KIR and HLA-C interactions promote differential dendritic cell maturation and is a major determinant of graft failure following kidney transplantation

Abstract

Background: HLA-C is an important ligand for killer immunoglobulin like receptors (KIR) that regulate natural killer (NK) cell function. Based on KIR specificity HLA-C molecules are allocated into two groups, HLA-C1 or HLA-C2; HLA-C2 is more inhibiting to NK cell function than HLA-C1. We studied the clinical importance of HLA-C genotypes on the long-term graft survival of 760 kidney transplants performed at our centre utilising a population based genetic study and cell culture model to define putative mechanisms.

Methods and findings: Genotyping was performed using conventional DNA PCR techniques and correlations made to clinical outcomes. We found that transplant recipients with HLA-C2 had significantly better long-term graft survival than transplant recipients with HLA-C1 (66% versus 44% at 10 years, log-rank p = 0.002, HR = 1.51, 95%CI = 1.16-1.97). In in-vitro NK and dendritic cell (DC) co-culture model we made several key observations that correlated with the population based genetic study. We observed that donor derived NK cells, on activation with IL-15, promoted differential HLA-C genotype dependent DC maturation. In NK-DC co-culture, the possession of HLA-C2 by DC was associated with anti-inflammatory cytokine production (IL-1RA/IL-6), diminished DC maturation (CD86, HLA-DR), and absent CCR7 expression. Conversely, possession of HLA-C1 by DC was associated with pro-inflammatory cytokine synthesis (TNF-α, IL-12p40/p70), enhanced DC maturation and up-regulation of CCR7 expression. By immunohistochemistry the presence of donor NK cells was confirmed in pre-transplant kidneys.

Conclusions: We propose that after kidney transplantation IL-15 activated donor derived NK cells interact with recipient DC with less activation of indirect allo-reactivity in HLA-C2 positive recipients than HLA-C1 positive recipients; this has implications for long-term graft survival. Early events following kidney transplantation involving NK-DC interaction via KIR and HLA-C immune synapse may have a central role in long-term kidney transplant outcomes.

Figure 1. Kaplan Meier survival curve showing death non-censored graft survival for the presence or absence of HLA-C2 allele in the recipient.

The presence of an HLA-C2 allele in the recipient was associated with a significant improvement in death non-censored graft survival (10-year survival: 65.7% versus 43.8%).

Figure 2. Kaplan Meier survival curve showing death censored graft survival for the presence or absence of HLA-C2 allele in the recipient.

The presence of an HLA-C2 allele in the recipient was associated with a significant improvement in death censored graft survival (10-year survival: 74.2% versus 54.3%).

Figure 3. Kaplan Meier survival curve showing patient survival for the presence or absence of HLA-C2 allele in the recipient.

The presence of an HLA-C2 allele in the recipient was associated with a significant improvement in patient survival (10-year survival: 88.5% versus 80.4%).

Figure 4. Immunohistochemistry slide demonstrating donor derived CD56 positive cells (anti-CD56 antibody staining brown in colour: arrow as indicator) in pre-transplant kidney biopsy tissue.

In brief, the method for development of this slide included dewaxing and antigen retrieval obtained by W-cap system (Bio-Optica) and staining with mouse monoclonal anti-CD56 antibody (IgG2b Novocastra) used at a dilution of 1∶50 and visualised with the EnVision detection system (DAKO). This image is representative of the observation made for biopsies taken from five different kidney transplants studied. Cell counts (degree of infiltration) were performed using light microscopy and counting 10 randomly selected high power fields at a magnification of 400× (area = 0.17 mm²). Mean (±SEM) of 3±2 CD56 positive cells were identified per high power field.

Figure 5. Comparisons are made for ΔMFI of CD86, HLA-DR and CCR7 expression between DCs with either HLA-C1 or HLA-C2 homozygous allele.

Data shown are ΔMFI of CD86, HLA-DR and CCR7 expressed by DC in NK-DC co-culture in the presence of 1 ng/ml IL-15 at cell ratios of either 1∶1 or 1∶5. ΔMFI are calculated as the difference of MFI for DC in co-culture versus DC in isolation i.e. spontaneous expression. In NK-DC co-culture, in the presence of IL-15, DC with HLA-C1 homozygous allele express more co-stimulation molecules, and MHC class II molecules than DC with HLA-C2 homozygous alleles. Furthermore expression of trafficking chemokine CCR7 is virtually exclusive to HLA-C1 homozygotes indicating their predominant role in T-cell immune priming in secondary lymphoid tissues. Data shown for 4 independent experiments performed in each group and * indicates statistical significance with p<0.05 by Mann Whitney U test.

Figure 6. Flow cytometer data illustrating the impact of IL-15 (1 ng/ml) treated NK-DC co-culture on the expression of DC maturation (HLA-DR FITC) and chemokine (CCR7 PE) markers, comparing responses for DCs with either HLA-C1 ( figures 6b, d, f ) or HLA-C2 ( figures 6c, e, g ) homozygous alleles.

(a) shows DC stained with isotype control for HLA-DR & CCR7, (b) & (c) shows background DC staining where DCs are in isolation in the presence of IL-15, (d) & (e) shows HLA-DR & CCR7 expression by DC in NK-DC co-culture at ratios of 1∶1 in the presence of IL-15, (f) & (g) shows DC markers in NK-DC co-culture at ratios 1∶5 in the presence of IL-15. This data clearly demonstrates that in NK-DC co-culture at ratios of 1∶1 in the presence of IL-15, HLA-C1 homozygous DCs undergo significantly greater maturation than HLA-C2 homozygous DCs and attain CCR7 chemokine expression required for trafficking to secondary lymphoid tissues. Results are representative of 4 experiments with 5,000 DC gated events captured.

Figure 7. Supernatants were collected from NK-DC co-culture experiments at cell ratios of 1∶1 treated with IL-15 and tested for cytokine synthesis using 25 cytokine multiplex bead immunoassay.

Data shown are for all the cytokines that were expressed in culture supernatants. Of these, comparisons were made between cytokine synthesis for DCs with either HLA-C1 homozygous allele or DCs with HLA-C2 homozygous alleles. Data shown excludes background synthesis by cells in isolation. These data indicates that DC with HLA-C1 alleles express significantly greater pro-inflammatory cytokines in NK-DC co-culture favouring immune maturation whereas DC with HLA-C2 alleles predominantly express anti-inflammatory cytokines that are inhibiting to DC maturation. Means ± SEM for 4 independent experiments in each group are shown and * indicates statistical significance with p<0.05 by Mann-Whitney U test.

Figure 8. Supernatants were collected from NK-DC co-culture experiments at cell ratios of 1∶5 treated with IL-15 and tested for cytokine synthesis using 25 cytokine multiplex bead immunoassay.

Data shown are for all the cytokines that were expressed in culture supernatants. Of these, comparisons were made between cytokine synthesis for DCs with either HLA-C1 homozygous allele or DCs with HLA-C2 homozygous alleles. Data shown excludes background synthesis by cells in isolation. These data indicates that DC with HLA-C1 alleles express significantly greater pro-inflammatory cytokines in NK-DC co-culture favouring immune maturation whereas DC with HLA-C2 alleles predominantly express anti-inflammatory cytokines that are inhibiting to DC maturation. Means ± SEM for 4 independent experiments in each group are shown and * indicates statistical significance with p<0.05 by Mann-Whitney U test.