functionality

Discussion

Methods

BKv-specific T-cell proliferation in response to BKv antigens

BKv-specific T-cell cytokine secretion in response to BKv antigens

BKv-specific T-cell cytotoxicity in response to BKv antigens

BKv-specific T-cell proliferation in response to heterospecific immunity provided by allogeneic CD4 T-cell help

• Statistical analysis

Abbreviations

Declarations

ACKNOWLEDGMENTS

DISCLOSURE

DATA AVAILABILITY STATEMENT

References

Supplementary Materials And Methods

Competing Interests

Supplementary Files

Status:

Version 1