Allogeneic CD4 T Cells Sustain Effective BK Polyomavirus-Specific CD8 T Cell Response in Kidney Transplant Recipients

Introduction BK polyomavirus-associated nephropathy (BKPyVAN) is a significant complication in kidney transplant recipients (KTRs), associated with a higher level of plasmatic BK polyomavirus (BKPyV) replication and leading to poor graft survival. Methods We prospectively followed-up with 100 KTRs with various degrees of BKPyV reactivation (no BKPyV reactivation, BKPyV-DNAuria, BKPyV-DNAemia, and biopsy-proven BKPyVAN [bp-BKPyVAN], 25 patients per group) and evaluated BKPyV-specific T cell functionality and phenotype. Results We demonstrate that bp-BKPyVAN is associated with a loss of BKPyV-specific T cell proliferation, cytokine secretion, and cytotoxic capacities. This severe functional impairment is associated with an overexpression of lymphocyte inhibitory receptors (programmed cell death 1 [PD1], cytotoxic T lymphocyte-associated protein 4, T cell immunoreceptor with Ig and ITIM domains, and T cell immunoglobulin and mucin domain-containing-3), highlighting an exhausted-like phenotype of BKPyV-specific CD4 and CD8 T cells in bp-BKPyVAN. This T cell dysfunction is associated with low class II donor-recipient human leukocyte antigen (HLA) divergence. In contrast, in the context of higher class II donor-recipient HLA (D/R-HLA) divergence, allogeneic CD4 T cells can provide help that sustains BKPyV-specific CD8 T cell responses. In vitro, allogeneic HLA-mismatched CD4 T cells rescue BKPyV-specific CD8 T cell responses. Conclusion Our findings suggest that in KTRs, allogeneic CD4 T cells can help to maintain an effective BKPyV-specific CD8 T cell response that better controls BKPyV replication in the kidney allograft and may protect against BKPyVAN.


(b)
BKPyV-specific T-cell functionality was evaluated in KTRs without BKPyV reactivation.

SUPPLEMENTARY MATERIAL -Figure S2. Assessment of the expression of inhibitory receptors on BKPyV-specific CD4 and CD8 T cells
We evaluated the expression of lymphocyte inhibitory receptors (programmed cell death 1 -PD1, cytotoxic T-lymphocyte-associated protein 4 -CTLA4, T cell immunoreceptor with Ig and ITIM domains -TIGIT, T-cell immunoglobulin and mucin-domain containing-3 -TIM3).
Cytokine-secreting BKPyV-specific CD4 and CD8 T cells (Boolean gate from IFNγand/or TNFα-secreting cells) were assessed for the expression of PD1 and/or CTLA4 and compared to non-responding T cells (without cytokine production).To evaluate the expression of lymphocyte inhibitory receptors (PD1 and CTLA4), PBMCs were stained with the following antibodies (PD1-BV650, Biotin CTLA4-BV711 Streptavidin).
Proliferative BKPyV-specific CD4 and CD8 T cells were assessed for the expression of PD1 and/or TIM3 and/or TIGIT and compared to non-responding T cells (non-proliferative T cells).

BKPyV-specific CD4 T cells in KTR groups
BKPyV-specific CD4 T cells were expressed as the number of cells per 10,000 CD4 T cells and then subjected to square root transformation to normalize the data distributions.the rules of the local ethics committee.Written informed consent was obtained from all patients.
The research was conducted independently of sex or gender considerations.
We evaluated clinical characteristics, kidney graft outcome, immunosuppressive regimen, plasma and urine BKPyV viral load, HLA mismatches between donor and recipient, and BKPyV-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 BKPyV viral loads and each patient's duration of BKPyV reactivation.Plasma and urine samples were obtained during routine visits to our center and during acute allograft failure.BKPyV screening was performed in accordance with international guidelines 1,2 by assessing plasma BKPyV load monthly in the first nine 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.Urinary BKPyV load was also determined during plasma screening for BKPyV.We quantified BKPyV DNA by BKPyVspecific real-time PCR (BKV R-GENE).
Glomerular filtration rate was estimated with the MDRD-4 formula (eGFR) in the first month after transplantation, 12 months post-transplantation or at BKPyVAN 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 when 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 BKPyVAN diagnosis or 12 months after transplantation.
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 × 10 7 cells/mL and used within 12 months after cryopreservation.
For BKPyV activation, PBMCs were incubated with two different BKPyV-specific antigens (LT-Ag and VP1).These overlapping BKPyV-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).We also used CEF peptides (pool of peptides from class I-restricted T-cell epitopes from cytomegalovirus, Epstein-Barr, and influenza viruses used at a final concentration of 1 μg/mL/peptide) as a global antiviral immune control.All activations were performed relative to a negative control (unstimulated cells) and a positive control (staphylococcal enterotoxin B -SEB, at a final concentration of 0.2 µg/mL).We assessed lymphocyte functionality by measuring proliferation, cytokine production, and cytotoxic capacities, 18 by flow cytometry (Figure S1).The lymphopenia of the KTRs resulted in low frequencies of BKPyV-specific T cells, as previously described, 11,50 and high rates of cell mortality during the freezing and thawing processes.We, therefore, assessed lymphocyte function for a limited number of KTRs in each group.Furthermore, we ensured that the analysis of BKPyV-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] total T cells for CD8 T cells, with a median of 590.5 [142.3-2,070]BKPyV-specific responding T cells.To confirm the specificity of the BKPyV-specific antigenic activation, we isolated BKPyV-specific CD8 T cells using BKPyV-specific pentamers from a subgroup of HLA-A2 or HLA-B7 KTRs.BKPyV-loaded pentamers were from ProImmune ® .CD8 T cells were stained as recommended by the manufacturer.
T-cell proliferation in response to BKPyV antigens was assessed by CFSE dilution.

BKPyV-specific T-cell proliferation in the presence of allogeneic CD4 T-cell help
We assessed lymphocyte proliferation in response to BKPyV peptides in the presence and absence of allogeneic CD4 T cells.PBMCs from thirteen KTRs with bp-BKPyVANwere stained with a fluorescent dye, depleted of autologous CD4 T cells by magnetic depletion (CD4 MicroBeads), and incubated for five days with BKPyV-specific peptide pools in the presence of autologous or allogeneic third-party CD4 T cells, at a ratio of one CD4 to five non-CD4 T cells.We distinguished PBMCs from allogeneic third-party cells using two fluorescent dyes amino-actinomycin D (7AAD) staining.Numbers indicate the percentage of 7AAD + target cells.7AAD: 7-amino-actinomycin D; BKPyV: BK polyomavirus; CEF: cytomegalovirus, Epstein-Barr virus, and influenza virus peptide pools; CFSE: carboxyfluorescein diacetate succinimidyl ester; IFNγ: interferon-γ; TNFα: tumor necrosis factor-α.
(n)    represents the number of patients.Two BKPyV-specific responses were analyzed for each patient after activation with two BKPyV-specific peptide pools (LT-Ag and VP1-peptides).BKPyV-DNuria: patients with BKPyV reactivation in urine; BKPyV-DNAemia: patients with plasma BKPyV reactivation; biopsy-proven BKPyVAN: patients with biopsy-proven BK polyomavirus-associated nephropathy; BKPyV: BK polyomavirus; IFNγ: Interferon-γ, LT-Ag: large tumor antigen; n: number of patients; TNFα: Tumor necrosis factor-α, TNFα & IFNγ: Coproduction of Interferon-γ and Tumor necrosis factor-α, VP1: viral protein 1. p-values indicate the significance of differences between groups in Kruskal-Wallis tests, followed by Dunn's tests for multiple comparisons.Scatter dot plots are shown with the median and interquartile range.*p < 0.05.SUPPLEMENTARY MATERIAL -Figure S4.Polyfunctionality of antiviral CD8 T cells (after CEF activation) in KTR groups CD8 T-cell functionality after CEF activation in the groups of KTRs, with an assessment of (a) proliferation, (b) cytotoxic activity, (c) IFNγ production, (d) TNFα production, and (e) coproduction of IFNγ and TNFα.CEF-specific CD8 T cells were expressed as the number of cells per 10,000 CD8 T cells and then subjected to natural logarithm or square root transformation to normalize the data distributions.(n) represents the number of patients.BKPyV-DNAuria: patients with BKPyV reactivation in urine; BKPyV-DNAemia: patients with plasma BKPyV reactivation; biopsy-proven BKPyVAN: patients with biopsy-proven BK polyomavirus-Barr virus, and influenza virus peptide pools; CFSE: carboxyfluorescein diacetate succinimidyl ester; IFNγ: interferon-γ; n: number of patients; 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.expression on BKPyV-specific T cells in the different groups of BKPyV reactivation The expression of inhibitory receptors (PD1, CTLA4) was evaluated on BKPyV-specific T cells in patients without BKPyV reactivation, BKPyV-DNAuria, BKPyV-DNAemia and bp-BKPyVAN groups, on CD 4 and CD8 T-cells.p-values indicate the significance of differences between groups in Mann-Whitney U tests.Scatter dot plots are shown with the median and interquartile range.*p < 0.05, **p < 0.005, ****p < 0.0001, n= number of patients.Comparison of identifying BKPyV-specific CD8 T cells (% of CD8 T cells) according to CFSE dilution or using BKPyV-specific pentamers (n=8).b-Functional assessment of BKPyVpentamers positive CD8 T cells: proliferation capacity (CFSE dilution) (n=5) or cytokine production (IFN or TNF) (n=2).c-Expression of PD1 and TIGIT in BKPyV-specific CD8 T cells stimulated with BKPyV peptides and identified by BKPyV-specific pentamers.The FASC gating strategy and a representative dot plot of the expression of TIGIT and PD1 in CD8 T-cells from a patient (HLA-A2) with a BKPyVAN are shown.The left panel shows the expression of TIGIT and PD1 in BKPyV-pentamers positive CD8, and the right panel shows their expression on BKPyV-pentamers negative CD8 T cells.d shows the percentage of TIGIT or PD1 expression on BKPyV-specific CD8 T cells identified by BKPyV-specific pentamers from 5 patients (HLA-A2 and/or HLA-B7) with a BKPyVAN.PD1 and TIGIT were more frequently observed in BKPyV pentamers positive CD8 T cells than in non-specific CD8 Correlation between class I D/R-HLA-divergence and class I HLA mismatches classified as 0, 1, or 2 mismatches in the BKv-viremia and BKvAN groups (a).Correlation between class II D/R-HLA-divergence and class II HLA mismatches classified as 0, 1, or 2 mismatches in the BKv-viremia and BKvAN groups (b).nb: number of class I or II HLA mismatches.c and d: Correlation between D/R-HLA-divergence and allogeneic T-cell proliferation.Allogeneic CD4 T cells were stained with CFSE and mixed with allogeneic PBMC depleted from CD4 T cells using anti-CD4 magnetic beads (Miltenyi).Proliferating T cells were evaluated by CFSE dilution at day 5. D/R-HLA-divergence (Grantham score) was calculated for each combination of donor and recipient and each HLA loci.The Grantham score (class I (HLA-A and HLA-B) or II (HLA-DR and HLA-DQ)) was compared to the square root of the percentage of proliferating CD4 T cells.D/R Class-II HLA divergence significantly correlates with the proliferation of allogeneic CD4 T cells but not with D/R Class-I HLA divergence.**** p<0.0001SUPPLEMENTARY MATERIAL -Table S1.Therapeutic management before and after bp-BKPyVANdiagnosis SUPPLEMENTARY MATERIAL -Table S2.Assessment of BKPyV and CEF-specific Tcell responses in the context of kidney transplantation (KTRs without BKPyV reactivation)

Table-S2. Assessment of BKPyV and CEF-specific T-cell responses in the context of kidney transplantation (KTRs without BKPyV reactivation) Assessment of antiviral T-cell response
BKPyV: BK polyomavirus; n: number of patients; CEF: pool of cytomegalovirus, Epstein Barr virus, and influenza virus peptides; cell nb: normalized cell number; NA: Not applicable.Continuous data are expressed as medians and interquartile ranges.For each patient, two BKPyV-specific responses were analyzed after stimulation with LT-Ag and VP1 peptides.