Tina Senff

Background/Objectives: Anal squamous cell carcinoma (SCCA) is an HPV-driven malignancy with rising incidence, yet systematic HPV genotyping across recurrences and metastases is lacking. We characterized the complete HPV genotype distribution across the disease course. Methods: We retrospectively analyzed 213 tumor specimens from 136 patients (82 female, 54 male) with histologically confirmed SCCA, spanning primary, recurrent, and metastatic disease (2009–2019 and from 2020 onwards). Patients contributed one or more specimens depending on the number of sampled disease events; specimens comprised 138 primary biopsies, 55 recurrence biopsies, 2 re-recurrence biopsies, and 18 distant metastases. HPV genotyping was conducted using a validated 28-type multiplex real-time PCR assay (Anyplex™ II HPV28, Seegene Inc.). Results: HPV DNA was detected in 179/213 specimens (84.0%; 95% CI 78.9–88.7) and 125/136 patients (91.9%; 95% CI 86.8–96.3). Across 269 HPV type detections, 24 distinct types were identified, with HPV 16 predominating both per-specimen (90.5%) and per-detection (60.2%) values. Non-HPV-16/18 high-risk types accounted for one in four high-risk detections (54/219; 24.7%; 95% CI 19.4–30.8). HPV positivity differed significantly by specimen type: 90.6% in primary biopsies versus 70.9% in recurrences (p = 0.001) and 72.2% in metastases (p = 0.038). Co-infections occurred in 24.6% of HPV-positive specimens (95% CI 18.5–31.6), with up to eight types. In metastases, the spectrum narrowed to 4 of 24 types; two harbored HPV 33 as the sole high-risk type. Conclusions: SCCA harbors a substantially broader HPV type spectrum than previously recognized. One in four high-risk detections lies beyond HPV 16/18, and high-risk types other than HPV 16 were the sole high-risk genotype detected in individual metastatic specimens. HPV positivity rates are significantly lower across specimen types, while the metastatic type spectrum converges on a few high-risk genotypes. Broad-spectrum HPV genotyping beyond types 16 and 18 warrants adoption as standard practice in SCCA diagnostics and surveillance, and potential stratification for HPV-directed therapies.

Nodal T-follicular helper cell lymphoma, angioimmunoblastic-type (AITL), is characterized by constitutional symptoms, advanced-stage disease, and generalized lymphadenopathy. A genetic hallmark of this lymphoma is the frequent occurrence of the RHOA mutation G17V in neoplastic cells, which is observed in around 60% of patients. Because RHOA is involved in both T-cell receptor downstream signalling and cell migration, we hypothesized that the characteristic presentation of AITL could be the result of enhanced tumor cell migration. Therefore, this study aimed to elucidate the impact of the RHOA variant G17V on the migration of neoplastic T cells. We transfected the T-cell lymphoma cell lines HH and HuT78 to stably express the RHOA-G17V variant. RHOA-G17V-expressing T cells did not exhibit enhanced motility compared to empty-vector-transfected cells in microchannels, a 3D collagen gel, or primary human lymphatic tissue. Cells of the HH cell line expressing RHOA-G17V had an increased number of cells with cleaved collagen compared with the empty-vector-transfected cells. Therefore, we hypothesized that the early spread of AITL tumor cells may be related to remodelling of the extracellular matrix. Accordingly, we observed a significant negative correlation between the relative area of collagen in histological sections from 18 primary AITL and the allele frequency of the RHOA-G17V mutation. In conclusion, our results suggest that the characteristic presentation of AITL with early, widespread dissemination of lymphoma cells is not the result of an enhanced migration capacity due to the RHOA-G17V mutation; instead, this feature may rather be related to extracellular matrix remodelling.

Invariant NK T (iNKT) cells are implicated in viral clearance; however, their role in hepatitis C virus (HCV) infection remains controversial. Here, iNKT cells were studied during different stages of HCV infection. iNKT cells from patients with acute HCV infection and people who inject drugs (PWID) with chronic or spontaneously resolved HCV infection were characterized by flow cytometry. In a longitudinal analysis during acute HCV infection, frequencies of activated CD38 ⁺ iNKT cells reproducibly declined in spontaneously resolving patients, whereas they were persistently elevated in patients progressing to chronic infection. During the first year of infection, the frequency of activated CD38 ⁺ or CD69 ⁺ iNKT cells strongly correlated with alanine transaminase levels with particularly pronounced correlations in spontaneously resolving patients. Increased frequencies of activated iNKT cells in chronic HCV infection were confirmed in cross-sectional analyses of PWID with chronic or spontaneously resolved HCV infection; however, no apparent functional differences were observed with various stimulation protocols. Our data suggest that iNKT cells are activated during acute hepatitis C and that activation is sustained in chronic infection. The correlation between the frequency of activated iNKT cells and alanine transaminase may point toward a role of iNKT cells in liver damage.

Evaluation and power of seroprevalence studies depend on the performed serological assays. The aim of this study was to assess four commercial serological tests from EUROIMMUN, DiaSorin, Abbott, and Roche as well as an in-house immunofluorescence and neutralization test for their capability to identify SARS-CoV-2 seropositive individuals in a high-prevalence setting. Therefore, 42 social and working contacts of a German super-spreader were tested. Consistent with a high-prevalence setting, 26 of 42 were SARS-CoV-2 seropositive by neutralization test (NT), and immunofluorescence test (IFT) confirmed 23 of these 26 positive test results (NT 61.9% and IFT 54.8% seroprevalence). Four commercial assays detected anti-SARS-CoV-2 antibodies in 33.3-40.5% individuals. Besides an overall discrepancy between the NT and the commercial assays regarding their sensitivity, this study revealed that commercial SARS-CoV-2 spike-based assays are better to predict the neutralization titer than nucleoprotein-based assays are.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affects millions of people and killed hundred-thousands of individuals. While acute and intermediate interactions between SARS-CoV-2 and the immune system have been studied extensively, long-term impacts on the cellular immune system remained to be analyzed. Here, we comprehensively characterized immunological changes in peripheral blood mononuclear cells in 49 COVID-19 convalescent individuals (CI) in comparison to 27 matched SARS-CoV-2 unexposed individuals (UI). Despite recovery from the disease for more than 2 months, CI showed significant decreases in frequencies of invariant NKT and NKT-like cells compared to UI. Concomitant with the decrease in NKT-like cells, an increase in the percentage of Annexin V and 7-AAD double positive NKT-like cells was detected, suggesting that the reduction in NKT-like cells results from cell death months after recovery. Significant increases in regulatory T cell frequencies, TIM-3 expression on CD4 and CD8 T cells, as well as PD-L1 expression on B cells were also observed in CI, while the cytotoxic potential of T cells and NKT-like cells, defined by GzmB expression, was significantly diminished. However, both CD4 and CD8 T cells of CI showed increased Ki67 expression and were fully capable to proliferate and produce effector cytokines upon TCR stimulation. Collectively, we provide the first comprehensive characterization of immune signatures in patients recovering from SARS-CoV-2 infection, suggesting that the cellular immune system of COVID-19 patients is still under a sustained influence even months after the recovery from disease.

We analysed SARS-CoV-2 specific antibody responses in 42 social and working contacts of a super-spreader from the Heinsberg area in Germany. Consistent with a high-prevalence setting 26 individuals had SARS-CoV-2 antibodies determined by in-house neutralisation testing. These results were compared with four commercial assays, suggesting limited sensitivity of the assays in such a high-prevalence setting. Although SARS-CoV-2 nucleocapsid-restricted tests showed a better sensitivity, spike-based assays had a stronger correlation with neutralisation capacity.

We whole-genome sequenced 55 SARS-CoV-2 isolates from Germany to investigate SARS-CoV-2 outbreaks in 2020 in the Heinsberg district and Düsseldorf. While the genetic structure of the Heinsberg outbreak indicates a clonal origin, reflecting superspreading dynamics from mid-February during the carnival season, distinct viral strains were circulating in Düsseldorf in March, reflecting the city’s international links. Limited detection of Heinsberg strains in the Düsseldorf area despite geographical proximity may reflect efficient containment and contact-tracing efforts.

Infection with the human CMV associates with phenotypic alterations in lymphocyte subsets. A highly reproducible finding in CMV-seropositive individuals is an expansion of NKG2C^pos NK cells. In this study, we analyzed if the altered NK cell compartment in CMV-seropositive human donors may affect CMV-specific CD8 T cells. Resting CMV-specific CD8 T cells were terminally differentiated and expressed high levels of the NKG2C ligand HLA-E. Activation of CMV-specific CD8 T cells with the cognate Ag further increased HLA-E expression. In line with a negative regulatory effect of NKG2C^pos NK cells on HLA-E^high CD8 T cells, depletion of NKG2C^pos NK cells enhanced Ag-specific expansion of CMV-specific CD8 T cells in vitro. In turn, the activation of NK cells in coculture with CMV-specific CD8 T cells promoted a selective loss of HLA-E^high CD8 T cells. To test if NKG2C^pos NK cells can target HLA-E^high CD8 T cells, Jurkat T cells with and without stabilized HLA-E on the surface were used. NKG2C^pos NK cells stimulated with HLA-E^high Jurkat cells released higher levels of Granzyme B compared with NKG2C^neg NK cells and NKG2C^pos NK cells stimulated with HLA-E^low Jurkat cells. Moreover, intracellular levels of caspase 3/7 were increased in HLA-E^high Jurkat cells compared with HLA-E^low Jurkat cells, consistent with higher rates of apoptosis in HLA-E^high T cells in the presence of NKG2C^pos NK cells. Our data show that NKG2C^pos NK cells interact with HLA-E^high CD8 T cells, which may negatively regulate the expansion of CMV-specific CD8 T cells upon activation. Key Points CD56^pos NKG2C^high NK cell individuals have lower frequencies of TEMRA CD8 T cells. Depletion of NKG2C^pos NK cells augments expansion of CMV-specific CD8 T cells. NKG2C^pos NK cells negatively regulate HLA-E^high CD8 T cells.

We whole-genome sequenced 55 SARS-CoV-2 isolates from Western Germany and investigated the genetic structure of SARS-CoV-2 outbreaks in the Heinsberg district and Düsseldorf. While the genetic structure of the Heinsberg outbreak indicates a clonal origin, reflective of superspreading dynamics during the carnival season, distinct viral strains are circulating in Düsseldorf, reflecting the city’s international links. Limited detection of Heinsberg strains in the Düsseldorf area despite geographical proximity may reflect efficient containment and contact tracing efforts.

`NK cell–mediated regulation of antigen-specific T cells can contribute to and exacerbate chronic viral infection, but the protective mechanisms against NK cell–mediated attack on T cell immunity are poorly understood. Here, we show that progranulin (PGRN) can reduce NK cell cytotoxicity through reduction of NK cell expansion, granzyme B transcription, and NK cell–mediated lysis of target cells. Following infection with the lymphocytic choriomeningitis virus (LCMV), PGRN levels increased — a phenomenon dependent on the presence of macrophages and type I IFN signaling. Absence of PGRN in mice ( Grn ^–/– ) resulted in enhanced NK cell activity, increased NK cell–mediated killing of antiviral T cells, reduced antiviral T cell immunity, and increased viral burden, culminating in increased liver immunopathology. Depletion of NK cells restored antiviral immunity and alleviated pathology during infection in Grn ^–/– mice. In turn, PGRN treatment improved antiviral T cell immunity. Taken together, we identified PGRN as a critical factor capable of reducing NK cell–mediated attack of antiviral T cells.