Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-15T13:52:56Z No. of bitstreams: 1 Luciana Souza de Aragão França Redução....pdf: 995559 bytes, checksum: ba08fbe35c691051eb1d16b7657f0655 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-15T13:53:13Z (GMT) No. of bitstreams: 1 Luciana Souza de Aragão França Redução....pdf: 995559 bytes, checksum: ba08fbe35c691051eb1d16b7657f0655 (MD5) / Made available in DSpace on 2016-02-15T13:53:13Z (GMT). No. of bitstreams: 1 Luciana Souza de Aragão França Redução....pdf: 995559 bytes, checksum: ba08fbe35c691051eb1d16b7657f0655 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / As alergias afetam cerca de 20 a 30% da população mundial e sua prevalência, bem como a gravidade dos sintomas, tem aumentado nas últimas décadas. As terapias existentes para as desordens do trato respiratório ocorrem por períodos prolongados, apresentam efeitos colaterais, muitas vezes não são efetivas para pacientes graves e dependem do afastamento do alérgeno. Uma alternativa para esses pacientes seria a indução de tolerância imunológica, através da terapia celular com células dendríticas pulsadas com o alérgeno. O presente trabalho objetivou avaliar o efeito de células dendríticas mielóides sensibilizadas in vitro com extrato de B. tropicalis em modelo murino de alergia respiratória. Em modelos experimentais de alergia respiratória, células T auxiliares (Th2) alérgeno-específica produzem citocinas que regulam a síntese de anticorpos IgE alérgeno-específicos e controlam a inflamação eosinofílica e a remodelação do tecido das vias aéreas, e esses parâmetros foram analisados neste trabalho. Células dendríticas tolerogênicas de camundongos A/J foram obtidas na presença de GM-CSF e dexametasona e caracterizadas por apresentarem baixa expressão de MHC-II e moléculas coestimulatórias, redução acentuada de PD-L2 e um perfil antiinflamatório de produção de citocina.A inoculação prévia de células dendríticas sensibilizadascom Blomia reduziu especificamente e significativamente o número total de leucócitos e o número de eosinófilos no fluido de lavagem broncoalveolar e reduziu a produção de IgE, em comparação à inoculação de células sensibilizadas com ovalbumina, e estes efeitos foram mais intensos com uma segunda inoculação de células tolerogênicas. Além disso, redução do infiltrado inflamatório pulmonar foi observada em animais que tinham sido tratados com células dendríticas, independente do antígeno utilizado na sensibilização das mesmas. Estes resultados indicam que células dendríticas geradas como descrito neste trabalho podem inibir uma resposta alérgica Th2. A especificidade do tratamento, entretanto, deve ser melhor investigada. / Allergies affect about 20-30% of world population and its prevalence and severity of symptoms has increased in recent decades. Existing therapies to respiratory tract disorders are extense, with side effects, not effective for severe patients and depending on the allergen removal. An alternative for these patients is the induction of immune tolerance by cell therapy with dendritic cells pulsed with the allergen. This study aimed to evaluate the effect of myeloid dendritic cells sensitized in vitro with B. tropicalis extract in a murine model of respiratory allergy. In experimental models of respiratory allergy, T helper cells (Th2) produce allergen-specific cytokines which regulate the synthesis of allergen-specific IgE antibodies and control eosinophilic inflammation and tissue remodeling of the airways, these parameters were evaluated in this study. Tolerogenic dendritic cells from A / J mice were obtained in the presence of GM-CSF and dexamethasone and were characterized by low MHC-II expression and costimulatory molecules, marked reduction of PD-L2 and antiinflammatory profile of cytokine production. Prior inoculation of sensitized dendritic cells with Blomiasignificantly and specifically reduced the total number of leukocytes and eosinophils in the bronchoalveolar lavage fluid and reduces IgE production compared to inoculation with cells sensitized with ovalbumin, and these effects were improved with a second inoculation of tolerogenic cells. Furthermore, reducing the pulmonary inflammatory infiltrate was seen in animals that had been treated with dendritic cells regardless of the antigen used for sensitization. These results indicate that dendritic cells generated as described here can inhibit an allergic Th2 response. The specificity of the treatment, however, should be examined.

Carrapatos são artrópodes hematófagos de distribuição cosmopolita que têm grande importância médica e veterinária devido ao efeito deletério direto causado por se fixarem e sugarem seus hospedeiros, como também por serem importantes vetores de doenças para o homem e para os animais domésticos. Sabendo que carrapatos permanecem fixos em seus hospedeiros por longos períodos de tempo sem serem rejeitados, é possível inferir que esses ácaros possuam um arsenal de mecanismos que atuem no controle da resposta imune do hospedeiro. De fato, diversos trabalhos têm demonstrado que carrapatos são capazes de modular a resposta imune de seus hospedeiros através de componentes presentes na saliva, que são inoculados durante o repasto sangüíneo. Assim, este trabalho procurou investigar se carrapatos exercem a modulação da resposta imune do hospedeiro através do recrutamento de células T regulatórias CD4+CD25+ (Tregs), com a intenção de conter uma resposta inflamatória / imune prejudicial à sua alimentação. Para isso, células isoladas de amostras de pele e linfonodos de camundongos BALB/c infestados com carrapatos Rhipicephalus sanguineus foram analisadas quanto à expressão das moléculas de superfície CD4, CD25, CTLA-4, CD45RB, GITR e CD103 (fenótipo de células Tregs), por citometria de fluxo. Adicionalmente, as células obtidas dos linfonodos foram avaliadas quanto à expressão de mensagem para o fator de transcrição Foxp3 (característico da função regulatória), por PCR quantitativo. Paralelamente, saliva de R. sanguineus foi inoculada na orelha de animais da mesma linhagem, a fim de se comparar o infiltrado celular com o obtido na pele dos camundongos infestados com carrapatos. Os resultados mostraram que as infestações não alteraram a percentagem de células T CD4+CD25+ nem a expressão de moléculas associadas ao fenótipo de células Tregs nas células infiltradas na lesão de fixação dos carrapatos ou nos linfonodos em comparação a camundongos controles. Também não se verificou aumento da expressão do gene para Foxp3 nos linfonodos em nenhum dos grupos analisados. Por outro lado, a inoculação de saliva na orelha de camundongos induziu um aumento significativo da população de células T CD4+, porém estas também não apresentavam fenótipo regulatório, sugerindo que o mecanismo de imunomodulação exercido pelos carrapatos sobre seus hospedeiros não é mediado por essas células. Resultados adicionais mostraram que a saliva de carrapatos reduziu significativamente a percentagem de células dendríticas nas orelhas dos camundongos, sugerindo que carrapatos podem estar modulando a resposta imune de seus hospedeiros por diminuírem o repovoamento da pele com células dendríticas, as quais são essenciais na vigilância imune dos tecidos periféricos. / Ticks are bloodsucking arthropods that feed on vertebrates and are responsible for serious global economic losses both through the effects of blood sucking and as vectors of pathogens. A tick?s bloodmeal lasts for several days, during which it remains fixed to the host and avoids rejection by local inflammatory and immunological reactions. This status is achieved by the escape mechanisms ticks have evolved. In fact, many studies have demonstrated that ticks modulate the host immune response through salivary compounds inoculated during their bloodmeals. This study investigated if during bloodfeeding ticks can recruit regulatory T cells in an attempt to modulate the host immune response and to control inflammatory responses that could be harmful to tick feeding. BALB/c mice were infested with Rhipicephalus sanguineus, the skin at feeding sites and the regional lymph nodes were collected, and the cells forming the local infiltrates were analyzed by flow cytometry for simultaneous expression of CD4, CD25, CTLA-4, CD45RB, GITR and CD103 molecules. Additionally, expression of mRNA for Foxp3 was measured in the lymph node cells. Tick saliva was also inoculated into the ears of BALB/c mice in order to compare the local cellular infiltrate with that elicited by artificial infestation. Control animals were sham infested. The results show that, relative to sham-infested tissues, tick infestations did not alter the percentage of CD4+CD25+ T cells present at the site of their attachment or in draining lymph nodes. Infestations also did not increase the expression of Foxp3 in skin and lymph nodes. On the other hand, saliva inoculated into the ear induced a significant increase in the number of CD4+ T lymphocytes recruited to the site of inoculation, although these cells did not express a regulatory phenotype. These results suggest that the modulation of the host immune response by ticks does not involve CD4+CD25+ T cells. Additional results showed that tick saliva reduced the percentage of dendritic cells in the skin of infested mice. This finding indicates that ticks may modulate the host immune response by diminishing the repopulation of skin with dendritic cells, which are essential for maintaining surveillance of peripheral tissues for incoming antigens.

Erkrankungen das allergischen Formenkreises (Asthma bronchiale, Atopische Dermatitis, Allergische Rhino-Konjunktivitis) sind in ständiger Zunahme begriffen. Für den in der Klinik tätigen Pädiater stellt das allergische Asthma bronchiale die wichtigste Erkrankung aus dieser Gruppe dar. Asthma ist die häufigste chronische Atemwegs-Erkrankung im Kindesalter und verursacht durch Medikation und Hospitalisation enorme volkswirtschaftliche Kosten. Kardinale Symptome von Asthma sind reversibler Bronchospasmus nach Exposition mit z.B. Allergenen (Atemwegs-Obstruktion), eine funktionell abnorme glatte Atemwegsmuskulatur, die durch eine verstärkte Kontraktilität nach unspezifischer Stimulation gekennzeichnet ist (Bronchiale Hyperreagibilität oder Atemwegs-Hyperreaktivität, AHR), und das Vorliegen einer chronischen Entzündung der kleinen und mittleren Atemwege (Atemwegs-Inflammation, AI). Derzeitig finden für die Behandlung von Asthma bronchiale lediglich die symptomatische Therapie der Obstruktion mit muskelrelaxierenden Medikamenten (Bedarfstherapie) und die unspezifische anti-entzündliche Therapie mit steroidalen Antiphlogistika (Dauertherapie) regelmäßige und breite Anwendung. Während die Mehrzahl der Patienten hiermit relativ beschwerdefrei eingestellt werden kann, gibt es aber auch Asthmatiker, die unter den Nebenwirkungen einer hochdosierten, systemischen Steroid-Dauertherapie leiden (steroid-pflichtiges Asthma), oder trotz der intensiven Anwendung von Kortikoiden nicht symptomfrei bleiben (steroid-resistentes Asthma). Für diese Patientengruppe fehlt bislang eine effektive, nebenwirkungsarme und spezifisch anti-asthmatische Therapie. Grundlage für die Etablierung innovativer Strategien für die Behandlung von Asthma kann jedoch nur die genaue Kenntnis der pathophysiologischen Mechanismen sein, die zur Ausbildung der klinischen Symptome führen. Allergischen Erkrankungen liegt eine fehlgeleitete Immunreaktion gegen Umweltstoffe, wie z.B. Tierhaarepithelien, Blütenpollen oder Lebensmittel zugrunde. Mittlerweile gilt als gesichert, dass ein Ungleichgewicht in der Antwort von T-Lymphozyten auf diese Antigene die wesentliche Grundlage für die Ausbildung des allergischen Phänotyps darstellt. Bei allergischen Patienten überwiegt die Produktion von sog. Th2-Zytokinen. Diese sind von T-Zellen produzierte Botenstoffe, die für die Induktion und Regulierung der wesentlichen pathognomonischen Mechanismen bei der allergischen Immunreaktion verantwortlich sind. Durch Interleukin (IL)-4 und IL-13 kommt es zur gesteigerten Produktion von allergen-spezifischen IgE-Antikörpern, die Grundlage für die Aktivierung von Mastzellen und die Entwicklung der allergischen Frühreaktion (early asthmatic reaction). Durch IL-5, IL-3 und GM-CSF werden eosinophile Zellen aktiviert und wandern in die Atemwege ein. Hier entwickelt sich das Bild einer chronischen Atemwegs-Entzündung, und die Folge ist die allergische Spätreaktion (late asthmatic reaction). Der genaue Mechanismus und die Interaktion von T-Zell-Zytokinen, IgE-Produktion und eosinophiler AI ist nicht völlig geklärt und Gegenstand der vorliegenden Arbeit. Für die immunologischen Untersuchungen von AI und AHR wurde die Maus als Modell gewählt, die durch ihre gut definierte Immunologie und die Vielzahl von verfügbaren immunologischen "Werkzeugen" wie z.B. Antikörper und genetisch homogenen Stämmen entscheidende Vorteile bietet. Die in jüngerer Zeit entwickelten genetisch manipulierten Mausstämme ermöglichen darüber hinaus die genaue Analyse der Rolle einzelner Faktoren in der Pathogenese einer Erkrankung, da ihr vollständiges Fehlen (Defizienz) oder ihre Überexpression (Transgenität) direkte Rückschlüsse auf ihre Funktion erlauben. Zunächst wurden unterschiedliche Modelle der allergischen Sensibilisierung und Atemwegs-Provokation mit Allergen etabliert und miteinander verglichen. Das erste Modell, die Atemwegs-Sensibilisierung mit ausschließlicher Gabe von Allergen als Aerosol, imitiert den natürlichen Sensibilisierungsweg über die inhalative Route beim asthmatischen Patienten. Dieser Modus führt zu geringer allergen-spezifischer IgE-Produktion, einer marginalen inflammatorischen Reaktion in den Atemwegen und zu unspezifischer AHR, messbar in vitro durch elektrische Feldstimulation von trachealen Segmenten. Eine ausgeprägte inflammatorische Komponente oder die Ausbildung von in vivo AHR wie bei asthmatischen Patienten fehlen jedoch in diesem Modell. Als zweites wurde das Modell der passiven Sensibilisierung mit allergen-spezifischem IgE gefolgt von Allergen-Provokationen der Atemwege etabliert. Dieses Modell erlaubt im Gegensatz zum vorherigen die Unterscheidung des Einflusses von IgE und Allergen-Provokation der Atemwege. Auch hier entwickelt sich eine nur geringe, aber für die Ausbildung der AHR erforderliche eosinophile AI, die über die in vitro Bestimmung messbar ist. Als drittes Modell wurde die systemische Sensibilisierung mit Allergen gefolgt von Allergen-Provokationen der Atemwege etabliert. In diesem Modell kommt es zu hoher IgE-Produktion, und es herrscht eine ausgeprägte, eosinophile AI vor, die durch spezifische Immunohistochemie darstellbar und quantifizierbar ist. Für die Messung der AHR wurden zwei unterschiedliche in vivo Methoden entwickelt, die invasive Bestimmung des Atemwegswiderstandes und die Ganzkörper-Plethysmographie am nicht narkotisierten Tier. Durch den Einsatz von monoklonalen Antikörpern und genetisch alterierten Mausstämmen wurden in den drei unterschiedlichen Modellen der Einfluss und die Interaktion der wesentlichen Parameter der allergischen Immunreaktion für die Entwicklung von AI und AHR definiert. In den ersten beiden Modellen (Atemwegs- und passive Sensibilisierung) wurde anhand von T-Zell- und B-Zell-defizienten Mausstämmen und durch Einsatz von Antikörpern gegen T-Zellen oder Zytokine gezeigt, dass für die Entwicklung von eosinophiler AI die T-Zell-vermittelte Produktion von IL-5, für die Entwicklung von in vitro AHR das Zusammenspiel von allergen-spezifischer IgE-Produktion und eosinophiler AI notwendig ist. Im Modell der systemischen Sensibilisierung konnte in Mäusen, die genetisch defizient für B-Zellen oder Mastzellen oder mit anti-IgE Antikörpern behandelt waren, eine eosinophile AI und in vivo AHR wie bei normalen Tieren ausgelöst werden. Im Gegensatz hierzu kam es bei IL-4- oder IL-5-defizienten Mäusen oder nach Behandlung mit anti-IL-5 Antikörpern weder zu inflammatorischen Reaktionen noch zu funktionellen Veränderungen in den Atemwegen. Es kann hieraus gefolgert werden, dass bei allergen-induzierter AHR mit nur gering ausgeprägter AI ein gegen das erhöhte IgE gerichteter Ansatz (z.B. anti-IgE Antikörper) erfolgreich bei der Behandlung von Asthma sein kann. Bei Vorliegen von massiver AI scheint eine gegen die eosinophile Infiltration gerichtete Strategie (z.B. anti-IL-4/5 Antikörper) jedoch erfolgversprechender zu sein. / Allergic diseases such as bronchial asthma, atopic dermatitis and allergic rhino-conjunctivitis are steadily increasing. Asthma is the most common chronic airway disease in childhood, and leads to enormous socio-economic problems due to medication and hospitalisation. Cardinal symptoms of asthma are reversible bronchospasm after exposure with allergen (Airway Obstruction), a functional abnormality of the smooth muscles of the airways, that is characterized by increased contractility following unspecific stimulation (Bronchial Hyperreagibility or Airway Hyperreactivity, AHR), and the presence of a chronic inflammation in the small and middle-sized airways (Airway Inflammation, AI). Currently, treatment of asthma includes symptomatic therapy of airway obstruction with muscle relaxing medications (reliever) and unspecific anti-inflammatory therapy with cortico steroids (controller). Whereas the majority of patients lifes relatively safe and uncompromitted with this kind of treatment, a minority of asthmatic patients suffer either under the side-effects of continuous systemic high-dose steroids (steroid-dependent asthma), or stay symptomatic despite intensive treatment with steroids (steroid-resistent asthma). For this subgroup of patients, an efective, specific and safe mode of anti-asthmatic therapy is still missing. Imperative for the formulation of any innovative strategies for the treatment of asthma is the thorough knowledge of the pathophysiological mechanisms leading to the development of the disease. Allergic diseases are the consequence of aberrant immune reactions against common environmental antigens, such as pollen, food proteins or animal fur. It is commonly accepted by now that a dysregualtion of the T cell responses against these antigens are the main reason for the development of an allergic disease. In the case of allergic patients, production of so-called Th2-cytokines is increased, whereas Th1-cytokine production is relatively low. Production of the Th2-cytokines interleukin (IL)-4 and IL-13 induces increased production of allergen-specific IgE antibodies, resulting in immediate type of hypersensitivity reactions (early asthmatic reaction). Th2-cytokines IL-5, IL-3 and GM-CSF activate and recruit eosinophilic cells in the airways, leading to chronic eosinophilic airway inflammation and AHR (late asthmatic reaction). The exact mechanisms and the interaction of T cell cytokine production, IgE-production and eosinophilic AI is not fully understood and objective of the present presentation. For the immunological characterization of the basic mechanisms leading to the development of allergen-induced AI and AHR, the mouse was chosen as a model animal. Different modes of allergic sensitization and airway allergen challenge were established and compared to one another: sensitization with exclusive delivery of allergen via the airways, mimicking the natural way of sensitization and leading to moderate IgE-production, marginal AI and unspecifc AHR that is detectable in vitro by elektric field stimulation of tracheal segments; passive sensitization with allergen-specific IgE followed by allergen airway challenges, allowing the careful studies of IgE-dependent effects on AI and AHR; and systemic sensitization with allergen followed by repeated airway allergen challenges, leading to high IgE production and a profound eosinophilic AI. For detection of AHR following this mode of sensitization, two different in vivo methods were developed, invasive measurement of airway resistance and whole-body plethysmography of non-anesthesized animals. In the first two protocols (airway and passive sensitization), it was shown utilizing T-cell- and B-cell-deficient mouse strains and monoclonal antibodies against T cells or T cell cytokines, that for the development of AI and AHR the combined interaction of T-cell-mediated production of IL-5 and allergen-specific IgE-production was required. In contrast, in the mode of systemic sensitization, development of eosinophilic AI and in vivo AHR was independent of any Ig production or the presence of B cells, whereas IL-4- or IL-5-deficient mice or mice treated with anti-IL-5 antibodies prior to airway challenges did not develop any functional or structural abnormalities of the airways. In conclusion, these data show that treatment strategies aiming against increased IgE production (anti-IgE antibodies) may be effective in clinical situations with only limited airway inflammatory responses. In contrast, in patients with massive and predominant eosinophilic AI, approaches against the inflammatory component of the disease (anti-IL-4, anti-IL-5 antibodies) may be more promising for more specific treatment of bronchial asthma.

Carrapatos são artrópodes hematófagos de distribuição cosmopolita que têm grande importância médica e veterinária devido ao efeito deletério direto causado por se fixarem e sugarem seus hospedeiros, como também por serem importantes vetores de doenças para o homem e para os animais domésticos. Sabendo que carrapatos permanecem fixos em seus hospedeiros por longos períodos de tempo sem serem rejeitados, é possível inferir que esses ácaros possuam um arsenal de mecanismos que atuem no controle da resposta imune do hospedeiro. De fato, diversos trabalhos têm demonstrado que carrapatos são capazes de modular a resposta imune de seus hospedeiros através de componentes presentes na saliva, que são inoculados durante o repasto sangüíneo. Assim, este trabalho procurou investigar se carrapatos exercem a modulação da resposta imune do hospedeiro através do recrutamento de células T regulatórias CD4+CD25+ (Tregs), com a intenção de conter uma resposta inflamatória / imune prejudicial à sua alimentação. Para isso, células isoladas de amostras de pele e linfonodos de camundongos BALB/c infestados com carrapatos Rhipicephalus sanguineus foram analisadas quanto à expressão das moléculas de superfície CD4, CD25, CTLA-4, CD45RB, GITR e CD103 (fenótipo de células Tregs), por citometria de fluxo. Adicionalmente, as células obtidas dos linfonodos foram avaliadas quanto à expressão de mensagem para o fator de transcrição Foxp3 (característico da função regulatória), por PCR quantitativo. Paralelamente, saliva de R. sanguineus foi inoculada na orelha de animais da mesma linhagem, a fim de se comparar o infiltrado celular com o obtido na pele dos camundongos infestados com carrapatos. Os resultados mostraram que as infestações não alteraram a percentagem de células T CD4+CD25+ nem a expressão de moléculas associadas ao fenótipo de células Tregs nas células infiltradas na lesão de fixação dos carrapatos ou nos linfonodos em comparação a camundongos controles. Também não se verificou aumento da expressão do gene para Foxp3 nos linfonodos em nenhum dos grupos analisados. Por outro lado, a inoculação de saliva na orelha de camundongos induziu um aumento significativo da população de células T CD4+, porém estas também não apresentavam fenótipo regulatório, sugerindo que o mecanismo de imunomodulação exercido pelos carrapatos sobre seus hospedeiros não é mediado por essas células. Resultados adicionais mostraram que a saliva de carrapatos reduziu significativamente a percentagem de células dendríticas nas orelhas dos camundongos, sugerindo que carrapatos podem estar modulando a resposta imune de seus hospedeiros por diminuírem o repovoamento da pele com células dendríticas, as quais são essenciais na vigilância imune dos tecidos periféricos. / Ticks are bloodsucking arthropods that feed on vertebrates and are responsible for serious global economic losses both through the effects of blood sucking and as vectors of pathogens. A tick?s bloodmeal lasts for several days, during which it remains fixed to the host and avoids rejection by local inflammatory and immunological reactions. This status is achieved by the escape mechanisms ticks have evolved. In fact, many studies have demonstrated that ticks modulate the host immune response through salivary compounds inoculated during their bloodmeals. This study investigated if during bloodfeeding ticks can recruit regulatory T cells in an attempt to modulate the host immune response and to control inflammatory responses that could be harmful to tick feeding. BALB/c mice were infested with Rhipicephalus sanguineus, the skin at feeding sites and the regional lymph nodes were collected, and the cells forming the local infiltrates were analyzed by flow cytometry for simultaneous expression of CD4, CD25, CTLA-4, CD45RB, GITR and CD103 molecules. Additionally, expression of mRNA for Foxp3 was measured in the lymph node cells. Tick saliva was also inoculated into the ears of BALB/c mice in order to compare the local cellular infiltrate with that elicited by artificial infestation. Control animals were sham infested. The results show that, relative to sham-infested tissues, tick infestations did not alter the percentage of CD4+CD25+ T cells present at the site of their attachment or in draining lymph nodes. Infestations also did not increase the expression of Foxp3 in skin and lymph nodes. On the other hand, saliva inoculated into the ear induced a significant increase in the number of CD4+ T lymphocytes recruited to the site of inoculation, although these cells did not express a regulatory phenotype. These results suggest that the modulation of the host immune response by ticks does not involve CD4+CD25+ T cells. Additional results showed that tick saliva reduced the percentage of dendritic cells in the skin of infested mice. This finding indicates that ticks may modulate the host immune response by diminishing the repopulation of skin with dendritic cells, which are essential for maintaining surveillance of peripheral tissues for incoming antigens.