Depleting myeloid-biased haematopoietic stem cells rejuvenates aged immunity.

Ageing of the immune system is characterized by decreased lymphopoiesis and adaptive immunity, and increased inflammation and myeloid pathologies1,2. Age-related changes in populations of self-renewing haematopoietic stem cells (HSCs) are thought to underlie these phenomena3. During youth, HSCs with balanced output of lymphoid and myeloid cells (bal-HSCs) predominate over HSCs with myeloid-biased output (my-HSCs), thereby promoting the lymphopoiesis required for initiating adaptive immune responses, while limiting the production of myeloid cells, which can be pro-inflammatory4. Ageing is associated with increased proportions of my-HSCs, resulting in decreased lymphopoiesis and increased myelopoiesis3,5,6. Transfer of bal-HSCs results in abundant lymphoid and myeloid cells, a stable phenotype that is retained after secondary transfer; my-HSCs also retain their patterns of production after secondary transfer5. The origin and potential interconversion of these two subsets is still unclear. If they are separate subsets postnatally, it might be possible to reverse the ageing phenotype by eliminating my-HSCs in aged mice. Here we demonstrate that antibody-mediated depletion of my-HSCs in aged mice restores characteristic features of a more youthful immune system, including increasing common lymphocyte progenitors, naive T cells and B cells, while decreasing age-related markers of immune decline. Depletion of my-HSCs in aged mice improves primary and secondary adaptive immune responses to viral infection. These findings may have relevance to the understanding and intervention of diseases exacerbated or caused by dominance of the haematopoietic system by my-HSCs.

A bivalent Adenovirus-Vectored Vaccine induces a robust humoral response, but does not protect cynomolgus macaques against a lethal challenge with Sudan virus.

The most recent Sudan virus (SUDV) outbreak in Uganda was first detected in September 2022 and resulted in 164 laboratory-confirmed cases and 77 deaths. There are no approved vaccines against SUDV. Here, we investigated the protective efficacy of ChAdOx1-biEBOV in cynomolgus macaques using a prime or a prime-boost regimen. ChAdOx1-biEBOV is a replication-deficient simian adenovirus vector encoding SUDV and Ebola virus (EBOV) glycoproteins (GPs). Intramuscular vaccination induced SUDV and EBOV GP-specific IgG responses and neutralizing antibodies. Upon challenge with SUDV, vaccinated animals showed signs of disease like those observed in control animals, and no difference in survival outcomes were measured among all three groups. Viral load in blood samples and in tissue samples obtained after necropsy were not significantly different between groups. Overall, this study highlights the importance of evaluating vaccines in multiple animal models and demonstrates the importance of understanding protective efficacy in both animal models and human hosts.

CD47 Blockade Leads to Chemokine-Dependent Monocyte Infiltration and Loss of B Cells from the Splenic Marginal Zone.

CD47 is an important innate immune checkpoint through its interaction with its inhibitory receptor on macrophages, signal-regulatory protein α (SIRPα). Therapeutic blockade of CD47-SIRPα interactions is a promising immuno-oncology treatment that promotes clearance of cancer cells. However, CD47-SIRPα interactions also maintain homeostatic lymphocyte levels. In this study, we report that the mouse splenic marginal zone B cell population is dependent on intact CD47-SIRPα interactions and blockade of CD47 leads to the loss of these cells. This depletion is accompanied by elevated levels of monocyte-recruiting chemokines CCL2 and CCL7 and infiltration of CCR2+Ly6Chi monocytes into the mouse spleen. In the absence of CCR2 signaling, there is no infiltration and reduced marginal zone B cell depletion. These data suggest that CD47 blockade leads to clearance of splenic marginal zone B cells.

Upregulation of CD47 Is a Host Checkpoint Response to Pathogen Recognition.

It is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. We now show that the very early innate response also has an immunosuppressive component. Infected cells upregulate the CD47 "don't eat me" signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (APC) functions. A CD47 mimic that acts as an essential virulence factor is encoded by all poxviruses, but CD47 expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that encode no mimic. CD47 upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (PRRs). Furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis C patients, upregulated CD47 on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. Indeed, results from antibody-mediated CD47 blockade experiments as well as CD47 knockout mice revealed an immunosuppressive role for CD47 during infections with lymphocytic choriomeningitis virus and Mycobacterium tuberculosis Since CD47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify CD47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents.IMPORTANCE Immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (PRRs). PRR binding sets off a cascade of events that activates immune responses. We now show that, in addition to activating immune responses, PRR signaling also initiates an immunosuppressive response, probably to limit inflammation. The importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the CD47 molecule, can lead to enhanced immune responses to any pathogen that triggers PRR signaling. Since most or all pathogens trigger PRRs, CD47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. Such immunotherapy could be done without a requirement for knowing the HLA type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile.

Immunotherapeutic Blockade of CD47 Inhibitory Signaling Enhances Innate and Adaptive Immune Responses to Viral Infection.

Paradoxically, early host responses to infection include the upregulation of the antiphagocytic molecule, CD47. This suggests that CD47 blockade could enhance antigen presentation and subsequent immune responses. Indeed, mice treated with anti-CD47 monoclonal antibody following lymphocytic choriomeningitis virus infections show increased activation of both macrophages and dendritic cells (DCs), enhancement of the kinetics and potency of CD8+ T cell responses, and significantly improved virus control. Treatment efficacy is critically dependent on both APCs and CD8+ T cells. In preliminary results from one of two cohorts of humanized mice infected with HIV-1 for 6 weeks, CD47 blockade reduces plasma p24 levels and restores CD4+ T cell counts. The results indicate that CD47 blockade not only enhances the function of innate immune cells but also links to adaptive immune responses through improved APC function. As such, immunotherapy by CD47 blockade may have broad applicability to treat a wide range of infectious diseases.

Robust growth of avirulent phase II Coxiella burnetii in bone marrow-derived murine macrophages.

Published data show that murine bone marrow-derived macrophages (BMDM) restrict growth of avirulent phase II, but not virulent phase I, Coxiella burnetii. Growth restriction of phase II bacteria is thought to result from potentiated recognition of pathogen-associated molecular patterns, which leads to production of inhibitory effector molecules. Past studies have used conditioned medium from L-929 murine fibroblasts as a source of macrophage-colony stimulating factor (M-CSF) to promote differentiation of bone marrow-derived myeloid precursors into macrophages. However, uncharacterized components of conditioned medium, such as variable amounts of type I interferons, can affect macrophage activation status and their permissiveness for infection. In the current study, we show that the C. burnetii Nine Mile phase II (NMII) strain grows robustly in primary macrophages from C57BL/6J mice when bone marrow cells are differentiated with recombinant murine M-CSF (rmM-CSF). Bacteria were readily internalized by BMDM, and replicated within degradative, LAMP1-positive vacuoles to achieve roughly 3 logs of growth over 6 days. Uninfected BMDM did not appreciably express CD38 or Egr2, markers of classically (M1) and alternatively (M2) activated macrophages, respectively, nor did infection change the lack of polarization. In accordance with an M0 phenotype, infected BMDM produced moderate amounts of TNF and nitric oxide. Similar NMII growth results were obtained using C57BL/6J myeloid progenitors immortalized with an estrogen-regulated Hoxb8 (ER-Hoxb8) oncogene. To demonstrate the utility of the ER-Hoxb8 system, myeloid progenitors from natural resistance-associated macrophage protein 1 (Nramp1) C57BL/6J knock-in mice were transduced with ER-Hoxb8, and macrophages were derived from immortalized progenitors using rmM-CSF and infected with NMII. No difference in growth was observed when compared to macrophages from wild type mice, indicating depletion of metal ions by the Nramp1 transporter does not negatively impact NMII growth. Results with NMII were recapitulated in primary macrophages where C57BL/6J Nramp1+ BMDM efficiently killed Salmonella enterica serovar Typhimurium. M-CSF differentiated murine macrophages from bone marrow and conditional ER-Hoxb8 myeloid progenitors will be useful ex vivo models for studying Coxiella-macrophage interactions.

Activated CD8+ T cells induce expansion of Vß5+ regulatory T cells via TNFR2 signaling.

Vß5(+) regulatory T cells (Tregs), which are specific for a mouse endogenous retroviral superantigen, become activated and proliferate in response to Friend virus (FV) infection. We previously reported that FV-induced expansion of this Treg subset was dependent on CD8(+) T cells and TNF-α, but independent of IL-2. We now show that the inflammatory milieu associated with FV infection is not necessary for induction of Vß5(+) Treg expansion. Rather, it is the presence of activated CD8(+) T cells that is critical for their expansion. The data indicate that the mechanism involves signaling between the membrane-bound form of TNF-α on activated CD8(+) T cells and TNFR2 on Tregs. CD8(+) T cells expressing membrane-bound TNF-α but no soluble TNF-α remained competent to induce strong Vß5(+) Treg expansion in vivo. In addition, Vß5(+) Tregs expressing only TNFR2 but no TNFR1 were still responsive to expansion. Finally, treatment of naive mice with soluble TNF-α did not induce Vß5(+) Treg expansion, but treatment with a TNFR2-specific agonist did. These results reveal a new mechanism of intercellular communication between activated CD8(+) T cell effectors and Tregs that results in the activation and expansion of a Treg subset that subsequently suppresses CD8(+) T cell functions.

IL-2-independent and TNF-α-dependent expansion of Vß5+ natural regulatory T cells during retrovirus infection.

Friend virus infection of mice induces the expansion and activation of regulatory T cells (Tregs) that dampen acute immune responses and promote the establishment and maintenance of chronic infection. Adoptive transfer experiments and the expression of neuropilin-1 indicate that these cells are predominantly natural Tregs rather than virus-specific conventional CD4(+) T cells that converted into induced Tregs. Analysis of Treg TCR Vß chain usage revealed a broadly distributed polyclonal response with a high proportionate expansion of the Vß5(+) Treg subset, which is known to be responsive to endogenous retrovirus-encoded superantigens. In contrast to the major population of Tregs, the Vß5(+) subset expressed markers of terminally differentiated effector cells, and their expansion was associated with the level of the antiviral CD8(+) T cell response rather than the level of Friend virus infection. Surprisingly, the expansion and accumulation of the Vß5(+) Tregs was IL-2 independent but dependent on TNF-α. These experiments reveal a subset-specific Treg induction by a new pathway.

Negative impact of IFN-γ on early host immune responses to retroviral infection.

The immune system is tasked with defending against a myriad of microbial infections, and its response to a given infectious microbe may be strongly influenced by coinfection with another microbe. It was shown that infection of mice with lactate dehydrogenase-elevating virus (LDV) impairs early adaptive immune responses to Friend virus (FV) coinfection. To investigate the mechanism of this impairment, we examined LDV-induced innate immune responses and found LDV-specific induction of IFN-α and IFN-γ. LDV-induced IFN-α had little effect on FV infection or immune responses, but unexpectedly, LDV-induced IFN-γ production dampened Th1 adaptive immune responses and enhanced FV infection. Two distinct effects were identified. First, LDV-induced IFN-γ signaling indirectly modulated FV-specific CD8+ T cell responses. Second, intrinsic IFN-γ signaling in B cells promoted polyclonal B cell activation and enhanced early FV infection, despite promotion of germinal center formation and neutralizing Ab production. Results from this model reveal that IFN-γ production can have detrimental effects on early adaptive immune responses and virus control.

Co-infection with the friend retrovirus and mouse scrapie does not alter prion disease pathogenesis in susceptible mice.

Prion diseases are fatal, transmissible neurodegenerative diseases of the central nervous system. An abnormally protease-resistant and insoluble form (PrP(Sc)) of the normally soluble protease-sensitive host prion protein (PrP(C)) is the major component of the infectious prion. During the course of prion disease, PrP(Sc) accumulates primarily in the lymphoreticular and central nervous systems. Recent studies have shown that co-infection of prion-infected fibroblast cells with the Moloney murine leukemia virus (Mo-MuLV) strongly enhanced the release and spread of scrapie infectivity in cell culture, suggesting that retroviral coinfection might significantly influence prion spread and disease incubation times in vivo. We now show that another retrovirus, the murine leukemia virus Friend (F-MuLV), also enhanced the release and spread of scrapie infectivity in cell culture. However, peripheral co-infection of mice with both Friend virus and the mouse scrapie strain 22L did not alter scrapie disease incubation times, the levels of PrP(Sc) in the brain or spleen, or the distribution of pathological lesions in the brain. Thus, retroviral co-infection does not necessarily alter prion disease pathogenesis in vivo, most likely because of different cell-specific sites of replication for scrapie and F-MuLV.

Virus-specific CD8+ T cells upregulate programmed death-1 expression during acute friend retrovirus infection but are highly cytotoxic and control virus replication.

It was recently reported that inhibitory molecules such as programmed death-1 (PD-1) were upregulated on CD8(+) T cells during acute Friend retrovirus infection and that the cells were prematurely exhausted and dysfunctional in vitro. The current study confirms that most activated CD8(+) T cells upregulated expression of PD-1 during acute infection and revealed a dichotomy of function between PD-1(hi) and PD-1(lo) subsets. More PD-1(lo) cells produced antiviral cytokines such as IFN-γ and TNF-α, whereas more PD-1(hi) cells displayed characteristics of cytotoxic effectors such as production of granzymes and surface expression of CD107a. Importantly, CD8(+) T cells mediated rapid in vivo cytotoxicity and were critical for control of acute Friend virus replication. Thus, direct ex vivo analyses and in vivo experiments revealed high CD8(+) T cell functionality and indicate that PD-1 expression during acute infection is not a marker of T cell exhaustion.

In vitro and in vivo analyses of regulatory T cell suppression of CD8+ T cells.

The study of regulatory T cells (Treg) requires methods for both in vivo and in vitro analyses, both of which have different limitations, but which complement each other to give a more complete picture of physiological function than either method alone. Our analyses have focused on Treg-mediated suppression of CD8(+) T cells, and in particular Tregs induced by viral infection. One of the unique characteristics of virus-induced Tregs is that they can suppress CD8(+) T cell function in vitro without the requirement for additional stimulation. This ability correlates with their suppressive capacity and activated status in vivo. Interestingly, while virus-induced Tregs suppress CD8(+) T cell function in vitro and in vivo, they do not suppress proliferation unless they are further activated in vitro.

Transient depletion of regulatory T cells in transgenic mice reactivates virus-specific CD8+ T cells and reduces chronic retroviral set points.

Although chronic infections with viruses such as HIV and hepatitis C virus have been associated with regulatory T cell (Treg)-mediated suppression of virus-specific CD8(+) T-cell activity, no causal relationship between Tregs and chronic viral set points has been established. Using transgenic mice in which Tregs can be selectively ablated, we now show that transient depletion of Tregs during a chronic retroviral infection allows exhausted CD8(+) T cells to regain antiviral functions, including secretion of cytokines, production of cytotoxic molecules, and virus-specific cytolytic activity. Furthermore, short-term Treg ablation resulted in long-term reductions in chronic virus loads. These results demonstrate that Treg-mediated immunosuppression can be a significant factor in the maintenance of chronic viral infections and that Treg-targeted immunotherapy could be a valuable component in therapeutic strategies to treat chronic infectious diseases.

Tissue-specific abundance of regulatory T cells correlates with CD8+ T cell dysfunction and chronic retrovirus loads.

Infection of mice with Friend virus induces the activation of CD4(+) regulatory T cells (Tregs) that suppress virus-specific CD8(+) T cells. This suppression leads to incomplete virus clearance and the establishment of virus persistence. We now show that Treg-mediated suppression of CD8(+) T cells is tissue specific, occurring in the spleen but not the liver. Regardless of infection status, there was a 5-fold lower proportion of Tregs in the liver than in the spleen, much lower absolute cell numbers, and the relatively few Tregs present expressed less CD25. Results indicated that reduced expression of CD25 on liver Tregs was due to microenvironmental factors including low levels of IL-2 production by CD4(+) Th cells in that tissue. Low CD25 expression on liver Tregs did not impair their ability to suppress CD8(+) T cells in vitro. Correlating with the decreased proportion of Tregs in the liver was a significantly increased proportion of virus-specific CD8(+) T cells compared with the spleen. The virus-specific CD8(+) T cells from the liver did not appear suppressed given that they produced both IFN-gamma and granzyme B, and they also showed evidence of recent cytolytic activity (CD107a(+)). The functional phenotype of the virus-specific CD8(+) T cells correlated with a 10-fold reduction of chronic Friend virus levels in the liver compared with the spleen. Thus, suppression of CD8(+) T cells by virus-induced Tregs occurs in a tissue-specific manner and correlates with profound effects on localized levels of chronic infection.

Retroviral immunology: lessons from a mouse model.

Friend virus (FV) is a murine retrovirus that causes acute disease leading to lethal erythroleukemia in most strains of mice. Strains of mice that mount strong and rapid immune responses can recover from acute infection, but nevertheless develop life-long chronic infections. The study of this infection has revealed the types of immune responses required for both recovery from the acute phase and the control of the chronic phase of infection. This knowledge has led to vaccines and therapeutics to prevent and treat infections and associated disease states. The FV model has provided insights into immunological mechanisms found to be relevant to human infections with retroviruses such as HIV-1 and HTLV-1.

Suppression of acute anti-friend virus CD8+ T-cell responses by coinfection with lactate dehydrogenase-elevating virus.

Friend virus (FV) and lactate dehydrogenase-elevating virus (LDV) are endemic mouse viruses that can cause long-term chronic infections in mice. We found that numerous mouse-passaged FV isolates also contained LDV and that coinfection with LDV delayed FV-specific CD8(+) T-cell responses during acute infection. While LDV did not alter the type of acute pathology induced by FV, which was severe splenomegaly caused by erythroproliferation, the immunosuppression mediated by LDV increased both the severity and the duration of FV infection. Compared to mice infected with FV alone, those coinfected with both FV and LDV had delayed CD8(+) T-cell responses, as measured by FV-specific tetramers. This delayed response accounted for the prolonged and exacerbated acute phase of FV infection. Suppression of FV-specific CD8(+) T-cell responses occurred not only in mice infected concomitantly with LDV but also in mice chronically infected with LDV 8 weeks prior to infection with FV. The LDV-induced suppression was not mediated by T regulatory cells, and no inhibition of the CD4(+) T-cell or antibody responses was observed. Considering that most human adults are carriers of chronically infectious viruses at the time of new virus insults and that coinfections with viruses such as human immunodeficiency virus and hepatitis C virus are currently epidemic, it is of great interest to determine how infection with one virus may impact host responses to a second infection. Coinfection of mice with LDV and FV provides a well-defined, natural host model for such studies.

Combined CD137 (4-1BB) and adjuvant therapy generates a developing pool of peptide-specific CD8 memory T cells.

In practice, vaccines should induce lasting and efficacious T cell immunity without promoting deleterious pathological consequences. To accomplish this goal we immunized mice with ovalbumin peptide, polyinosinic-polycytidylic and anti-CD137. Vaccinated mice retained a massive functional CD8 T cell memory pool in lymphoid and non-lymphoid tissues for >1 year. The memory T cells clonally expanded, produced substantial amounts of IFNgamma, and responded vigorously to vesicular stomatitis virus infection. To understand how the vaccine might function, we showed that the antigen-specific T cells must bear CD137 in order for optimal priming to occur. Thus, anti-CD137 agonist mAb directly stimulated peptide-specific CD8 T cells and conditioned them to survive. In contrast, CD137-deficient CD8 T cells did not survive despite CD137 expression by antigen presenting cells. Taken together, the data indicate that CD137 and adjuvant combined therapy is an efficacious vaccine strategy for immunization with non-replicating inert antigen.

Interfacing T-cell effector and regulatory function through CD137 (4-1BB) co-stimulation.

Historically, co-stimulation has been regarded as a prerequisite for T-cell activation. A lack of co-stimulation results in peripheral T-cell tolerance, which manifests as unresponsiveness or death through apoptosis. Recent findings, however, suggest that eliciting co-stimulation can also induce tolerance. Reconciling these diametrically opposed results is certainly of academic importance but more importantly transcends basic immunology and impacts present and future clinical trials that are centered on modulating T-cell co-stimulation. This review will focus on CD137 (4-1BB) and propose a mechanism of action in which CD137-primed CD8 T cells express effector function and also inhibit CD4 T-cell activation. This model suggests that the same CD8 T cells possess antitumor effector function interfaced with regulatory capacity, which ultimately leads to concomitant inhibition of tumorigenesis and autoimmune disease.

4-1BB and OX40 dual costimulation synergistically stimulate primary specific CD8 T cells for robust effector function.

CD40, 4-1BB, and OX40 are costimulatory molecules belonging to the TNF/nerve growth factor superfamily of receptors. We examined whether simultaneous costimulation affected the responses of T cells using several different in vivo tracking models in mice. We show that enforced dual costimulation through 4-1BB and OX40, but not through CD40, induced profound specific CD8 T cell clonal expansion. In contrast, the response of specific CD4 T cells to dual costimulation was additive rather than synergistic. The synergistic response of the specific CD8 T cells persevered for several weeks, and the expanded effector cells resided throughout lymphoid and nonlymphoid tissue. Dual costimulation through 4-1BB and OX40 did not increase BrdU incorporation nor an increase in the number of rounds of T cell division in comparison to single costimulators, but rather enhanced accumulation in a cell-intrinsic manner. Mechanistically speaking, we show that CD8 T cell clonal expansion and effector function did not require T help, but accumulation in (non)lymphoid tissue was predominantly CD4 T cell dependent. To determine whether this approach would be useful in a physiological setting, we demonstrated that dual costimulation mediated rejection of an established murine sarcoma. Importantly, effector function directed toward established tumors was CD8 T cell dependent while being entirely CD4 T cell independent, and the timing of enforced dual costimulation was exquisitely regulated. Collectively, these data suggest that simultaneous dual costimulation through 4-1BB and OX40 induces a massive burst of CD8 T cell effector function sufficient to therapeutically treat established tumors even under immunocompromising conditions.

Effector CD8 T cells possess suppressor function after 4-1BB and Toll-like receptor triggering.

To better understand how innate and adaptive immune responses interact with each other, we combined 4-1BB T cell costimulation with specific adjuvants to determine whether these treatments would influence specific T cell expansion and function in vivo. In the presence of 4-1BB ligation and Toll-like receptor 3 (TLR)3 and/or TLR4 triggering, CD8 T cell clonal expansion and survival was augmented profoundly. Specific T cells primed in vivo with TLR ligands responded normally to in vitro recall stimulus, but, surprisingly, copriming with 4-1BB costimulation significantly impaired the recall response even though many more specific effector T cells were rescued in vivo. Here, we demonstrate that the rescued CD8 T cells suppressed CD4 T cell proliferation via a type beta transforming growth factor-dependent mechanism. Thus, 4-1BB and TLR ligands induce survival of specific effector CD8 T cells with suppressive recall potential, which may explain the dual role that 4-1BB activation plays in mediating tumor clearance and prevention of autoimmune disease.