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.

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.

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.

Systemic tumor necrosis factor generated during lethal Plasmodium infections impairs dendritic cell function.

Dendritic cells (DCs) initiate innate and adaptive immune responses including those against malaria. Although several studies have shown that DC function is normal during malaria, other studies have shown compromised function. To establish why these studies had different findings, we examined DCs from mice infected with two lethal species of parasite, Plasmodium berghei or P. vinckei, and compared them to DCs from nonlethal P. yoelii 17XNL or P. chabaudi infections. These studies found that DCs from only the lethal infections became uniformly mature 7 days after infection and were functionally impaired as they were unable to endocytose latex particles, secrete IL-12, or present OVA to transgenic OTII T cells. These changes coincided with a peak in levels of systemic TNF-alpha. Because TNF-alpha is known to mature DCs, we used TNF-KO mice to determine the role of this cytokine in the loss of DC function. In the TNF-KO mice, phenotype, Ag presentation, and IL-12 secretion by DCs were restored to normal following both lethal infections. This study shows that the systemic production of TNF-alpha contributes to poor DC function during lethal infections. These studies may explain, at least in part, immunosuppression that is associated with malaria.

Plasmodium yoelii can ablate vaccine-induced long-term protection in mice.

Malaria is a serious cause of morbidity and mortality for people living in endemic areas, but unlike many other infections, individuals exposed to the parasite do not rapidly become resistant to subsequent infections. High titers of Ab against the 19-kDa C-terminal fragment of the merozoite surface protein-1 can mediate complete protection in model systems; however, previous studies had not determined whether this vaccine generated long-term protection. In this study, we report that functional memory cells generated by merozoite surface protein-1, per se, do not offer any protection. This is because the parasite induces deletion of vaccine-specific memory B cells as well as long-lived plasma cells including those specific for bystander immune responses. Our study demonstrates a novel mechanism by which Plasmodium ablates immunological memory of vaccines, which would leave the host immuno-compromised.

Dendritic cells and follicular dendritic cells express a novel ligand for CD38 which influences their maturation and antibody responses.

CD38 is a cell surface molecule with ADP-ribosyl cyclase activity, which is predominantly expressed on lymphoid and myeloid cells. CD38 has a significant role in B-cell function as some anti-CD38 antibodies can deliver potent growth and differentiation signals, but the ligand that delivers this signal in mice is unknown. We used a chimeric protein of mouse CD38 and human immunogobulin G (IgG) (CD38-Ig) to identify a novel ligand for murine CD38 (CD38L) on networks of follicular dendritic cells (FDCs) as well as dendritic cells (DCs) in the spleen. Flow-cytometry found that all DC subsets expressed cytoplasmic CD38L but only fresh ex vivo CD11c+ CD11b- DCs had cell surface CD38L. Anti-CD38 antibody blocked the binding of CD38-Ig to CD38L, confirming the specificity of detection. CD38-Ig immuno-precipitated ligands of 66 and 130 kDa. Functional studies found that CD38-Ig along with anti-CD40 and anti-major histocompatibility complex (MHC) class II antibody provided maturation signals to DCs in vitro. When CD38-Ig was administered in vivo with antigen, IgG2a responses were significantly reduced, suggesting that B and T cells expressing CD38 may modulate the isotype of antibodies produced through interaction with CD38L on DCs. CD38-Ig also expanded FDC networks when administered in vivo. In conclusion, this study has identified a novel ligand for CD38 which has a role in functional interactions between lymphocytes and DCs or FDCs.