Characterization of the SARS-CoV-2 antibody landscape in Norway in the late summer of 2022: high seroprevalence in all age groups with patterns of primary Omicron infection in children and hybrid immunity in adults.

BACKGROUND: According to Norwegian registries, 91% of individuals ≥ 16 years had received ≥ 1 dose of COVID-19 vaccine by mid-July 2022, whereas less than 2% of children < 12 years were vaccinated. Confirmed COVID-19 was reported for 27% of the population, but relaxation of testing lead to substantial underreporting. We have characterized the humoral immunity to SARS-CoV-2 in Norway in the late summer of 2022 by estimating the seroprevalence and identifying antibody profiles based on reactivity to Wuhan or Omicron-like viruses in a nationwide cross-sectional collection of residual sera, and validated our findings using cohort sera. METHODS: 1,914 anonymized convenience sera and 243 NorFlu-cohort sera previously collected from the Oslo-area with reported infection and vaccination status were analyzed for antibodies against spike, the receptor-binding domain (RBD) of the ancestral Wuhan strain and Omicron BA.2 RBD, and nucleocapsid (N). Samples were also tested for antibodies inhibiting RBD-ACE2 interaction. Neutralization assays were performed on subsets of residual sera against B.1, BA.2, XBB.1.5 and BQ.1.1. RESULTS: The national seroprevalence estimate from vaccination and/or infection was 99.1% (95% CrI 97.0-100.0%) based on Wuhan (spike_W and RBD_W) and RBD_BA2 antibodies. Sera from children < 12 years had 2.2 times higher levels of antibodies against RBD_BA2 than RBD_W and their seroprevalence estimate showed a 14.4 percentage points increase when also including anti-RBD_BA2 antibodies compared to Wuhan-antibodies alone. 50.3% (95% CI 45.0-55.5%) of residual sera from children and 38.1% (95% CI 36.0-40.4%) of all residual sera were positive for anti-N-antibodies. By combining measurements of binding- and ACE2-RBD-interaction-inhibiting antibodies, reactivity profiles indicative of infection and vaccination history were identified and validated using cohort sera. Residual sera with a profile indicative of hybrid immunity were able to neutralize newer Omicron variants XBB.1.5 and BQ.1.1. CONCLUSIONS: By late summer of 2022, most of the Norwegian population had antibodies to SARS-CoV-2, and almost all children had been infected. Antibody profiles indicated that children mostly had experienced a primary Omicron infection, while hybrid immunity was common among adults. The finding that sera displaying hybrid immunity could neutralize newer Omicron variants indicates that Wuhan-like priming of the immune response did not have a harmful imprinting effect and that infections induce cross-reacting antibodies against future variants.

Seroprevalence of SARS-CoV-2 and humoral immune responses to COVID-19 mRNA vaccines among people who use drugs - in the light of tailored mitigating strategies.

BACKGROUND: During the initial wave of the COVID-19 pandemic, there was a surprisingly low incidence of SARS-CoV-2 among People Who Use Drugs (PWUD) in Oslo, Norway, despite their heightened vulnerability regarding risk of infection and severe courses of the disease.This study aims to investigate the seroprevalence of SARS-CoV-2 antibodies among PWUD, their antibody responses to relevant virus infections and COVID-19 mRNA vaccines, and their vaccination coverage compared to the general population. METHODS: Conducted as a prospective cohort study, data was collected from residents in six institutions for homeless PWUD and users of a low-threshold clinic for opioid agonist treatment. Ninety-seven participants were recruited for SARS-CoV-2 seroprevalence analysis. Additional two participants with known positive SARS-CoV-2 test results were recruited for further analyses. Twenty-five participants completed follow-up. Data included questionnaires, nasal swabs and blood samples. Data on vaccination coverage was obtained from the National Vaccine Register. Serologic methods included detection of antibodies to relevant virus proteins, neutralizing antibodies to SARS-CoV-2, antibodies to the full-length spike protein, and receptor-binding domain from SARS-CoV-2. RESULTS: Among PWUD, antibodies to SARS-CoV-2 were detected in 2 out of 97 samples before vaccines against SARS-CoV-2 were available, comparable to a 2.8% frequency in population-based screening. Levels of serum antibodies to seasonal coronaviruses and Epstein-Barr-Virus (EBV) in PWUD were similar to population-based levels. After the second vaccine dose, binding and neutralizing antibody levels to SARS-CoV-2 in PWUD were comparable to controls. Eighty-four of PWUD received at least one dose of COVID-19 mRNA vaccine, compared to 89% in the general population. CONCLUSION: Results indicate that PWUD did not exhibit increased SARS-CoV-2 seroprevalence or elevated serum antibodies to seasonal coronaviruses and EBV. Moreover, vaccine responses in PWUD were comparable to controls, suggesting that vaccination is effective in conferring protection against SARS-CoV-2 also in this population.

Incidence and outcome of COVID-19 following vaccine and hybrid immunity in patients on immunosuppressive therapy: identification of protective post-immunisation anti-RBD antibody levels in a prospective cohort study.

OBJECTIVES: To assess incidence, severity and predictors of COVID-19, including protective post-vaccination levels of antibodies to the receptor-binding domain of SARS-CoV-2 spike protein (anti-RBD), informing further vaccine strategies for patients with immune-mediated inflammatory diseases (IMIDs) on immunosuppressive medication. METHODS: IMIDs on immunosuppressives and healthy controls (HC) receiving SARS-CoV-2 vaccines were included in this prospective observational study. COVID-19 and outcome were registered and anti-RBD antibodies measured 2-5 weeks post-immunisation. RESULTS: Between 15 February 2021 and 15 February 2023, 1729 IMIDs and 350 HC provided blood samples and self-reported COVID-19. The incidence of COVID-19 was 66% in patients and 67% in HC, with re-infection occurring in 12% of patients. Severe COVID-19 was recorded in 22 (2%) patients and no HC. No COVID-19-related deaths occurred. Vaccine-induced immunity gave higher risk of COVID-19 (HR 5.89 (95% CI 4.45 to 7.80)) than hybrid immunity. Post-immunisation anti-RBD levels <6000 binding antibody units/mL were associated with an increased risk of COVID-19 following three (HR 1.37 (95% CI 1.08 to 1.74)) and four doses (HR 1.28 (95% CI 1.02 to 1.62)), and of COVID-19 re-infection (HR 4.47 (95% CI 1.87 to 10.67)). CONCLUSION: Vaccinated patients with IMID have a low risk of severe COVID-19. Hybrid immunity lowers the risk of infection. High post-immunisation anti-RBD levels protect against COVID-19. These results suggest that knowledge on COVID-19 history, and assessment of antibody levels post-immunisation can help individualise vaccination programme series in high-risk individuals. TRIAL REGISTRATION NUMBER: NCT04798625.

Humoral vaccine response and breakthrough infections in kidney transplant recipients during the COVID-19 pandemic: a nationwide cohort study.

Background: Kidney transplant recipients (KTRs) experienced reduced SARS-CoV-2 vaccine response and were at increased risk of severe COVID-19. It is unknown if level of vaccine induced anti-receptor binding domain IgG (anti-RBD IgG) correlates with protection from and survival following COVID-19. We aimed to evaluate the effect of vaccine response on risk of breakthrough infections (BTI) and COVID-19 death in KTRs. Methods: We performed a nationwide study, examining the competing risk of SARS-CoV-2 infection, COVID-19 related/unrelated death, and vaccine efficacy as assessed by level of anti-RBD IgG response 4-10 weeks after each vaccination. The study included all KTR in Norway alive and with a functioning graft on February 20th, 2020, and events after November 11th, 2022 were right-censored. A pre-pandemic reference-cohort from January 1st 2019 to January 1st 2020 was included to evaluate excess mortality. The study was conducted at Oslo University Hospital, Rikshospitalet, Norway. Findings: The study included 3607 KTRs (59 [48-70] years) with a functioning graft at February 20th, 2020, who received (median [IQR]) 4 [3-4] vaccines (range 2-6, 99% mRNA). Anti-RBD IgG was measured in 12 701 serum samples provided by 3213 KTRs. Vaccine response was assessed 41 [31-57] days after vaccination. A total of 1090 KTRs were infected with SARS-CoV-2, 1005 (92%) were BTI, and vaccine response did not protect against BTI. The hazard ratio for COVID-19 related death 40 days post-infection was 1.71 (95% CI: 1.14, 2.56) comparing vaccine response levels (≥5 vs. ≥5000 BAU/mL). No excess non-COVID-19 mortality was registered in KTRs surviving SARS-CoV-2 infection compared to a 2019 pre-pandemic reference. Interpretation: Our findings suggested that SARS-CoV-2 mRNA vaccine response did not predict protection against infection, but prevention of fatal disease progression in KTRs and greater vaccine response further reduced the risk of COVID-19 death. No excess non-COVID-19 mortality was seen during the pandemic. Funding: CEPI and internal funds.

Prevalent and immunodominant CD8 T cell epitopes are conserved in SARS-CoV-2 variants.

The emergence of SARS-CoV-2 variants of concern (VOC) is driven by mutations that mediate escape from neutralizing antibodies. There is also evidence that mutations can cause loss of T cell epitopes. However, studies on viral escape from T cell immunity have been hampered by uncertain estimates of epitope prevalence. Here, we map and quantify CD8 T cell responses to SARS-CoV-2-specific minimal epitopes in blood drawn from April to June 2020 from 83 COVID-19 convalescents. Among 37 HLA ligands eluted from five prevalent alleles and an additional 86 predicted binders, we identify 29 epitopes with an immunoprevalence ranging from 3% to 100% among individuals expressing the relevant HLA allele. Mutations in VOC are reported in 10.3% of the epitopes, while 20.6% of the non-immunogenic peptides are mutated in VOC. The nine most prevalent epitopes are conserved in VOC. Thus, comprehensive mapping of epitope prevalence does not provide evidence that mutations in VOC are driven by escape of T cell immunity.

Four SARS-CoV-2 vaccine doses or hybrid immunity in patients on immunosuppressive therapies: a Norwegian cohort study.

Background: Data on response and safety of repeated vaccinations and hybrid immunity in patients with immune-mediated inflammatory diseases on immunosuppressive therapy is needed to further develop vaccination strategies in this vulnerable population. This study aimed to evaluate hybrid immunity and humoral immune response and safety of four SARS-CoV-2 vaccine doses in patients with immune-mediated inflammatory diseases on immunosuppressive therapy. Methods: This prospective observational Norwegian study of vaccine response to COVID-19 (Nor-vaC) included adult patients aged 18 years and older with immune-mediated inflammatory diseases (rheumatoid arthritis, spondyloarthritis, psoriatic arthritis, Crohn's disease, or ulcerative colitis) on immunosuppressive therapy, who had received four SARS-CoV-2 vaccine doses (vaccine group) or three vaccine doses followed by COVID-19 (hybrid group), and healthy controls receiving three vaccine doses (control group). Patients were recruited from the Division of Rheumatology at Diakonhjemmet Hospital, Oslo, and the Department of Gastroenterology at Akershus University Hospital, Lørenskog. Patients who had COVID-19 before the third vaccine dose, and patients with allergies or intolerances to elements of the vaccine were excluded. Antibodies to the receptor-binding domain of SARS-CoV-2 spike protein (anti-RBD antibodies) were assessed 2-4 weeks following vaccination or COVID-19. This study is registered at Clinialtrials.gov, NCT04798625. Findings: Between Nov 12, 2021, and April 19, 2022, 1458 participants with immune-mediated inflammatory diseases provided post-vaccination samples at 2-4 weeks following a third vaccine dose. After 544 participants were excluded, 715 (78%) of the remaining 914 participants received the fourth dose of the vaccine, and of these, 536 (75%) provided post-vaccination samples 2-4 weeks after their fourth vaccination (vaccine group). 199 (22%) of the 914 had COVID-19 after their third dose of the vaccine and of these, 167 (84%) provided samples (hybrid group). 256 of the eligible 703 patients had rheumatoid arthritis, 107 had spondyloarthritis, 115 had psoriatic arthritis, 130 had Crohn's disease, and 95 had ulcerative colitis). Median age was 56 years [IQR 45-65], 398 (57%) were women, and 305 (43%) were men. Patients in the vaccine group had higher anti-RBD antibody concentrations following the fourth vaccine dose (median 6192 BAU/ml [IQR 2878-11 243]) than after the third dose (median 5087 BAU/ml [1250-9081]; p< 0·0001), but lower antibody concentrations than the control group following the third dose (median 7595 BAU/ml [5916-12 001]; p< 0·0001). Antibody concentrations were higher in the patients in the hybrid group (23 548 BAU/ml [IQR 11 440-35 935]) than in the vaccine group (p<0·0001). No difference was found in antibody concentrations between the fourth dose of BNT162b2 (full-dose) and mRNA-1273 (half-dose). Patients and controls had a comparable safety profile after both three and four vaccine doses. Interpretation: Vaccine boosters improve humoral immune responses and are safe in patients with immune-mediated inflammatory diseases on immunosuppressive therapy, and administration should be considered regularly in this patient group. Hybrid immunity with omicron induces a strong humoral response suggesting longer intervals between booster doses in this patient group. Funding: The South-Eastern Norway Regional Health Authority, The Coalition for Epidemic Preparedness Innovations, Akershus University Hospital.

Immune responses to SARS-CoV-2 vaccines in celiac disease.

BACKGROUND AND AIMS: SARS-CoV-2 infection and development of the disease COVID-19 is a serious threat to our society. Effective vaccines have now entered the market, but most patient populations were not included in the registration clinical trials. There is evidence that patients with celiac disease (CeD) have reduced effect of vaccines such as the hepatitis B vaccine. Hence, we investigated the humoral response to SARS-CoV-2 vaccines (Chadox1, Comirnaty and Spikevax) in CeD patients and healthy controls. METHODS: CeD patients from a patient registry at Oslo University Hospital were invited to donate serum samples before and after vaccination. We sent out 1537 invitations and received paired samples from 85 individuals. These were compared with similar samples from 238 healthy controls. Sera were analyzed for antibodies to the Spike protein from SARS-CoV2 and the receptor-binding domain. The results where then converted into binding antibody units (BAU)/ml to compare. RESULTS: Prevaccination samples showed that very few patients had been earlier exposed to Sars-CoV2 and the antibody levels were low. Postvaccination analysis showed overlap of antibody levels between CeD and healthy controls. On average, the CeD patient group had 5555.0 BAU/ml (330.1 SD) while the average in healthy controls was 5419 (184.7 SD). CONCLUSION: The humoral response to SARS-CoV-2 vaccines in CeD patients is similar to that observed in healthy controls.

People who use drugs show no increase in pre-existing T-cell cross-reactivity toward SARS-CoV-2 but develop a normal polyfunctional T-cell response after standard mRNA vaccination.

People who use drugs (PWUD) are at a high risk of contracting and developing severe coronavirus disease 2019 (COVID-19) and other infectious diseases due to their lifestyle, comorbidities, and the detrimental effects of opioids on cellular immunity. However, there is limited research on vaccine responses in PWUD, particularly regarding the role that T cells play in the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we show that before vaccination, PWUD did not exhibit an increased frequency of preexisting cross-reactive T cells to SARS-CoV-2 and that, despite the inhibitory effects that opioids have on T-cell immunity, standard vaccination can elicit robust polyfunctional CD4+ and CD8+ T-cell responses that were similar to those found in controls. Our findings indicate that vaccination stimulates an effective immune response in PWUD and highlight targeted vaccination as an essential public health instrument for the control of COVID-19 and other infectious diseases in this group of high-risk patients.

Immunogenicity and Safety of Standard and Third-Dose SARS-CoV-2 Vaccination in Patients Receiving Immunosuppressive Therapy.

OBJECTIVE: Immunogenicity and safety following receipt of the standard SARS-CoV-2 vaccination regimen in patients with immune-mediated inflammatory diseases (IMIDs) are poorly characterized, and data after receipt of the third vaccine dose are lacking. The aim of the study was to evaluate serologic responses and adverse events following the standard 2-dose regimen and a third dose of SARS-CoV-2 vaccine in IMID patients receiving immunosuppressive therapy. METHODS: Adult patients receiving immunosuppressive therapy for rheumatoid arthritis, spondyloarthritis, psoriatic arthritis, Crohn's disease, or ulcerative colitis, as well as healthy adult controls, who received the standard 2-dose SARS-CoV-2 vaccination regimen were included in this prospective observational study. Analyses of antibodies to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein were performed prior to and 2-4 weeks after vaccination. Patients with a weak serologic response, defined as an IgG antibody titer of ≤100 arbitrary units per milliliter (AU/ml) against the receptor-binding domain of the full-length SARS-Cov-2 spike protein, were allotted a third vaccine dose. RESULTS: A total of 1,505 patients (91%) and 1,096 healthy controls (98%) had a serologic response to the standard regimen (P < 0.001). Anti-RBD antibody levels were lower in patients (median 619 AU/ml interquartile range [IQR] 192-4,191) than in controls (median 3,355 AU/ml [IQR 896-7,849]) (P < 0.001). The proportion of responders was lowest among patients receiving tumor necrosis factor inhibitor combination therapy, JAK inhibitors, or abatacept. Younger age and receipt of messenger RNA-1273 vaccine were predictors of serologic response. Of 153 patients who had a weak response to the standard regimen and received a third dose, 129 (84%) became responders. The vaccine safety profile among patients and controls was comparable. CONCLUSION: IMID patients had an attenuated response to the standard vaccination regimen as compared to healthy controls. A third vaccine dose was safe and resulted in serologic response in most patients. These data facilitate identification of patient groups at risk of an attenuated vaccine response, and they support administering a third vaccine dose to IMID patients with a weak serologic response to the standard regimen.

HLA-DRB3*01:01 exhibits a dose-dependent impact on HPA-1a antibody levels in HPA-1a-immunized women.

HLA-DRB3*01:01 is a predisposing factor for human platelet antigen 1a (HPA-1a) immunization, which is responsible for most cases of fetal and neonatal alloimmune thrombocytopenia. The aim of this study was to investigate if the HLA-DRB3*01:01 allele imposes a dose-dependent effect on anti-HPA-1a levels and neonatal platelet counts. One hundred and thirty HPA-1a-immunized women were divided into 3 groups: HLA-DRB3*01:01 negative, HLA-DRB3*01:01 hemizygous or heterozygous, and HLA-DRB3*01:01 homozygous. The dose of the HLA-DRB3*01:01 allele was determined by sequencing exon 2 of the HLA-DRB3 gene followed by HLA-DRB3 and HLA-DRB1 typing of selected samples. Anti-HPA-1a levels at time of delivery and neonatal platelet counts were compared among groups. There was a significant dose-dependent effect of the HLA-DRB3*01:01 allele on anti-HPA-1a levels (global P value [P global] = .0032). Median (range) anti-HPA-1a levels were 1.5 IU/mL (0.0-19.0 IU/mL), 21.1 IU/mL (0.0-1967 IU/mL), and 43.7 IU/mL (1.0-980 IU/mL) in women with 0, 1, and 2 copies of the HLA-DRB3*01:01 allele, respectively. There was also a significant, but opposite, dose-dependent effect of the mother's HLA-DRB3*01:01 allele on the platelet count of the newborn (P global = .0155). Median (range) neonatal platelet counts were 241 × 109/L (59 × 109/L to 393 × 109/L), 107 × 109/L (4 × 109/L to 387 × 109/L) and 32 × 109/L (4 × 109/L to 352 × 109/L) for newborns of mothers with 0, 1, and 2 copies of the HLA-DRB3*01:01 allele, respectively. Thus, the HLA-DRB3*01:01 allele exhibits a dose-dependent impact on maternal anti-HPA-1a levels in HPA-1a-immunized women.

Identification of MHC Class Ib Ligands for Stimulatory and Inhibitory Ly49 Receptors and Induction of Potent NK Cell Alloresponses in Rats.

Early studies indicate that rats may have a repertoire of MHC class Ib-reactive Ly49 stimulatory receptors capable of mounting memory-like NK cell alloresponses. In this article, we provide molecular and functional evidence for this assumption. Pairs of Ly49 receptors with sequence similarities in the lectin-like domains, but with opposing signaling functions, showed specificity for ligands with class Ia-like structural features encoded from the first telomeric MHC class Ib gene cluster, RT1-CE, which is syntenic with the H2-D/H2-L/H2-Q cluster in mice. The activating Ly49s4 receptor and its inhibitory counterparts, Ly49i4 and Ly49i3, reacted with all allelic variants of RT1-U, whereas Ly49s5 and Ly49i5 were specific for RT1-Eu NK cell cytolytic responses were predictably activated and inhibited, and potent in vivo NK alloresponses were induced by repeated MHC class Ib alloimmunizations. Additional Ly49-class Ib interactions, including RT1-Cl with the Ly49s4/Ly49i4/Ly49i3 group of receptors, were characterized using overexpressed receptor/ligand pairs, in vitro functional assays, and limited mutational analyses. Obvious, as well as subtle, Ly49-class Ib interactions led to ligand-induced receptor calibration and NK subset expansions in vivo. Together, these studies suggest that in vivo NK alloresponses are controlled by pleomorphic Ly49-class Ib interactions, some of which may not be easily detectable in vitro.

Frontline Science: A hyporesponsive subset of rat NK cells negative for Ly49s3 and NKR-P1B are precursors to the functionally mature NKR-P1B+ subset.

Rat NK cells are divided into major subsets expressing either Ly49 receptors or the inhibitory NKR-P1B receptor in conjunction with NKG2A/C/E receptors. A minor subset of NKp46+ cells lacking expression of both Ly49 receptors and NKR-P1B is present in blood and spleen and is associated with decreased functional competence. We hypothesized that this subset may represent precursors to Ly49+ and/or NKR-P1B+ NK cells. When cultured in vitro in IL-2 and IL-15 or adoptively transferred to syngeneic hosts, a portion of NKR-P1B-Ly49s3- cells transformed to express NKR-P1B, but very little Ly49s3. Acquisition of NKR-P1B by NKR-P1B-Ly49s3- cells coincided with increased degranulation. In addition, although NKR-P1B-Ly49s3- cells highly proliferate, proliferative activity was reduced upon acquisition of NKR-P1B at comparable levels to bona fide NKR-P1B+ NK cells. A fraction of NKR-P1B-Ly49s3- cells remained negative for NKR-P1B, both in vitro and after adoptive transfer in vivo. Most NKR-P1B-Ly49s3- cells expressed the transcription factor Eomesodermin and NK cell markers, indicating that these cells represent conventional NK cells. Our findings suggest that the NKR-P1B-Ly49s3- NK cells are precursors to NKR-P1B single-positive cells and that functional competence is acquired upon expression of NKR-P1B.

Degranulation Response in Cytotoxic Rat Lymphocytes Measured with a Novel CD107a Antibody.

Measuring degranulation through CD107a expression has become an advantageous tool for testing the functional capacity of cytotoxic cells. Such functional studies have been hampered in the rat by the lack of a suitable anti-rat CD107a antibody. In this study, we report a novel hybridoma generated by immunizing Armenian inbred hamsters with transfected Chinese hamster ovary cells expressing CD107a. The SIM1 clone exhibited specific reactivity with CD107a and measured degranulation from natural killer (NK) cells stimulated with target cells or mAb crosslinking of their activating receptors. Degranulation in IL-2-activated NK cells could also be measured, when using low effector to target ratios. SIM1 also stained activated CD8, but not CD4 T cells. This report characterizes the degranulation response in cytotoxic rat cells with a new antibody against rat CD107a.

Functional characterization of a conserved pair of NKR-P1 receptors expressed by NK cells and T lymphocytes in liver and gut.

Natural killer cell receptor protein 1 (NKR-P1) molecules are C-type lectin-like receptors modulating cellular responses toward target cells expressing C-type lectin-like related (Clr) molecules. Although the function of the prototypic rat NKR-P1A receptor and its inhibitory counterpart NKR-P1B are known, little is known about NKR-P1F and NKR-P1G apart from their promiscuity for Clr ligands. Here we generated mAbs against both receptors for phenotypic and functional analyses in rat tissues. NKR-P1F induced redirected lysis and robust Ca(2+) signaling in NK cells, which were prevented by simultaneous engagement of NKR-P1G. NKR-P1G also inhibited NK-cell lysis of Clr transfectants. NKR-P1F was expressed by most NK cells and NKR-P1A(+) T cells in all tissues analyzed, and by many NKR-P1A(-) intestinal T cells, while NKR-P1G was expressed by subsets of these cells with highest prevalence in gut and liver. In the intraepithelial compartment, the proportion of NKR-P1A(+) and NKR-P1F(+) cells was high at birth and thereafter declined, while NKR-P1B(+) and NKR-P1G(+) cells increased with age. Expression levels were also modulated by cytokines, with an increase of NKR-P1B and NKR-P1G induced by inflammatory cytokines, and a reduction of NKR-P1A by TGF-ß. The physiological impact of NKR-P1 receptors might thus be dependent on age, tissue, and inflammatory status.

The tetraspanin CD53 modulates responses from activating NK cell receptors, promoting LFA-1 activation and dampening NK cell effector functions.

NK cells express several tetraspanin proteins, which differentially modulate NK cell activities. The tetraspanin CD53 is expressed by all resting NK cells and was previously shown to decrease NK cell cytotoxicity upon ligation. Here, we show that CD53 ligation reduced degranulation of rat NK cells in response to tumour target cells, evoked redirected inhibition of killing of Fc-bearing targets, and reduced the IFN-γ response induced by plate-bound antibodies towards several activating NK cell receptors (Ly49s3, NKR-P1A, and NKp46). CD53 induced activation of the ß2 integrin LFA-1, which was further enhanced upon co-stimulation with activating NK cell receptors. Concordant with a role for CD53 in increasing NK cell adhesiveness, CD53 ligation induced a strong homotypic adhesion between NK cells. Further, the proliferative capacity of NK cells to a suboptimal dose of IL-2 was enhanced by CD53 ligation. Taken together, these data suggest that CD53 may shift NK cell responses from effector functions towards a proliferation phase.

The role of alloresponsive Ly49+ NK cells in rat islet allograft failure in the presence and absence of cytomegalovirus.

There are still many factors to discover to explain the low success rates of islet allografts. In this study, we demonstrate that specific subpopulations of alloreactive NK cells may be involved in the failure of islet allografts. By performing allotransplantation in rats (n = 13), we observed peripheral expansion and infiltration of alloreactive Ly49i2(+) NK cells in the grafts. An effective strategy in rats to enhance the expansion of Ly49i2(+) NK cells is performing a rat cytomegalovirus infection (n = 6). Cytomegalovirus infection was associated with an early expansion of the Ly49i2(+) NK cells and accelerated islet graft failure. The Ly49i2(+) NK cells are both alloreactive and involved in virus clearance. The expansion of this subpopulation could not be blocked by cyclosporin A immunosuppression. Also alloreactive KLRH1(+) NK cells infiltrated the grafts, but nonalloreactive NKR-P1B(+) cells were not observed in the islet allografts. Perforin staining of the infiltrating NK cells demonstrated the cytotoxic capacity of these cells. Our data suggest a role for this NK subpopulation in rat islet allograft destruction.

Identification of an MHC class I ligand for the single member of a killer cell lectin-like receptor family, KLRH1.

Natural killer cells are able to recognize and kill target cells according to differences in MHC class I expression. In rodents, the Ly49 receptors are primarily responsible for this MHC differentiation. We previously described the cloning of a novel C-type lectin-like receptor, KLRH1, encoded in the NK complex adjacent to the Ly49 genes and expressed by subsets of NK and NKT cells. MHC influence on selection of KLRH1(+) NK cells in congenic strains suggested that KLRH1 may have an MHC ligand, although we were unable to identify any such ligand. In this study, we have used a sensitive reporter system and Fc fusion protein to demonstrate that KLRH1 binds specifically to the classical MHC class I molecule RT1-A2 of the RT1(n) haplotype. Cytolytic activity of KLRH1-transfected RNK-16 cells was also inhibited by target cells expressing RT1-A2(n). Thus, KLRH1 represents a novel family of MHC allele-specific inhibitory receptors expressed by NK cells.

Rat mesenchymal stromal cells inhibit T cell proliferation but not cytokine production through inducible nitric oxide synthase.

Mesenchymal stromal cells (MSC) have important immunomodulatory properties, they inhibit T lymphocyte allo-activation and have been used to treat graft-versus-host disease. How MSC exert their immunosuppressive functions is not completely understood but species specific mechanisms have been implicated. In this study we have investigated the mechanisms for rat MSC mediated inhibition of T lymphocyte proliferation and secretion of inflammatory cytokines in response to allogeneic and mitogenic stimuli in vitro. MSC inhibited the proliferation of T cells in allogeneic mixed lymphocyte reactions and in response to mitogen with similar efficacy. The anti-proliferative effect was mediated by the induced expression of nitric oxide (NO) synthase and production of NO by MSC. This pathway was required and sufficient to fully suppress lymphocyte proliferation and depended on proximity of MSC and target cells. Expression of inducible NO synthase by MSC was induced through synergistic stimulation with tumor necrosis factor α and interferon γ secreted by activated lymphocytes. Conversely, MSC had a pronounced inhibitory effect on the secretion of these cytokines by T cells which did not depend on NO synthase activity or cell contact, but was partially reversed by addition of the cyclooxygenase (COX) inhibitor indomethacin. In conclusion, rat MSC use different mechanisms to inhibit proliferative and inflammatory responses of activated T cells. While proliferation is suppressed by production of NO, cytokine secretion appears to be impaired at least in part by COX-dependent production of prostaglandin E(2).

A novel NKR-P1B(bright) NK cell subset expresses an activated CD25(+)CX(3)CR1(+)CD62L(-)CD11b(-)CD27(-) phenotype and is prevalent in blood, liver, and gut-associated lymphoid organs of rats.

The inhibitory NKR-P1B receptor identifies a subset of rat splenic NK cells that is low in Ly49 receptors but enriched for CD94/NKG2 receptors. We report in this study a novel NKR-P1B(bright) NK subpopulation that is prevalent in peripheral blood, liver, and gut-associated lymphoid organs and scarce in the spleen, peripheral lymph nodes, bone marrow, and lungs. This NKR-P1B(bright) NK subset displays an activated phenotype, expressing CD25, CD93, CX(3)CR1 and near absence of CD62-L, CD11b, and CD27. Functionally, NKR-P1B(bright) NK cells are highly responsive in terms of IFN-γ production and exert potent cytolytic activity. They show little spontaneous proliferation, are reduced in numbers upon in vivo activation with polyinosinic:polycytidylic acid, and have poor survival in ex vivo cytokine cultures. Our findings suggest that NKR-P1B(bright) NK cells are fully differentiated effector cells that rapidly die upon further activation. The identification of this novel rat NK cell subset may facilitate future translational research of the role of distinct NK cell subsets under normal physiological conditions and during ongoing immune responses.

Phylogenetic and functional conservation of the NKR-P1F and NKR-P1G receptors in rat and mouse.

Two clusters of rat Nkrp1 genes can be distinguished based on phylogenetic relationships and functional characteristics. The proximal (centromeric) cluster encodes the well-studied NKR-P1A and NKR-P1B receptors and the distal cluster, the largely uncharacterized, NKR-P1F and NKR-P1G receptors. The inhibitory NKR-P1G receptor is expressed only by the Ly49s3(+) NK cell subset as detected by RT-PCR, while the activating NKR-P1F receptor is detected in both Ly49s3(+) and NKR-P1B(+) NK cells. The mouse NKR-P1G ortholog is expressed by both NKR-P1D(-) and NKR-P1D(+) NK cells in C57BL/6 mice. The rat and mouse NKR-P1F and NKR-P1G receptors demonstrate a striking, cross-species conservation of specificity for Clr ligands. NKR-P1F and NKR-P1G reporter cells reacted with overlapping panels of tumour cell lines and with cells transiently transfected with rat Clr2, Clr3, Clr4, Clr6 and Clr7 and mouse Clrc, Clrf, Clrg and Clrd/x, but not with Clr11 or Clrb, which serve as ligands for NKR-P1 from the proximal cluster. These data suggest that the conserved NKR-P1F and NKR-P1G receptors function as promiscuous receptors for a rapidly evolving family of Clr ligands in rodent NK cells.