Boosting the VZV-Specific Memory B and T Cell Response to Prevent Herpes Zoster After Kidney Transplantation.

Background: Solid organ transplant recipients are at high risk to develop (complicated) herpes zoster (HZ). Booster vaccination could prevent HZ. However, end-stage renal disease (ESRD) patients show poor immunological responses to vaccinations. We studied the effect of a live attenuated VZV booster vaccine on VZV-specific B and T cell memory responses in ESRD patients and healthy controls. NL28557.000.09, www.toetsingonline.nl. Methods: VZV-seropositive patients, aged ≥50 years, awaiting kidney transplantation, were vaccinated with Zostavax®. Gender and age-matched VZV-seropositive potential living kidney donors were included as controls. VZV-specific IgG titers were measured before, at 1, 3 and 12 months post-vaccination. VZV-specific B and T cell responses before, at 3 months and 1 year after vaccination were analysed by flow-cytometry and Elispot, respectively. Occurrence of HZ was assessed at 5 years post-vaccination. Results: 26 patients and 27 donors were included. Median VZV-specific IgG titers were significantly higher at all time-points post-vaccination in patients (mo 1: 3104 IU/ml [1967-3825], p<0.0001; mo 3: 2659 [1615-3156], p=0.0002; mo 12: 1988 [1104-2989], p=0.01 vs. pre: 1397 [613-2248]) and in donors (mo 1: 2981 [2126-3827], p<0.0001; mo 3: 2442 [2014-3311], p<0.0001; mo 12: 1788 [1368-2460], p=0.0005 vs. pre: 1034 [901-1744]. The patients' IgG titers were comparable to the donors' at all time-points. The ratio VZV-specific B cells of total IgG producing memory B cells had increased 3 months post-vaccination in patients (0.85 [0.65-1.34] vs. pre: 0.56 [0.35-0.81], p=0.003) and donors (0.85 [0.63-1.06] vs. pre: 0.53 [0.36-0.79], p<0.0001) and remained stable thereafter in donors. One year post-vaccination, the percentage of CD4+ central memory cells had increased in both patients (0.29 [0.08-0.38] vs. 0.12 [0.05-0.29], p=0.005) and donors (0.12 [0.03-0.37] vs. 0.09 [0.01-0.20], p=0.002) and CD4+ effector memory cells had increased in donors (0.07 [0.02-0.14] vs. 0.04 [0.01-0.12], p=0.007). Only 1 patient experienced HZ, which was non-complicated. Conclusion: VZV booster vaccination increases VZV-specific IgG titers and percentage VZV-specific memory T-cells for at least 1 year both in ESRD patients and healthy controls. VZV-specific memory B cells significantly increased in patients up to 3 months after vaccination. Prophylactic VZV booster vaccination prior to transplantation could reduce HZ incidence and severity after transplantation.

Natural regulatory T cells from patients with end-stage renal disease can be used for large-scale generation of highly suppressive alloantigen-specific Tregs.

Natural occurring regulatory T cells (nTregs) have the potential to offer a targeted approach of immunosuppression and are the cell type of interest for inducing tolerance in kidney transplantation. End-stage renal disease (ESRD) profoundly affects the composition and function of circulating T cells but little is known with respect to how nTreg potential is affected. To address this, nTregs of patients with ESRD (on dialysis or not) and healthy individuals were isolated, expanded using allogeneic mature monocyte-derived dendritic cells followed by anti-CD3/anti-CD28-coated beads and the different nTregs were extensively characterized by the demethylation status of the Treg-specific demethylated region within FOXP3 and expression of typical nTreg markers. Additionally, the suppressive capacity as well as cytokine producing cells were analyzed for allogeneic mature monocyte-derived dendritic cell-expanded nTregs. Compared to age- and gender-matched healthy individuals, similar frequencies of nTregs were present within the circulation of patients with ESRD either on dialysis or not. The isolated nTregs could be equally well or even better expanded using allogeneic mature monocyte-derived dendritic cells and extensive characterization did not reveal significant differences. The demethylation status of the Treg-specific demethylated region was maintained or even further promoted as was the expression of markers characteristic for nTregs. Moreover, suppressive capacity and the cytokine profile of allogeneic mature monocyte-derived dendritic cell-expanded nTregs was similar to that of healthy individuals. Thus, circulating nTregs of patients with ESRD can effectively be expanded to stable allo antigen-specific nTregs with potential clinical applicability.

Humoral and cellular response after varicella vaccination in VZV IgG seronegative kidney transplant candidates.

BACKGROUND: In immunocompromised patients, primary infection with VZV may have a disastrous clinical course. Vaccination of VZV-seronegative patients on the waiting list for renal transplantation may prevent severe disease. However, the immunologic response of end-stage renal disease patients to peptide vaccines is far from optimal. Our question was whether end-stage renal disease patients with undetectable VZV-IgG levels were able to mount an adequate humoral and cellular response to a live attenuated varicella vaccine. METHODS: Kidney transplant candidates with undetectable VZV levels were vaccinated twice with a live attenuated varicella vaccine at an interval of 6weeks. VZV IgG levels were analysed till 2years after vaccination. The VZV-specific T-cell reactivity was determined prior to vaccination and after transplantation. RESULTS: Seventy-seven percent (40/52) of the vaccinees reached positive VZV-IgG levels after vaccination (responders). Eighty-two percent (9/11) showed an increase in VZV-specific CD4+ memory T-cells (both central and effector memory cells). The percentage VZV-specific CD8+ memory T-cells did not increase. None of the non-responders suffered from primary VZV after transplantation. No severe vaccine-related adverse events were reported, only spontaneously resolving local skin irritation. CONCLUSION: The live attenuated varicella vaccine evokes positive VZV IgG-levels and VZV-specific memory T-cells in VZV-seronegative potential kidney transplant candidates.

Herpes zoster after lung transplantation boosts varicella zoster virus-specific adaptive immune responses.

BACKGROUND: Varicella zoster virus (VZV)-specific memory T cells are significantly lower in transplant recipients than in controls. In addition, VZV-specific immunoglobulin G titers are significantly lower after than before transplantation. Data on the incidence and timing of herpes zoster (HZ) after lung transplantation are limited. This study had two aims: first, we investigated the incidence and severity of HZ after lung transplantation; second, we determined the systemic VZV-specific T-cell and B-cell memory responses before and after HZ. METHODS: The records of 119 patients who underwent transplantation were analyzed for post-transplant HZ. The VZV-specific B-cell and T-cell memory responses of 5 patients before and after HZ were compared with 5 patients without HZ by enzyme-linked immunospot assay and flow cytometry, respectively. RESULTS: HZ was clinically diagnosed and confirmed by polymerase chain reaction on blister fluids and/or plasma in 17 transplant recipients. Uncomplicated cutaneous HZ was present in 12 patients, and 5 patients had disseminated HZ, of whom 1 died. The incidence of HZ after transplantation (38.2 cases/1,000 patient-years) was significantly higher than the age-matched healthy population (7-8 cases/1,000 patient-years). The frequency of VZV-specific immunoglobulin G-producing B cells (p = 0.06) and the percentage of VZV-specific CD4 and CD8 memory T cells increased after HZ to higher frequencies than in patients without HZ (p = 0.03). This was mainly attributed to VZV-reactive effector memory CD4 T cells (p = 0.02) and central memory (p = 0.02) and effector memory (p = 0.03) CD8 T cells. CONCLUSIONS: Lung transplant recipients are highly prone to develop HZ with severe complications. Despite deep immunosuppression, HZ boosted their systemic VZV-specific B-cell and T-cell memory responses.

Allogeneic Mature Human Dendritic Cells Generate Superior Alloreactive Regulatory T Cells in the Presence of IL-15.

Expansion of Ag-specific naturally occurring regulatory T cells (nTregs) is required to obtain sufficient numbers of cells for cellular immunotherapy. In this study, different allogeneic stimuli were studied for their capacity to generate functional alloantigen-specific nTregs. A highly enriched nTreg fraction (CD4(+)CD25(bright)CD127(-) T cells) was alloantigen-specific expanded using HLA-mismatched immature, mature monocyte-derived dendritic cells (moDCs), or PBMCs. The allogeneic mature moDC-expanded nTregs were fully characterized by analysis of the demethylation status within the Treg-specific demethylation region of the FOXP3 gene and the expression of both protein and mRNA of FOXP3, HELIOS, CTLA4, and cytokines. In addition, the Ag-specific suppressive capacity of these expanded nTregs was tested. Allogeneic mature moDCs and skin-derived DCs were superior in inducing nTreg expansion compared with immature moDCs or PBMCs in an HLA-DR- and CD80/CD86-dependent way. Remarkably, the presence of exogenous IL-15 without IL-2 could facilitate optimal mature moDC-induced nTreg expansion. Allogeneic mature moDC-expanded nTregs were at low ratios (<1:320), potent suppressors of alloantigen-induced proliferation without significant suppression of completely HLA-mismatched, Ag-induced proliferation. Mature moDC-expanded nTregs were highly demethylated at the Treg-specific demethylation region within the FOXP3 gene and highly expressed of FOXP3, HELIOS, and CTLA4. A minority of the expanded nTregs produced IL-10, IL-2, IFN-γ, and TNF-α, but few IL-17-producing nTregs were found. Next-generation sequencing of mRNA of moDC-expanded nTregs revealed a strong induction of Treg-associated mRNAs. Human allogeneic mature moDCs are highly efficient stimulator cells, in the presence of exogenous IL-15, for expansion of stable alloantigen-specific nTregs with superior suppressive function.

Systemic varicella zoster virus reactive effector memory T-cells impaired in the elderly and in kidney transplant recipients.

Varicella zoster virus (VZV) infections cause varicella and subsequently herpes zoster upon reactivation. Immune-compromised individuals and the elderly are at high risk of developing herpes zoster due to waning of VZV-specific T-cell immunity. In the present study, a novel functional T-cell assay was developed to test the correlation between age and VZV-specific T-cell responses in peripheral blood from healthy individuals. Secondly, VZV-specific T-cell responses from renal transplant recipients were compared with healthy individuals. Monocytes were differentiated into mature monocyte-derived dendritic cells (moDCs) and were infected with VZV. T-cells were co-cultured with autologous moDCs infected with VZV and subjected to flowcytometric analysis to identify the phenotype (i.e., naïve [NA: CCR7(+) CD45RO(-) ], central [CM: CCR7(+) CD45RO(+) ] and effector memory [EM: CCR7(-) CD45RO(+) ] T-cells) and the frequency of VZV-reactive T-cell subsets by intra-cellular IFN-γ flowcytometry. In contrast to NA and CM T-cells, the frequency of VZV-reactive CD4 and CD8 EM T-cells was inversely correlated with age (P = 0.0007 and P = 0.01). No difference was found in the percentage of VZV-reactive CD4 NA, CM and EM T-cells between transplant recipients and controls. However, the percentage of VZV-reactive CD8 EM T-cells was significantly lower in transplant recipients compared to controls (P = 0.02). In conclusion, moDCs infected with VZV are efficient antigen presenting cells applicable to enumerate and characterize the phenotype and differentiation status of the systemic VZV-specific T-cell response ex-vivo. The data suggest that VZV-reactive EM T-cells are impaired in the elderly and renal transplant recipients.

The granzyme B and interferon-gamma enzyme-linked immunospot assay as alternatives for cytotoxic T-lymphocyte precursor frequency after renal transplantation.

BACKGROUND: The interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) assay has gained increased popularity as a surrogate marker of cytotoxic T-lymphocyte (CTL) activity. However, the functional activity of CTL might be a more relevant surrogate marker of CTL. Therefore, the authors wondered whether the granzyme B (GrB) ELISPOT assay is a better marker for determining the number of CTL than the IFN-gamma ELISPOT assay. METHOD.: Peripheral blood mononuclear cells (PBMC) from 19 kidney transplant patients were stimulated with donor cells or third-party cells. The authors determined the CTL precursor frequency (CTLpf) and simultaneously measured the number of IFN-gamma- and GrB-producing cells (pc) by ELISPOT assay. RESULTS: In all three different assays, the reactivity to donor cells was significant lower than the reactivity to third-party cells: CTLpf, median: 9 versus 60/10(6) PBMC (P=0.0002); number of IFN-gamma pc: 10 versus 90/10(6) PBMC (P=0.0001); number of GrB pc: 60 versus 205/10(6) PBMC (P=0.05). When the authors compared the CTLpf after third-party stimulation with the corresponding ELISPOT results, they found a positive correlation between the CTLpf and the number of IFN-gamma pc (r(s)=0.47, P=0.05). No correlation was found between the CTLpf and the number of GrB pc (r(s)=0.23, P=0.36). However, when they compared the donor-specific CTLpf with the corresponding ELISPOT results, no correlation with the ELISPOT for IFN-gamma (r(s)=0.10, P=0.69) or GrB (r(s)=-0.24, P=0.34) was found. CONCLUSIONS: The authors feel that the CTLpf, as a measure of the actual endpoint of cytolytic activity and independent of the pathway of killing, remains the "gold standard" for determining donor-specific cytolytic activity after clinical organ transplantation.

The frequency of interferon-gproducing cells reflects alloreactivity against minor histocompatibility antigens.

BACKGROUND: In human leukocyte antigen (HLA)-identical living-related renal transplant recipients, no donor-specific alloreactivity can be detected in regular tests (mixed lymphocyte culture, helper T-lymphocyte precursor frequencies, cytotoxic T-lymphocyte precursor frequencies) to identify patients responding to minor histocompatibility antigens (mHag). We questioned whether a more sensitive method like the Elispot-assay could be more appropriate. METHODS AND RESULTS: Peripheral blood mononuclear cells (PBMC) from 16 HLA-identical living-related kidney transplant patients 3 months (range, 2 weeks to 5 months) after transplantation were tested for the frequency of interferon (IFN)-gamma producing cells by the Elispot-assay. PBMC from the recipient were stimulated with irradiated donor PBMC and corrected for backward stimulation. Donor-specific IFN-gamma producing cells (pc) in the range of 5 to 115 per million PBMC (median, 30 per million PBMC) were found. To evaluate the frequency of spot forming cells in time, PBMC from six patients who received an HLA-identical renal transplant were stimulated with irradiated donor PBMC before, approximately 3 months after, and 12 months after transplantation. Four patients who received an HLA-mismatched living-related kidney served as positive control. In the HLA-identical group, frequencies in the range of 0 to 10 IFN-gamma pc per million PBMC were found before transplantation, 0 to 30 per million PBMC 3 months after transplantation, and 0 to 45 per million PBMC 12 months after transplantation. In the HLA-mismatched group, significantly higher numbers were found: 10 to 480 IFN-gamma pc per million PBMC before, 20 to 360 per million PBMC at 3 months, and 30 to 590 per million PBMC 12 months after transplantation. CONCLUSION: Under immunosuppressive therapy, IFN-gamma pc specific for donor mHag can be found after HLA-identical living-related renal transplantation.