Consulting Associate Professor, Institute for Immunity, Transplantation and Infection
The immune system is a network of specialized cell types and tissues that communicates via cytokines and direct contact, to orchestrate specific types of defensive responses. Until recently, we could only study immune responses in a piecemeal, highly focused fashion, on major components like antibodies to the pathogen. But recent advances in technology and in our understanding of the many components of the system, innate and adaptive, have made possible a broader approach, where both the multiple responding cells and cytokines in the blood are measured. This systems immunology approach to a vaccine response or an infection gives us a more holistic picture of the different parts of the immune system that are mobilized and should allow us a much better understanding of the pathways and mechanisms of such responses, as well as to predict vaccine efficacy in different populations well in advance of efficacy studies. Here we summarize the different technologies and methods and discuss how they can inform us about the differences between diseases and vaccines, and how they can greatly accelerate vaccine development.
View details for DOI 10.1016/j.vaccine.2015.06.117
View details for Web of Science ID 000362306100006
View details for PubMedID 26232539
Cytomegalovirus (CMV) is a ?-herpesvirus present in a latent form in most people worldwide. In immunosuppressed individuals, CMV can reactivate and cause serious clinical complications, but the effect of the latent state on healthy people remains elusive. We undertook a systems approach to understand the differences between seropositive and negative subjects and measured hundreds of immune system components from blood samples including cytokines and chemokines, immune cell phenotyping, gene expression, ex vivo cell responses to cytokine stimuli, and the antibody response to seasonal influenza vaccination. As expected, we found decreased responses to vaccination and an overall down-regulation of immune components in aged individuals regardless of CMV status. In contrast, CMV-seropositive young adults exhibited enhanced antibody responses to influenza vaccination, increased CD8(+) T cell sensitivity, and elevated levels of circulating interferon-? compared to seronegative individuals. Experiments with young mice infected with murine CMV also showed significant protection from an influenza virus challenge compared with uninfected animals, although this effect declined with time. These data show that CMV and its murine equivalent can have a beneficial effect on the immune response of young, healthy individuals, which may explain the ubiquity of CMV infection in humans and many other species.
View details for DOI 10.1126/scitranslmed.aaa2293
View details for PubMedID 25834109
There is considerable heterogeneity in immunological parameters between individuals, but its sources are largely unknown. To assess the relative contribution of heritable versus non-heritable factors, we have performed a systems-level analysis of 210 healthy twins between 8 and 82 years of age. We measured 204 different parameters, including cell population frequencies, cytokine responses, and serum proteins, and found that 77% of these are dominated (>50% of variance) and 58% almost completely determined (>80% of variance) by non-heritable influences. In addition, some of these parameters become more variable with age, suggesting the cumulative influence of environmental exposure. Similarly, the serological responses to seasonal influenza vaccination are also determined largely by non-heritable factors, likely due to repeated exposure to different strains. Lastly, in MZ twins discordant for cytomegalovirus infection, more than half of all parameters are affected. These results highlight the largely reactive and adaptive nature of the immune system in healthy individuals.
View details for DOI 10.1016/j.cell.2014.12.020
View details for Web of Science ID 000347923200007
Females have generally more robust immune responses than males for reasons that are not well-understood. Here we used a systems analysis to investigate these differences by analyzing the neutralizing antibody response to a trivalent inactivated seasonal influenza vaccine (TIV) and a large number of immune system components, including serum cytokines and chemokines, blood cell subset frequencies, genome-wide gene expression, and cellular responses to diverse in vitro stimuli, in 53 females and 34 males of different ages. We found elevated antibody responses to TIV and expression of inflammatory cytokines in the serum of females compared with males regardless of age. This inflammatory profile correlated with the levels of phosphorylated STAT3 proteins in monocytes but not with the serological response to the vaccine. In contrast, using a machine learning approach, we identified a cluster of genes involved in lipid biosynthesis and previously shown to be up-regulated by testosterone that correlated with poor virus-neutralizing activity in men. Moreover, men with elevated serum testosterone levels and associated gene signatures exhibited the lowest antibody responses to TIV. These results demonstrate a strong association between androgens and genes involved in lipid metabolism, suggesting that these could be important drivers of the differences in immune responses between males and females.
View details for DOI 10.1073/pnas.1321060111
View details for Web of Science ID 000329614500068
View details for PubMedID 24367114
Despite the importance of the immune system in many diseases, there are currently no objective benchmarks of immunological health. In an effort to identifying such markers, we used influenza vaccination in 30 young (20-30 years) and 59 older subjects (60 to >89 years) as models for strong and weak immune responses, respectively, and assayed their serological responses to influenza strains as well as a wide variety of other parameters, including gene expression, antibodies to hemagglutinin peptides, serum cytokines, cell subset phenotypes and in vitro cytokine stimulation. Using machine learning, we identified nine variables that predict the antibody response with 84% accuracy. Two of these variables are involved in apoptosis, which positively associated with the response to vaccination and was confirmed to be a contributor to vaccine responsiveness in mice. The identification of these biomarkers provides new insights into what immune features may be most important for immune health.
View details for DOI 10.1038/msb.2013.15
View details for PubMedID 23591775
During seasonal influenza epidemics, disease burden is shouldered predominantly by the very young and the elderly. Elderly individuals are particularly affected, in part because vaccine efficacy wanes with age. This has been linked to a reduced ability to induce a robust serum antibody response. Here, we show that this is due to reduced quantities of vaccine-specific antibodies, rather than a lack of antibody avidity or affinity. We measured levels of vaccine-specific plasmablasts by ELISPOT 1 week after immunization of young and elderly adults with inactivated seasonal influenza vaccine. Plasmablast-derived polyclonal antibodies (PPAbs) were generated from bulk-cultured B cells, while recombinant monoclonal antibodies (re-mAbs) were produced from single plasmablasts. The frequency of vaccine-specific plasmablasts and the concentration of PPAbs were lower in the elderly than in young adults, whereas the yields of secreted IgG per plasmablast were not different. Differences were not detected in the overall vaccine-specific avidity or affinity of PPAbs and re-mAbs between the 2 age groups. In contrast, reactivity of the antibodies induced by the inactivated seasonal influenza vaccine toward the 2009 pandemic H1N1 virus, which was not present in the vaccine, was higher in the elderly than in the young. These results indicate that the inferior antibody response to influenza vaccination in the elderly is primarily due to reduced quantities of vaccine-specific antibodies. They also suggest that exposure history affects the cross-reactivity of vaccination-induced antibodies.
View details for DOI 10.1172/JCI57834
View details for Web of Science ID 000293495500022
View details for PubMedID 21785218
The immune system recognizes diverse melanoma antigens. However, tumors can evade the immune response, therefore growing and progressing. It has been reported that galectin-3 and galectin-1 can induce apoptosis of activated lymphocytes. However, there is strong evidence indicating that the regulation of galectins function in the human tumor microenvironment is a complex process that is influenced by diverse biological circumstances. Here, we have investigated 33 biopsies (eight primary and 25 metastases) from 24 melanoma patients (15-72 years old) and describe the correlation between the expression of galectin-3 or galectin-1 and the level of apoptosis of tumor-associated lymphocytes using immunohistochemistry and an in situ nick translation assay. The range of galectin-3-positive tumor cells varied between 0% and 93% and that of galectin-1-positive tumor cells varied between 5% and 97%. In addition, 23 +/- 27% of tumor-associated lymphocytes were apoptotic. Although our results show a correlation between galectin-3 expression and apoptosis of tumor-associated lymphocytes, we could not find such correlation with galectin-1. Considering the complex process of cancer immunoediting, various interacting factors must be considered.
View details for DOI 10.2353/ajpath.2006.050971
View details for Web of Science ID 000237325700025
View details for PubMedID 16651632
Common variable immune deficiency (CVID) is the most common symptomatic primary immune deficiency, affecting ~1 in 25,000 persons. These patients suffer from impaired antibody responses, autoimmunity, and susceptibility to lymphoid cancers. To explore the cellular basis for these clinical phenotypes, we conducted high-throughput DNA sequencing of immunoglobulin heavy chain gene rearrangements from 93 CVID patients and 105 control subjects and sorted naïve and memory B cells from 13 of the CVID patients and 10 of the control subjects. The CVID patients showed abnormal VDJ rearrangement and abnormal formation of complementarity-determining region 3 (CDR3). We observed a decreased selection against antibodies with long CDR3s in memory repertoires and decreased variable gene replacement, offering possible mechanisms for increased patient autoreactivity. Our data indicate that patient immunodeficiency might derive from both decreased diversity of the naïve B cell pool and decreased somatic hypermutation in memory repertoires. The CVID patients also exhibited an abnormal clonal expansion of unmutated B cells relative to the controls. Although impaired B cell germinal center activation is commonly viewed as causative in CVID, these data indicate that CVID B cells diverge from controls as early as the pro-B stage, cell and suggest possible explanations for the increased incidence of autoimmunity, immunodeficiency, and lymphoma CVID patients.
View details for DOI 10.1126/scitranslmed.aab1216
View details for PubMedID 26311730
This study sought to determine whether a simple score combining indexes of right ventricular (RV) function and right atrial (RA) size would offer good discrimination of outcome in patients with pulmonary arterial hypertension (PAH).Identifying a simple score of outcome could simplify risk stratification of patients with PAH and potentially lead to improved tailored monitoring or therapy.We recruited patients from both Stanford University (derivation cohort) and VU University Medical Center (validation cohort). The composite endpoint for the study was death or lung transplantation. A Cox proportional hazard with bootstrap CI adjustment model was used to determine independent correlates of death or transplantation. A predictive score was developed using the beta coefficients of the multivariable models.For the derivation cohort (n = 95), the majority of patients were female (79%), average age was 43 ± 11 years, mean pulmonary arterial pressure was 54 ± 14 mm Hg, and pulmonary vascular resistance index was 25 ± 12 Wood units m(2). Over an average follow-up of 5 years, the composite endpoint occurred in 34 patients, including 26 deaths and 8 patients requiring lung transplant. On multivariable analysis, RV systolic dysfunction grade (hazard ratio [HR]: 3.4 per grade; 95% confidence interval [CI]: 2.0 to 7.8; p < 0.001), severe RA enlargement (HR: 3.0; 95% CI: 1.3 to 8.1; p = 0.009), and systemic blood pressure <110 mm Hg (HR: 3.3; 95% CI: 1.5 to 9.4; p < 0.001) were independently associated with outcome. A right heart (RH) score constructed on the basis of these 3 parameters compared favorably with the National Institutes of Health survival equation (0.88; 95% CI: 0.79 to 0.94 vs. 0.60; 95% CI: 0.49 to 0.71; p < 0.001) but was not statistically different than the REVEAL (Registry to Evaluate Early and Long-Term PAH Disease Management) score c-statistic of 0.80 (95% CI: 0.69 to 0.88) with p = 0.097. In the validation cohort (n = 87), the RH score remained the strongest independent correlate of outcome.In patients with prevalent PAH, a simple RH score may offer good discrimination of long-term outcome.
View details for DOI 10.1016/j.jcmg.2014.12.029
View details for PubMedID 25981508
Weaker immune responses are often observed in males compared to females. Since female hormones have proinflammatory properties and androgens have potent immunomodulatory effects, this sexual dimorphism in the immune response seems to be hormone dependent. Despite our current knowledge about the effect of sex hormones on immune cells, definition of the factors driving the sex differences in immunoclinical outcomes, such as the diminished response to infection and vaccination observed in men or the higher rates of autoimmunity observed in females, remains elusive. Recently, systems approaches to immune function have started to suggest a way toward establishing this connection. Such studies promise to improve our understanding of the mechanisms underlying the sexual dimorphism observed in the human immune system.
View details for DOI 10.1586/14760584.2015.966694
View details for Web of Science ID 000350052800012
View details for PubMedID 25278153
A study of genome-wide gene expression in major depressive disorder (MDD) was undertaken in a large population-based sample to determine whether altered expression levels of genes and pathways could provide insights into biological mechanisms that are relevant to this disorder. Gene expression studies have the potential to detect changes that may be because of differences in common or rare genomic sequence variation, environmental factors or their interaction. We recruited a European ancestry sample of 463 individuals with recurrent MDD and 459 controls, obtained self-report and semi-structured interview data about psychiatric and medical history and other environmental variables, sequenced RNA from whole blood and genotyped a genome-wide panel of common single-nucleotide polymorphisms. We used analytical methods to identify MDD-related genes and pathways using all of these sources of information. In analyses of association between MDD and expression levels of 13?857 single autosomal genes, accounting for multiple technical, physiological and environmental covariates, a significant excess of low P-values was observed, but there was no significant single-gene association after genome-wide correction. Pathway-based analyses of expression data detected significant association of MDD with increased expression of genes in the interferon ?/? signaling pathway. This finding could not be explained by potentially confounding diseases and medications (including antidepressants) or by computationally estimated proportions of white blood cell types. Although cause-effect relationships cannot be determined from these data, the results support the hypothesis that altered immune signaling has a role in the pathogenesis, manifestation, and/or the persistence and progression of MDD.Molecular Psychiatry advance online publication, 3 December 2013; doi:10.1038/mp.2013.161.
View details for DOI 10.1038/mp.2013.161
View details for PubMedID 24296977
Generalized immune activation during HIV infection is associated with an increased risk of cardiovascular disease, neurocognitive disease, osteoporosis, metabolic disorders, and physical frailty. The mechanisms driving this immune activation are poorly understood, particularly in individuals effectively treated with antiretroviral medications. We hypothesized that viral characteristics such as sequence diversity may play a role in driving HIV-associated immune activation. We therefore sequenced proviral DNA isolated from peripheral blood mononuclear cells from HIV-infected individuals on fully suppressive antiretroviral therapy. We performed phylogenetic analyses, calculated viral diversity and divergence in the env and pol genes, and determined coreceptor tropism and the frequency of drug resistance mutations. Comprehensive immune profiling included quantification of immune cell subsets, plasma cytokine levels, and intracellular signaling responses in T cells, B cells, and monocytes. These antiretroviral-treated HIV-infected individuals exhibited a wide range of diversity and divergence in both env and pol genes. However, proviral diversity and divergence in env and pol, coreceptor tropism, and the level of drug resistance did not significantly correlate with markers of immune activation. A clinical history of virologic failure was also not significantly associated with levels of immune activation, indicating that a past history of virologic failure does not inexorably lead to increased immune activation as long as suppressive antiretroviral medications are provided. Overall, this study demonstrates that latent viral diversity is unlikely to be a major driver of persistent HIV-associated immune activation.Chronic immune activation, which is associated with cardiovascular disease, neurologic disease, and early aging, is likely to be a major driver of morbidity and mortality in HIV-infected individuals. Although treatment of HIV with antiretroviral medications decreases the level of immune activation, levels do not return to normal. The factors driving this persistent immune activation, particularly during effective treatment, are poorly understood. In this study, we investigated whether characteristics of the latent, integrated HIV provirus that persists during treatment are associated with immune activation. We found no relationship between latent viral characteristics and immune activation in treated individuals, indicating that qualities of the provirus are unlikely to be a major driver of persistent inflammation. We also found that individuals who had previously failed treatment, but were currently effectively treated, did not have significantly increased levels of immune activation, providing hope that past treatment failures do not have a lifelong "legacy" impact.
View details for DOI 10.1128/JVI.01257-14
View details for Web of Science ID 000338924400037
Immunosenescence, defined as the age-associated dysregulation and dysfunction of the immune system, is characterized by impaired protective immunity and decreased efficacy of vaccines. An increasing number of immunological, clinical and epidemiological studies suggest that persistent Cytomegalovirus (CMV) infection is associated with accelerated aging of the immune system and with several age-related diseases. However, current evidence on whether and how human CMV (HCMV) infection is implicated in immunosenescence and in age-related diseases remains incomplete and many aspects of CMV involvement in immune aging remain controversial. The attendees of the 4th International Workshop on "CMV & Immunosenescence", held in Parma, Italy, 25-27th March, 2013, presented and discussed data related to these open questions, which are reported in this commentary.
View details for DOI 10.1016/j.exger.2014.03.020
View details for Web of Science ID 000335929300008
View details for PubMedID 24703889
Elderly humans show decreased humoral immunity to pathogens and vaccines, yet the effects of aging on B cells are not fully known. Chronic viral infection by CMV is implicated as a driver of clonal T cell proliferations in some aging humans, but whether CMV or EBV infection contributes to alterations in the B cell repertoire with age is unclear. We have used high-throughput DNA sequencing of IGH gene rearrangements to study the BCR repertoires over two successive years in 27 individuals ranging in age from 20 to 89 y. Some features of the B cell repertoire remain stable with age, but elderly subjects show increased numbers of B cells with long CDR3 regions, a trend toward accumulation of more highly mutated IgM and IgG Ig genes, and persistent clonal B cell populations in the blood. Seropositivity for CMV or EBV infection alters B cell repertoires, regardless of the individual's age: EBV infection correlates with the presence of persistent clonal B cell expansions, whereas CMV infection correlates with the proportion of highly mutated Ab genes. These findings isolate effects of aging from those of chronic viral infection on B cell repertoires and provide a baseline for understanding human B cell responses to vaccination or infectious stimuli.
View details for DOI 10.4049/jimmunol.1301384
View details for Web of Science ID 000329224000006
System-wide approaches are now being applied to study vaccine responses, whose mechanisms of action, and failure, are not well understood. These works have repeatedly shown vaccine response to be an orchestrated process involving multiple arms of immunity most noticeable sensing and innate components. Prediction of vaccine responses based on system-wide measures is achievable, but challenges remain for robust population wide predictions based only on pre-vaccination measures, especially in partially efficacious vaccines such as influenza. This is especially true in older adults, who are often less responsive to vaccination and exhibit high level of variation compared to young in many components of immunity. Despite this increase in variation, most of the studies on aging use group averages of immune phenotypes to model immune system behavior. Using systems approaches, it is possible to exploit this variation to form distinguishable clusters of phenotypes within and across individuals to discover underlying immune states.
View details for DOI 10.1016/j.coi.2013.07.009
View details for Web of Science ID 000325237500019
View details for PubMedID 23953808
Advances in technology and data analysis have made it possible to take a new look at human immunology. These advances run the gamut from systems biology approaches, which are likely in the vanguard of how we can start "to put the pieces together" of immune function, to a deeper understanding of specific diseases and vaccines and the immune repertoire. In our own experience, we have also found that asking simple questions about human immunity has often given us very surprising answers, causing a rethink of established dogma. Thus, we have developed a new perspective on the nature of the ?? TCR repertoire and also the likely role of T-cell repertoire (TCR) cross-reactivity in generating T memory independent of specific antigen interactions. These findings show that human immunology is not just a necessary step for "translating" basic immunology to treat diseases or develop better vaccines, but is also an important complement to the inbred mouse model.
View details for DOI 10.1101/sqb.2013.78.022905
View details for PubMedID 24638855
HLA-E is a non-classical MHC molecule whose expression by tumour cells has been recently reported in several human cancer types. We studied HLA-E expression in colorectal cancer patients, its clinical significance and prognostic value, as well as characterized its expression in colorectal cancer cell lines. We analysed HLA-E expression at the transcript level by qRT-PCR in micro-dissected samples and at the protein level by semiquantitative immunohistochemistry on paraffin-embedded tissue sections from 42 biopsies of colorectal cancer patients. We observed that HLA-E transcript and protein are spontaneously overexpressed in a significant proportion of colorectal tumour biopsies, as compared to normal mucosae. We also found a negative correlation between HLA-E expression and the CD57+ cells infiltrate. Moreover, we analysed HLA-E expression in several colorectal cancer cell lines and demonstrated that IFN-gamma upregulates the expression of membrane HLA-E in vitro. Interestingly, we demonstrated that colorectal cancer cell lines overexpressing HLA-E at the cell surface inhibited NK-mediated cell lysis. Although IFN-gamma regulatory role needs further investigation, we provide evidence suggesting that this cytokine, within the tumour microenvironment, could promote HLA-E translocation to the surface of tumour epithelial cells. Furthermore, we showed that upregulation of HLA-E could be a marker of shorter disease-free survival in Dukes' C patients and we suggest that this molecule renders tumours less susceptible to immune attack.
View details for Web of Science ID 000253507600014
View details for PubMedID 18292941
Sixteen melanoma patients (1 stage IIC, 8 stage III, and 7 stage IV) were treated in a Phase I study with a vaccine (DC/Apo-Nec) composed of autologous dendritic cells (DCs) loaded with a mixture of apoptotic/necrotic allogeneic melanoma cell lines (Apo-Nec), to evaluate toxicity and immune responses. Also, IL-10 1082 genotype was analyzed in an effort to predict disease progression.PBMC were obtained after leukapheresis and DCs were generated from monocytes cultured in the presence of GM-CSF and IL-4 in serum-free medium. Immature DCs were loaded with gamma-irradiated Apo-Nec cells and injected id without adjuvant. Cohorts of four patients were given four vaccines each with 5, 10, 15, or 20 x 106 DC/Apo-Nec cell per vaccine, two weeks apart. Immune responses were measured by ELISpot and tetramer analysis. Il-10 genotype was measured by PCR and corroborated by IL-10 production by stimulated PBMC.Immature DCs efficiently phagocytosed melanoma Apo-Nec cells and matured after phagocytosis as evidenced by increased expression of CD83, CD80, CD86, HLA class I and II, and 75.2 +/- 16% reduction in Dextran-FITC endocytosis. CCR7 was also up-regulated upon Apo-Nec uptake in DCs from all patients, and accordingly DC/Apo-Nec cells were able to migrate in vitro toward MIP-3 beta. The vaccine was well tolerated in all patients. The DTH score increased significantly in all patients after the first vaccination (Mann-Whitney Test, p < 0.05). The presence of CD8+T lymphocytes specific to gp100 and Melan A/MART-1 Ags was determined by ELISpot and tetramer analysis in five HLA-A*0201 patients before and after vaccination; one patient had stable elevated levels before and after vaccination; two increased their CD8 + levels, one had stable moderate and one had negligible levels. The analysis of IL-10 promoter -1082 polymorphism in the sixteen patients showed a positive correlation between AA genotype, accompanied by lower in vitro IL-10 production by stimulated PBMC, and faster melanoma progression after lymph nodes surgery (p = 0.04). With a mean follow-up of 49.5 months post-surgery, one stage IIC patient and 7/8 stage III patients remain NED but 7/7 stage IV patients have progressed.We conclude that DC/Apo-Nec vaccine is safe, well tolerated and it may induce specific immunity against melanoma Ags. Patients with a low-producing IL-10 polymorphism appear to have a worst prognosis.Clinicaltrials.gov (NHI) NCT00515983.
View details for DOI 10.1186/1479-5876-6-6
View details for Web of Science ID 000253863300001
View details for PubMedID 18221542
In the present study, we demonstrate, in rigorous fashion, that human monocyte-derived immature dendritic cells (DCs) can efficiently cross-present tumor-associated antigens when co-cultured with a mixture of human melanoma cells rendered apoptotic/necrotic by gamma irradiation (Apo-Nec cells).We evaluated the phagocytosis of Apo-Nec cells by FACS after PKH26 and PKH67 staining of DCs and Apo-Nec cells at different times of coculture. The kinetics of the process was also followed by electron microscopy. DCs maturation was also studied monitoring the expression of specific markers, migration towards specific chemokines and the ability to cross-present in vitro the native melanoma-associated Ags MelanA/MART-1 and gp100.Apo-Nec cells were efficiently phagocytosed by immature DCs (iDC) (55 +/- 10.5%) at 12 hs of coculture. By 12-24 hs we observed digested Apo-Nec cells inside DCs and large empty vacuoles as part of the cellular processing. Loading with Apo-Nec cells induced DCs maturation to levels achieved using LPS treatment, as measured by: i) the decrease in FITC-Dextran uptake (iDC: 81 +/- 5%; DC/Apo-Nec 33 +/- 12%); ii) the cell surface up-regulation of CD80, CD86, CD83, CCR7, CD40, HLA-I and HLA-II and iii) an increased in vitro migration towards MIP-3beta. DC/Apo-Nec isolated from HLA-A*0201 donors were able to induce >600 pg/ml IFN-gamma secretion of CTL clones specific for MelanA/MART-1 and gp100 Ags after 6 hs and up to 48 hs of coculture, demonstrating efficient cross-presentation of the native Ags. Intracellular IL-12 was detected in DC/Apo-Nec 24 hs post-coculture while IL-10 did not change.We conclude that the use of a mixture of four apoptotic/necrotic melanoma cell lines is a suitable source of native melanoma Ags that provides maturation signals for DCs, increases migration to MIP-3beta and allows Ag cross-presentation. This strategy could be exploited for vaccination of melanoma patients.
View details for DOI 10.1186/1479-5876-5-19
View details for Web of Science ID 000246389600001
View details for PubMedID 17448240