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8 ABSTRACT Historically pig is allocated to a group of animals which use certain parts of their small intestine to acquire a fully developed primary B cell reperoire. Development of such primary repertoire is independent on the antigen presence and resembles the primary lymphopoietic activity of avian bursa of Fabricius. However, some findings concernig the pig's alignment in the above mentioned group suggest otherwise. This graduation thesis is focused on the investigtion of lymphocyte populations and subpopulations in the small intestine of germ-free and conventional piglets. The aim is to determine whether the percentage amounts of lymphocyte populations is dependent on the intestinal colonization. Using Flow Cytometry the significant differences between individual samples were assesed allowing us to conclude which parts of the small intestine could possibly be used for the development of B cell repertoire. Moreover, the status of isotype switching of B lymphocytes isolated from different intestinal parts was determined by the means of PCR analysis. Our data suggest that the small intestine colonization has a crucial role in development of all the main lymphocyte populations as well as some of their subpopulations. The greatest influence of colonization was observed concerning B lymphocytes and their...

As pregas vocais (PV) são estruturas únicas, altamente especializadas na vibração para a produção sonora. Em grande parte, decorrente da estruturação em camadas da lâmina própria (LP). Essa estruturação não está presente ao nascimento, somente após anos de uso vibratório e fonatório da PV é que a LP está finalmente estruturada. As pregas ventriculares (PVT) não são, habitualmente, estruturas vibratórias na produção sonora, e possuem a LP menos organizada em estratos, sendo menos especializadas para a vibração. Até o presente momento, não se tem conhecimento do que ocorre com os constituintes da LP de PVTs de pessoas que as utilizam como fonte produtora de voz. No presente estudo, foram comparados os constituintes colágenos e as fibras elásticas da LP de PVTs de indivíduos que as utilizam como principal fonte vibratória na produção de voz com o grupo controle. Foram selecionados seis indivíduos que utilizavam pelo menos uma das PVTs como fonte de vibração para a produção sonora por ao menos seis anos. Delas, colheu-se pequeno fragmento (0,5 cm2), que após processamento histológico, as fibras colágenas foram coradas com Picrosirus Red e as fibras elásticas com Weigert resorcina-fucsina. Foram obtidas 54 imagens da camada mais superficial da LP de cada PVT para cada coloração. Após a aquisição das imagens, as fibras colágenas tipo I e tipo III, colágenas totais e fibras elásticas foram quantificadas utilizando-se o software Image-Pro Plus, e comparadas com as PVTs dos controles. A análise estatística foi realizada por meio do teste T de Student para amostras não pareadas. A porcentagem de colágeno total na camada mais superficial da LP de PVT utilizada como fonte vibratória para a produção de som foi significativamente maior em relação aos controles. O mesmo ocorreu com a quantidade de colágeno tipo I. Não houve diferenças na quantidade de colágeno tipo III e de fibras elásticas. Como conclusão, pode-se afirmar que a utilização da PVT como fonte vibratória produtora de som leva ao aumento da quantidade de fibras colágenas totais e do tipo I na camada mais superficial da sua LP / The vocal folds (VF) are unique structures, highly specialized in vibrating for sound production. This specialization is mainly due to a layered structure of the lamina propria (LP). This layered structure is not present at birth, and develops only after a several years of phonation. The LP of the mature vocal fold consists of three layers. The ventricular folds (VTF) are not originally vibrating structures for sound production, and in its LP the layers are poorly organized. It is not known what happens to the constituents of the LP in the VTF in subjects that use VTF vibration as a source of voice production. In the present study, the distribution and quantity of collagen and elastic fibers of the lamina propria from VTF of patients that use it as the main source of vibration for voice production were compared with the VTF from control subjects. Six individuals that used at least one of the VTF as source of vibration for sound production for minimum of six years were selected. A small fragment of VTF (0.5 cm2) used as vibration source of sound production was collected from each subject. The samples were processed for histological analysis. Collagen fibers were stained with Picrosirus Red and elastic fibers were stained with Weigert\'s Resorcin-Fuchsin. A total of 54 images were obtained from the superficial layer of the LP from each VTF for each stain. After image acquisition, collagen type I, III, total collagen and elastic fibers were quantified and compared with the VTF from the control group. Quantification was done using Image-Pro Plus software. Statistics were performed using an unpaired Student T test. The amount of total collagen in the most superficial layer of LP when the VTF was used as the source of vibration for the production of sound was significantly higher when compared to controls. The same result was seen for the amount of type I collagen in both groups. There was no difference in the quantity of type III collagen and elastic fibers between the two groups. Vibration of the VTF as a source of sound, for at least six years, leads to an increase in the amount of total collagen fibers and an increase in type I collagen, but does not increase the amount of type III collagen and elastic fibers in the most superficial layer of LP. These results may help elucidate the unique development of the lamina propria of the vocal fold

The development of mucosal dendritic cells (DCs) in cattle is poorly understood and an analysis of myeloid cells in the bovine small intestine is required to increase our knowledge in this area. The phenotype, frequency and distribution of mucosal myeloid and lymphoid lamina propria leukocytes (LPL) and intraepithelial leukocytes (IEL) in the ileum and jejunum of newborn calves (3-5 weeks old) were analyzed using flow cytometry and immunohistochemistry (IHC). LPL and IEL were isolated through the use of chemical and enzymatic incubations. Costaining with a CD45-specific monoclonal antibody allowed us to exclude all non-leukocytic cells from our analysis of IEL and LPL. The morphology of CD45+CD11c+MHC Class II+ cells isolated from the lamina propria (LP) of ileum and jejunum showed myeloid characteristics, validating the use of CD11c and MHC Class II co-expression to identify myeloid cells. Regional differences in the frequency and number of leukocytes isolated from the IEL and LP compartments of the ileum and jejunum were analyzed in newborn calves. The CD11cHiCD14+ and CD335+ NK cell populations were significantly more abundant in the ileum than the jejunum. IHC was then used to identify the distribution of myeloid cells within the intestine. This analysis confirmed the presence of a variety of myeloid cell populations within the LP. Furthermore, CD11c+ cells were uniquely distributed within the jejunal, but not the ileal IEL compartment. In contrast, CD11b+ cells were present in the ileal, but absent from the jejunal, IEL compartment. A comparison of myeloid cell populations isolated from jejunum and blood dentified distinct mucosal DC populations, such as CD11c+CD13+ cells, which were present in he jejunum but absent from blood. The phenotype, frequency and distribution of IEL and LPL in the ileum and jejunum of weaned calves (6 months old) were then investigated. Significant regional differences were observed when comparing mucosal T cell populations with CD8+ and γδ T cells more abundant in the ileum and CD4+ T cells more abundant in the jejunum. Proportionally, there were no significant differences between the frequency and number of myeloid populations in the two regions. IHC was, once again, used to confirm these unique distributions of cells within each region. CD11b+ cells were present in the LP of both the ileum and jejunum, although a small number of CD11b+ cells were found in the ileal epithelium. CD4+ T cells were restricted to the LP, while CD8+ and γδ T cells were restricted to the IEL compartment. Significant age-related changes were observed when comparing mucosal leukocyte populations in the ileum and jejunum of newborn and 6 month old calves. In the ileum there was an age-related enrichment of CD8+ and γδ T cells, while in the jejunum there was enrichment in CD4+ and CD8+ T cells. In contrast, total myeloid (CD11c+MHC Class II+) cells number remained unchanged but there was a significant age-related enrichment of DC subpopulations (CD13, CD26, CD205). In conclusion, the ileum and jejunum of the newborn calf was populated by diverse myeloid subpopulations, some of which were distinct from myeloid subpopualtions identified in blood. Furthermore, the total number of CD11cHiMHC Class II+ myeloid cells isolated from a 10 cm segment of intestine did not change with age. If neonatal DCs are functionally equivalent to DCs present in weaned calves then the neonatal mucosal immune system appears to have an equivalent capacity to acquire and present antigens acquired from diet, commensal microflora, or pathogens. The one limitation to this conclusion may be the marked difference in the distribution of intraepithelial DC and macrophage distribution when comparing newborn and weaned calves.

The development of mucosal dendritic cells (DCs) in cattle is poorly understood and an analysis of myeloid cells in the bovine small intestine is required to increase our knowledge in this area. The phenotype, frequency and distribution of mucosal myeloid and lymphoid lamina propria leukocytes (LPL) and intraepithelial leukocytes (IEL) in the ileum and jejunum of newborn calves (3-5 weeks old) were analyzed using flow cytometry and immunohistochemistry (IHC). LPL and IEL were isolated through the use of chemical and enzymatic incubations. Costaining with a CD45-specific monoclonal antibody allowed us to exclude all non-leukocytic cells from our analysis of IEL and LPL. The morphology of CD45+CD11c+MHC Class II+ cells isolated from the lamina propria (LP) of ileum and jejunum showed myeloid characteristics, validating the use of CD11c and MHC Class II co-expression to identify myeloid cells. Regional differences in the frequency and number of leukocytes isolated from the IEL and LP compartments of the ileum and jejunum were analyzed in newborn calves. The CD11cHiCD14+ and CD335+ NK cell populations were significantly more abundant in the ileum than the jejunum. IHC was then used to identify the distribution of myeloid cells within the intestine. This analysis confirmed the presence of a variety of myeloid cell populations within the LP. Furthermore, CD11c+ cells were uniquely distributed within the jejunal, but not the ileal IEL compartment. In contrast, CD11b+ cells were present in the ileal, but absent from the jejunal, IEL compartment. A comparison of myeloid cell populations isolated from jejunum and blood dentified distinct mucosal DC populations, such as CD11c+CD13+ cells, which were present in he jejunum but absent from blood. The phenotype, frequency and distribution of IEL and LPL in the ileum and jejunum of weaned calves (6 months old) were then investigated. Significant regional differences were observed when comparing mucosal T cell populations with CD8+ and γδ T cells more abundant in the ileum and CD4+ T cells more abundant in the jejunum. Proportionally, there were no significant differences between the frequency and number of myeloid populations in the two regions. IHC was, once again, used to confirm these unique distributions of cells within each region. CD11b+ cells were present in the LP of both the ileum and jejunum, although a small number of CD11b+ cells were found in the ileal epithelium. CD4+ T cells were restricted to the LP, while CD8+ and γδ T cells were restricted to the IEL compartment. Significant age-related changes were observed when comparing mucosal leukocyte populations in the ileum and jejunum of newborn and 6 month old calves. In the ileum there was an age-related enrichment of CD8+ and γδ T cells, while in the jejunum there was enrichment in CD4+ and CD8+ T cells. In contrast, total myeloid (CD11c+MHC Class II+) cells number remained unchanged but there was a significant age-related enrichment of DC subpopulations (CD13, CD26, CD205). In conclusion, the ileum and jejunum of the newborn calf was populated by diverse myeloid subpopulations, some of which were distinct from myeloid subpopualtions identified in blood. Furthermore, the total number of CD11cHiMHC Class II+ myeloid cells isolated from a 10 cm segment of intestine did not change with age. If neonatal DCs are functionally equivalent to DCs present in weaned calves then the neonatal mucosal immune system appears to have an equivalent capacity to acquire and present antigens acquired from diet, commensal microflora, or pathogens. The one limitation to this conclusion may be the marked difference in the distribution of intraepithelial DC and macrophage distribution when comparing newborn and weaned calves.

The development of mucosal dendritic cells (DCs) in cattle is poorly understood and an analysis of myeloid cells in the bovine small intestine is required to increase our knowledge in this area. The phenotype, frequency and distribution of mucosal myeloid and lymphoid lamina propria leukocytes (LPL) and intraepithelial leukocytes (IEL) in the ileum and jejunum of newborn calves (3-5 weeks old) were analyzed using flow cytometry and immunohistochemistry (IHC). LPL and IEL were isolated through the use of chemical and enzymatic incubations. Costaining with a CD45-specific monoclonal antibody allowed us to exclude all non-leukocytic cells from our analysis of IEL and LPL. The morphology of CD45+CD11c+MHC Class II+ cells isolated from the lamina propria (LP) of ileum and jejunum showed myeloid characteristics, validating the use of CD11c and MHC Class II co-expression to identify myeloid cells. Regional differences in the frequency and number of leukocytes isolated from the IEL and LP compartments of the ileum and jejunum were analyzed in newborn calves. The CD11cHiCD14+ and CD335+ NK cell populations were significantly more abundant in the ileum than the jejunum. IHC was then used to identify the distribution of myeloid cells within the intestine. This analysis confirmed the presence of a variety of myeloid cell populations within the LP. Furthermore, CD11c+ cells were uniquely distributed within the jejunal, but not the ileal IEL compartment. In contrast, CD11b+ cells were present in the ileal, but absent from the jejunal, IEL compartment. A comparison of myeloid cell populations isolated from jejunum and blood dentified distinct mucosal DC populations, such as CD11c+CD13+ cells, which were present in he jejunum but absent from blood. The phenotype, frequency and distribution of IEL and LPL in the ileum and jejunum of weaned calves (6 months old) were then investigated. Significant regional differences were observed when comparing mucosal T cell populations with CD8+ and γδ T cells more abundant in the ileum and CD4+ T cells more abundant in the jejunum. Proportionally, there were no significant differences between the frequency and number of myeloid populations in the two regions. IHC was, once again, used to confirm these unique distributions of cells within each region. CD11b+ cells were present in the LP of both the ileum and jejunum, although a small number of CD11b+ cells were found in the ileal epithelium. CD4+ T cells were restricted to the LP, while CD8+ and γδ T cells were restricted to the IEL compartment. Significant age-related changes were observed when comparing mucosal leukocyte populations in the ileum and jejunum of newborn and 6 month old calves. In the ileum there was an age-related enrichment of CD8+ and γδ T cells, while in the jejunum there was enrichment in CD4+ and CD8+ T cells. In contrast, total myeloid (CD11c+MHC Class II+) cells number remained unchanged but there was a significant age-related enrichment of DC subpopulations (CD13, CD26, CD205). In conclusion, the ileum and jejunum of the newborn calf was populated by diverse myeloid subpopulations, some of which were distinct from myeloid subpopualtions identified in blood. Furthermore, the total number of CD11cHiMHC Class II+ myeloid cells isolated from a 10 cm segment of intestine did not change with age. If neonatal DCs are functionally equivalent to DCs present in weaned calves then the neonatal mucosal immune system appears to have an equivalent capacity to acquire and present antigens acquired from diet, commensal microflora, or pathogens. The one limitation to this conclusion may be the marked difference in the distribution of intraepithelial DC and macrophage distribution when comparing newborn and weaned calves.

As pregas vocais (PV) são estruturas únicas, altamente especializadas na vibração para a produção sonora. Em grande parte, decorrente da estruturação em camadas da lâmina própria (LP). Essa estruturação não está presente ao nascimento, somente após anos de uso vibratório e fonatório da PV é que a LP está finalmente estruturada. As pregas ventriculares (PVT) não são, habitualmente, estruturas vibratórias na produção sonora, e possuem a LP menos organizada em estratos, sendo menos especializadas para a vibração. Até o presente momento, não se tem conhecimento do que ocorre com os constituintes da LP de PVTs de pessoas que as utilizam como fonte produtora de voz. No presente estudo, foram comparados os constituintes colágenos e as fibras elásticas da LP de PVTs de indivíduos que as utilizam como principal fonte vibratória na produção de voz com o grupo controle. Foram selecionados seis indivíduos que utilizavam pelo menos uma das PVTs como fonte de vibração para a produção sonora por ao menos seis anos. Delas, colheu-se pequeno fragmento (0,5 cm2), que após processamento histológico, as fibras colágenas foram coradas com Picrosirus Red e as fibras elásticas com Weigert resorcina-fucsina. Foram obtidas 54 imagens da camada mais superficial da LP de cada PVT para cada coloração. Após a aquisição das imagens, as fibras colágenas tipo I e tipo III, colágenas totais e fibras elásticas foram quantificadas utilizando-se o software Image-Pro Plus, e comparadas com as PVTs dos controles. A análise estatística foi realizada por meio do teste T de Student para amostras não pareadas. A porcentagem de colágeno total na camada mais superficial da LP de PVT utilizada como fonte vibratória para a produção de som foi significativamente maior em relação aos controles. O mesmo ocorreu com a quantidade de colágeno tipo I. Não houve diferenças na quantidade de colágeno tipo III e de fibras elásticas. Como conclusão, pode-se afirmar que a utilização da PVT como fonte vibratória produtora de som leva ao aumento da quantidade de fibras colágenas totais e do tipo I na camada mais superficial da sua LP / The vocal folds (VF) are unique structures, highly specialized in vibrating for sound production. This specialization is mainly due to a layered structure of the lamina propria (LP). This layered structure is not present at birth, and develops only after a several years of phonation. The LP of the mature vocal fold consists of three layers. The ventricular folds (VTF) are not originally vibrating structures for sound production, and in its LP the layers are poorly organized. It is not known what happens to the constituents of the LP in the VTF in subjects that use VTF vibration as a source of voice production. In the present study, the distribution and quantity of collagen and elastic fibers of the lamina propria from VTF of patients that use it as the main source of vibration for voice production were compared with the VTF from control subjects. Six individuals that used at least one of the VTF as source of vibration for sound production for minimum of six years were selected. A small fragment of VTF (0.5 cm2) used as vibration source of sound production was collected from each subject. The samples were processed for histological analysis. Collagen fibers were stained with Picrosirus Red and elastic fibers were stained with Weigert\'s Resorcin-Fuchsin. A total of 54 images were obtained from the superficial layer of the LP from each VTF for each stain. After image acquisition, collagen type I, III, total collagen and elastic fibers were quantified and compared with the VTF from the control group. Quantification was done using Image-Pro Plus software. Statistics were performed using an unpaired Student T test. The amount of total collagen in the most superficial layer of LP when the VTF was used as the source of vibration for the production of sound was significantly higher when compared to controls. The same result was seen for the amount of type I collagen in both groups. There was no difference in the quantity of type III collagen and elastic fibers between the two groups. Vibration of the VTF as a source of sound, for at least six years, leads to an increase in the amount of total collagen fibers and an increase in type I collagen, but does not increase the amount of type III collagen and elastic fibers in the most superficial layer of LP. These results may help elucidate the unique development of the lamina propria of the vocal fold

Philosophiae Doctor - PhD / Histological observations revealed that oral epithelium originated from a single ectodermal layer. As the ectoderm grew so it differentiated into squamous epithelium. The first features of squamous differentiation were noticed at 8 weeks in utero in areas where keratinized mucosae were developing, and these were the changing of cuboidal to cylindrical basal cells and the subsequent growth of prickle cells from these cylindrical basal cells. The prickle cells merged with the existing primitive cells and at no stage could a separate squamous epithelial layer I such as the stratum tritermedium of the epidermis I be observed inside the mouth. At 12 weeks in utero squamous differentiation had reached a stage where acidophilic layers appeared in certain regions on the epithelial layer. The time of appearance of these layers varied from case to case. At this stage most of the primitive characteristics had disappeared from the keratinizing epithelium. Unlike the periderm of the skin which was shed into the amniotic fluid, shedding of primitive epithelial cells from the keratinizing squamous epithelium was not noticeable. Thence, the growth of keratinizing epithelium was followed by an increase of acidophilic layers, the appearance of keratohyaline granules in cells and, in some instances, full keratinization. The latter I however I was almost exclusively confined to the vermilion border of the lip. The squamous epithelium of the lining mucosa, which is unkeratinized I developed at a much slower tempo. It retained its cuboidal-shaped basal cells and the primitive features of the overlying cells were lost only at about 4- 5 months in utero I when squamous differentiation set in. At no stage was the squamous differentiation a prominent feature. At junctions between keratinized and unkeratinized epithelia and epidermis the epithelium exhibited features of both types of epithelia that were being joined. This was especially noticeable at the junction between vermilion epithelium and epidermis, where part of the vermilion epithelium displayed a prominent intermediate type of layer. Similarly, acidophilic layers of keratinizing epithelium merged imperceptibly with the walls of cells of unkeratinizing epithelium, creating a small region of an unkeratinizing type of epithelium with keratinized cells. Thus the development of the oral epithelium is through differentiation and renewal of epithelial cells: the ectodermal layer developes into an epithelial layer which is recognised by its squamous appearance. The subsequent growth is by constant renewal of this differentiated epithelium. The pattern of epithelial development I the appearance of the junctional epithelia and the manner in which acidophilic layers merge with unkeratinized epithelial cells I indicate a unity between these epithelia. According to these developmental features, the epithelium of the mouth and epidermis can be classified into less differentiated and better differentiated, but with a commonbackground for these epithelia. When the formation and the established appearance of keratin in the mouth and on the skin was compared histologically I ultrastructurally and histochemically I a unity between these features became apparent. Ultrastructurally it appeared that keratin consisted basically of 2 cytoplasmic constituents: tonofilaments and a fine granular substance. The tonofilaments were gathered at first into bundles and then broken up into finer tonofibrils. These finer fibrils mixed with a granular ground substance to form a homogenous granular filamentous material. This product can be regarded as a pre-keratin. With the addition of a keratohyaline layer to the process I keratin was formed, Apart from the keratohyaline granules several additional changes took place in cells concerned in this process I whether keratin was formed or not. These changes were flattening of cells, extensive interdigitation between cell walls, disappearance of micro-villi I loss of structure in desmosomes I thickening of cell walls and the disappearance of glycogen from cells. Some of these features were displayed in each of the types of epithelium examined here.

Macrophages are leukocytes of the innate immune system that display great phenotypic plasticity to mediate diverse functions. The ontogeny of tissue resident macrophages has been debated in recent decades. It is now recognised that tissue macrophages can be replenished from embryonically-derived precursors, and/or monocyte intermediates in a tissue specific manner. Interferon Regulatory Factor 5 (IRF5) is a transcription factor that promotes a pro-inflammatory phenotype in macrophages in vitro and in vivo. Indeed, IRF5 contributes to the pathogenesis of experimental inflammatory arthritis, lupus, and obesity via recruitment and activation of effector cells. Research described here as part of this thesis, involves the profiling of the intestinal Mononuclear Phagocyte system to investigate the role of IRF5 in the development of monocyte-derived macrophages in the Colonic Lamina Propria (cLP) which are exclusively replenished by adult Ly6C^hi monocytes. Using Mixed Bone Marrow Chimaeras (MBMCs) we showed that in shared environment Wild-Type (WT) cLP macrophages dominated IRF5-deficient (Irf5^-/-) cLP macrophages in both steady state and inflammation. The development of in vitro bone marrow derived macrophages, and the reconstitution of the haematopoietic compartment in bone marrow of MBMCs were not significantly affected by IRF5 deficiency. IRF5 promoted the accumulation of WT monocytes in the cLP of MBMCs in a process possibly dependent on the CCL2/CCR2 axis. Furthermore, IRF5 expression committed Ly6C^hi monocytes to a pro-inflammatory macrophage fate in the inflamed cLP, characterised by protein expression of the cytokines IL1β, and TNFα, and the expression of Ccl4 and Ccl8 transcripts, whilst loss of IRF5 favoured accumulation of CD11b⁺ IRF4-dependent Dendritic Cells. Of significance, IRF5 expression might have prevented further differentiation of inflammatory macrophages into tissue-resident macrophages, thus supporting an inflammatory state. Irf5-/- mice were protected from Helicobacter hepaticus + αIL10R colitis. Intriguingly, protection from colitis may also be conferred by the presence of Irf5-/- haematopoietic cells, evidenced by WT:Irf5-/- MBMCs . Modulation of IRF5 activity may therefore be a viable therapeutic strategy. RNA sequencing identified that C1q, Cd81, and Ccl8 were upregulated in WT macrophages from MBMC, which may prove therapeutic targets.

<p>T lymphocytes regulate the initiation, duration, and magnitude of adaptive immune responses and function as effector cells in cell mediated immunity. To become immunologically competent they must generate functional antigen receptors. This process takes place in the thymus and requires somatic recombination of T cell receptor (TCR) genes. It is mediated by the endonucleases recombination activating gene-1 (RAG1) and RAG2. Although the thymus regresses at puberty, T cells are present throughout life implying that other tissues must provide the proper milieu for T cell development. This thesis describes extrathymic T cell maturation in man. RAG1, RAG2, and the preTα-chain (pTα), which is exclusively utilized in developing T cells, were used as markers for TCR gene rearrangement. Two new exons (1A and 1B) encoding sequences in the 5’ untranslated region (5’UTR) of mRNA were discovered in the human RAG1 gene. The previously described 5’UTR exon (renamed 1C) was located between the new exons and exon 2, the latter containing the entire coding sequence. We found that small intestinal lymphocytes of the T cell lineage expressed the new exons in three different splice forms. RAG1 mRNA containing the 1C exon was not expressed in small intestinal lymphocytes. In contrast, splice forms containing the 1A exon were not expressed in thymocytes. RAG1 and pTα mRNA expressing lymphocytes were seen both within the epithelium and in lamina propria. Thymocyte-like CD2⁺CD7⁺CD3^-, CD4⁺CD8⁺, CD1a⁺, and IL7-R+ lymphocytes were identified in the small intestinal mucosa. CD2⁺CD7⁺CD3^- cells had the highest expression levels of mRNA for RAG1 and pTα, suggesting that the small intestinal mucosa is indeed a site for T cell maturation. Small intestinal T lymphocytes were also shown to kill via the Fas/FasL pathway in a TCR/CD3 independent manner and via the perforin/granzyme pathway in a TCR/CD3 dependent manner. The Fas/FasL-mediated cytotoxicity may reflect an ongoing selection process of extrathymically maturated T cells. </p><p>The nasopharyngeal tonsil is the major inductive site for immune reactions against inhaled antigens. Previous demonstration of RAG1 expression in tonsillar B cells was interpreted as antigen driven receptor revision. The present study confirms the expression of RAG1 in B cells. We also found that RAG1, RAG2, and pTa mRNAs were expressed in lymphocytes of the T cell lineage. A small population of cells with the immature phenotype CD2+CD7+CD3- was demonstrated. This population had the highest expression levels of mRNA for RAG1, RAG2, pTα and terminal deoxynucleotidyl transferase. All four splice-forms of RAG1 mRNA were expressed. RAG1 and pTα mRNA expressing cells were mainly located in the proximity of the surface epithelium and in the outer rim of the follicles. These results suggest that the nasopharyngeal tonsil is a site where extrathymic T cell development and antigen driven TCR revision are occurring in parallel. </p><p>Celiac disease (CD) is a small intestinal enteropathy characterized by permanent intolerance to gluten. Gluten reactive intestinal T cells are central in the pathogenesis and CD can be regarded as a failure to maintain tolerance to this food antigen. Expression of the RAG1 1A/2 splice form was significantly decreased in small intestinal T cell subsets of CD patients suggesting that impaired TCR gene rearrangement could contribute to failure of maintain tolerance in CD. </p><p>Together, these findings show that both small intestinal and nasopharyngeal tonsillar lymphocytes of T cell lineage have the molecular machinery for antigen receptor rearrangement and that thymocyte-like lymphocytes are present in both tissues. Thus these organs are likely sites of T lymphocyte ontogeny as well as for secondary T cell receptor rearrangement in man. </p>