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The critical role of commensal microbiota in regulating the host immune response has been established. In addition, it is known that host–microbial interactions are bidirectional, and this interplay is tightly regulated to prevent chronic inflammatory disease. Although many studies have focused on the role of classic T cell subsets, unconventional lymphocytes such as NKT cells and innate lymphoid cells also contribute to the regulation of homeostasis at mucosal surfaces and influence the composition of the intestinal microbiota. In this review, we discuss the mechanisms involved in the cross-regulation between NKT cells, innate lymphoid cells, and the gut microbiota. Moreover, we highlight how disruptions in homeostasis can lead to immune-mediated disorders.

The first malaria vaccine has been recently approved for children living in malaria-endemic areas. While this is long-awaited and welcome news, the modest efficacy of the vaccine highlights several areas that require further attention. Here, we describe the likely impact of the vaccine and where clinical and basic discovery research will still be required.

Malaria in pregnancy (MIP) causes poor birth outcomes, but its impact on neurocognitive development has not been well characterized. Between 2012 and 2014, we enrolled 307 mother–infant pairs and monitored 286 infants for neurocognitive development using the Malawi Developmental Assessment Tool at 6, 12, and 24 months of age. MIP was diagnosed from peripheral blood and placental specimens. Cord blood cytokine levels were assessed for a subset of neonates. Predictors of neurodevelopment were examined using mixed-effect logistic regression for developmental delay. Among the participants, 78 mothers (25.4%) had MIP, and 45 infants (15.7%) experienced severe neurocognitive delay. MIP was not associated with differences in cord blood cytokine levels or neurocognitive development. Preterm birth, low birthweight, increasing maternal education level, and increasing interleukin 6 levels were associated significantly with delay. The results highlight the prevalence of severe delay and a need for broad access to early childhood support in this setting.

OBJECTIVES HIV-exposed uninfected (HEU) infants exhibit altered vaccine responses and an increased mortality compared with HIV-unexposed infants. Here, vaccine responses in HEU and HIV-unexposed cord blood monocytes (CBMs) were assessed following Bacillus Calmette--Guerin (BCG) treatment. DESIGN Innate responses to in-vitro BCG treatment were assessed through transcriptional profiling using CBMs obtained from a Nigerian cohort of HIV-infected and uninfected women. METHODS HIV-unexposed (n = 9) and HEU (n = 10) infant CBMs were treated with BCG and transcriptionally profiled with the Nanostring nCounter platform. Differential expression and pathway enrichment analyses were performed, and transcripts were identified with enhanced or dampened BCG responses. RESULTS Following BCG stimulation, several pathways associated with inflammatory gene expression were upregulated irrespective of HIV exposure status. Both HIV-unexposed and HEU monocytes increased expression of several cytokines characteristic of innate BCG responses, including IL1β, TNFα, and IL-6. Using differential expression analysis, we identified genes significantly upregulated in HEU compared with HIV-unexposed monocytes including monocyte chemokine CCL7 and anti-inflammatory cytokine TNFAIP6. In contrast, genes significantly upregulated in HIV-unexposed compared with HEU monocytes include chemokine CCL3 and cytokine IL23A, both of which influence anti-mycobacterial T-cell responses. Finally, two genes, which regulate prostaglandin production, CSF2 and PTGS2, were also more significantly upregulated in the HIV-unexposed cord blood indicating that inflammatory mediators are suppressed in the HEU infants. CONCLUSION HEU monocytes exhibit altered induction of several key innate immune responses, providing mechanistic insights into dysregulated innate response pathways that can be therapeutically targeted to improve vaccine responses in HEU infants.

Immune dysfunction is a hallmark of chronic HCV infection and viral clearance with direct antivirals recover some of these immune defects. TCRVγ9Vδ2 T cell dysfunction in treated HCV patients however is not well studied and was the subject of this investigation. Peripheral blood cells from patients who had achieved sustained virologic response (SVR) or those who had relapsed after interferon-free therapy were phenotyped using flow cytometry. Functional potential of Vγ9Vδ2-T cells was tested by measuring proliferation in response to aminobisphosphonate Zoledronic acid, and cytotoxicity against HepG2 hepatoma cell line. TCR sequencing was performed to analyze impact of HCV infection on Vδ2-T cell repertoire. Vγ9Vδ2 cells from patients were activated and therapy resulted in reduction of CD38 expression on these cells in SVR group. Relapsed patients had Vδ2 cells with persistently activated and terminally differentiated cytotoxic phenotype (CD38(+)CD45RA(+)CD27(−)CD107a(+)). Irrespective of outcome with therapy, majority of patients had persistently poor Vδ2-T cell proliferative response to Zoledronate along with lower expression of CD56, which identifies anti-tumor cytotoxic subset, relative to healthy controls. There was no association between the number of antigen reactive Vγ2-Jγ1.2 TCR rearrangements at baseline and levels of proliferation indicating non-response to Zoledronate is not due to depletion of phosphoantigen responding chains. Thus, HCV infection results in circulating Vγ9Vδ2-T cells with a phenotype equipped for immediate effector function but poor cytokine response and expansion in response to antigen, a functional defect that may have implications for susceptibility for carcinogenesis despite HCV cure.

Background. Plasmodium falciparum placental infection primes the fetal immune system and alters infant immunity. Mechanisms leading to these outcomes are not completely understood. We focused on V gamma 2V delta 2 cells, which are part of the immune response against many pathogens, including P. falciparum. These unconventional lymphocytes respond directly to small, nonpeptidic antigens, independent of major histocompatibility complex presentation. We wondered whether placental malaria, which may increase fetal exposure to P. falciparum metabolites, triggers a response by neonatal V gamma 2V delta 2 lymphocytes that can be a marker for the extent of fetal exposure to malarial antigens. Methods. Cord blood mononuclear cells were collected from 15 neonates born to mothers with P. falciparum infection during pregnancy (8 with placental malaria) and 25 unexposed neonates. V gamma 2V delta 2cell phenotype, repertoire, and proliferative responses were compared between newborns exposed and those unexposed to P. falciparum. Results. Placental malaria-exposed neonates had increased proportions of central memory V gamma 2V delta 2 cells in cord blood, with an altered V?2 chain repertoire ex vivo and after stimulation. Conclusion. Our results suggest that placental malaria affects the phenotype and repertoire of neonatal V gamma 2V delta 2 lymphocytes. Placental malaria may lower the capacity for subsequent V gamma 2V delta 2 cell responses and impair the natural resistance to infectious diseases or the response to pediatric vaccination.

Objective Determine whether reconstitution of Vγ2Vδ2 T cells in patients with HIV is due to new cell synthesis with recovery of the T-cell receptor repertoire or proliferative expansion of residual cells from the time of treatment initiation. Design Perform a cross-sectional analysis of the T-cell receptor complexity of Vγ2 chain in patients treated for HIV, natural virus suppressors who control viremia to undetectable levels, patients with chronic low-level viremia in the absence of therapy, and uninfected controls. Apply quantitative methods for repertoire analysis to assess the degree of Vδ2 repertoire loss or reconstitution. Methods T-cell receptor Vγ2 chain DNA clones (up to 300 per patient sample) were sequenced and aligned to enumerate the antigen-reactive subset with Vγ2-Jγ1.2 rearrangements. Predominant shared (public) sequences in each patient were compared to a reference library of public sequences from uninfected controls to assess the extent of similarity. Repertoire comparisons were quantified through bioinformatics testing. Results Patients with prolonged virus suppression due to antiretroviral therapy reconstituted the Vγ2 T-cell repertoire to near-normal levels. Natural virus suppressors were similar to the treatment group. Severe defects in the Vγ2 T-cell receptor repertoire were observed in patients with chronic viremia despite the absence of overt disease. Conclusion Prolonged HIV suppression with antiretroviral therapy leads to reconstitution of the Vγ2Vδ2 T-cell subset deleted in HIV disease. Direct evidence for repair of the T-cell receptor repertoire supports a view that treatment-associated immune reconstitution is due to new cell synthesis and not to expansion of residual cell populations.

Compared with adults, the circulating Vγ2Vδ2 T-cell population in cord blood is present at low levels and does not show the strong bias for Vγ2-Jγ1.2 rearrangements. These features may be a result of limited exposure to stimulatory phosphoantigens, lack of T-cell-derived interleukin-2 (IL-2) or both. In cord blood mononuclear cell cultures, a single round of stimulation, using aminobisphosphonates to elevate phosphoantigen levels, resulted in expansion of adult-like Vγ2 chains and accumulation of memory cells with cytotoxic potential. Selection was similar using IL-2 or myeloid-derived IL-15. The Vγ2Vδ2 T cells present in neonates are capable of generating potent immune responses even when relying on IL-15.

A successful pregnancy depends on the maintenance of tolerance at the fetal–maternal interface; strong inflammation in the placental bed is generally associated with adverse fetal outcomes. Among the mechanisms that foster tolerance and limit inflammation, the fetal immune system favors Th2 or regulatory responses over Th1 responses. The unintended consequence of this functional program is high susceptibility to infections. Human Vδ2 T cells mount innate-like responses to a broad range of microorganisms and are poised for Th1 responses before birth. In infants they likely play a key role in protection against pathogens by exerting early Th1 effector functions, improving function of other innate cells, and promoting Th1 polarization of adaptive responses. However, their propensity to release Th1 mediators may require careful regulation during fetal life to avoid exaggerated proinflammatory responses. We investigated molecules with the potential to act as a rheostat for fetal Vδ2 cells. Programmed death 1 (PD1) is a negative regulator of T cell responses and a determinant of tolerance, particularly at the fetal–maternal interface. Neonatal Vδ2 cells upregulate PD1 shortly after activation and, unlike their adult counterparts, express this molecule for at least 28 d. Engagement of PD1 by one of its ligands, PDL1, effectively dampens TCR-mediated responses (TNF-α production and degranulation) by neonatal Vδ2 cells and may thus help maintain their activity within safe limits. PD1 expression by neonatal Vδ2 cells is inversely associated with promoter DNA methylation. Prolonged PD1 expression may be part of a functional program to control Vδ2 cell inflammatory responses during fetal life.

Oral Abstract We hypothesize that placental malaria causes chronic in utero inflam-mation with compensatory production of IL-10 and induction of Tregs. After birth,cytokine levels normalize, but Tregs may be maintained and downregulate effectiveimmune responses to malaria resulting in increased risk of malaria during infancy.We are currently conductingflow cytometric studies on cord blood to further explorethese hypotheses. Our results might inform the design and implementation of prenatalinterventions to protect the health of pregnant women, newborns and infants from malaria.

A major subset of human peripheral blood γδ T cells expresses the Vγ2Vδ2 T cell receptor and responds to malignant or infectious diseases. We noted significant differences in the numbers of Vγ2Vδ2 T cells in blood samples from healthy Caucasian CA or African American (AA) donors. On average, CA donors had 3.71% ± 4.37% Vδ2 cells (as a percentage of total lymphocytes) compared with 1.18% ± 2.14% Vδ2 cells for AA donors (p < 0.0001). Age and race had the greatest impact on Vδ2 cell levels; the effect of age was similar for both racial groups. The Vδ2 cell population was dominated, for both donor groups, by cells expressing the Vγ2-Jγ1.2 Vδ2 T cell receptor, an apparent result of strong positive selection and there was substantial overlap in the public Vγ2 clonotypes from both racial groups. Mechanisms for selection and amplification of Vδ2 cells are nearly identical for both groups, despite the significant difference in baseline levels. These data show that appropriate controls, matched for age and race, may be required for clinical studies of Vγ2Vδ2 T cells in infectious disease or cancer and raise important questions about the mechanisms regulating the levels of circulating Vδ2 cells.

To evaluate Vgamma2Vdelta2 T cells in a group of HIV-infected patients who suppress HIV replication without antiretroviral therapy (natural viral suppressors, NVSs).: It is a cross-sectional study.We compared Vgamma2Vdelta2 T-cell frequency, T-cell repertoire, and responses to isopentenyl pyrophosphate stimulation between NVSs (n = 21) and HIV-uninfected controls (n = 27) and between NVSs and HIV-infected patients taking HAART with suppressed viral replication (HIV-P; n = 25).NVSs had a mean frequency of 1.06 +/- 0.82% CD3Vdelta2+ cells among total lymphocytes, which was significantly higher than both control groups (HIV-negative: 0.50 +/- 0.53%, P = 0.042; HIV-P: 0.34 +/- 0.37%, P = 0.002). The proportion of Vgamma2 chains correlating with the Vgamma2-Jgamma1.2 rearrangement was reduced among NVSs compared with HIV-negative controls (0.57 +/- 0.06 vs. 0.32 +/- 0.04; P = 0.016) but was increased compared with HIV-P patients (0.32 +/- 0.04 vs. 0.22 +/- 0.03; P = 0.03). NVSs had a similar baseline frequency of CD27/CD45RA effector cells (19.6 +/- 4.2%) compared with HIV-negative controls (20.8 +/- 12.9%; P = 0.35).The altered gammadelta T-cell receptor repertoire among NVS was consistent with the known effect of HIV-1 on these cells. Uniquely among all HIV-infected groups, NVS reconstituted the gammadelta T-cell population, eventually reaching levels significantly above controls. This capacity to recover gammadelta T-cell numbers and function distinguishes individuals who control HIV-1 with and without HAART.

Rhesus macaques infected with the WE strain of lymphocytic choriomeningitis virus (LCMV-WE) serve as a model for human infection with Lassa fever virus. To identify the earliest events of acute infection, rhesus macaques were monitored immediately after lethal infection for changes in peripheral blood mononuclear cells (PBMCs). Changes in CD3, CD4, CD8 and CD20 subsets did not vary outside the normal fluctuations of these blood cell populations; however, natural killer (NK) and gammadelta T cells increased slightly on day 1 and then decreased significantly after two days. The NK subsets responsible for the decrease were primarily CD3-CD8+ or CD3-CD16+ and not the NKT (primarily CD3+CD56+) subset. Macaques infected with a non-virulent arenavirus, LCMV-Armstrong, showed a similar drop in circulating NK and gammadelta T cells, indicating that this is not a pathogenic event. V(3)9 T cells, representing the majority of circulating gammadelta T cells in rhesus macaques, displayed significant apoptosis when incubated with LCMV in cell culture; however, the low amount of cell death for virus-co-cultured NK cells was insufficient to account for the observed disappearance of this subset. Our observations in primates are similar to those seen in LCMV-infected mice, where decreased circulating NK cells were attributed to margination and cell death. Thus, the disappearance of these cells during acute hemorrhagic fever in rhesus macaques may be a cytokine-induced lymphopenia common to many virus infections.

Infectious diseases during pregnancy can impact the development of fetal immunity, leading to reduced neonatal resistance to infection and decreased responses to pediatric vaccines. Plasmodium falciparum causes placental infection in low parity pregnant women and is among the pathogens that affect fetal immunity. Recognizing the relationship between malaria and gammadelta T lymphocytes in adults, we asked whether neonatal gammadelta T cells would be altered in malaria-endemic regions as a marker for changes in fetal immunity. Our initial studies compared cord blood gammadelta T cells from deliveries to HIV- mothers in Jos (Nigeria) where malaria is endemic, or in Rome (Italy). We noted substantial differences in the Vgamma2 repertoire for cord blood collected in Jos or Rome; differences were consistent with a negative selection mechanism operating on the fetal Vgamma2 chain repertoire in neonates from Jos. A specific disruption affected the fraction of gammadelta T cells that we expect will respond to Bacille Calmette-Guerin (BCG). Fetal gammadelta T cell depletion might be a mechanism for impaired neonatal immunity and lowered responses to pediatric vaccines.

Monocyte differentiation into dendritic cells (DCs) depends on microenvironmental conditions. In this study, the capacity of human monocytes to differentiate into mature DCs and their ability to induce an antiviral immune response was investigated in HIV-infected patients. In healthy subjects, monocytes differentiate into CD1a DCs in the presence of granulocyte macrophage colony-stimulating factor and interleukin (IL)-4 and matured in the presence of lipopolysaccharide. Here, we found that in 30% and 45% of HIV-infected white and African subjects, respectively, monocytes gave rise to a homogeneous CD1a DC population. In the patients who gave rise only to the CD1a DCs, this population spontaneously produced IL-10 but not IL-12, and induced a T helper 2-like immune response when cultured with human T cells isolated from cord blood mononuclear cells. In patients with monocytes differentiated into CD1a DCs, a high percentage of HIV-specific CD4 T cells producing IL-4 were seen in the peripheral blood. Furthermore, differentiation of monocytes into DCs with CD1a phenotype correlated with low CD4 T-cell counts and high viral loads in HIV-infected subjects. These results suggest that the differentiation of monocytes into CD1a DCs may be a phenotypic marker associated with progression of the disease. © 2007 Lippincott Williams & Wilkins, Inc.

The V gamma 2 V delta 2 T cell subset responds to Bacille Calmette-Guerin (BCG) immunization in macaques and may be a component of protective immunity against tuberculosis. We characterized the effects of BCG on the V gamma 2 V delta 2 T cell receptor repertoire by comparing the starting population of V gamma 2 chains in cynomolgus macaques with the repertoire found after priming or booster immunization with BCG. The starting repertoire was dominated by public V gamma 2 chain sequences that were found repeatedly among unrelated animals. Primary exposure to BCG triggered expansion of cells expressing public V gamma 2 chains and booster immunization was often associated with contraction of these same subsets. Thus, BCG-reactive V gamma 2 chains were present at high frequency in the repertoire of mycobacteria-naïve macaques and they comprised the major response to primary or booster immunization. Normal selection processes that created the naïve V gamma 2 repertoire in macaques, also encoded the capacity for rapid responses to mycobacteria. The unusual composition of a normal V gamma 2 repertoire helps to explain the powerful gammadelta T cell responses to BCG immunization.

Vaccinia virus (VV) is an effective vaccine and vector but has evolved multiple mechanisms for evading host immunity. We characterized the interactions of VV (TianTan and New York City Board of Health strains) with human gammadelta T cells because of the role they play in immune control of this virus. Exposure to VV failed to trigger proliferative responses in gammadelta T cells from unprimed individuals, but it was an unexpected finding that VV blocked responses to model antigens by the Vgamma2Vdelta2 T cell subset. Infectious or ultraviolet light-inactivated VV inhibited proliferative Vgamma2Vdelta2 T cell responses to phosphoantigens and tumor cells, prevented cytolysis of Daudi B cells, and reduced cytokine production. Inhibiting Vgamma2Vdelta2 T cells may be a mechanism for evading host immunity and increasing VV virulence. Increased VV replication or expression in the absence of gammadelta T cell responses might contribute to its potency as a vaccine against poxvirus and recombinant antigens.

Interactions between natural killer (NK) cells and dendritic cells (DC) affect maturation and function of both cell populations, including NK cell killing of DC (editing), which is important for controlling the quality of immune responses. We also know that antigen-stimulated Vγ2Vδ2 T cells co-stimulate NK cells via 4-1BB to enhance the killing of tumour cell lines but we do not know what regulates 4-1BB expression or whether other NK effector functions including DC killing, might also be influenced by NK–γδ T-cell cross-talk. Here we show that antigen-stimulated γδ T cells co-stimulate NK cells through inducible T-cell co-stimulator (ICOS)– ICOS ligand (ICOSL) and this signal increases NK cell killing of autologous DC. Effects of NK–γδ T-cell co-culture, which could be reproduced with soluble ICOS-Fc fusion protein, included increased CD69 and 4-1BB expression, interferon-γ, tumour necrosis factor-α, macrophage inflammatory protein-1β, I-309, RANTES and sFas ligand production, as well as elevated mRNA levels for co-stimulatory receptors OX40 (TNFRSF4) and GITR (TNFRSF18). Hence, ICOS–ICOSL co-stimulation of NK by Vγ2Vδ2 T cells had broad effects on NK phenotype and effector functions. The NK–γδ T-cell cross-talk links innate and antigen-specific lymphocyte responses in the control of cytotoxic effector function and DC killing.

Lymphocytes expressing a T cell receptor (TCR) composed of Vgamma9 and Vdelta2 chains represent a minor fraction of human thymocytes. Extrathymic selection throughout post-natal life causes the proportion of cells with a Vgamma9-JP rearrangement to increase and elevates the capacity for responding to non-peptidic phosphoantigens. Extrathymic selection is so powerful that phosphoantigen-reactive cells comprise about 1 in 40 circulating memory T cells from healthy adults and the subset can be expanded rapidly upon infection or in response to malignancy. Skewing of the gamma delta TCR repertoire is accompanied by selection for public gamma chain sequences such that many unrelated individuals overlap extensive in their circulating repertoire. This type of selection implies the presence of a monomorphic antigen-presenting molecule that is an object of current research but remains incompletely defined. While selection on a monomorphic presenting molecule may seem unusual, similar mechanisms shape the alpha beta T cell repertoire including the extreme examples of NKT or mucosal-associated invariant T cells (MAIT) and the less dramatic amplification of public Vbeta chain rearrangements driven by individual MHC molecules and associated with resistance to viral pathogens. Selecting and amplifying public T cell receptors whether alpha beta or gamma delta, are important steps in developing an anticipatory TCR repertoire. Cell clones expressing public TCR can accelerate the kinetics of response to pathogens and impact host survival.

Human immunodeficiency virus (HIV) type 1 dysregulates γδ T cells as part of an immune evasion mechanism. Nearly three decades of research defined the effects of HIV on γδ T cells and how this impacts disease. With highly effective antiretroviral therapy providing virus suppression and longer survival, we expected a return to normal for γδ T cells. This is not the case. Even in patients with CD4 T cell reconstitution, normal γδ T cell levels and function are not recovered. The durable damage to Vδ2 T cells is paralleled by defects in NK, CD8 T cells, and dendritic cells. Whether these consequences of HIV stem from similar or distinct mechanisms are not known and effective means for recovering the full range of cellular immunity have not been discovered. These unanswered questions receive too little attention in the overall program of efforts to cure HIV this disease. Approved drugs capable of increasing Vδ2 T cell function are being tested in clinical trials for cancer and hold promise for restoring normal function in patients with HIV disease. The impetus for conducting clinical trials will come from understanding the significance of γδ T cells in HIV disease and what might be gained from targeted immunotherapy. This review traces the history and current progress of AIDS-related research on γδ T cells. We emphasize the damage to γδ T cells that persists despite effective virus suppression. These chronic immune deficits may be linked to the comorbidities of AIDS (cancer, cardiovascular disease, metabolic disease, and others) and will hinder efforts to eradicate HIV by cytotoxic T or NK cell killing. Here, we focus on one subset of T cells that may be critical in the pathogenesis of HIV and an attractive target for new immune-based therapies.

Circulating Vγ2Vδ2 T-cell populations in healthy human beings are poised for rapid responses to bacterial or viral pathogens. We asked whether Vγ2Vδ2 T cells use the Toll-like receptor (TLR) family to recognize pathogen-associated molecular pattern molecules and to regulate cell functions. Analysis of expanded Vγ2Vδ2 T-cell lines showed the abundant presence of TLR2 mRNA, implying that these receptors are important for cell differentiation or function. However, multiple efforts to detect TLR2 protein on the cell surface or in cytoplasmic compartments gave inconsistent results. Functional assays confirmed that human Vγ2Vδ2 T cells could respond to the TLR2 agonist (S)-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys4-OH trihydrochloride (Pam3Cys), but the response required coincident stimulation through the γδ T-cell receptor (TCR). Dually stimulated cells produced higher levels of cytoplasmic or cell-free gamma interferon and showed increased expression of the lysosome-associated membrane protein CD107a on the cell surface. A functional TLR2 that requires coincident TCR stimulation may increase the initial potency of Vγ2Vδ2 T-cell responses at the site of infection and promote the rapid development of subsequent acquired antipathogen immunity.

To the Editor—Hartjen et al compared γδ T-cell levels and phenotypes in human immunodeficiency virus (HIV)–infected patients. Their letter describes differences from our published results [1] and questions whether the HIV-associated changes in Vδ2 T cells reported by us [2] are found in most of HIV-infected patients. The control group in the study by Hartjen et al, represented in Figure 1C of their report, has a mean value of approximately 1% Vδ2 T cells among total lymphocytes in blood. This value is on the low end of reported levels for Vδ2 cells among healthy donors in any of our previous studies [2] or in a study of sex and age effects on Vδ2 T cells [3]. If we use a median value from the Hartjen et al data, Vδ2 levels for their control group would be

Lymphocyte subset reference values used to monitor infectious diseases, including HIV/AIDS, tuberculosis, malaria, or other immunological disorders in healthy children in Cameroon, are lacking. Values for Caucasian cohorts are already being utilized for clinical decisions but could be inappropriate for African populations. We report here the immunological profile for children aged from birth through 6 years in Cameroon and also compare our values to data from other African and Caucasian populations. In a cohort of 352 healthy children, aged 0 to 6 years, the relative and absolute numbers of T-cell subsets, B cells, and NK lymphocytes were determined from peripheral blood collected in EDTA tubes. Samples were stained with BD Multitest reagents in Trucount tubes and analyzed by using CellQuest-Pro and FlowJo software. We evaluated about 23 different lymphocyte subsets in which the absolute number and percentage values differed significantly (P< 0.05) with age and peaked between 6 and 12 months. B-cell values were higher compared to reported values from developed countries. Differences in activated and differentiated T cells were observed in subjects between 1 and 6 years of age. The absolute CD8+T-cell count and the CD4+/CD8+ratio seem to depend on gender. Normal lymphocyte subsets values among children from Cameroon differ from reported values in Caucasian and some African populations. The differences observed could be due to genetic and environmental factors coupled with the methodology used. These values could be used as initial national reference guidelines as more data are assembled.

Background In lymphocyte subsets, expression of CD56 (neural cell adhesion molecule-1) correlates with cytotoxic effector activity. For cells bearing the Vγ2Vδ2 T cell receptor, isoprenoid pyrophosphate stimulation leads to uniform activation and proliferation, but only a fraction of cells express CD56 and display potent cytotoxic activity against tumor cells. Our goal was to show whether CD56 expression was regulated stochastically, similar to conventional activation antigens, or whether CD56 defined a lineage of cells committed to the cytotoxic phenotype. Results Tracking individual cell clones defined by their Vγ2 chain CDR3 region sequences, we found that CD56 was expressed on precursor cytotoxic T cells already present in the population irrespective of their capacity to proliferate after antigen stimulation. Public T cell receptor sequences found in the CD56+ subset from one individual might appear in the CD56- subset of another donor. The commitment of individual clones to CD56+ or CD56- lineages was stable for each donor over a 1 year interval. Conclusion The ability to express CD56 was not predicted by TCR sequence or by the strength of signal received by the TCR. For γδ T cells, cytotoxic effector function is acquired when cytotoxic precursors within the population are stimulated to proliferate and express CD56. Expression of CD56 defines a committed lineage to the cytotoxic phenotype.

Gammadelta (gammadelta) T cells expressing the Vgamma2-Jgamma1.2Vdelta2 (Vgamma9-JPVdelta2, alternate nomenclature) T cell receptor (TCR) constitute the major peripheral blood population of gammadelta T cells in adult humans and are specifically depleted during human immunodeficiency virus (HIV) disease. Vgamma2-Jgamma1.2Vdelta2 T cells provide a convenient model for assessing the impact of antiretroviral therapy on cell populations that are not susceptible to direct infection because they do not express CD4 and depletion occurs by indirect mechanisms. We obtained longitudinal PBMC samples from 16 HIV-infected individuals who enrolled in the Multicenter AIDS Cohort Study (MACS) and were starting highly active antiretroviral therapy (HAART). Vgamma2-Jgamma1.2Vdelta2 T cells were depleted in these individuals as a result of HIV infection. Despite evidence for clinical benefits of HAART, the Vgamma2-Jgamma1.2Vdelta2 T cell repertoire did not recover after HAART initiation irrespective of treatment duration. These studies highlight important defects among cell subsets lost due to indirect effects of HIV.

HIV infection causes rapid and lasting defects in the population of Vgamma2Vdelta2 T cells. To fully describe the impact of HIV, we examined PBMC samples from HIV+ patients receiving highly active antiretroviral therapy, who had displayed prolonged viral control and CD4 counts above 300 cells/mm3. We observed lower frequencies of CD27-/CD45RA- Vgamma2Vdelta2 cells in HIV+ individuals when compared with controls, coupled with an increased proportion of CD45RA+ cells. These changes were common among 24 HIV+ patients and were not related to CD4 cell count or viral RNA burden. Vgamma2 cells from HIV+ individuals had lower expression of Granzyme B and displayed reduced cytotoxicity against Daudi targets after in vitro stimulation. There was increased expression of FasR (CD95) on Vgamma2 cells from HIV+ PBMC that may be a mechanism for depletion of Vgamma2 cells during disease. In addition to the well-characterized defects in the Vgamma2 repertoire and functional responses to phosphoantigen, the proportion of CD27-/CD45RA- Vgamma2Vdelta2 T cells after isopentenyl pyrophosphate stimulation was reduced sharply in HIV+ donors versus controls. Thus, HIV infection has multiple impacts on the circulating Vgamma2Vdelta2 T cell population that combine to reduce the potential effector activity in terms of tumor cytotoxicity. Changes in Vgamma2Vdelta2 T cells, along with concomitant effects on NK and NKT cells that also contribute to tumor surveillance, may be important factors for elevating the risk of malignancy during AIDS.

A single injection of ML29 reassortant vaccine for Lassa fever induces low, transient viremia, and low or moderate levels of ML29 replication in tissues of common marmosets depending on the dose of the vaccination. The vaccination elicits specific immune responses and completely protects marmosets against fatal disease by induction of sterilizing cell-mediated immunity. DNA array analysis of human peripheral blood mononuclear cells from healthy donors exposed to ML29 revealed that gene expression patterns in ML29-exposed PBMC and control, media-exposed PBMC, clustered together confirming safety profile of the ML29 in non-human primates. The ML29 reassortant is a promising vaccine candidate for Lassa fever.

Human Vgamma2Vdelta2 T cells exhibit T cell receptor-dependent, MHC-unrestricted recognition of antigen and play important roles in tumor and pathogen immunity. To characterize antigen recognition by the Vgamma2Vdelta2 TCR, we used the combined approach of spectratyping and CDR3 sequence analysis that measures changes in the TCR repertoire before and after stimulation with a phosphoantigen (isopentenyl pyrophosphate) or an irradiated tumor cell line (Daudi B lymphoma). Here we describe common Vgamma2 chains that are substantially involved in the response to both phosphoantigens and tumor cells. The recognition properties of common Vgamma2 chains explains the observation that Vgamma2Vdelta2 T cells expanded by phosphoantigen stimulation specifically recognize and kill some but not all tumor cell lines. Our studies further justify efforts to stimulate tumor immunity by administering low molecular weight phosphoantigens and boosting the frequency and tumor effector functions of circulating Vgamma2Vdelta2 T cells.

Vg2Vd2 T cells are the predominant peripheral blood subset of – T cells and recognize tumor cells in a TCR-dependent, MHC-unrestricted manner. Following HIV infection there is a rapid and targeted depletion of circulating, anti-tumor Vy2Vd2 T cells with concomitant increases in B cell lymphoma incidence. We hypothesized that the specific depletion of Vy2Vd2 T cells after HIV compromises an important tumor surveillance system and directly contributes to the increased incidence of B cell lymphoma observed in HIV-positive cohorts. To test this hypothesis we collected tumor cell lines derived from clinical cases of AIDS-related lymphoma and tested them for susceptibility to Vy2Vd2 cytotoxicity and ability to stimulate Vy2Vd2 T cell proliferation from PBMC in vitro. Several tumor cell lines including Daudi, KSY1 and IBL4 were recognized and lysed by polyclonal Vy2Vd2 T cell effectors in a dose-dependent manner. We screened several irradiated AIDS-derived tumor lines in vitro for the capacity to stimulate Vy2Vd2 T cells and identified IBL4 as a novel tumor stimulator for the Vy2Vd2 subset that skewed the Vy2 repertoire toward longer chain lengths without affecting the overall distribution of Vy2 chain lengths and without obvious tumor-specific CDR3 sequences commonality among responding blood donors. These data provide important insight into tumor recognition by Vy2Vd2 T cells and justify chemotherapeutic approaches using alkylphosphate stimulation to boost the frequency and tumor effector function of circulating Vy2Vd2 lymphocytes.

The control of maternal immunity during pregnancy and the impact of maternal infections on the fetal immune system are not well characterized. We initiated studies on pregnant women in Nigeria and their offspring, with a goal of characterizing maternal immunity and how it affects the developing fetus. We concentrated on – T cells, in particular on the Vg2-Jg1.2+ subset, a lymphocyte population responsible for rapid and robust Th1 responses to multiple pathogens and tumors. Otherwise healthy pregnant women in Nigeria (n = 22) had average Vg2-Jg1.2 frequencies significantly lower than a cohort of healthy control subjects gathered in North America and 6 of 22 (27%) women had severely depressed values. Among HIV-infected pregnant women in Nigeria the Vg2-Jg1.2+ subset was also low, but was similar to HIV+ adults from Baltimore. We also asked examined the – T cell receptor repertoire in cord blood mononuclear cells from deliveries to both HIV+ and HIV-neg Nigerian women. Most cord blood populations were naive and had very low representation of the Vg2-Jg1.2+ subset. However, 3 of 13 specimens (23%) were unusual in having a – T cell receptor repertoire that was more similar to adult values and unlike the highly naive cord blood cells seen in specimens from North American or European deliveries. These data reinforced our previous observations from cord blood specimens in the Ivory Coast, where we first noted the appearance of unusually mature cord blood cell populations with high functional responses to – T cell antigens. Knowing that – T cells are impacted not only by HIV, but also by malaria and tuberculosis, we are initiating a study to uncover possible environmental exposures during pregnancy and how these experiences imprint on the fetal immune system. At present, our focus is on malaria due to the high risk for infection in Nigeria, the known devastating consequences for babies born to infected mothers, and the interaction of HIV and malaria resulting in accelerated disease.

The mature gamma/delta T cell receptor repertoire arises by a process of positive selection and amplification of cells expressing the Vg2Vd2 TCR, with further skewing to TCR with the Vg2-Jg1.2 rearrangement. Once formed, the Vg2 repertoire is stable in healthy adults showing more than 50% sequence conservation over intervals of up to 7 years. The Vg2-Jg1.2 chains are among the most conserved elements in healthy individuals and even the balance among naive and memory cell types is maintained. This situation is altered by HIV infection. We showed previously that HIV disease was associated with the targeted loss of cells expressing the Vg2-Jg1.2 rearrangement. In longitudinal studies with specimens from the MACS cohort, we showed that peripheral blood Vg2Vd2 T cell has a high frequency of staining with the vital dye 7-AAD, in most specimens the Vg2Vd2 T cells were twice as likely to be 7-AAD positive compared with CD4+ T cells. The lower viability of circulating Vg2Vd2 T cells was reflected in the repertoire characteristics. We observed a high degree of repertoire conservation in HIV+ individuals, with values consistent with the lower range found in healthy adults. However, stability was confined largely to Vg2 cells with rearrangements using J segments other than Jg1.2. In most cases, any Jg1.2 sequences that were found in HIV+ individuals were not seen in subsequent specimens collected at 6 monthly intervals. The pattern of instability among only the Vg2-Jg1.2+ cells, shows that the effect of HIV on repertoire is a process of rapid turn-over among cells that may be reacting to viral antigens while cells without known responses to HIV (Vg2 with all other Jg rearrangements) are relatively unaffected. Even in this population of cells that are not susceptible to direct infection, owing to the lack of CD4 receptor, depletion is confined to the virus reactive cells.

Vaccinia virus (VV) is an effective vaccine and vector but has evolved multiple mechanisms for evading host immunity. We characterized the interactions of VV (TianTan and New York City Board of Health strains) with human gammadelta T cells because of the role they play in immune control of this virus. Exposure to VV failed to trigger proliferative responses in gammadelta T cells from unprimed individuals, but it was an unexpected finding that VV blocked responses to model antigens by the Vgamma2Vdelta2 T cell subset. Infectious or ultraviolet light-inactivated VV inhibited proliferative Vgamma2Vdelta2 T cell responses to phosphoantigens and tumor cells, prevented cytolysis of Daudi B cells, and reduced cytokine production. Inhibiting Vgamma2Vdelta2 T cells may be a mechanism for evading host immunity and increasing VV virulence. Increased VV replication or expression in the absence of gammadelta T cell responses might contribute to its potency as a vaccine against poxvirus and recombinant antigens.

HIV infection leads to a rapid and complete loss of cells expressing the Vg2-Jg1.2 chain of the gd T cell receptor. This cell subset is generally measured by spectratyping and is defined as Vg2 chains encoded by sequences between 990 and 996 nucleotides in length. In a previous cross-sectional study (Bordon, et al. JID 189:1482, 2004) we demonstrated a relationship between the duration of therapy and recovery of Vg2 sequences between 990–996 nucleotides. Now using longitudinal specimens and extensive analysis of Vg2 chains by DNA sequencing, we show that prolonged HAART does indeed reconstitute the repertoire of 990–996 length Vg2 chains, but they are no longer restricted to using the Jg1.2 segment. Instead, we observe Jg1.1 and Jg2.3 sequences with unusual N regions sequences that are rare in healthy individuals. In this unique example, we show that HIV infection exhausts the population of T cell clones capable of expressing the Vg2Jg1.2 chain, but that strong selection for Vg2 chains in the range of 990–996 nucleotides, forces the expansion of a previously minor population. Within the Vg2Vd2 T cell population, prolonged therapy enabled a functional reconstitution of the Vg2 repertoire. Even though repertoire selection for Vg2 is largely independent of thymic function, there was no evidence that T cell clones could be replaced from hematopoietic stem cells, despite the continued strong selection for Vg2 chains of the appropriate length. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

HIV infection results in an early and profound loss of Vg2Vd2 T cells, and this is the only T cell receptor-specific depletion that is common to all individuals with HIV/ AIDS. A similar pattern of Vg2Vd2 T cell depletion occurs in mycobacterium infection and during malaria. We hypothesize that the loss of Vg2Vd2 T cells, irrespective of the primary cause for this loss, results in disease acceleration during HIV+ tuberculosis or HIV+ malaria coinfections and also leads to increased incidence of cancer in the context of HIV/AIDS.

Gammadelta (γδ) T cells account for 1–10% of CD3+ lymphocytes in the peripheral blood and mostly express a heterodimeric T cell receptor (TCR) with Vγ2+ and Vδ2+ chains. Although the Vγ2/Vδ2 subset is defined by the shared expression of common TCR gene segments, these TCRs are highly diverse due to characteristic N nucleotide insertion and deletion at the complementarity-determining region 3 (CDR3) of both γand δ-chains. Vγ2/Vδ2 T cells recognize alkylphosphates that are ubiquitous intermediates in isoprenoid biosynthesis and tumor cells derived from hematopoietic malignancies in a non MHCrestricted, TCR-dependent manner. Previous work from our lab demonstrated that a model alkylphosphate, isopentenylpyrophosphate (IPP), specifically selects Jγ1.2+ chains and selectively skews the Vγ2 repertoire toward longer chain lengths. We assumed that Vγ2/Vδ2 recognition of alkylphosphates and tumor cells was common and hypothesized that Daudi B cells, the model tumor target for Vγ2/Vδ2 T cells, would similarly promote the outgrowth of Vγ2/Vδ2 lymphocytes with longer, Jγ1.2 Vγ2 TCRs. Peripheral blood mononuclear cells (PBMC) from 6 donors were stimulated in vitro with interleukin-2 (IL2) alone, IL2 and IPP, or IL2 and irradiated (120 Gy) Daudi tumor cells. The frequency of Vγ2/Vδ2 lymphocytes increased from 5.8 ± 7.8% on Day 0 to 5.5 ± 5.9%, 34 ± 32%, 47 ± 27% after 2 weeks in culture with IL2, IL2+IPP or IL2+Daudi, respectively. RNA was extracted before and after stimulation, Vγ2 and ζδ2 chains were amplified from reverse transcribed cDNA, and spectratype analysis was performed to assess changes in the distribution of Vg2 CDR3 lengths. IPP and Daudi similarly skewed the Vγ2 repertoire toward longer chain lengths, while not affecting the overall distribution of Vδ2 chain lengths. Comparison of Vγ2 CDR3 sequences from three donors suggest that recognition of IPP and Daudi is mediated by two distinct subsets of Vγ2/Vδ2 T cells, thus overturning the prevailing model for gd T cell recognition of tumors. Collectively, these experiments help clarify the role of Vγ2 CDR3 specificity in alkylphosphate and tumor recognition and demonstrate that discrete subsets of Vγ2/Vδ2 T cells mediate alkylphosphate and tumor responsiveness. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Approximately 1–10% of circulating CD3+ cells in the blood express the gamma delta (γδ) T cell receptor (TCR) and, of these γδ T cells, the majority express the Vγ2/Vδ2 receptor. In HIV-infection, there is a targeted destruction of Vγ2-Jγ1.2/Vδ2+ T cells. This is the only TCR-specific change common to all individuals infected with HIV. Because Vγ2/Vδ2 are potently cytotoxic for tumor cells, loss of these cells may be part of the mechanism that promotes AIDs-related malignancies. Tumor recognition by γδ T cells may require a 60 kDa heat shock protein (HSP60) on Daudi Burkitt's lymphoma cells. However, HSP60 recognition may not be mediated by the TCR but by another receptor on γδ T cells. Since γδ T cells also respond to microbial infection, this additional activatory receptor may be in the toll-like family of receptors that recognize pathogen-associated molecular patterns. To explore this recognition, we treated isopentenyl pyrophosphate (IPP) expanded Vγ2/Vδ2 with the TLR2 agonist PAM3Cys and analyzed the activation of an effector phenotype by measuring IFN-γ secretion and cell killing. Daudi cell killing appears to be enhanced by the addition of the TLR2 agonist. Intracellular staining of γδ T cells after a two-hour incubation with PAM3Cys and anti-γδ TCR antibody revealed as much as a ten-fold increase in IFN-g over that produced by anti-γδ TCR antibody stimulation alone. Therefore, it seems that TLR2 does play a role in Daudi cell killing and signaling through this receptor works synergistically with TCR signaling to induce an early TH1-type immune response by IFN-γ production. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

HIV infection increases the susceptibility to new M. tuberculosis (Mtb) infections, the risk of reactivating latent infections and the risk of rapid TB progression. γδ T cells, in particular the Vγ2Jγ1.2 subset, are thought to be part of the innate immune response to both HIV and Mtb. Importantly, both HIV and Mtb perturb gd T cells homeostasis, causing a profound and highly specific depletion of the Vγ2Jγ1.2 subset.

Circulating Vgamma2/Vdelta2 T cells in human and non-human primates respond to small molecular weight non-peptidic phosphoantigens in a major histocompatibility complex (MHC)-unrestricted manner. These responses are encoded by the Vgamma2/Jgamma1.2 chain of the T-cell receptor and are positively selected during early development to create a biased repertoire in adults. We characterized the Vgamma2 chain in cynomolgus macaques (Macaca fascicularis) to develop a non-human primate model for studying the effects of infection and therapy on the circulating Vgamma2/Vdelta2 T-cell subset. The cynomolgus macaque Vgamma2 chain was highly homologous to the Vgamma2 chain from human beings and rhesus macaques (Macaca mulatta), though we noted conserved substitutions in critical residues within the CDR3 for both macaque species. Despite these substitutions, Vgamma2/Vdelta2+ T cells from cynomolgus monkeys exhibited polyclonal responses to two different phosphoantigens. Proliferative responses were observed with both isopentenylpyrophosphate and alendronate, but stronger interferon-gamma secretory responses were observed with isopentenylpyrophosphate. In vitro stimulation and expansion led to selective outgrowth of the Vgamma2/Jgamma1.2 subset, with a marked shift in the Vgamma2 spectratype. As a result of the less biased starting repertoire for Vgamma2, the cynomolgus macaque constitutes a sensitive model for examining the effects of in vitro or in vivo treatments on the Vgamma2/Vdelta2 T-cell population. Our studies establish the value of cynomolgus macaques as a model for Vgamma2/Vdelta2 T-cell responses to non-peptidic antigens, and further evidence the remarkable evolutionary conservation of this unusual, phosphoantigen-responsive T-cell subset that is found only in primate species.

Arenaviruses are transmitted from rodents to human beings by blood or mucosal exposure. The most devastating arenavirus in terms of human disease is Lassa fever virus, causing up to 300,000 annual infections in West Africa. We used a model for Lassa fever in which Rhesus macaques were infected with a related virus, lymphocytic choriomeningitis virus (LCMV). Our goals were to determine the outcome of infection after mucosal inoculation and later lethal challenge, to characterize protective immune responses, and to test cross-protection between a virulent (LCMV-WE) and an avirulent (LCMV-ARM) strain of virus. Although intravenous infections in the monkey model were uniformly lethal, intragastric infections recapitulated the spectrum of clinical outcomes seen in human exposure to Lassa fever virus: death, recovery from disease, and most often, subclinical infection. Plaque neutralization, ELISA, lymphocyte proliferation, and chromium-release assays were used to monitor humoral and cellular immune responses. Cross protection between the two strains was observed. The three out of seven monkeys that experienced protection were also the three with the strongest cell-mediated immunity.

In humans, the circulating pool of mycobacteria-reactive Vγ9Vδ2+ T cells is expanded with age and may contribute to Mycobacterium tuberculosis immunosurveillance. We observed that two subsets of Vγ9Vδ2+ T cells could be identified on the basis of CD27 expression in immunocompetent adults, showing that functionally differentiated γδ T cells have lost CD27 expression. In contrast, the CD27−CD45RA−Vγ9Vδ2+ T cell subset of effector cells was absent in cord blood cells from healthy newborns and lacking in the peripheral blood from HIV-infected patients. Moreover, circulating Vγ9Vδ2+ T cell effectors were significantly reduced in patients with acute pulmonary tuberculosis, resulting in a reduced frequency of IFN-γ-producing cells after stimulation with nonpeptidic mycobacterial ligands. These observations indicate that monitoring and boosting γδ T cell effectors could be clinically relevant both in immunocompromised hosts and during active tuberculosis disease.

CD4 T cell reconstitution in patients with HIV is associated with increased responses to conventional vaccines and improved resistance to opportunistic infections. However, prolonged therapy fails to replenish a capacity for immune control over HIV and patients who interrupt therapy inevitably rebound to pre-treatment viral burden in plasma. Only elite patients, termed NVS or natural virus suppressors (1), control viremia to undetectable levels in the absence of therapy. Our earlier studies on NVS patients showed that this group was distinguished from all other HIV patients by the presence of normal levels of CD56+ V 2V 2 T cells in blood (2) reflecting reconstitution of the T cell receptor repertoire through new cell synthesis (3). The CD56 marker identifies cytotoxic effector lymphocytes including NK, CD8, T and NKT subsets, but it’s function remains unknown. We have now shown persistent depression of CD56 expression on CD8 T cells from HIV+ individuals except for NVS patients where the levels approach those found in normal controls. The CD56+ CD8 T cell subset is highly responsive to stimulus, expresses high levels of perforin/granzyme and the small population remaining in HIV patients on therapy, also express the immune exhaustion marker TIM-3, indicating cells may be lost through an apoptosis mechanism. The persisting defect in CD56 expression for 2 T cells subsets is consistent with a lack of cytolytic effector function and is present in patients with complete virus suppression for many years due to treatment, long after the T cell population is refreshed by new cell synthesis. The block to lytic effector function may explain why treated patients fail to eradicate viral reservoirs; this mechanism is a key target for new therapies designed to cure HIV disease. 1. M. M. Sajadi, A. Heredia, N. Le, N. T. Constantine, R. R. Redfield, HIV-1 natural viral suppressors: control of viral replication in the absence of therapy. AIDS 21, 517 (2007). 2. D. J. Riedel et al. Natural viral suppressors of HIV-1 have a unique capacity to maintain gamma delta T cells. AIDS 23, 1955 (2009). 3. S. Chaudhry, C. Cairo, V. Venturi, C. D. Pauza, The gamma delta T cell receptor repertoire is reconstituted in HIV patients after prolonged antiretroviral therapy. AIDS epub ahead of print, (2013).