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Hantaviruses cause the acute zoonotic diseases hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Infected patients show strong systemic inflammation and immune cell activation. NK cells are highly activated in HFRS, suggesting that also other innate lymphoid cells (ILCs) might be responding to infection. Here, we characterized peripheral ILC responses, and measured plasma levels of soluble factors and plasma viral load, in 17 Puumala virus (PUUV)-infected HFRS patients. This revealed an increased frequency of ILC2 in patients, in particular the ILC2 lineage-committed c-Kitlo ILC2 subset. Patients' ILCs showed an activated profile with increased proliferation and displayed altered expression of several homing markers. How ILCs are activated during viral infection is largely unknown. When analyzing PUUV-mediated activation of ILCs in vitro we observed that this was dependent on type I interferons, suggesting a role for type I interferons—produced in response to virus infection–in the activation of ILCs. Further, stimulation of naïve ILC2s with IFN-β affected ILC2 cytokine responses in vitro, causing decreased IL-5 and IL-13, and increased IL-10, CXCL10, and GM-CSF secretion. These results show that ILCs are activated in HFRS patients and suggest that the classical antiviral type I IFNs are involved in shaping ILC functions. Author summary: Hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) are acute zoonotic diseases caused by orthohantaviruses. Hantavirus initially infect the lung and then spread systemically, primarily infecting endothelial cells. The innate lymphoid cell (ILC) family consists of ILC1, ILC2, ILC3, and natural killer (NK) cells. While NK cells are well studied in viral infections, less is known regarding the other ILCs. Hantavirus-infected patients show highly activated immune cells, including NK cells, indicating that also other innate lymphoid cells (ILCs) might be affected. We discovered that Puumala virus (PUUV)-infected HFRS patients show increased frequency of ILC2s in circulation, and that ILCs are activated with an altered homing receptor expression pattern. Little is known regarding how ILCs are activated during viral infection. When analyzing, in vitro, for mechanisms behind activation we observed a PUUV-induced type I IFN-dependent activation of ILCs and that IFN-β affects ILC2 cytokine responses. These findings suggest that ILC functions are affected by classical antiviral type I IFNs. [ABSTRACT FROM AUTHOR]

Objective: Human hantavirus infections can cause haemorrhagic fever with renal syndrome (HFRS). The pathogenic mechanisms are not fully understood, nor if they affect the humoral immune system. The objective of this study was to investigate humoral immune responses to hantavirus infection and to correlate them to the typical features of HFRS: thrombocytopenia and transient kidney dysfunction. Methods: We performed a comprehensive characterisation of longitudinal antiviral B‐cell responses of 26 hantavirus patients and combined this with paired clinical data. In addition, we measured extracellular adenosine triphosphate (ATP) and its breakdown products in circulation and performed in vitro stimulations to address its effect on B cells. Results: We found that thrombocytopenia was correlated to an elevated frequency of plasmablasts in circulation. In contrast, kidney dysfunction was indicative of an accumulation of CD27−IgD− B cells and CD27−/low plasmablasts. Finally, we provide evidence that high levels of extracellular ATP and matrix metalloproteinase 8 can contribute to shedding of CD27 during human hantavirus infection. Conclusion: Our findings demonstrate that thrombocytopenia and kidney dysfunction associate with distinctly different effects on the humoral immune system. Moreover, hantavirus‐infected individuals have significantly elevated levels of extracellular ATP in circulation. [ABSTRACT FROM AUTHOR]

While the relationship of protective HLA class I alleles and HIV progression is well defined, the interaction of HLA-mediated protection and CD8 T-cell exhaustion is less well characterized. To gain insight in the influence of HLA-B*57:01 on the deterioration of CD8 T-cell responses during HIV infection in the absence of antiretroviral treatment, we compared HLA-B*57:01-restricted HIV-specific CD8 T-cell responses to responses restricted by other HLA class I alleles longitudinally following control of peak viremia. Detailed characterization of polyfunctionality, differentiation phenotypes, transcription factor and inhibitory receptor expression revealed progression of CD8 T-cell exhaustion over the course of the infection in both patient groups. However, early effects on the phenotype of the total CD8 T-cell population were apparent only in HLA-B*57-negative patients. The HLA-B*57:01-restricted, HIV-1 epitope-specific CD8 T-cell responses showed beneficial functional patterns and significantly lower frequencies of inhibitory receptor expression, i.e. PD-1 and PD-1/TIGIT co-expression, within the first year of infection. Co40 expression of PD-1 and TIGIT was correlated to clinical markers of disease progression and declining percentages of the T-bet(hi)/Eomes(dim) CD8 T-cell population. In accordance with clinical and immunological deterioration in the HLA-B*57:01 group, the difference in PD-1 and TIGIT receptor expression did not persist to later stages of the disease. [ABSTRACT FROM AUTHOR]

T cells are pivotal in the immune defense against cancers and infectious agents. To mount an effector response against cancer cells, T cells need to migrate to the cancer-site, engage in contacts with cancer cells, and perform their effector functions. Adoptive T cell therapy is an effective strategy as treatment of complications such as relapse or opportunistic infections after hematopoietic stem cell transplantations. This requires a sufficient amount of cells that are able to expand and respond to tumor or viral antigens. The cytokines interleukin (IL)-2 and IL-7 drive T cell differentiation, proliferation, and survival and are commonly used to expand T cells ex vivo. Here, we have used microchipbased live-cell imaging to follow the migration of individual T cells, their interactions with allogeneic monocytes, cell division, and apoptosis for extended periods of time; something that cannot be achieved by commonly used methods. Our data indicate that cells grown in IL-7+IL-2 had similar migration and contact dynamics as cells grown in IL-2 alone. However, the addition of IL-7 decreased cell death creating a more viable cell population, which should be beneficial when preparing cells for immunotherapy. [ABSTRACT FROM AUTHOR]

CD8+ T cell exhaustion represents a major hallmark of chronic HIV infection. Two key transcription factors governing CD8+ T cell differentiation, T-bet and Eomesodermin (Eomes), have previously been shown in mice to differentially regulate T cell exhaustion in part through direct modulation of PD-1. Here, we examined the relationship between these transcription factors and the expression of several inhibitory receptors (PD-1, CD160, and 2B4), functional characteristics and memory differentiation of CD8+ T cells in chronic and treated HIV infection. The expression of PD-1, CD160, and 2B4 on total CD8+ T cells was elevated in chronically infected individuals and highly associated with a T-betdimEomeshi expressional profile. Interestingly, both resting and activated HIV-specific CD8+ T cells in chronic infection were almost exclusively T-betdimEomeshi cells, while CMV-specific CD8+ T cells displayed a balanced expression pattern of T-bet and Eomes. The T-betdimEomeshi virus-specific CD8+ T cells did not show features of terminal differentiation, but rather a transitional memory phenotype with poor polyfunctional (effector) characteristics. The transitional and exhausted phenotype of HIV-specific CD8+ T cells was longitudinally related to persistent Eomes expression after antiretroviral therapy (ART) initiation. Strikingly, these characteristics remained stable up to 10 years after ART initiation. This study supports the concept that poor human viral-specific CD8+ T cell functionality is due to an inverse expression balance between T-bet and Eomes, which is not reversed despite long-term viral control through ART. These results aid to explain the inability of HIV-specific CD8+ T cells to control the viral replication post-ART cessation. [ABSTRACT FROM AUTHOR]

HIV-specific CD8+ T cells demonstrate an exhausted phenotype associated with increased expression of inhibitory receptors, decreased functional capacity, and a skewed transcriptional profile, which are only partially restored by antiretroviral treatment (ART). Expression levels of the inhibitory receptor, T cell immunoglobulin and ITIM domain (TIGIT), the co-stimulatory receptor CD226 and their ligand PVR are altered in viral infections and cancer. However, the extent to which the TIGIT/CD226/PVR-axis is affected by HIV-infection has not been characterized. Here, we report that TIGIT expression increased over time despite early initiation of ART. HIV-specific CD8+ T cells were almost exclusively TIGIT+, had an inverse expression of the transcription factors T-bet and Eomes and co-expressed PD-1, CD160 and 2B4. HIV-specific TIGIThi cells were negatively correlated with polyfunctionality and displayed a diminished expression of CD226. Furthermore, expression of PVR was increased on CD4+ T cells, especially T follicular helper (Tfh) cells, in HIV-infected lymph nodes. These results depict a skewing of the TIGIT/CD226 axis from CD226 co-stimulation towards TIGIT-mediated inhibition of CD8+ T cells, despite early ART. These findings highlight the importance of the TIGIT/CD226/PVR axis as an immune checkpoint barrier that could hinder future 'cure' strategies requiring potent HIV-specific CD8+ T cells. [ABSTRACT FROM AUTHOR]

HLA-B*5701 is the host factor most strongly associated with slow HIV-1 disease progression, although rates can vary within this group. Underlying mechanisms are not fully understood but likely involve both immunological and virological dynamics. The present study investigated HIV-1 in vivo evolution and epitope-specific CD8+ T cell responses in six HLA-B*5701 patients who had not received antiretroviral treatment, monitored from early infection for up to 7 years. The subjects were classified as highrisk progressors (HRPs) or low-risk progressors (LRPs) based on baseline CD4+ T cell counts. Dynamics of HIV-1 Gag p24 evolution and multifunctional CD8+ T cell responses were evaluated by high-resolution phylogenetic analysis and polychromatic flow cytometry, respectively. In all subjects, substitutions occurred more frequently in flanking regions than in HLA-B*5701- restricted epitopes. In LRPs, p24 sequence diversity was significantly lower; sequences exhibited a higher degree of homoplasy and more constrained mutational patterns than HRPs. The HIV-1 intrahost evolutionary rate was also lower in LRPs and followed a strict molecular clock, suggesting neutral genetic drift rather than positive selection. Additionally, polyfunctional CD8 T cell responses, particularly to TW10 and QW9 epitopes, were more robust in LRPs, who also showed significantly higher interleukin- 2 (IL-2) production in early infection. Overall, the findings indicate that HLA-B*5701 patients with higher CD4 counts at baseline have a lower risk of HIV-1 disease progression because of the interplay between specific HLA-linked immune responses and the rate and mode of viral evolution. The study highlights the power of a multidisciplinary approach, integrating high-resolution evolutionary and immunological data, to understand mechanisms underlying HIV-1 pathogenesis. [ABSTRACT FROM AUTHOR]

Objective: Human hantavirus infections can cause haemorrhagic fever with renal syndrome (HFRS). The pathogenic mechanisms are not fully understood, nor if they affect the humoral immune system. The objective of this study was to investigate humoral immune responses to hantavirus infection and to correlate them to the typical features of HFRS: thrombocytopenia and transient kidney dysfunction., Methods: We performed a comprehensive characterisation of longitudinal antiviral B-cell responses of 26 hantavirus patients and combined this with paired clinical data. In addition, we measured extracellular adenosine triphosphate (ATP) and its breakdown products in circulation and performed in vitro stimulations to address its effect on B cells., Results: We found that thrombocytopenia was correlated to an elevated frequency of plasmablasts in circulation. In contrast, kidney dysfunction was indicative of an accumulation of CD27 - IgD - B cells and CD27 -/low plasmablasts. Finally, we provide evidence that high levels of extracellular ATP and matrix metalloproteinase 8 can contribute to shedding of CD27 during human hantavirus infection., Conclusion: Our findings demonstrate that thrombocytopenia and kidney dysfunction associate with distinctly different effects on the humoral immune system. Moreover, hantavirus-infected individuals have significantly elevated levels of extracellular ATP in circulation., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Innate immune cells like monocytes patrol the vasculature and mucosal surfaces, recognize pathogens, rapidly redistribute to affected tissues and cause inflammation by secretion of cytokines. We previously showed that monocytes are reduced in blood but accumulate in the airways of patients with Puumala virus (PUUV) caused hemorrhagic fever with renal syndrome (HFRS). However, the dynamics of monocyte infiltration to the kidneys during HFRS, and its impact on disease severity are currently unknown. Here, we examined longitudinal peripheral blood samples and renal biopsies from HFRS patients and performed in vitro experiments to investigate the fate of monocytes during HFRS. During the early stages of HFRS, circulating CD14-CD16+ nonclassical monocytes (NCMs) that patrol the vasculature were reduced in most patients. Instead, CD14+CD16- classical (CMs) and CD14+CD16+ intermediate monocytes (IMs) were increased in blood, in particular in HFRS patients with more severe disease. Blood monocytes from patients with acute HFRS expressed higher levels of HLA-DR, the endothelial adhesion marker CD62L and the chemokine receptors CCR7 and CCR2, as compared to convalescence, suggesting monocyte activation and migration to peripheral tissues during acute HFRS. Supporting this hypothesis, increased numbers of HLA-DR+, CD14+, CD16+ and CD68+ cells were observed in the renal tissues of acute HFRS patients compared to controls. In vitro, blood CD16+ monocytes upregulated CD62L after direct exposure to PUUV whereas CD16- monocytes upregulated CCR7 after contact with PUUV-infected endothelial cells, suggesting differential mechanisms of activation and response between monocyte subsets. Together, our findings suggest that NCMs are reduced in blood, potentially via CD62L-mediated attachment to endothelial cells and monocytes are recruited to the kidneys during HFRS. Monocyte mobilization, activation and functional impairment together may influence the severity of disease in acute PUUV-HFRS., Competing Interests: The authors have declared that no competing interests exist.

HIV-specific CD8 + T cells demonstrate an exhausted phenotype associated with increased expression of inhibitory receptors, decreased functional capacity, and a skewed transcriptional profile, which are only partially restored by antiretroviral treatment (ART). Expression levels of the inhibitory receptor, T cell immunoglobulin and ITIM domain (TIGIT), the co-stimulatory receptor CD226 and their ligand PVR are altered in viral infections and cancer. However, the extent to which the TIGIT/CD226/PVR-axis is affected by HIV-infection has not been characterized. Here, we report that TIGIT expression increased over time despite early initiation of ART. HIV-specific CD8 + T cells were almost exclusively TIGIT + , had an inverse expression of the transcription factors T-bet and Eomes and co-expressed PD-1, CD160 and 2B4. HIV-specific TIGIT hi cells were negatively correlated with polyfunctionality and displayed a diminished expression of CD226. Furthermore, expression of PVR was increased on CD4 + T cells, especially T follicular helper (Tfh) cells, in HIV-infected lymph nodes. These results depict a skewing of the TIGIT/CD226 axis from CD226 co-stimulation towards TIGIT-mediated inhibition of CD8 + T cells, despite early ART. These findings highlight the importance of the TIGIT/CD226/PVR axis as an immune checkpoint barrier that could hinder future "cure" strategies requiring potent HIV-specific CD8 + T cells.