Joke M. Zuijderwijk

Immunohistochemical detection of somatostatin receptor subtypes sst1 and sst2A in human somatostatin receptor positive tumors.

Although in situ hybridization has been used to examine the distribution of messenger RNA for somatostatin receptor subtypes (sst) in human tumors, the cellular localization of sst1 and sst2A receptors has not been reported. In this study, we describe the cellular localization of human sst1 and sst2A receptor proteins in both cryostat- and paraffin-embedded sections of 25 human tumor tissues using two recently developed polyclonal antibodies. Six somatostatin (SS) receptor (SSR) positive tumors (two gastrinomas, three carcinoids, one pheochromocytoma) and one SSR negative tumor (renal cell carcinoma), selected by positive and negative SSR autoradiography, respectively, were studied by both immunohistochemistry and Western blot analysis. The six SSR positive tumors expressed sst2A, while 4 of 5 expressed sst1 as well. The SSR negative tumor did not express either sst1 or sst2A. Western blot analysis of wheat germ agglutinin purified membrane proteins confirmed the presence of the sst1 and sst2A glycosylated receptors. The paraffin-embedded sections gave best information with respect to the subcellular localization. Sst1 immunoreactivity was observed both on the membrane and in the cytoplasm, while sst2A showed predominantly membrane-associated immunoreactivity. This subcellular distribution of sst1 or sst2A receptors was confirmed in paraffin-embedded sections of 8 additional intestinal carcinoids, 5 gastrinomas and 5 pheochromocytomas. Sst1 receptors were detected in 7 out of 8 carcinoids, in all gastrinomas, and in 4 out of 5 pheochromocytomas, while 6 out of 8 carcinoids, all gastrinomas, and 3 out of 5 pheochromocytomas expressed sst2A receptors. In conclusion, sst1 and sst2A receptors show a differential subcellular localization in human SSR positive tumors. The use of SSR subtype selective antibodies to detect the subcellular distribution of SSR subtypes in individual tumor cells is an important step forward to understand more about the pathophysiological role of the different SSR subtypes in human tumors.

The direct and indirect allogeneic presentation pathway during acute rejection after human cardiac transplantation

Alloreactive T cells may be activated via a direct or an indirect antigen presentation pathway. We questioned whether the frequency of interferon (IFN)‐γ producing cells determined by enzyme‐linked immunospot (ELISPOT) assay is an effective tool to monitor the direct and/or indirect presentation pathway. Secondly, we wondered whether early and late acute rejection (AR) are associated with both pathways. Before (n = 15), during (n = 18) and after (n = 16) a period of AR, peripheral blood mononuclear cell (PBMC) samples were tested from 13 heart transplant recipients. The direct presentation pathway was always present. The number of IFN‐γ producing cells reactive to this pathway increased significantly (P = 0·04) during AR and the number decreased (P = 0·005) after AR therapy. In contrast, the indirect allogeneic presentation pathway was present in only eight of 18 AR samples. When the indirect presentation pathway was detectable, it increased significantly during AR. Five of eight of these AR occurred more than 6 months after transplantation. The ELISPOT assay, enumerating alloreactive IFN‐γ producing cells, is a valuable tool to determine the reactivity via both the direct and the indirect presentation pathway. The direct presentation pathway always plays a role in AR, while the indirect pathway contributes especially to late AR.

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)-&ggr; producing cells by the Elispot-assay. PBMC from the recipient were stimulated with irradiated donor PBMC and corrected for backward stimulation. Donor-specific IFN-&ggr; 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-&ggr; 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-&ggr; 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-&ggr; pc specific for donor mHag can be found after HLA-identical living-related renal transplantation.

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. J. Med. Virol. 84:2018–2025, 2012.

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+CD25brightCD127− 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.

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.

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.

IMPAIRED VARICELLA ZOSTER VIRUS SPECIFIC MEMORY T-CELL RESPONSES IN KIDNEY TRANSPLANT PATIENTS: 976

Introduction: Primary infection with varicella zoster virus (VZV) causes varicella, and VZV reactivation results in herpes zoster. Compared to healthy individuals, the incidence and severity of herpes zoster is higher in transplant recipients. VZV-specific memory T-cells are considered to prevent VZV reactivation. DCs are essential to present the cognate VZV antigens to T-cells. We questioned whether mature monocyte-derived dendritic cells (moDCs) from renal transplant patients are more susceptible to VZV than those from healthy controls, and whether phenotype and frequency of circulating VZV-specific T-cells and VZV-specific IgG titres in kidney transplant recipients are comparable with controls. Methods: Patients and controls had experienced varicella during childhood and had no signs of herpes zoster. CD3+ T-cells and monocytes (CD14+) were isolated from PBMC from adult kidney transplant recipients, 2 years after transplantation and on Tacro and MMF therapy, and healthy gender and age matched volunteers served as controls. Monocytes were differentiated into mature moDCs and infected with VZV. T-cells were co-cultured with autologous VZV-infected moDCs 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 (i.e. IFN-g+). Results: The VZV-specific IgG titres were comparable between transplant recipients and healthy individuals. In whole blood the number of CD4 (p=0.006) and CD8 (p=0.03) T-cells were significantly lower in transplant recipients compared to healthy individuals. The proportion of CD4 and CD8 cells within the CD3+ T-cells were comparable between transplant recipients and controls. Also, the proportion NA, CM and EM within the CD4 and CD8 cells were comparable between transplant recipients and controls. There was no difference in grade of VZV infection between moDCs from transplant recipients and controls. No difference was found in the percentage of VZV-specific CD4 NA, CM and EM T-cells between transplant recipients and controls. However, the percentage of VZV-specific CD8 memory T-cells was lower in patients compared to controls (p=0.07). This impairment was significant in the CD8 EM T-cell pool (p=0.05). Remarkably, lower number of transplant patients (5 of 11) had circulating VZV-specific CD8 EM T-cells compared to controls (10 of 13). In contrast to the CD8 CM T-cells (rs=0.42, p=0.16), the frequency of VZV-specific CD8 EM T-cells population was inversely correlated with the age of healthy individuals (rs=-0.53, p=0.06), suggesting that transplant patients have ‘aged’ VZV-specific CD8 EM T-cells. Conclusion: Mature moDCs from transplant recipients and healthy individuals can be similarly infected by VZV. The VZV related complications after transplantation results from an impaired VZV-specific memory T-cell response. Prophylactic VZV vaccination before transplantation might boost the patient’s memory T-cell repertoire and thereby reduces the morbidity associated with herpes zoster after transplantation.