Soluble immune checkpoints as correlates for HIV persistence and T cell function in people with HIV on antiretroviral therapy.

Introduction: In people with HIV (PWH) both off and on antiretroviral therapy (ART), the expression of immune checkpoint (IC) proteins is elevated on the surface of total and HIV-specific T-cells, indicating T-cell exhaustion. Soluble IC proteins and their ligands can also be detected in plasma, but have not been systematically examined in PWH. Since T-cell exhaustion is associated with HIV persistence on ART, we aimed to determine if soluble IC proteins and their ligands also correlated with the size of the HIV reservoir and HIV-specific T-cell function. Methods: We used multiplex bead-based immunoassay to quantify soluble programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), T cell immunoglobulin domain and mucin domain 3 (TIM-3), PD-1 Ligand 1 (PD-L1) and PD-1 Ligand 2 (PD-L2) in plasma from PWH off ART (n=20), on suppressive ART (n=75) and uninfected controls (n=20). We also quantified expression of membrane-bound IC and frequencies of functional T-cells to Gag and Nef peptide stimulation on CD4+ and CD8+ T-cells using flow cytometry. The HIV reservoir was quantified in circulating CD4+ T-cells using qPCR for total and integrated HIV DNA, cell-associated unspliced HIV RNA and 2LTR circles. Results: Soluble (s) PD-L2 level was higher in PWH off and on ART compared to uninfected controls. Higher levels of sPD-L2 correlated with lower levels of HIV total DNA and higher frequencies of gag-specific CD8+ T-cells expressing CD107a, IFNγ or TNFα. In contrast, the concentration of sLAG-3 was similar in uninfected individuals and PWH on ART, but was significantly elevated in PWH off ART. Higher levels of sLAG-3 correlated with higher levels of HIV total and integrated DNA, and lower frequency of gag-specific CD4+ T cells expressing CD107a. Similar to sLAG-3, levels of sPD-1 were elevated in PWH off ART and normalized in PWH on ART. sPD-1 was positively correlated with the frequency of gag-specific CD4+ T cells expressing TNF-a and the expression of membrane-bound PD-1 on total CD8+ T-cells in PWH on ART. Discussion: Plasma soluble IC proteins and their ligands correlate with markers of the HIV reservoir and HIV-specific T-cell function and should be investigated further in in large population-based studies of the HIV reservoir or cure interventions in PWH on ART.

A Peptide-Based PD1 Antagonist Enhances T-Cell Priming and Efficacy of a Prophylactic Malaria Vaccine and Promotes Survival in a Lethal Malaria Model.

The blockade of programmed cell death-1 (PD1) and its ligand PDL1 has been proven to be a successful immunotherapy against several cancers. Similar to cancer, PD1 contributes to the establishment of several chronic infectious diseases, including malaria. While monoclonal antibodies (mAbs) targeting checkpoint receptors are revolutionary in cancer treatment, the immune-related adverse events (irAEs) may prevent their utilization in prophylactic and therapeutic treatments of infectious diseases. The irAEs are, in part, due to the prolonged half-life of mAbs resulting in prolonged activation of the immune system. As an alternative modality to mAbs, peptides represent a viable option because they possess a shorter pharmacokinetic half-life and offer more formulation and delivery options. Here, we report on a 22-amino acid immunomodulatory peptide, LD01, derived from a Bacillus bacteria. When combined prophylactically with an adenovirus-based or irradiated sporozoite-based malaria vaccine, LD01 significantly enhanced antigen-specific CD8+ T cell expansion. Therapeutically, LD01 treatment of mice infected with a lethal malaria strain resulted in survival that was associated with lower numbers of FOXP3+Tbet+CD4+ regulatory T cells. Taken together, our results demonstrate that LD01 is a potent immunomodulator that acts upon the adaptive immune system to stimulate T cell responses both prophylactically and therapeutically.

Progression of Disease Within 24 Months in Follicular Lymphoma Is Associated With Reduced Intratumoral Immune Infiltration.

PURPOSE: Understanding the immunobiology of the 15% to 30% of patients with follicular lymphoma (FL) who experience progression of disease within 24 months (POD24) remains a priority. Solid tumors with low levels of intratumoral immune infiltration have inferior outcomes. It is unknown whether a similar relationship exists between POD24 in FL. PATIENTS AND METHODS: Digital gene expression using a custom code set-five immune effector, six immune checkpoint, one macrophage molecules-was applied to a discovery cohort of patients with early- and advanced-stage FL (n = 132). T-cell receptor repertoire analysis, flow cytometry, multispectral immunofluorescence, and next-generation sequencing were performed. The immune infiltration profile was validated in two independent cohorts of patients with advanced-stage FL requiring systemic treatment (n = 138, rituximab plus cyclophosphamide, vincristine, prednisone; n = 45, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone), with the latter selected to permit comparison of patients experiencing a POD24 event with those having no progression at 5 years or more. RESULTS: Immune molecules showed distinct clustering, characterized by either high or low expression regardless of categorization as an immune effector, immune checkpoint, or macrophage molecule. Low programmed death-ligand 2 (PD-L2) was the most sensitive/specific marker to segregate patients with adverse outcomes; therefore, PD-L2 expression was chosen to distinguish immune infiltrationHI (ie, high PD-L2) FL biopsies from immune infiltrationLO (ie, low PD-L2) tumors. Immune infiltrationHI tissues were highly infiltrated with macrophages and expanded populations of T-cell clones. Of note, the immune infiltrationLO subset of patients with FL was enriched for POD24 events (odds ratio [OR], 4.32; c-statistic, 0.81; P = .001), validated in the independent cohorts (rituximab plus cyclophosphamide, vincristine, prednisone: OR, 2.95; c-statistic, 0.75; P = .011; and rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone: OR, 7.09; c-statistic, 0.88; P = .011). Mutations were equally proportioned across tissues, which indicated that degree of immune infiltration is capturing aspects of FL biology distinct from its mutational profile. CONCLUSION: Assessment of immune-infiltration by PD-L2 expression is a promising tool with which to help identify patients who are at risk for POD24.

Immune checkpoint blockade in infectious diseases.

The upregulation of immune checkpoint molecules, such as programmed cell death protein 1 (PD1) and cytotoxic T lymphocyte antigen 4 (CTLA4), on immune cells occurs during acute infections, such as malaria, as well as during chronic persistent viral infections, including HIV and hepatitis B virus. These pathways are important for preventing immune-driven pathology but can also limit immune-mediated clearance of the infection. The recent success of immune checkpoint blockade in cancer therapy suggests that targeting these pathways would also be effective for preventing and treating a range of infectious diseases. Here, we review our current understanding of immune checkpoint pathways in the pathogenesis of infectious diseases and discuss the potential for therapeutically targeting these pathways in this setting.

ELISPOT Assay to Measure Peptide-specific IFN-γ Production.

Interferon-gamma (IFN-γ) is crucial for immunity against intracellular pathogens and for tumor control. It is produced predominantly by natural killer (NK) and natural killer T cells (NKT) as well as by antigen-specific Th1 CD4+ and CD8+ effector T cells. When investigating immune responses against pathogens and cancer cells, measuring antigen-specific cytokine-responses by cells of adaptive immunity offers an advantage over total non-specific cytokine responses. Significantly, the measurement of antigen-specific IFN-γ responses against pathogens or cancer cells, when compared to a treatment group, provides a quantitative measure of how well the treatment works. Measuring antigen-specific IFN-γ responses involves culture of the cells being considered (CD4+ or CD8+ T cells) with antigen presenting cells (APC) and a specific peptide from the target pathogen or cancer cell compared to control cultures without a peptide. After a suitable timeframe, the cytokine released is measured by an ELISPOT assay. The difference in the number of cells secreting IFN-γ, with and without peptide, is a measure of antigen-specific IFN-γ responses. This assay can be applied to other cytokines such as IL-10.

Programmed Death-1 Ligand 2-Mediated Regulation of the PD-L1 to PD-1 Axis Is Essential for Establishing CD4(+) T Cell Immunity.

Many pathogens, including Plasmodium spp., exploit the interaction of programmed death-1 (PD-1) with PD-1-ligand-1 (PD-L1) to "deactivate" T cell functions, but the role of PD-L2 remains unclear. We studied malarial infections to understand the contribution of PD-L2 to immunity. Here we have shown that higher PD-L2 expression on blood dendritic cells, from Plasmodium falciparum-infected individuals, correlated with lower parasitemia. Mechanistic studies in mice showed that PD-L2 was indispensable for establishing effective CD4(+) T cell immunity against malaria, because it not only inhibited PD-L1 to PD-1 activity but also increased CD3 and inducible co-stimulator (ICOS) expression on T cells. Importantly, administration of soluble multimeric PD-L2 to mice with lethal malaria was sufficient to dramatically improve immunity and survival. These studies show immuno-regulation by PD-L2, which has the potential to be translated into an effective treatment for malaria and other diseases where T cell immunity is ineffective or short-lived due to PD-1-mediated signaling.

Mice lacking Programmed cell death-1 show a role for CD8(+) T cells in long-term immunity against blood-stage malaria.

Even after years of experiencing malaria, caused by infection with Plasmodium species, individuals still have incomplete immunity and develop low-density parasitemia on re-infection. Previous studies using the P. chabaudi (Pch) mouse model to understand the reason for chronic malaria, found that mice with a deletion of programmed cell death-1 (PD-1KO) generate sterile immunity unlike wild type (WT) mice. Here we investigated if the mechanism underlying this defect during acute immunity also impacts on long-term immunity. We infected WT and PD-1KO mice with Pch-malaria and measured protection as well as immune responses against re-infections, 15 or 20 weeks after the original infection had cleared. WT mice showed approximately 1% parasitemia compared to sterile immunity in PD-1KO mice on re-infection. An examination of the mechanisms of immunity behind this long-term protection in PD-1KO mice showed a key role for parasite-specific CD8(+) T cells even when CD4(+) T cells and B cells responded to re-infection. These studies indicate that long-term CD8(+) T cell-meditated protection requires consideration for future malaria vaccine design, as part of a multi-cell type response.

Malaria drives T cells to exhaustion.

Malaria is a significant global burden but after >30 years of effort there is no vaccine on the market. While the complex life cycle of the parasite presents several challenges, many years of research have also identified several mechanisms of immune evasion by Plasmodium spp. Recent research on malaria, has investigated the programmed cell death-1 (PD-1) pathway which mediates exhaustion of T cells, characterized by poor effector functions and recall responses and in some cases loss of the cells by apoptosis. Such studies have shown exhaustion of CD4(+) T cells and an unappreciated role for CD8(+) T cells in promoting sterile immunity against blood stage malaria. This is because PD-1 mediates up to a 95% reduction in numbers and functional capacity of parasite-specific CD8(+) T cells, thus masking their role in protection. The role of T cell exhaustion during malaria provides an explanation for the absence of sterile immunity following the clearance of acute disease which will be relevant to future malaria-vaccine design and suggests the need for novel therapeutic solutions. This review will thus examine the role of PD-1-mediated T cell exhaustion in preventing lasting immunity against malaria.

PD-1 dependent exhaustion of CD8+ T cells drives chronic malaria.

Malaria is a highly prevalent disease caused by infection by Plasmodium spp., which infect hepatocytes and erythrocytes. Blood-stage infections cause devastating symptoms and can persist for years. Antibodies and CD4(+) T cells are thought to protect against blood-stage infections. However, there has been considerable difficulty in developing an efficacious malaria vaccine, highlighting our incomplete understanding of immunity against this disease. Here, we used an experimental rodent malaria model to show that PD-1 mediates up to a 95% reduction in numbers and functional capacity of parasite-specific CD8(+) T cells. Furthermore, in contrast to widely held views, parasite-specific CD8(+) T cells are required to control both acute and chronic blood-stage disease even when parasite-specific antibodies and CD4(+) T cells are present. Our findings provide a molecular explanation for chronic malaria that will be relevant to future malaria-vaccine design and may need consideration when vaccine development for other infections is problematic.

Long-term antibody memory induced by synthetic peptide vaccination is protective against Streptococcus pyogenes infection and is independent of memory T cell help.

Streptococcus pyogenes (group A Streptococcus [GAS]) is a leading human pathogen associated with a diverse array of mucosal and systemic infections. Vaccination with J8, a conserved region synthetic peptide derived from the M-protein of GAS and containing only 12 aa from GAS, when conjugated to diphtheria toxoid, has been shown to protect mice against a lethal GAS challenge. Protection has been previously shown to be Ab-mediated. J8 does not contain a dominant GAS-specific T cell epitope. The current study examined long-term Ab memory and dissected the role of B and T cells. Our results demonstrated that vaccination generates specific memory B cells (MBC) and long-lasting Ab responses. The MBC response can be activated following boost with Ag or limiting numbers of whole bacteria. We further show that these memory responses protect against systemic infection with GAS. T cell help is required for activation of MBC but can be provided by naive T cells responding directly to GAS at the time of infection. Thus, individuals whose T cells do not recognize the short synthetic peptide in the vaccine will be able to generate a protective and rapid memory Ab response at the time of infection. These studies significantly strengthen previous findings, which showed that protection by the J8-diphtheria toxoid vaccine is Ab-mediated and suggest that in vaccine design for other organisms the source of T cell help for Ab responses need not be limited to sequences from the organism itself.

Malaria infection alters the expression of B-cell activating factor resulting in diminished memory antibody responses and survival.

Malaria is a major cause of morbidity worldwide with reports of over 200-500 million infected individuals and nearly 1 million deaths each year. Antibodies have been shown to play a critical role in controlling the blood stage of this disease; however, in malaria-endemic areas antibody immunity is slow to develop despite years of exposure to Plasmodium spp. the causative parasite. Using rodent Plasmodium yoelii YM, we provide evidence that malarial infections result in a decrease in the proportion of DCs that express the B-cell survival factor, BAFF, resulting in a decreased ability of these DCs to support memory B-cell differentiation into antibody secreting cells (ASCs) and/or the survival of ASCs. Further, compared with infected WT mice, ASC numbers were significantly increased in malaria-infected transgenic mice that either overexpressed BAFF or mice with BAFF-independent B-cell survival (B-cell-restricted TRAF3 deletion). Remarkably, BAFF-overexpressing mice were protected from lethal malaria infections, indicating the significance of the role BAFF plays in determining the outcome of malaria infections. These findings describe a previously unappreciated mechanism by which Plasmodium spp. can depress the generation of protective antibody responses.

The mammalian PYHIN gene family: phylogeny, evolution and expression.

BACKGROUND: Proteins of the mammalian PYHIN (IFI200/HIN-200) family are involved in defence against infection through recognition of foreign DNA. The family member absent in melanoma 2 (AIM2) binds cytosolic DNA via its HIN domain and initiates inflammasome formation via its pyrin domain. AIM2 lies within a cluster of related genes, many of which are uncharacterised in mouse. To better understand the evolution, orthology and function of these genes, we have documented the range of PYHIN genes present in representative mammalian species, and undertaken phylogenetic and expression analyses. RESULTS: No PYHIN genes are evident in non-mammals or monotremes, with a single member found in each of three marsupial genomes. Placental mammals show variable family expansions, from one gene in cow to four in human and 14 in mouse. A single HIN domain appears to have evolved in the common ancestor of marsupials and placental mammals, and duplicated to give rise to three distinct forms (HIN-A, -B and -C) in the placental mammal ancestor. Phylogenetic analyses showed that AIM2 HIN-C and pyrin domains clearly diverge from the rest of the family, and it is the only PYHIN protein with orthology across many species. Interestingly, although AIM2 is important in defence against some bacteria and viruses in mice, AIM2 is a pseudogene in cow, sheep, llama, dolphin, dog and elephant. The other 13 mouse genes have arisen by duplication and rearrangement within the lineage, which has allowed some diversification in expression patterns. CONCLUSIONS: The role of AIM2 in forming the inflammasome is relatively well understood, but molecular interactions of other PYHIN proteins involved in defence against foreign DNA remain to be defined. The non-AIM2 PYHIN protein sequences are very distinct from AIM2, suggesting they vary in effector mechanism in response to foreign DNA, and may bind different DNA structures. The PYHIN family has highly varied gene composition between mammalian species due to lineage-specific duplication and loss, which probably indicates different adaptations for fighting infectious disease. Non-genomic DNA can indicate infection, or a mutagenic threat. We hypothesise that defence of the genome against endogenous retroelements has been an additional evolutionary driver for PYHIN proteins.

Dendritic cells: the Trojan horse of malaria?

Malaria, caused by Plasmodium spp., is responsible for over 200 million infections worldwide and 650,000 deaths annually. Until recently, it was thought that blood-stage parasites survived and replicated in hepatocytes and red blood cells exclusively. We recently showed that blood-stage parasites could infect, survive and replicate within plasmacytoid dendritic cells of the spleen and that these cells could release infective parasites. Here we discuss the implications of this novel niche in the spleen.

Are plasmacytoid dendritic cells the misguided sentinels of malarial immunity?

Dendritic cells (DCs), the sentinels of immunity, reside in almost every organ of the body. These cells are responsible for initiating immune responses against infectious agents. DCs are divided into different subsets based on their biological functions, with plasmacytoid DCs (pDCs) and conventional DCs (cDCs) being two major populations. The ability of DCs to protect against malaria infection was recently questioned when pDCs were reported to be a reservoir for rodent Plasmodium spp. in the spleen. This opinion article explores how the occupation of pDCs by the parasite may corrupt immunity against malaria.

Rodent blood-stage Plasmodium survive in dendritic cells that infect naive mice.

Plasmodium spp. parasites cause malaria in 300 to 500 million individuals each year. Disease occurs during the blood-stage of the parasite's life cycle, where the parasite is thought to replicate exclusively within erythrocytes. Infected individuals can also suffer relapses after several years, from Plasmodium vivax and Plasmodium ovale surviving in hepatocytes. Plasmodium falciparum and Plasmodium malariae can also persist after the original bout of infection has apparently cleared in the blood, suggesting that host cells other than erythrocytes (but not hepatocytes) may harbor these blood-stage parasites, thereby assisting their escape from host immunity. Using blood stage transgenic Plasmodium berghei-expressing GFP (PbGFP) to track parasites in host cells, we found that the parasite had a tropism for CD317(+) dendritic cells. Other studies using confocal microscopy, in vitro cultures, and cell transfer studies showed that blood-stage parasites could infect, survive, and replicate within CD317(+) dendritic cells, and that small numbers of these cells released parasites infectious for erythrocytes in vivo. These data have identified a unique survival strategy for blood-stage Plasmodium, which has significant implications for understanding the escape of Plasmodium spp. from immune-surveillance and for vaccine development.

A novel synthetic adjuvant enhances dendritic cell function.

The lipid core peptide (LCP) is a novel, synthetic, self-adjuvanted vaccine delivery system that neatly incorporates the adjuvant, carrier and antigenic peptides of a vaccine into a single molecular entity. This system has been previously shown to efficiently deliver vaccines and induce immunity. Because adjuvants target sentinels of the immune response, such as dendritic cells (DCs), that are widely distributed throughout the body to initiate specific immune responses, we investigated the effects of the adjuvant on DCs. Here we show that LCP targets vaccines to DCs and induces their activation.

What have we learnt from mouse models for the study of malaria?

Malaria is a serious cause of morbidity and mortality and yet a vaccine is not available. Studies have used animal models to understand the pathogenesis of disease and a large amount of data on parasite biology, immune regulation and disease processes have been gained from these studies. Moreover, these models have been used for pre-clinical testing of various drugs and vaccines. Here, we discuss the features of various mouse models used to study the immunobiology of malaria and test pre-clinical vaccines and conclude that animal models have a role in the study of malaria but the experimental conditions used for testing must reflect the environment of infected individuals.

Soluble CD38 significantly prolongs the lifespan of memory B-cell responses.

The development and maintenance of memory B cells (MBC) is dependent on germinal centres (GC) with follicular dendritic cell (FDC) networks. We have previously shown that FDC networks within GC of the spleen express a novel ligand for CD38 and that the administration of soluble CD38 induces an expansion of these cellular structures. We therefore used adoptive transfer studies to investigate whether the expansion of FDC networks with soluble CD38 affected the generation and maintenance of antigen-specific MBC. These studies found that the administration of soluble CD38 significantly extended the period after which MBC could be activated and that the frequencies of these cells also were increased. In conclusion, soluble CD38 appears to significantly extend the lifespan of antibody memory by increasing the numbers of MBC.

What really happens to dendritic cells during malaria?

As dendritic cells (DCs) initiate all adaptive and some innate immune responses, it is not surprising that DC function during malaria is the subject of intensive investigations. However, the results of these investigations have so far been controversial. Here, we discuss various aspects of these studies, including the influence of the species and strain of Plasmodium on DC function, the effects of Plasmodium infection on the activation of CD8(+) T cells by DCs, the effects of haemozoin and the effects of Plasmodium infections on DC Toll-like-receptor signalling.