Simon Metenou

At Homeostasis Filarial Infections Have Expanded Adaptive T Regulatory but Not Classical Th2 Cells

Despite the well-documented immune suppression associated with human helminth infections, studies characterizing the immune response at the single-cell level are scanty. We used multiparameter flow cytometry to characterize the type of effector (Th1, Th2, and Th17) and regulatory (natural T regulatory cells [nTregs] and adaptive Treg cells [aTreg/type 1 regulatory cells (Tr1s)]) CD4+ and CD8+ T cells in filaria-infected (Fil+) and -uninfected (Fil−) individuals at homeostasis (in the absence of stimulation). Frequencies of CD4+ lymphocytes spontaneously producing IL-4, IL-10, and IL-17A were significantly higher in Fil+, as were those of IL-10+/IL-4+ double-producing CD4+ cells. Interestingly, frequencies of Th17 and aTreg/Tr1s but not classical Th1 or Th2 cells were significantly increased in Fil+ compared to Fil− individuals. Although the frequency of nTreg was increased in Fil+, IL-10 was overwhelmingly produced by CD4+CD25− cells. Moreover, the concentration of IL-10 produced spontaneously in vitro strongly correlated with the integrated geometric mean fluorescence intensity of IL-10–producing aTreg/Tr1s in Fil+. Together, these data show that at steady state, IL-10–producing aTreg/Tr1 as well as nTreg and effector Th17 CD4+ cells are expanded in vivo in human filarial infections. Moreover, we have established baseline ex vivo frequencies of effector and Tregs at homeostasis at a population level.

Filarial Infection Suppresses Malaria-Specific Multifunctional Th1 and Th17 Responses in Malaria and Filarial Coinfections

The mechanisms underlying the modulation of both the malaria-specific immune response and the course of clinical malaria in the context of concomitant helminth infection are poorly understood. We used multiparameter flow cytometry to characterize the quality and the magnitude of malaria-specific T cell responses in filaria-infected and -uninfected individuals with concomitant asymptomatic Plasmodium falciparum malaria in Mali. In comparison with filarial-uninfected subjects, filarial infection was associated with higher ex vivo frequencies of CD4+ cells producing IL-4, IL-10, and IL-17A (p = 0.01, p = 0.001, and p = 0.03, respectively). In response to malaria Ag stimulation, however, filarial infection was associated with lower frequencies of CD4+ T cells producing IFN-γ, TNF-α, and IL-17A (p < 0.001, p = 0.04, and p = 0.04, respectively) and with higher frequencies of CD4+IL10+T cells (p = 0.0005). Importantly, filarial infection was associated with markedly lower frequencies of malaria Ag-specific Th1 (p < 0.0001), Th17 (p = 0.012), and “TNF-α” (p = 0.0008) cells, and a complete absence of malaria-specific multifunctional Th1 cells. Filarial infection was also associated with a marked increase in the frequency of malaria-specific adaptive regulatory T/Tr1 cells (p = 0.024), and the addition of neutralizing anti–IL-10 Ab augmented the amount of Th1-associated cytokine produced per cell. Thus, among malaria-infected individuals, concomitant filarial infection diminishes dramatically the frequencies of malaria-specific Th1 and Th17 T cells, and alters the quality and magnitude of malaria-specific T cell responses.

Impact of filarial infections on coincident intracellular pathogens: Mycobacterium tuberculosis and Plasmodium falciparum

Purpose of reviewTo examine the consequences of the immune modulation seen in chronic filarial infection on responses to intracellular pathogens (and their antigens) that are often co-endemic with filarial infections, namely Plasmodium and Mycobacterium tuberculosis. Recent findingsMuch of the recent data on filaria/mycobacteria or filaria/Plasmodium co-infection has focused on the modulation of mycobacteria-specific or malaria-specific responses by chronic filarial infection. As such, filarial infections very clearly alter the magnitude and quality of the mycobacteria-specific or malaria-specific cytokine responses, responses that have been typically associated with control of these intracellular pathogens. SummaryAlthough phylogenetically distinct, mycobacteria and Plasmodium spp. often share the same geographical niche with filarial infections. The complex interplay between filarial parasites that are associated with immunomodulation and those microbial pathogens that require a proinflammatory or unmodulated response for their control is easily demonstrable ex vivo, but whether this interplay affects disease outcome in tuberculosis or malaria remains an open question.

Filariasis attenuates anemia and proinflammatory responses associated with clinical malaria: a matched prospective study in children and young adults.

Background Wuchereria bancrofti (Wb) and Mansonella perstans (Mp) are blood-borne filarial parasites that are endemic in many countries of Africa, including Mali. The geographic distribution of Wb and Mp overlaps considerably with that of malaria, and coinfection is common. Although chronic filarial infection has been shown to alter immune responses to malaria parasites, its effect on clinical and immunologic responses in acute malaria is unknown. Methodology/Principal Findings To address this question, 31 filaria-positive (FIL+) and 31 filaria-negative (FIL−) children and young adults, matched for age, gender and hemoglobin type, were followed prospectively through a malaria transmission season. Filarial infection was defined by the presence of Wb or Mp microfilariae on calibrated thick smears performed between 10 pm and 2 am and/or by the presence of circulating filarial antigen in serum. Clinical malaria was defined as axillary temperature ≥37.5°C or another symptom or sign compatible with malaria infection plus the presence of asexual malaria parasites on a thick blood smear. Although the incidence of clinical malaria, time to first episode, clinical signs and symptoms, and malaria parasitemia were comparable between the two groups, geometric mean hemoglobin levels were significantly decreased in FIL− subjects at the height of the transmission season compared to FIL+ subjects (11.4 g/dL vs. 12.5 g/dL, p<0.01). Plasma levels of IL-1ra, IP-10 and IL-8 were significantly decreased in FIL+ subjects at the time of presentation with clinical malaria (99, 2145 and 49 pg/ml, respectively as compared to 474, 5522 and 247 pg/ml in FIL− subjects). Conclusions/Significance These data suggest that pre-existent filarial infection attenuates immune responses associated with severe malaria and protects against anemia, but has little effect on susceptibility to or severity of acute malaria infection. The apparent protective effect of filarial infection against anemia is intriguing and warrants further study in a larger cohort.

Expanded Numbers of Circulating Myeloid Dendritic Cells in Patent Human Filarial Infection Reflect Lower CCR1 Expression

APC dysfunction has been postulated to mediate some of the parasite-specific T cell unresponsiveness seen in patent filarial infec-tion. We have shown that live microfilariae of Brugia malayi induce caspase-dependent apoptosis in human monocyte-derived dendritic cells (DCs) in vitro. This study addresses whether apoptosis observed in vitro extends to patent filarial infections in humans and is reflected in the number of circulating myeloid DCs (mDCs; CD11c−CD123lo) in peripheral blood of infected microfilaremic individuals. Utilizing flow cytometry to identify DC subpopulations (mDCs and plasmacytoid DCs [pDCs]) based on expression of CD11c and CD123, we found a significant increase in numbers of circulating mDCs (CD11c+CD123lo) in filaria-infected individuals compared with uninfected controls from the same filaria-endemic region of Mali. Total numbers of pDCs, monocytes, and lymphocytes did not differ between the two groups. To investigate potential causes of differences in mDC numbers between the two groups, we assessed chemokine receptor expression on mDCs. Our data indicate that filaria-infected individuals had a lower percentage of circulating CCR1+ mDCs and a higher percentage of circulating CCR5+ mDCs and pDCs. Finally, live microfilariae of B. malayi were able to downregulate cell-surface expression of CCR1 on monocyte-derived DCs and diminish their calcium flux in response to stimulation by a CCR1 ligand. These findings suggest that microfilaria are capable of altering mDC migration through downregulation of expression of some chemokine receptors and their signaling functions. These observations have major implications for regulation of immune responses to these long-lived parasites.

Interferon regulatory factor modulation underlies the bystander suppression of malaria antigen‐driven IL‐12 and IFN‐γ in filaria‐malaria co‐infection

In areas where polyparasitism is highly prevalent, the impact of multiple parasites on the host response is underestimated. In particular, the presence of helminth infection coincident with malaria profoundly alters the production of malaria‐specific IFN‐γ, IL‐12p70, CXCL9, CXCL10 and CXCL11, cytokines/chemokines known to be critical in mediating malaria‐specific immunity. In order to elucidate the mechanisms underlying the suppression of malaria‐specific cytokines/chemokines, we assessed the expression of malaria‐specific IL‐12Rβ1, IL‐12Rβ2 and interferon regulatory factor (IRF)‐1 in blood obtained from 18 filaria‐infected (Fil+) and 17 filaria‐uninfected (Fil−) individuals in a filaria‐malaria co‐endemic region of Mali. We found that Fil+ individuals had significantly lower RNA expression of IRF‐1 but not IL‐12Rβ1 or IL‐12Rβ2 in response to malaria antigen stimulation. We also measured the frequency of IL‐12‐producing DCs from these subjects and found that Fil+ subjects had lower frequencies of IL‐12+ mDCs after malaria antigen stimulation than did the Fil− subjects. Modeling these data in vitro, we found that mDCs pre‐exposed to live microfilariae not only produced significantly lower levels of CXCL‐9, CXCL‐10, IL‐12p35, IL‐12p40, IL‐12p19 and CXCL‐11 following stimulation with malaria antigen but also markedly downregulated the expression of IRF‐1, IRF‐2 and IRF‐3 compared with microfilaria‐unexposed mDCs. siRNA‐inhibition of irf‐1 in mDCs downregulated the production of IL‐12p70 through repression of IL‐12p35. Our data demonstrate that the modulation of IRFs seen in filarial (and presumably other tissue‐invasive helminths) infection underlies the suppression of malaria‐specific cytokines/chemokines that play a crucial role in immunity to malaria.

Eosinophil-Associated Processes Underlie Differences in Clinical Presentation of Loiasis Between Temporary Residents and Those Indigenous to Loa-Endemic Areas

BACKGROUND Loa loa has emerged as an important public health problem due to the occurrence of immune-mediated severe posttreatment reactions following ivermectin distribution. Also thought to be immune-mediated are the dramatic differences seen in clinical presentation between infected temporary residents (TR) and individuals native to endemic regions (END). METHODS All patients diagnosed with loiasis at the National Institutes of Health between 1976 and 2012 were included. Patients enrolled in the study underwent a baseline clinical and laboratory evaluation and had serum collected and stored. Stored pretreatment serum was used to measure filaria-specific antibody responses, eosinophil-related cytokines, and eosinophil granule proteins. RESULTS Loa loa infection in TR was characterized by the presence of Calabar swelling (in 82% of subjects), markedly elevated eosinophil counts, and increased filaria-specific immunoglobulin G (IgG) levels; these findings were thought to reflect an unmodulated immune response. In contrast, END showed strong evidence for immune tolerance to the parasite, with high levels of circulating microfilariae, few clinical symptoms, and diminished filaria-specific IgG. The striking elevation in eosinophil counts among the TR group was accompanied by increased eosinophil granule protein levels (associated with eosinophil activation and degranulation) as well as elevated levels of eosinophil-associated cytokines. CONCLUSIONS These data support the hypothesis that differing eosinophil-associated responses to the parasite may be responsible for the marked differences in clinical presentations between TR and END populations with loiasis.

Analyses of 123 Peripheral Human Immune Cell Subsets: Defining Differences with Age and between Healthy Donors and Cancer Patients not Detected in Analysis of Standard Immune Cell Types

Recent advances in human immunology have led to the identification of novel immune cell subsets and the biological function of many of these subsets has now been identified. The recent US Food and Drug Administration approval of several immunotherapeutics for the treatment of a variety of cancer types and the results of ongoing immunotherapy clinical studies requires a more thorough interrogation of the immune system. We report here the use of flow cytometry-based analyses to identify 123 immune cell subsets of peripheral blood mononuclear cells. The use of these panels defines multiple differences in younger (< 40 years) vs. older (≥ 40 years) individuals and between aged-matched apparently healthy individuals and metastatic cancer patients, aspects not seen in the analysis of the following standard immune cell types: CD8, CD4, natural killer, natural killer-T, regulatory T, myeloid derived suppressor cells, conventional dendritic cells (DCs), plasmacytoid DCs and B cells. The use of these panels identifying 123 immune cell subsets may aid in the identification of patients who may benefit from immunotherapy, either prior to therapy or early in the immunotherapeutic regimen, for the treatment of cancer or other chronic or infectious diseases.

Highly heterogeneous, activated and short-lived regulatory T cells during chronic filarial infection

The mechanisms underlying the increase in the numbers of regulatory T (Treg) cells in chronic infection settings remain unclear. Here we have delineated the phenotype and transcriptional profiles of Treg cells from 18 filarial‐infected (Fil+) and 19 filarial‐uninfected (Fil−) subjects. We found that the frequencies of Foxp3+ Treg cells expressing CTLA‐4, GITR, LAG‐3, and IL‐10 were significantly higher in Fil+ subjects compared with that in Fil− subjects. Foxp3‐expressing Treg‐cell populations in Fil+ subjects were also more heterogeneous and had higher expression of IL‐10, CCL‐4, IL‐29, CTLA‐4, and TGF‐β than Fil− subjects, each of these cytokines having been implicated in immune suppression. Moreover, Foxp3‐expressing Treg cells from Fil+ subjects had markedly upregulated expression of activation‐induced apoptotic genes with concomitant downregulation of those involved in cell survival. To determine whether the expression of apoptotic genes was due to Treg‐cell activation, we found that the expression of CTLA‐4, CDk8, RAD50, TNFRSF1A, FOXO3, and RHOA were significantly upregulated in stimulated cells compared with unstimulated cells. Taken together, our results suggest that in patent filarial infection, the expanded Treg‐cell populations are heterogeneous, short‐lived, activated, and express higher levels of molecules known to modulate immune responsiveness, suggesting that filarial infection is associated with high Treg‐cell turnover.

Phase I, dose-escalation, clinical trial of MVA-Brachyury-TRICOM vaccine demonstrating safety and brachyury-specific T cell responses.

Meeting abstracts Brachyury is a tumor-associated antigen and transcription factor that drives the epithelial-to-mesenchymal transition in human carcinomas. This Phase I study assessed whether patients with advanced cancer or chordoma, a rare tumor of the notochord that overexpresses brachyury, can