NKp44/HLA-DP-dependent regulation of CD8 effector T cells by NK cells.

Although natural killer (NK) cells are recognized for their modulation of immune responses, the mechanisms by which human NK cells mediate immune regulation are unclear. Here, we report that expression of human leukocyte antigen (HLA)-DP, a ligand for the activating NK cell receptor NKp44, is significantly upregulated on CD8+ effector T cells, in particular in human cytomegalovirus (HCMV)+ individuals. HLA-DP+ CD8+ T cells expressing NKp44-binding HLA-DP antigens activate NKp44+ NK cells, while HLA-DP+ CD8+ T cells not expressing NKp44-binding HLA-DP antigens do not. In line with this, frequencies of HLA-DP+ CD8+ T cells are increased in individuals not encoding for NKp44-binding HLA-DP haplotypes, and contain hyper-expanded CD8+ T cell clones, compared to individuals expressing NKp44-binding HLA-DP molecules. These findings identify a molecular interaction facilitating the HLA-DP haplotype-specific editing of HLA-DP+ CD8+ T cell effector populations by NKp44+ NK cells and preventing the generation of hyper-expanded T cell clones, which have been suggested to have increased potential for autoimmunity.

The role of interleukin-21 in COVID-19 vaccine-induced B cell-mediated immune responses in patients with kidney disease and kidney transplant recipients.

T-cell-mediated help to B cells is required for the development of humoral responses, in which the cytokine interleukin (IL)-21 is key. Here, we studied the mRNA-1273 vaccine-induced SARS-CoV-2-specific memory T-cell IL-21 response, memory B cell response, and immunoglobulin (Ig)G antibody levels in peripheral blood at 28 days after the second vaccination by ELISpot and the fluorescent bead-based multiplex immunoassay, respectively. We included 40 patients with chronic kidney disease (CKD), 34 patients on dialysis, 63 kidney transplant recipients (KTR), and 47 controls. We found that KTR, but not patients with CKD and those receiving dialysis, showed a significantly lower number of SARS-CoV-2-specific IL-21 producing T cells than controls (P < .001). KTR and patients with CKD showed lower numbers of SARS-CoV-2-specific IgG-producing memory B cells when compared with controls (P < .001 and P = .01, respectively). The T-cell IL-21 response was positively associated with the SARS-CoV-2-specific B cell response and the SARS-CoV-2 spike S1-specific IgG antibody levels (both Pearson r = 0.5; P < .001). In addition, SARS-CoV-2-specific B cell responses were shown to be IL-21 dependent. Taken together, we show that IL-21 signaling is important in eliciting robust B cell-mediated immune responses in patients with kidney disease and KTR.

Th1-dominant cytokine responses in kidney patients after COVID-19 vaccination are associated with poor humoral responses.

Cytokines are regulators of the immune response against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, the contribution of cytokine-secreting CD4+ and CD8+ memory T cells to the SARS-CoV-2-specific humoral immune response in immunocompromised kidney patients is unknown. Here, we profiled 12 cytokines after stimulation of whole blood obtained 28 days post second 100 µg mRNA-1273 vaccination with peptides covering the SARS-CoV-2 spike (S)-protein from patients with chronic kidney disease (CKD) stage 4/5, on dialysis, kidney transplant recipients (KTR), and healthy controls. Unsupervised hierarchical clustering analysis revealed two distinct vaccine-induced cytokine profiles. The first profile was characterized by high levels of T-helper (Th)1 (IL-2, TNF-α, and IFN-γ) and Th2 (IL-4, IL-5, IL-13) cytokines, and low levels of Th17 (IL-17A, IL-22) and Th9 (IL-9) cytokines. This cluster was dominated by patients with CKD, on dialysis, and healthy controls. In contrast, the second cytokine profile contained predominantly KTRs producing mainly Th1 cytokines upon re-stimulation, with lower levels or absence of Th2, Th17, and Th9 cytokines. Multivariate analyses indicated that a balanced memory T cell response with the production of Th1 and Th2 cytokines was associated with high levels of S1-specific binding and neutralizing antibodies mainly at 6 months after second vaccination. In conclusion, seroconversion is associated with the balanced production of cytokines by memory T cells. This emphasizes the importance of measuring multiple T cell cytokines to understand their influence on seroconversion and potentially gain more information about the protection induced by vaccine-induced memory T cells.

Antibody and T-Cell Responses 6 Months After Coronavirus Disease 2019 Messenger RNA-1273 Vaccination in Patients With Chronic Kidney Disease, on Dialysis, or Living With a Kidney Transplant.

BACKGROUND: The immune response to COVID-19 vaccination is inferior in kidney transplant recipients (KTRs) and to a lesser extent in patients on dialysis or with chronic kidney disease (CKD). We assessed the immune response 6 months after mRNA-1273 vaccination in kidney patients and compared this to controls. METHODS: A total of 152 participants with CKD stages G4/5 (eGFR <30 mL/min/1.73 m2), 145 participants on dialysis, 267 KTRs, and 181 controls were included. SARS-CoV-2 Spike S1 specific IgG antibodies were measured using fluorescent bead-based multiplex-immunoassay, neutralizing antibodies to ancestral, Delta, and Omicron (BA.1) variants by plaque reduction, and T-cell responses by interferon-γ release assay. RESULTS: At 6 months after vaccination, S1-specific antibodies were detected in 100% of controls, 98.7% of CKD G4/5 patients, 95.1% of dialysis patients, and 56.6% of KTRs. These figures were comparable to the response rates at 28 days, but antibody levels waned significantly. Neutralization of the ancestral and Delta variants was detected in most participants, whereas neutralization of Omicron was mostly absent. S-specific T-cell responses were detected at 6 months in 75.0% of controls, 69.4% of CKD G4/5 patients, 52.6% of dialysis patients, and 12.9% of KTRs. T-cell responses at 6 months were significantly lower than responses at 28 days. CONCLUSIONS: Although seropositivity rates at 6 months were comparable to rates at 28 days after vaccination, significantly decreased antibody levels and T-cell responses were observed. The combination of low antibody levels, reduced T-cell responses, and absent neutralization of the newly emerging variants indicates the need for additional boosts or alternative vaccination strategies in KTRs. CLINICAL TRIALS REGISTRATION: NCT04741386.

Alternative strategies to increase the immunogenicity of COVID-19 vaccines in kidney transplant recipients not responding to two or three doses of an mRNA vaccine (RECOVAC): a randomised clinical trial.

BACKGROUND: An urgent need exists to improve the suboptimal COVID-19 vaccine response in kidney transplant recipients (KTRs). We aimed to compare three alternative strategies with a control single dose mRNA-1273 vaccination: a double vaccine dose, heterologous vaccination, and temporary discontinuation of mycophenolate mofetil or mycophenolic acid. METHODS: This open-label randomised trial, done in four university medical centres in the Netherlands, enrolled KTRs without seroconversion after two or three doses of an mRNA vaccine. Between Oct 20, 2021, and Feb 2, 2022, 230 KTRs were randomly assigned block-wise per centre by a web-based system in a 1:1:1 manner to receive 100 µg mRNA-1273, 2 × 100 µg mRNA-1273, or Ad26.COV2-S vaccination. In addition, 103 KTRs receiving 100 µg mRNA-1273, were randomly assigned 1:1 to continue (mycophenolate mofetil+) or discontinue (mycophenolate mofetil-) mycophenolate mofetil or mycophenolic acid treatment for 2 weeks. The primary outcome was the percentage of participants with a spike protein (S1)-specific IgG concentration of at least 10 binding antibody units per mL at 28 days after vaccination, assessed in all participants who had a baseline measurement and who completed day 28 after vaccination without SARS-CoV-2 infection. Safety was assessed as a secondary outcome in all vaccinated patients by incidence of solicited adverse events, acute rejection or other serious adverse events. This trial is registered with ClinicalTrials.gov, NCT05030974 and is closed. FINDINGS: Between April 23, 2021, and July 2, 2021, of 12 158 invited Dutch KTRs, 3828 with a functioning kidney transplant participated in a national survey for antibody measurement after COVID-19 vaccination. Of these patients, 1311 did not seroconvert after their second vaccination and another 761 not even after a third. From these seronegative patients, 345 agreed to participate in our repeated vaccination study. Vaccination with 2 × mRNA-1273 or Ad26.COV2-S was not superior to single mRNA-1273, with seroresponse rates of 49 (68%) of 72 (95% CI 56-79), 46 (63%) of 73 (51-74), and 50 (68%) of 73 (57-79), respectively. The difference with single mRNA-1273 was -0·4% (-16 to 15; p=0·96) for 2 × mRNA-1273 and -6% (-21 to 10; p=0·49) for Ad26.COV2-S. Mycophenolate mofetil- was also not superior to mycophenolate mofetil+, with seroresponse rates of 37 (80%) of 46 (66-91) and 31 (67%) of 46 (52-80), and a difference of 13% (-5 to 31; p=0·15). Local adverse events were more frequent after a single and double dose of mRNA-1273 than after Ad26.COV2-S (65 [92%] of 71, 67 [92%] of 73, and 38 [50%] of 76, respectively; p<0·0001). No acute rejection occurred. There were no serious adverse events related to vaccination. INTERPRETATION: Repeated vaccination increases SARS-CoV-2-specific antibodies in KTRs, without further enhancement by use of a higher dose, a heterologous vaccine, or 2 weeks discontinuation of mycophenolate mofetil or mycophenolic acid. To achieve a stronger response, possibly required to neutralise new virus variants, repeated booster vaccination is needed. FUNDING: The Netherlands Organization for Health Research and Development and the Dutch Kidney Foundation.

Fungal sensing by dectin-1 directs the non-pathogenic polarization of TH17 cells through balanced type I IFN responses in human DCs.

The non-pathogenic TH17 subset of helper T cells clears fungal infections, whereas pathogenic TH17 cells cause inflammation and tissue damage; however, the mechanisms controlling these distinct responses remain unclear. Here we found that fungi sensing by the C-type lectin dectin-1 in human dendritic cells (DCs) directed the polarization of non-pathogenic TH17 cells. Dectin-1 signaling triggered transient and intermediate expression of interferon (IFN)-ß in DCs, which was mediated by the opposed activities of transcription factors IRF1 and IRF5. IFN-ß-induced signaling led to integrin αvß8 expression directly and to the release of the active form of the cytokine transforming growth factor (TGF)-ß indirectly. Uncontrolled IFN-ß responses as a result of IRF1 deficiency induced high expression of the IFN-stimulated gene BST2 in DCs and restrained TGF-ß activation. Active TGF-ß was required for polarization of non-pathogenic TH17 cells, whereas pathogenic TH17 cells developed in the absence of active TGF-ß. Thus, dectin-1-mediated modulation of type I IFN responses allowed TGF-ß activation and non-pathogenic TH17 cell development during fungal infections in humans.

IL-10-Secreting CD8+ T Cells Specific for Human Cytomegalovirus (HCMV): Generation, Maintenance and Phenotype.

HCMV-specific CD8+ T-cells are potent anti-viral effector cells in HCMV infected individuals, but evidence from other viral infections suggests that CD8+ T-cells can also produce the immunomodulatory cytokine IL-10. In this work we show that there are HCMV-specific IL-10 CD8+ T-cell responses in a cohort of individuals aged 23-76 years of age, predominantly directed against the HCMV proteins known to be expressed during latent infections as well as towards the proteins US3 and pp71. The analysis of HCMV-specific responses established during primary infection has shown that the IL-10 responses to US3 and pp71 HCMV proteins are detectable in the first weeks post infection, but not the responses to latency-associated proteins, and this IL-10 response is produced by both CD8+ and CD4+ T-cells. Phenotyping studies of HCMV-specific IL-10+ CD8+ T-cells show that these are CD45RA+ effector memory cells and co-express CD28 and CD57, however, the expression of the inhibitory receptor PD-1 varied from 90% to 30% between donors. In this study we have described for the first time the HCMV-specific IL-10 CD8+ T-cell responses and have demonstrated their broad specificity and the potential immune modulatory role of the immune response to HCMV latent carriage and periodic reactivation.

SARS-CoV-2 Spike-specific IFN-γ T-cell Response After COVID-19 Vaccination in Patients With Chronic Kidney Disease, on Dialysis, or Living With a Kidney Transplant.

Studies have shown that coronavirus disease 2019 (COVID-19) vaccination is associated with a lower humoral response in vulnerable kidney patients. Here, we investigated the T-cell response following COVID-19 vaccination in kidney patients compared with controls. Methods: Patients with chronic kidney disease (CKD) stage G4/5 [estimated glomerular filtration rate <30 mL/min/1.73 m2], on dialysis, or living with a kidney transplant and controls received 2 doses of the mRNA-1273 COVID-19 vaccine. Peripheral blood mononuclear cells were isolated at baseline and 28 d after the second vaccination. In 398 participants (50% of entire cohort; controls n = 95, CKD G4/5 n = 81, dialysis n = 78, kidney transplant recipients [KTRs] n = 144)' SARS-CoV-2-specific T cells were measured using an IFN-γ enzyme-linked immune absorbent spot assay. Results: A significantly lower SARS-CoV-2-specific T-cell response was observed after vaccination of patients on dialysis (54.5%) and KTRs (42.6%) in contrast to CDK G4/5 (70%) compared with controls (76%). The use of calcineurin inhibitors was associated with a low T-cell response in KTRs. In a subset of 20 KTRs, we observed waning of the cellular response 6 mo after the second vaccination, which was boosted to some extent after a third vaccination, although T-cell levels remained low. Conclusion: Our data suggest that vaccination is less effective in these patient groups, with humoral nonresponders also failing to mount an adequate cellular response, even after the third vaccination. Given the important role of T cells in protection against disease and cross-reactivity to SARS-CoV-2 variants, alternative vaccination strategies are urgently needed in these high-risk patient groups.

Transcriptional profiling of human Vδ1 T cells reveals a pathogen-driven adaptive differentiation program.

γδ T cells are generally considered innate-like lymphocytes, however, an "adaptive-like" γδ compartment has now emerged. To understand transcriptional regulation of adaptive γδ T cell immunobiology, we combined single-cell transcriptomics, T cell receptor (TCR)-clonotype assignment, ATAC-seq, and immunophenotyping. We show that adult Vδ1+ T cells segregate into TCF7+LEF1+Granzyme Bneg (Tnaive) or T-bet+Eomes+BLIMP-1+Granzyme B+ (Teffector) transcriptional subtypes, with clonotypically expanded TCRs detected exclusively in Teffector cells. Transcriptional reprogramming mirrors changes within CD8+ αß T cells following antigen-specific maturation and involves chromatin remodeling, enhancing cytokine production and cytotoxicity. Consistent with this, in vitro TCR engagement induces comparable BLIMP-1, Eomes, and T-bet expression in naive Vδ1+ and CD8+ T cells. Finally, both human cytomegalovirus and Plasmodium falciparum infection in vivo drive adaptive Vδ1 T cell differentiation from Tnaive to Teffector transcriptional status, alongside clonotypic expansion. Contrastingly, semi-invariant Vγ9+Vδ2+ T cells exhibit a distinct "innate-effector" transcriptional program established by early childhood. In summary, adaptive-like γδ subsets undergo a pathogen-driven differentiation process analogous to conventional CD8+ T cells.

The RECOVAC Immune-response Study: The Immunogenicity, Tolerability, and Safety of COVID-19 Vaccination in Patients With Chronic Kidney Disease, on Dialysis, or Living With a Kidney Transplant.

BACKGROUND: In kidney patients COVID-19 is associated with severely increased morbidity and mortality. A comprehensive comparison of the immunogenicity, tolerability, and safety of COVID-19 vaccination in different cohorts of kidney patients and a control cohort is lacking. METHODS: This investigator driven, prospective, controlled multicenter study included 162 participants with chronic kidney disease (CKD) stages G4/5 (eGFR < 30 mL/min/1.73m2), 159 participants on dialysis, 288 kidney transplant recipients, and 191 controls. Participants received 2 doses of the mRNA-1273 COVID-19 vaccine (Moderna). The primary endpoint was seroconversion. RESULTS: Transplant recipients had a significantly lower seroconversion rate when compared with controls (56.9% versus 100%, P < 0.001), with especially mycophenolic acid, but also, higher age, lower lymphocyte concentration, lower eGFR, and shorter time after transplantation being associated with nonresponder state. Transplant recipients also showed significantly lower titers of neutralizing antibodies and T-cell responses when compared with controls. Although a high seroconversion rate was observed for participants with CKD G4/5 (100%) and on dialysis (99.4%), mean antibody concentrations in the CKD G4/5 cohort and dialysis cohort were lower than in controls (2405 [interquartile interval 1287-4524] and 1650 [698-3024] versus 3186 [1896-4911] BAU/mL, P = 0.06 and P < 0.001, respectively). Dialysis patients and especially kidney transplant recipients experienced less systemic vaccination related adverse events. No specific safety issues were noted. CONCLUSIONS: The immune response following vaccination in patients with CKD G4/5 and on dialysis is almost comparable to controls. In contrast, kidney transplant recipients have a poor response. In this latter, patient group development of alternative vaccination strategies are warranted.

CD8 and CD4 T Cell Populations in Human Kidneys.

BACKGROUND: At border sites, and in internal organs, tissue resident memory T cells (TRM) contribute to the immune barrier against pathogens like viruses, bacteria, fungi, and cancer. However, information on the presence and function of these cells in the human kidney is scant. In order to better understand the T cell-mediated immunological defense in this organ, we aimed to determine phenotypic and functional aspects of CD8 and CD4 T cells present in healthy and allograft kidney tissue. METHODS: Using multichannel flow cytometry, we assessed the phenotype and function of T cells in healthy renal tissue samples (n = 5) and kidney allograft tissue (n = 7) and compared these aspects to T cells in peripheral blood from healthy controls (n = 13). RESULTS: Kidney tissue samples contained substantial amounts of CD8 and CD4 T cells. In contrast to the circulating cells, kidney T cells frequently expressed CD69 and CD103, and were more often actively cycling. Furthermore, nearly all kidney T cells expressed CXCR3, and often expressed CXCR6 compared to T cells in the circulation. Markedly, kidney T cells produced greater quantities of IFNγ than circulating cells and were frequently polyfunctional. CONCLUSION: Functional T cells with the characteristic traits of TRM reside in human kidney tissues. These cells are more often actively cycling and frequently express CXCR3 and CXCR6.

Human CXCR5+ PD-1+ CD8 T cells in healthy individuals and patients with hematologic malignancies.

Immune checkpoint blockade (ICB) has revolutionized cancer therapy, but varying response rates illustrate the need for biomarkers of response. Studies in mice have identified a subset of CD8 T cells that is essential for response to PD-1 ICB. These CD8 T cells co-express CXCR5, PD-1 and Tcf1, and provide effector T cells upon PD-1 ICB. It is unknown whether similar T cells play a role in PD-1 ICB in humans. We studied human peripheral blood and lymph nodes (LNs) for the frequency, phenotype, and functionality of CXCR5+ PD-1+ CD8 T cells. We find that CXCR5+ PD-1+ CD8 T cells are memory-like cells, express Tcf1, and lack expression of effector molecules. CXCR5+ PD-1+ CD8 T cells produce cytokines upon stimulation, but have limited proliferative capacity. We studied patients with hematologic malignancies with varying response rates to PD-1 ICB. Specifically in chronic lymphocytic leukemia, in which PD-1 ICB does not induce clinical responses, CXCR5+ PD-1+ CD8 T cells show loss of the memory phenotype and increased effector differentiation. In conclusion, we identified CXCR5+ PD-1+ CD8 T cells in human peripheral blood and LN, which could play a similar role during PD-1 ICB. Future studies should analyze CXCR5+ PD-1+ CD8 T cells during PD-1 ICB and their importance for therapeutic response.

The effect of high-salt diet on t-lymphocyte subpopulations in healthy males-A pilot study.

Animal studies show that high-salt diet affects T-cell subpopulations, but evidence in humans is scarce and contradictory. This pilot study investigated the effect of a 2-week high-salt diet on T-cell subpopulations (ie, γδ T cells, Th17 cells, and regulatory T cells) in five healthy males. The mean (SD) age of the participants was 33 (2) years, with normal body mass index, kidney function, and baseline blood pressure. In terms of phenotype, there was an isolated increase of CD69 expression in Vδ1 T cells (P = .04), which is an early activation marker. There were no statistically significant changes or trends in any of the other tested markers or in the Th17 or regulatory T-cell subsets. The increase in CD69 was strongly correlated to increases in 24-hour urinary sodium excretion (r = .93, P = .02). These results of this pilot may motivate the use of longer dietary salt interventions in future studies on salt and adaptive immune cells.

Human Lymph Node Stromal Cells Have the Machinery to Regulate Peripheral Tolerance during Health and Rheumatoid Arthritis.

BACKGROUND: In rheumatoid arthritis (RA) the cause for loss of tolerance and anti-citrullinated protein antibody (ACPA) production remains unidentified. Mouse studies showed that lymph node stromal cells (LNSCs) maintain peripheral tolerance through presentation of peripheral tissue antigens (PTAs). We hypothesize that dysregulation of peripheral tolerance mechanisms in human LNSCs might underlie pathogenesis of RA. METHOD: Lymph node (LN) needle biopsies were obtained from 24 RA patients, 23 individuals positive for RA-associated autoantibodies but without clinical disease (RA-risk individuals), and 14 seronegative healthy individuals. Ex vivo human LNs from non-RA individuals were used to directly analyze stromal cells. Molecules involved in antigen presentation and immune modulation were measured in LNSCs upon interferon γ (IFNγ) stimulation (n = 15). RESULTS: Citrullinated targets of ACPAs were detected in human LN tissue and in cultured LNSCs. Human LNSCs express several PTAs, transcription factors autoimmune regulator (AIRE) and deformed epidermal autoregulatory factor 1 (DEAF1), and molecules involved in citrullination, antigen presentation, and immunomodulation. Overall, no clear differences between donor groups were observed with exception of a slightly lower induction of human leukocyte antigen-DR (HLA-DR) and programmed cell death 1 ligand (PD-L1) molecules in LNSCs from RA patients. CONCLUSION: Human LNSCs have the machinery to regulate peripheral tolerance making them an attractive target to exploit in tolerance induction and maintenance.

Tissue-resident mucosal-associated invariant T (MAIT) cells in the human kidney represent a functionally distinct subset.

Mucosal-associated invariant T (MAIT) cells are innate-like T-cells that recognize bacterial riboflavin metabolites. They are present in human blood but are abundant at barrier sites, including the liver, lungs, and kidneys, where they possess a CD69+ /CD103+/- tissue-resident phenotype. In renal tissue, MAIT cells likely defend against the ascending uropathogens responsible for urinary tract infections (UTIs), which are common, especially among renal transplant recipients (RTRs). Nevertheless, the functional role for MAIT cells in renal tissue and the influence of renal transplantation on MAIT cells remains unclear. Using multiparameter flow cytometry and the MR1-tetramer, we characterized MAIT cell phenotype and function in healthy renal tissue (n = 6), renal transplants explanted after allograft failure (n = 14) and in blood from healthy controls (n = 20) and RTRs before and 1-year after transplantation (n = 21). MAIT cells in renal tissue constitute a distinct CD69+ CD103+/- population that displays typical phenotypic features of tissue-resident T-cells and is skewed toward IL-2, GM-CSF, and IL-17A production upon stimulation. The circulating MAIT cell population was not decreased in number in RTRs pre- or post-transplantation. Tissue-resident MAIT cells in the kidney represent a functionally distinct population. This shows how MAIT cells in the kidney may be involved in the protection against microorganisms.

Assessing Anti-HCMV Cell Mediated Immune Responses in Transplant Recipients and Healthy Controls Using a Novel Functional Assay.

HCMV infection, reinfection or reactivation occurs in 60% of untreated solid organ transplant (SOT) recipients. Current clinical approaches to HCMV management include pre-emptive and prophylactic antiviral treatment strategies. The introduction of immune monitoring to better stratify patients at risk of viraemia and HCMV mediated disease could improve clinical management. Current approaches quantify T cell IFNγ responses specific for predominantly IE and pp65 proteins ex vivo, as a proxy for functional control of HCMV in vivo. However, these approaches have only a limited predictive ability. We measured the IFNγ T cell responses to an expanded panel of overlapping peptide pools specific for immunodominant HCMV proteins IE1/2, pp65, pp71, gB, UL144, and US3 in a cohort of D+R- kidney transplant recipients in a longitudinal analysis. Even with this increased antigen diversity, the results show that while all patients had detectable T cell responses, this did not correlate with control of HCMV replication in some. We wished to develop an assay that could directly measure anti-HCMV cell-mediated immunity. We evaluated three approaches, stimulation of PBMC with (i) whole HCMV lysate or (ii) a defined panel of immunodominant HCMV peptides, or (iii) fully autologous infected cells co-cultured with PBMC or isolated CD8+ T cells or NK cells. Stimulation with HCMV lysate often generated non-specific antiviral responses while stimulation with immunodominant HCMV peptide pools produced responses which were not necessarily antiviral despite strong IFNγ production. We demonstrated that IFNγ was only a minor component of secreted antiviral activity. Finally, we used an antiviral assay system to measure the effect of whole PBMC, and isolated CD8+ T cells and NK cells to control HCMV in infected autologous dermal fibroblasts. The results show that both PBMC and especially CD8+ T cells from HCMV seropositive donors have highly specific antiviral activity against HCMV. In addition, we were able to show that NK cells were also antiviral, but the level of this control was highly variable between donors and not dependant on HCMV seropositivity. Using this approach, we show that non-viraemic D+R+ SOT recipients had significant and specific antiviral activity against HCMV.

Clinical consequences of primary CMV infection after renal transplantation: a case-control study.

The impact of primary cytomegalovirus infection (pCMV) on renal allograft function and histology is controversial. We evaluated the influence on incidence of acute rejection, allograft loss, allograft function and interstitial fibrosis/tubular atrophy (IF/TA). Retrospective case-control study, recipients transplanted between 2000 and 2014. Risk of acute rejection and allograft loss for those who experienced pCMV infection compared with those who did not, within an exposure period of two months after transplantation. Besides, its influence on allograft function and histology at one to three years after transplantation. Of 113 recipients experienced pCMV infection, 306 remained CMV seronegative. pCMV infection in the exposure period could not be proven as increasing the risk for acute rejection [HR = 2.18 (95% CI 0.80-5.97) P = 0.13] or allograft loss [HR = 1.11 (95%CI 0.33-3.72) P = 0.87]. Combination of pCMV infection and acute rejection posed higher hazard for allograft loss than acute rejection alone [HR = 3.69 (95% CI 1.21-11.29) P = 0.02]. eGFR(MDRD) values did not significantly differ at years one [46 vs. 50], two [46 vs. 51] and three [46 vs. 52]. No association between pCMV infection and IF/TA could be demonstrated [OR = 2.15 (95%CI 0.73-6.29) P = 0.16]. pCMV infection was not proven to increase the risk for acute rejection or allograft loss. However, it increased the risk for rejection-associated allograft loss. In remaining functioning allografts, it was not significantly associated with decline in function nor with presence of IF/TA.

Tissue-resident memory T cells invade the brain parenchyma in multiple sclerosis white matter lesions.

Multiple sclerosis is a chronic inflammatory, demyelinating disease, although it has been suggested that in the progressive late phase, inflammatory lesion activity declines. We recently showed in the Netherlands Brain Bank multiple sclerosis-autopsy cohort considerable ongoing inflammatory lesion activity also at the end stage of the disease, based on microglia/macrophage activity. We have now studied the role of T cells in this ongoing inflammatory lesion activity in chronic multiple sclerosis autopsy cases. We quantified T cells and perivascular T-cell cuffing at a standardized location in the medulla oblongata in 146 multiple sclerosis, 20 neurodegenerative control and 20 non-neurological control brain donors. In addition, we quantified CD3+, CD4+, and CD8+ T cells in 140 subcortical white matter lesions. The location of CD8+ T cells in either the perivascular space or the brain parenchyma was determined using CD8/laminin staining and confocal imaging. Finally, we analysed CD8+ T cells, isolated from fresh autopsy tissues from subcortical multiple sclerosis white matter lesions (n = 8), multiple sclerosis normal-appearing white matter (n = 7), and control white matter (n = 10), by flow cytometry. In normal-appearing white matter, the number of T cells was increased compared to control white matter. In active and mixed active/inactive lesions, the number of T cells was further augmented compared to normal-appearing white matter. Active and mixed active/inactive lesions were enriched for both CD4+ and CD8+ T cells, the latter being more abundant in all lesion types. Perivascular clustering of T cells in the medulla oblongata was only found in cases with a progressive disease course and correlated with a higher percentage of mixed active/inactive lesions and a higher lesion load compared to cases without perivascular clusters in the medulla oblongata. In all white matter samples, CD8+ T cells were located mostly in the perivascular space, whereas in mixed active/inactive lesions, 16.3% of the CD8+ T cells were encountered in the brain parenchyma. CD8+ T cells from mixed active/inactive lesions showed a tissue-resident memory phenotype with expression of CD69, CD103, CD44, CD49a, and PD-1 and absence of S1P1. They upregulated markers for homing (CXCR6), reactivation (Ki-67), and cytotoxicity (GPR56), yet lacked the cytolytic enzyme granzyme B. These data show that in chronic progressive multiple sclerosis cases, inflammatory lesion activity and demyelinated lesion load is associated with an increased number of T cells clustering in the perivascular space. Inflammatory active multiple sclerosis lesions are populated by CD8+ tissue-resident memory T cells, which show signs of reactivation and infiltration of the brain parenchyma.

Divergent chemokine receptor expression and the consequence for human IgG4 B cell responses.

IgG4 antibodies are unique to humans. IgG4 is associated with tolerance during immunotherapy in allergy, but also with pathology, as in pemphigus vulgaris and IgG4-related disease. Its induction is largely restricted to nonmicrobial antigens, and requires repeated or prolonged antigenic stimulation, for reasons poorly understood. An important aspect in generating high-affinity IgG antibodies is chemokine receptor-mediated migration of B cells into appropriate niches, such as germinal centers. Here, we show that compared to IgG1 B cells, circulating IgG4 B cells express lower levels of CXCR3, CXCR4, CXCR5, CCR6, and CCR7, chemokine receptors involved in GC reactions and generation of long-lived plasma cells. This phenotype was recapitulated by in vitro priming of naive B cells with an IgG4-inducing combination of TFH /TH2 cytokines. Consistent with these observations, we found a low abundance of IgG4 B cells in secondary lymphoid tissues in vivo, and the IgG4 antibody response is substantially more short-lived compared to other IgG subclasses in patient groups undergoing CD20+ B cell depletion therapy with rituximab. These results prompt the hypothesis that factors needed to form IgG4 B cells restrain at the same time the induction of a robust migratory phenotype that could support a long-lived IgG4 antibody response.