Fetal programming and lactation: modulating gene expression in response to undernutrition during intrauterine life.

BACKGROUND: Adverse environmental conditions during intrauterine life, known as fetal programming, significantly contribute to the development of diseases in adulthood. Fetal programming induced by factors like maternal undernutrition leads to low birth weight and increases the risk of cardiometabolic diseases. METHODS: We studied a rat model of maternal undernutrition during gestation (MUN) to investigate gene expression changes in cardiac tissue using RNA-sequencing of day 0-1 litters. Moreover, we analyzed the impact of lactation at day 21, in MUN model and cross-fostering experiments, on cardiac structure and function assessed by transthoracic echocardiography, and gene expression changes though qPCR. RESULTS: Our analysis identified specific genes with altered expression in MUN rats at birth. Two of them, Agt and Pparg, stand out for being associated with cardiac hypertrophy and fibrosis. At the end of the lactation period, MUN males showed increased expression of Agt and decreased expression of Pparg, correlating with cardiac hypertrophy. Cross-fostering experiments revealed that lactation with control breastmilk mitigated these expression changes reducing cardiac hypertrophy in MUN males. CONCLUSIONS: Our findings highlight the interplay between fetal programming, gene expression, and cardiac hypertrophy suggesting that lactation period is a potential intervention window to mitigate the effects of fetal programming. IMPACT: Heart remodeling involves the alteration of several groups of genes and lactation period plays a key role in establishing gene expression modification caused by fetal programming. We could identify expression changes of relevant genes in cardiac tissue induced by undernutrition during fetal life. We expose the contribution of the lactation period in modulating the expression of Agt and Pparg, relevant genes associated with cardiac hypertrophy. This evidence reveal lactation as a crucial intervention window for preventing or countering fetal programming.

IL-18-induced HIF-1α in ILC3s ameliorates the inflammation of C. rodentium-induced colitis.

Group 3 innate lymphoid cells (ILC3s) are vital for defending tissue barriers from invading pathogens. Hypoxia influences the production of intestinal ILC3-derived cytokines by activating HIF. Yet, the mechanisms governing HIF-1α in ILC3s and other innate RORγt+ cells during in vivo infections are poorly understood. In our study, transgenic mice with specific Hif-1a gene inactivation in innate RORγt+ cells (RAG1KO HIF-1α▵Rorc) exhibit more severe colitis following Citrobacter rodentium infection, primarily due to the inability to upregulate IL-22. We find that HIF-1α▵Rorc mice have impaired IL-22 production in ILC3s, while non-ILC3 innate RORγt+ cells, also capable of producing IL-22, remain unaffected. Furthermore, we show that IL-18, induced by Toll-like receptor 2, selectively triggers IL-22 in ILC3s by transcriptionally upregulating HIF-1α, revealing an oxygen-independent regulatory pathway. Our results highlight that, during late-stage C. rodentium infection, IL-18 induction in the colon promotes IL-22 through HIF-1α in ILC3s, which is crucial for protection against this pathogen.

A melting pot of Roman dogs north of the Alps with high phenotypic and genetic diversity and similar diets.

Several dog skeletons were excavated at the Roman town of Augusta Raurica and at the military camp of Vindonissa, located in the northern Alpine region of Switzerland (Germania Superior). The relationships between them and the people, the nature of their lives, and the circumstances of their deaths are unclear. In order to gain insight into this dog population, we collected 31 dogs deposited almost simultaneously in two wells (second half of the third century CE), three dogs from burial contexts (70-200 CE and third to fifth century CE) at Augusta Raurica, and two dogs from burial contexts at Vindonissa (ca. first century CE). We detected a mixed population of young and adult dogs including small, medium and large sized individuals. Three small dogs had conspicuous phenotypes: abnormally short legs, and one with a brachycephalic skull. Stable isotope analysis of a subset of the dogs showed that their diets were omnivorous with a substantial input of animal proteins and little variation, except one with a particularly low δ15N value, indicating a diet low in animal proteins. Partial mitochondrial DNA sequences from 25 dogs revealed eight haplotypes within canine haplogroup A (11 dogs; 44%; 5 haplotypes), C (8 dogs; 32%; 1 haplotype), D (4 dogs, 16%; 1 haplotype) and B (2 dogs, 8%; 1 haplotype). Based on shotgun sequencing, four Roman mitogenomes were assembled, representing sub-haplogroups A1b3, A1b2 and C2. No canine pathogens were identified, weakening the assumption of infectious disease as a cause for dog disposal. The genetic and morphological diversity observed in dogs of Augusta Raurica and Vindonissa is similar to modern dog diversity.

Nuclear envelope disruption triggers hallmarks of aging in lung alveolar macrophages.

Aging is characterized by gradual immune dysfunction and increased disease risk. Genomic instability is considered central to the aging process, but the underlying mechanisms of DNA damage are insufficiently defined. Cells in confined environments experience forces applied to their nucleus, leading to transient nuclear envelope rupture (NER) and DNA damage. Here, we show that Lamin A/C protects lung alveolar macrophages (AMs) from NER and hallmarks of aging. AMs move within constricted spaces in the lung. Immune-specific ablation of lamin A/C results in selective depletion of AMs and heightened susceptibility to influenza virus-induced pathogenesis and lung cancer growth. Lamin A/C-deficient AMs that persist display constitutive NER marks, DNA damage and p53-dependent senescence. AMs from aged wild-type and from lamin A/C-deficient mice share a lysosomal signature comprising CD63. CD63 is required to limit damaged DNA in macrophages. We propose that NER-induced genomic instability represents a mechanism of aging in AMs.

Bacteria-instructed B cells cross-prime naïve CD8+ T cells triggering effective cytotoxic responses.

In addition to triggering humoral responses, conventional B cells have been described in vitro to cross-present exogenous antigens activating naïve CD8+ T cells. Nevertheless, the way B cells capture these exogenous antigens and the physiological roles of B cell-mediated cross-presentation remain poorly explored. Here, we show that B cells capture bacteria by trans-phagocytosis from previously infected dendritic cells (DC) when they are in close contact. Bacterial encounter "instructs" the B cells to acquire antigen cross-presentation abilities, in a process that involves autophagy. Bacteria-instructed B cells, henceforth referred to as BacB cells, rapidly degrade phagocytosed bacteria, process bacterial antigens and cross-prime naïve CD8+ T cells which differentiate into specific cytotoxic cells that efficiently control bacterial infections. Moreover, a proof-of-concept experiment shows that BacB cells that have captured bacteria expressing tumor antigens could be useful as novel cellular immunotherapies against cancer.

[Epidemiology of leptospirosis in the wetlands of Southern Spain]. / Epidemiología de la leptospirosis en los humedales del sur de España.

OBJECTIVE: To find out whether the leptospirosis incidence rate among red swamp crayfish collectors in the harvesting season is higher than in the general population, and to identify risk factors and assess the direct and indirect health costs associated with leptospirosis seroconversion. METHOD: This study was carried out between 1 July 2017 and 31 March 2018 in the municipality of Isla Mayor (Seville, Spain). It took the form of a prospective cohort study (exposed population: swamp crayfish collectors; non-exposed population: general population). The population was invited to take part in a prevalence study to be conducted using the ELISA qualitative technique, and informed consent was obtained from those who agreed. Negative serology cases were then included in the cohort study. Both cohorts were monitored clinically and symptomatic cases were serology tested. A second serum sample was taken from the swamp crayfish collectors at the end of the monitoring period to detect asymptomatic cases. Serovars were confirmed by microscopic agglutination testing. A bivariate descriptive analysis was carried out and cumulative incidence and relative risk were calculated, with positive serology being taken as the dependent variable. RESULTS: A total of 278 people were included in the study, of whom 92 made up the swamp crayfish collectors cohort and 186 the general population cohort. Women made up 46.8% of the sample, but only 29.3% of the collectors cohort. The mean age was 45.1 (±16.4) years. Nine cases of seroconversion were detected: eight among swamp crayfish collectors and one in the general population. Overall cumulative incidence was therefore 3.2%: 8.7% in the exposed group and 0.5% in the non-exposed group. Relative risk was 16.2% (95% confidence interval: 2.1-127.4). The total cost of medical assistance and illness-related losses associated with leptospirosis was 1568€/case. CONCLUSIONS: Leptospirosis in Isla Mayor is strongly associated with red swamp crayfish collecting. It's incidence here is much higher than that reported in studies published in other countries.

CD4 T-Cell Subsets and the Pathophysiology of Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is an umbrella term for the chronic immune-mediated idiopathic inflammation of the gastrointestinal tract, manifesting as Crohn's disease (CD) or ulcerative colitis (UC). IBD is characterized by exacerbated innate and adaptive immunity in the gut in association with microbiota dysbiosis and the disruption of the intestinal barrier, resulting in increased bacterial exposure. In response to signals from microorganisms and damaged tissue, innate immune cells produce inflammatory cytokines and factors that stimulate T and B cells of the adaptive immune system, and a prominent characteristic of IBD patients is the accumulation of inflammatory T-cells and their proinflammatory-associated cytokines in intestinal tissue. Upon antigen recognition and activation, CD4 T-cells differentiate towards a range of distinct phenotypes: T helper(h)1, Th2, Th9, Th17, Th22, T follicular helper (Tfh), and several types of T-regulatory cells (Treg). T-cells are generated according to and adapt to microenvironmental conditions and participate in a complex network of interactions among other immune cells that modulate the further progression of IBD. This review examines the role of the CD4 T-cells most relevant to IBD, highlighting how these cells adapt to the environment and interact with other cell populations to promote or inhibit the development of IBD.

Pathophysiology of Inflammatory Bowel Disease: Innate Immune System.

Inflammatory bowel disease (IBD), comprising Crohn's disease (CD) and ulcerative colitis (UC), is a heterogeneous state of chronic intestinal inflammation with no exact known cause. Intestinal innate immunity is enacted by neutrophils, monocytes, macrophages, and dendritic cells (DCs), and innate lymphoid cells and NK cells, characterized by their capacity to produce a rapid and nonspecific reaction as a first-line response. Innate immune cells (IIC) defend against pathogens and excessive entry of intestinal microorganisms, while preserving immune tolerance to resident intestinal microbiota. Changes to this equilibrium are linked to intestinal inflammation in the gut and IBD. IICs mediate host defense responses, inflammation, and tissue healing by producing cytokines and chemokines, activating the complement cascade and phagocytosis, or presenting antigens to activate the adaptive immune response. IICs exert important functions that promote or ameliorate the cellular and molecular mechanisms that underlie and sustain IBD. A comprehensive understanding of the mechanisms underlying these clinical manifestations will be important for developing therapies targeting the innate immune system in IBD patients. This review examines the complex roles of and interactions among IICs, and their interactions with other immune and non-immune cells in homeostasis and pathological conditions.

Analysis of Monocyte Recruitment During Inflammation by Intravital Imaging.

Monocytes play essential roles in the inflammatory and anti-inflammatory processes that take place during an immune response, acting both within the vascular network and interstitially. Monocytes are activated, mobilized, and recruited in response to an inflammatory stimulus or different forms of tissue injury. The recruitment of circulating monocytes to the inflamed tissue is essential to resolving the injury.Monocyte recruitment is a multistep process that begins with a decrease in rolling velocity, is followed by adhesion to the endothelium and crawling over the luminal vessel surface, and culminates in monocyte transmigration into the surrounding tissue. Intravital microscopy is a powerful visualization tool for the study of leukocyte behavior and function, intercellular interactions, cell trafficking, and recruitment in pathological and physiological conditions. This modality is therefore widely used for the detailed analysis of the immune response to multiple insults and the molecular mechanisms underlying monocyte interactions within the vascular system in vivo. This chapter describes a protocol for the use of intravital microscopy to analyze monocyte recruitment from the blood vessel to the inflammatory site.

Epidemiología de la leptospirosis en los humedales del sur de España / Epidemiology of leptospirosis in the wetlands of Southern Spain

Objetivo: Conocer si la incidencia de leptospirosis en los capturadores de cangrejo rojo durante el periodo de captura es superior a la de la población general, así como identificar factores de riesgo y estimar los costes sanitarios directos e indirectos asociados a los casos de seroconversión de dicha enfermedad.Método: Este estudio se realizó desde el 1 de julio de 2017 hasta el 31 de marzo de 2018. Se llevó a cabo un estudio de cohortes prospectivo (población expuesta: capturadores de cangrejo rojo; población no expuesta: población general) en el municipio de Isla Mayor (Sevilla, España). Previo consentimiento informado, se invitó a la población al estudio de prevalencia mediante la técnica cualitativa ELISA. Los que tuvieron serología negativa fueron incluidos en el estudio de cohortes. Ambas cohortes se siguieron clínicamente y a los casos sintomáticos se les realizó serología. A los capturadores de cangrejo rojo se les tomó una segunda muestra de suero al final del seguimiento para detectar asintomáticos. La serovariedad se confirmó mediante aglutinación microscópica. Se realizó un análisis descriptivo bivariado y se calcularon la incidencia acumulada y el riesgo relativo. La serología positiva se tomó como variable dependiente.Resultados:Se incluyeron en el estudio 278 personas, de las que 92 constituían la cohorte de capturadores y 186 la de población general. El 46,8% de la muestra eran mujeres, aunque entre los capturadores de cangrejo rojo estas solo representaban el 29,3%. La edad media de la muestra fue de 45,1 (± 16,4) años. Se detectaron nueve seroconversiones: ocho en capturadores de cangrejo rojo y una en población general. Por lo tanto, la incidencia acumulada fue de 8,7% en capturadores de cangrejo rojo y de 0,5% en población general, siendo el riesgo relativo de 16,2 (intervalo de confianza del 95%: 2,1-127,4). El coste total de la asistencia sanitaria y de las pérdidas por enfermedad asociadas a la leptospirosis fue de 1568 € por caso(AU)

Objective: To find out whether the leptospirosis incidence rate among red swamp crayfish collectors in the harvesting season is higher than in the general population, and to identify risk factors and assess the direct and indirect health costs associated with leptospirosis seroconversion. Method: This study was carried out between 1 July 2017 and 31 March 2018 in the municipality of Isla Mayor (Seville, Spain). It took the form of a prospective cohort study (exposed population: swamp crayfish collectors; non-exposed population: general population). The population was invited to take part in a prevalence study to be conducted using the ELISA qualitative technique, and informed consent was obtained from those who agreed. Negative serology cases were then included in the cohort study. Both cohorts were monitored clinically and symptomatic cases were serology tested. A second serum sample was taken from the swamp crayfish collectors at the end of the monitoring period to detect asymptomatic cases. Serovars were confirmed by microscopic agglutination testing. A bivariate descriptive analysis was carried out and cumulative incidence and relative risk were calculated, with positive serology being taken as the dependent variable. Results: A total of 278 people were included in the study, of whom 92 made up the swamp crayfish collectors cohort and 186 the general population cohort. Women made up 46.8% of the sample, but only 29.3% of the collectors cohort. The mean age was 45.1 (±16.4) years. Nine cases of seroconversion were detected: eight among swamp crayfish collectors and one in the general population. Overall cumulative incidence was therefore 3.2%: 8.7% in the exposed group and 0.5% in the non-exposed group. Relative risk was 16.2% (95% confidence interval: 2.1-127.4). The total cost of medical assistance and illness-related losses associated with leptospirosis was 1568€/case. (AU)

Recent Advances in Intermediate Filaments-Volume 1.

We would like to make readers of the second edition of the Special Issue from the International Journal of Molecular Sciences on the Recent Advances in Intermediate Filaments aware of the content of the first edition on this same topic [...].

ß2-Integrin Adhesion Regulates Dendritic Cell Epigenetic and Transcriptional Landscapes to Restrict Dendritic Cell Maturation and Tumor Rejection.

Dendritic cells (DC), the classic antigen-presenting cells of the immune system, switch from an adhesive, phagocytic phenotype in tissues, to a mature, nonadhesive phenotype that enables migration to lymph nodes to activate T cells and initiate antitumor responses. Monocyte-derived DCs are used in cancer immunotherapy, but their clinical efficacy is limited. Here, we show that cultured bone marrow-derived DCs (BM-DC) expressing dysfunctional ß2-integrin adhesion receptors displayed enhanced tumor rejection capabilities in B16.OVA and B16-F10 melanoma models. This was associated with an increased CD8+ T-cell response. BM-DCs expressing dysfunctional ß2-integrins or manipulated to disrupt integrin adhesion or integrin/actin/nuclear linkages displayed spontaneous maturation in ex vivo cultures (increased costimulatory marker expression, IL12 production, and 3D migration capabilities). This spontaneous maturation was associated with an altered DC epigenetic/transcriptional profile, including a global increase in chromatin accessibility and H3K4me3/H3K27me3 histone methylation. Genome-wide analyses showed that H3K4me3 methylation was increased on DC maturation genes, such as CD86, Il12, Ccr7, and Fscn1, and revealed a role for a transcription factor network involving Ikaros and RelA in the integrin-regulated phenotype of DCs. Manipulation of the integrin-regulated epigenetic landscape in wild-type ex vivo-cultured BM-DCs enhanced their functionality in tumor rejection in vivo. Thus, ß2-integrin-mediated adhesion to the extracellular environment plays an important role in restricting DC maturation and antitumor responses through regulation of the cellular epigenetic and transcriptional landscape. Targeting ß2-integrins could therefore be a new strategy to improve the performance of current DC-based cancer immunotherapies.

Innate Lymphoid Cells in Intestinal Homeostasis and Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a heterogeneous state of chronic intestinal inflammation of unknown cause encompassing Crohn's disease (CD) and ulcerative colitis (UC). IBD has been linked to genetic and environmental factors, microbiota dysbiosis, exacerbated innate and adaptive immunity and epithelial intestinal barrier dysfunction. IBD is classically associated with gut accumulation of proinflammatory Th1 and Th17 cells accompanied by insufficient Treg numbers and Tr1 immune suppression. Inflammatory T cells guide innate cells to perpetuate a constant hypersensitivity to microbial antigens, tissue injury and chronic intestinal inflammation. Recent studies of intestinal mucosal homeostasis and IBD suggest involvement of innate lymphoid cells (ILCs). These lymphoid-origin cells are innate counterparts of T cells but lack the antigen receptors expressed on B and T cells. ILCs play important roles in the first line of antimicrobial defense and contribute to organ development, tissue protection and regeneration, and mucosal homeostasis by maintaining the balance between antipathogen immunity and commensal tolerance. Intestinal homeostasis requires strict regulation of the quantity and activity of local ILC subpopulations. Recent studies demonstrated that changes to ILCs during IBD contribute to disease development. A better understanding of ILC behavior in gastrointestinal homeostasis and inflammation will provide valuable insights into new approaches to IBD treatment. This review summarizes recent research into ILCs in intestinal homeostasis and the latest advances in the understanding of the role of ILCs in IBD, with particular emphasis on the interaction between microbiota and ILC populations and functions.

Lamin A/C and the Immune System: One Intermediate Filament, Many Faces.

Nuclear envelope lamin A/C proteins are a major component of the mammalian nuclear lamina, a dense fibrous protein meshwork located in the nuclear interior. Lamin A/C proteins regulate nuclear mechanics and structure and control cellular signaling, gene transcription, epigenetic regulation, cell cycle progression, cell differentiation, and cell migration. The immune system is composed of the innate and adaptive branches. Innate immunity is mediated by myeloid cells such as neutrophils, macrophages, and dendritic cells. These cells produce a rapid and nonspecific response through phagocytosis, cytokine production, and complement activation, as well as activating adaptive immunity. Specific adaptive immunity is activated by antigen presentation by antigen presenting cells (APCs) and the cytokine microenvironment, and is mainly mediated by the cellular functions of T cells and the production of antibodies by B cells. Unlike most cell types, immune cells regulate their lamin A/C protein expression relatively rapidly to exert their functions, with expression increasing in macrophages, reducing in neutrophils, and increasing transiently in T cells. In this review, we discuss and summarize studies that have addressed the role played by lamin A/C in the functions of innate and adaptive immune cells in the context of human inflammatory and autoimmune diseases, pathogen infections, and cancer.

Immunobiology of Atherosclerosis: A Complex Net of Interactions.

Cardiovascular disease is the leading cause of mortality worldwide, and atherosclerosis the principal factor underlying cardiovascular events. Atherosclerosis is a chronic inflammatory disease characterized by endothelial dysfunction, intimal lipid deposition, smooth muscle cell proliferation, cell apoptosis and necrosis, and local and systemic inflammation, involving key contributions to from innate and adaptive immunity. The balance between proatherogenic inflammatory and atheroprotective anti-inflammatory responses is modulated by a complex network of interactions among vascular components and immune cells, including monocytes, macrophages, dendritic cells, and T, B, and foam cells; these interactions modulate the further progression and stability of the atherosclerotic lesion. In this review, we take a global perspective on existing knowledge about the pathogenesis of immune responses in the atherosclerotic microenvironment and the interplay between the major innate and adaptive immune factors in atherosclerosis. Studies such as this are the basis for the development of new therapies against atherosclerosis.

Lamin A/C deficiency in CD4+ T-cells enhances regulatory T-cells and prevents inflammatory bowel disease.

The mechanisms by which lamin A/C in CD4+ T-cells control intestinal homeostasis and can cause inflammatory bowel disease (IBD) are unknown. Here, we explore lamin A/C in a mouse model of IBD. Adoptive transfer to Rag1-/- mice of Lmna-/- CD4+ T-cells, which have enhanced regulatory T-cells (Treg) differentiation and function, induced less severe IBD than wild-type T-cells. Lamin A/C deficiency in CD4+ T-cells enhanced transcription of the Treg master regulator FOXP3, thus promoting Treg differentiation, and reduced Th1 polarization, due to epigenetic changes in the Th1 master regulator T-bet. In mesenteric lymph nodes, retinoic acid (RA) released by CD103+ dendritic cells downregulated lamin A/C in CD4+ T-cells, enhancing Treg differentiation. However, non-RA-producing CD103- dendritic cells predominated in peripheral lymph nodes, facilitating lamin A/C expression in CD4+ T-cells and therefore Th1 differentiation. Our findings establish lamin A/C as a key regulator of Th differentiation in physiological conditions and show it as a potential immune-regulatory target in IBD. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series.

Horse domestication revolutionized warfare and accelerated travel, trade, and the geographic expansion of languages. Here, we present the largest DNA time series for a non-human organism to date, including genome-scale data from 149 ancient animals and 129 ancient genomes (≥1-fold coverage), 87 of which are new. This extensive dataset allows us to assess the modern legacy of past equestrian civilizations. We find that two extinct horse lineages existed during early domestication, one at the far western (Iberia) and the other at the far eastern range (Siberia) of Eurasia. None of these contributed significantly to modern diversity. We show that the influence of Persian-related horse lineages increased following the Islamic conquests in Europe and Asia. Multiple alleles associated with elite-racing, including at the MSTN "speed gene," only rose in popularity within the last millennium. Finally, the development of modern breeding impacted genetic diversity more dramatically than the previous millennia of human management.

Disruption of the CCL1-CCR8 axis inhibits vascular Treg recruitment and function and promotes atherosclerosis in mice.

The CC chemokine 1 (CCL1, also called I-309 or TCA3) is a potent chemoattractant for leukocytes that plays an important role in inflammatory processes and diseases through binding to its receptor CCR8. Here, we investigated the role of the CCL1-CCR8 axis in atherosclerosis. We found increased expression of CCL1 in the aortas of atherosclerosis-prone fat-fed apolipoprotein E (Apoe)-null mice; moreover, in vitro flow chamber assays and in vivo intravital microscopy demonstrated an essential role for CCL1 in leukocyte recruitment. Mice doubly deficient for CCL1 and Apoe exhibited enhanced atherosclerosis in aorta, which was associated with reduced plasma levels of the anti-inflammatory interleukin 10, an increased splenocyte Th1/Th2 ratio, and a reduced regulatory T cell (Treg) content in aorta and spleen. Reduced Treg recruitment and aggravated atherosclerosis were also detected in the aortas of fat-fed low-density lipoprotein receptor-null mice treated with CCR8 blocking antibodies. These findings demonstrate that disruption of the CCL1-CCR8 axis promotes atherosclerosis by inhibiting interleukin 10 production and Treg recruitment and function.

An In Vivo Mouse Model to Measure Naïve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells.

Quantification of naïve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and naïve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.