Peripheral natural killer cell and allo-stimulated T-cell function in kidney transplant recipients associate with cancer risk and immunosuppression-related complications

Abstract

Reducing immunosuppression has been proposed as a means of preventing cancer in kidney transplant recipients but this can precipitate graft rejection. Here we tested whether anti-tumor natural killer (NK) cell and allo-responsive T-cell function in kidney transplant recipients may predict cancer risk and define risk of rejection. NK cell function was measured by the release of lactate dehydrogenase and T-cell allo-response by interferon-γ quantification using a panel of reactive T-cell enzyme-linked immunospot (ELISPOT) in 56 kidney transplant recipients with current or past cancer and 26 kidney transplant recipients without cancer. NK function was significantly impaired and the allo-response was significantly lower in kidney transplant recipients with cancer. With prospective follow-up, kidney transplant recipients with poor NK cell function had a hazard ratio of 2.1 (95% confidence interval 0.97-5.00) for the combined end point of metastatic cancer, cancer-related death, or septic death. Kidney transplant recipients with low interferon-γ release were also more likely to reach this combined end point. Thus, posttransplant monitoring of allo-immunity and NK cell function is useful for assessing the risk of over immunosuppression for the development of malignancy and/or death from cancer or sepsis.

Figure 1. Dichotomised Natural Killer (NK) Cell Function in Kidney Transplant Recipients (KTR)

The depicted NK cell function is dichotomised on the median amount of Lactate Dehydrogenase (LDH) released when KTR peripheral blood mononuclear cells are co-cultured with K562 at a ratio of 20:1, i.e. High NK function (black) / Low NK function (grey). (A) Bar graph of 62 KTR. The 22 KTR with current cancer have a greater proportion of KTR with low NK cell function (68%) compared with the 19 KTR with past cancer (26%) and 21 KTR with no cancer (33%) with a Chi Squared p-value of 0.014. (B) A Kaplan-Meier survival curve depicting the dichotomised data of the 41 KTR with current and past cancer, developing metastatic cancer or dying of immunosuppressive related diseases (i.e. cancer or sepsis) over 500 days. KTR with low NK cell function (grey line) has lower survival proportions at 365 days (32% vs 71%) and lower overall survival with a Hazard Ratio [95% Confidence Interval] = 2.1 [0.97–5.00], log-rank p=0.063, when compared to KTR with high NK cell function (black line).

Figure 2. Allo-stimulated Interferon-γ Release from Kidney Transplant Recipients (KTR)

Allo-response was measured by co-culturing 1×10⁵ allogeneic B cells with 3×10⁵ peripheral blood mononuclear cells (PBMC) from KTR and quantitating Interferon-γ released from cells (spots) via Enzyme Linked Immuno-SPOT (ELISPOT). The Intra-assay coefficient of variation (C.V.) was determined to be 31%. (A) Box plot representing median (range) numbers of spots from 77 KTR. The KTR with current (n=29) and past (n=24) cancer both have decreased IFN-γ allo-responses when compared to KTR with no cancer (n=24, p<0.001) using a Kruskal-Wallis non-parametric test. (B) A receiver operator characteristic (ROC) curve for the 53 KTR who had current or past cancer who went on to developed metastatic cancer or died of immunosuppressive drug related diseases (i.e. cancer or sepsis) over the median (range) of days followed = 303 (8–1712). The ROC has an accuracy of 69% (p=0.025) with trade-off of 280 spots per 3×10⁵ PBMC (spots) leading to a sensitivity of 53% and specificity of 79%. The 280 spots trade-off was identified using a single value that had both the highest likelihood ratio and Youden’s index. (C) A Kaplan-Meier survival curve for metastatic cancer or immunosuppressive related death (cancer or sepsis) free days dichotomised on the trade-off value of 280 spots, over 500 days. KTR with <280 spots (grey line) have lower survival rates (23% vs 61%, at 500 days), log-rank test p=0.050, when compared to KTR with >280 spots (black line).

Figure 3. Immunosuppressive Drug Regimen and Cancer Associations to Natural Killer (NK) cell function in Kidney Transplant Recipients (KTR)

A panel of bar plots depicting NK cell function dichotomised on the median NK cell function of 62 KTR, with various drug regimens and with either current cancer (Cancer) or past/no cancer (No Cancer). (A) KTR on triple therapy (3 Drugs) does not differ from their 1–2 Drug counterparts. However, KTR with cancer have a greater proportion of KTR with low NK cell function than the KTR with no cancer when on 1–2 Drugs (63% Vs. 19%, p=0.029) but does not reach statistical significance when on 3 Drugs (83% Vs. 35%, p=0.073). (B) KTR on Mycophenolate (MMF)-based regimens does not differ from their non-MMF based counterparts. However, there was a greater proportion of KTR with cancer that have low NK function compared to KTR with no cancer, which is only statistically significant when KTR were not on MMF-based regimens (67% Vs. 18%, p=0.021). (C) KTR on Calcineurin Inhibitor (CNI)-based regimens does not differ from their non-CNI counterparts. While KTR with cancer on CNI and not on CNI have a greater proportion of KTR with low NK function (70% and 67%, respectively) these do not statistically differ from their non-cancer counterparts (30% and 36%, respectively) with (p=0.056 and p=0.116, respectively). All statistical analysis performed using Fisher’s exact tests.

Figure 4. Immunosuppressive Drug Regimen and Cancer Associations to Allo-stimulated Interferon-γ Release in Kidney Transplant Recipients (KTR)

A panel of box plots depicting median (range) of allo-stimulated interferon-γ (IFN-γ) release from 77 KTR with various drug regimens and with either current cancer (Cancer) or past/no cancer (No Cancer). (A) IFN-γ release from KTR on triple therapy (3 Drugs) does not differ from their 1–2 drug counterparts. However, KTR with cancer had less allo-stimulated IFN-γ release than the KTR with no cancer for those on 3 drugs (p=0.001). (B) KTR on Mycophenolate (MMF)-based regimens does not differ from their non-MMF based counterparts. However, KTR with cancer has lower IFN-γ release than KTR with no cancer KTR are on MMF-based regimens (p=0.014). (C) KTR on Calcineurin Inhibitor (CNI)-based regimens does not differ from their non-CNI counterparts. However, KTR with cancer have less allo-stimulated IFN-γ release than the KTR with no cancer for those on CNI-based regimens (p=0.014). All statistical analysis performed using Mann-Whitney tests.

Figure 5. Associations of Kidney Graft Function and proteinuria (>500mg/day) to Natural Killer (NK) Cell Function and Allo-stimulated Interferon-γ Release in Kidney Transplant Recipients (KTR)

(A–B) Bar graphs depicting proportions of KTR with high (black) and low (grey) NK cell function dichotomised on median NK cell function. (A) Bar graph of 62 KTR. Estimated glomerular filtration rate (eGFR) does not associate to dichotomised NK cell function (p=0.276), using Chi Squared analysis. (B) Bar graph depicting 57 KTR. There is a larger proportion of KTR with high NK function (85%) with proteinuria (>500mg/day) compared to KTR with lower or no detectable proteinuria (48%, p=0.026) using a Fisher’s exact test. (C–D) Box plots depicting the median (range) of allo-stimulated Interferon-γ (IFN-γ) release (Spots) from 3×10⁵ Peripheral Blood Mononuclear Cells (PBMC) measured by Enzyme Linked Immun-SPOT (ELISPOT) in KTR. (C) For 77 KTR tested there are no differences between the different groups of eGFR and amount of IFN-γ release (p=0.860), using a Kruskal-Wallis test. (D) Out of 71 KTR there is no difference between those KTR with proteinuria (n=18) and those without (n=53, p=0.473), using a Mann-Whitney test.