Gostaríamos de agradecer a Aaron Selya para discussão útil. Este trabalho foi financiado pelo NIH conceder AI29575, CA108835, AI077097 de JS, uma bolsa-piloto da Johns Hopkins Malaria Research Institute e do Departamento de Defesa conceder PC 040972 para MO

1. Fazendo, HLA-A2-Ig baseada AAPC <ol><li> Prepare tampão borato estéril <ol><li> Dissolver o ácido bórico em água para fazer 0,1 M. Ajustar o pH para 7,0. </li><li> Filtrar através de filtro estéril 0.22μm e armazenar a 4 ° C. </li></ol></li><li> Prepare tampão de lavagem estéril Bead <ol><li> Tome 956ml PBS magnésio w / o ou cálcio. </li><li> Adicionar 30ml de soro humano AB. </li><li> Adicionar EDTA 2mM concentração final. </li><li> Adicionar 0.1gsodium azida. </li><li> Filtrar com um filtro de 0,22 mM estéril e armazenar a 4 ° C. </li></ol></li><li> Tomar 1ml de contas Invitrogen M-450 Epoxy do estoque, aproximadamente 400 milhões de contas (contas pode ser contado por hemocitômetro), e colocado em um, frasco de vidro estéril parafuso superior. </li><li> Colocar o frasco contra um ímã Dynal MPC-1, enquanto as contas aderir à parede do frasco, retire o sobrenadante por aspiração. Lavar contas uma vez com 1ml de tampão borato. </li><li> Contas ressuspender em uma mistura de tampão borato 1ml e 20 mcg de HLA-A2-Ig dímero e 20 mg de anti-CD28 humano mAb (Clone 9.3). </li><li> Proteína para a conjugação talão: coloque o frasco de vidro sobre rotador e gire a 4 ° C por 24 horas. </li><li> Colocar o tubo no MPC-1 ímã e remover todos tampão borato. </li><li> Lave as contas duas vezes com tampão de lavagem 1ml Bead. </li><li> Incubar as contas em tampão de lavagem 1ml Bead, gire a 4 ° C por 24 horas. Porque tampão de lavagem contém Bead humana soro AB, ele irá bloquear todos os sites de proteína residual vinculativo sobre as contas. </li><li> Remover o sobrenadante do frasco de vidro, substitua por 1 ml tampão de lavagem fresca Bead. </li></ol> 2. Controle de qualidade dos AAPC e peptídeo de carga, armazenamento <ol><li> Adicionar ~ 5 × 10 5 contas para FACS 100μl tampão de lavagem em tubos FACS, e mancha com 1μl de anti-mouse IgG1-PE (reconhecer a parte Fc de HLA-A2-Ig) e 1μl de anti-mouse IgG2a-FITC (reconhecer a parte Fc de anti-CD28 mAb). Após coloração por 20 minutos em FACS tampão de lavagem, lave novamente e ler o resultado coloração imediatamente por citômetro de fluxo (Figura 4). </li><li> Loading peptídeo para as contas: lavar as contas duas vezes em frasco de vidro com 1 ml estéril PBS. Ressuspender com 1ml de PBS estéril, em seguida, adicionar 10μl de peptídeo CMV (1mg/ml) </li><li> Contar as contas por hemocitômetro, e rotular o frasco com data e concentração. </li><li> AAPC está pronto para uso após pelo menos 3 dias de incubação peptídeo a 4 ° C, para dar tempo suficiente de ligação de peptídeos para o dímero HLA-A2-Ig. As esferas podem ser armazenados a 4 ° C e permanecer funcional por pelo menos seis meses. </li></ol> 3. CTL isolamento humano <ol><li> Coletar ~ 100ml de sangue periférico fresco de doadores HLA-A2 saudável e positiva em 10 de sódio BD tubos Vacutainer Heparina. Use uma agulha calibre 21 ou maior para evitar a hemólise. </li><li> Centrifugar a 300xg por 10 minutos em temperatura ambiente </li><li> Remova cuidadosamente a camada de plasma topo por aspiração. O plasma pode ser utilizado como suplemento para o meio de cultura. </li><li> Substituir o plasma coletado com PBS estéril e transferir o sangue para um balão de cultura estéreis T75 ou tubos cônicos de 50ml. Misturar o sangue com PBS bem pipetando cima e para baixo. </li><li> Uma vez que todo o sangue é coletado, prepare mais quatro tubos de 50ml cónico e adicionar 15ml de Ficoll-Paque Plus. </li><li> Lentamente sobreposição de 30 35ml de células do sangue em cima do Ficoll de cada tubo. Manter a interface entre as distintas Ficoll e células do sangue. </li><li> Centrifugar a 500xg por 20 minutos em temperatura ambiente. Ligue o freio &quot;off&quot; ea aceleração tão baixo quanto possível manter uma relação clara entre as camadas. </li><li> Com uma pipeta serológico, aspirar cuidadosamente a camada de PBMC e recolher o CMSP em um tubo cônico de 50ml fresco. Quando todas as PBMC são colhidas adicionar 30ml de PBS e giram em 400xg por 10 min. Desprezar o sobrenadante e lavar mais uma vez com PBS 30ml para remover todos os Ficoll residual. </li><li> Proceder para isolamento de células CD8 + T usando Miltenyi humana CD8 + T kit de isolamento de células de acordo com o protocolo do fabricante. Este kit altamente enriquece de células CD8 + (geralmente&gt; 95%), esgotando CD8 - células. </li><li> Contar as células T CD8 +. A pureza esperada deve ser superior a 95%. Para confirmar essa utilização 2x10 5 células e realizar uma análise CD4/3/8 FACS. O CTL restante pode ser utilizado de imediato para a estimulação antígeno-específica AAPC ou eles podem ser congelados para uso futuro. </li></ol> 4. In vitro sistema de cultivo baseado AAPC <ol><li> Prepare meio de cultura <ol><li> Para TF (T fator de crescimento celular, feita no laboratório 4) 2X meio de cultura: meio RPMI completo, mais 5% de doadores autólogos plasma e 8% T fator de crescimento celular. </li><li&gt; Plasma de doadores autólogos pode ser substituído por inativado pelo calor humano AB soro. </li></ol></li><li> Ressuspender CTL 1 milhão em 8ml de TF meio de cultura 2X mais 8ml de meio RPMI completo, adicionar 1 × 10 AAPC 6, misture bem. </li><li> Use uma pipeta multi-canal para as células em placa de 96 poços fundo U placas de cultura de tecidos. (160 mL por poço) </li><li> Células de cultura em 37 ° C, 5% CO 2 incubater durante 7 dias. Alimentar as células no dia 4 com 80 mL bem TF / médio 2X. </li><li> As células estão prontas para serem colhidas no dia 7. Após a colheita, coloque as células contra o ímã e remova a AAPC de idade. </li><li> Especificidade do antígeno pode ser determinada através da coloração tetrâmero acordo com o protocolo do fabricante. Coloração fenótipo e coloração de citocinas intracelulares são realizadas de acordo com nossos 3 estudo anterior. </li><li> As células podem ser colhidas com replated AAPC novamente sob as mesmas condições. Número de células e especificidade do antígeno é esperado um aumento após a estimulação repetida. </li></ol> 5. Resultados representativos: Um exemplo de AAPC após conjugação HLA A2-Ig e anti-CD28 é mostrado na Figura 4. Conjugação de proteínas de sucesso é evidente uma clara mudança de coloração do anticorpo correspondente. Enquanto a freqüência de CMV CTL específicas no sangue periférico é tipicamente 0,5-1%, após uma única semana de AAPC mediada estimulação, a especificidade pode chegar a 55-93% (Figura 2 e 3). A expansão da CTL antígeno específico pode ser muito variável entre os diferentes doadores, mas os resultados são reprodutíveis dentro do mesmo doador. Por extrapolação, a expansão de células CMV específicos podem ser milhares de vezes em comparação com os níveis de precursor diretamente ex vivo (dados não mostrados). Coloração de citocinas intracelulares (Figura 3) mostra que essas CTL expandidas são polifuncionais, ao invés de exausto, depois de cultura de células prolongada e proliferação significativa. <img src="/files/ftp_upload/2801/2801fig1.jpg" alt="Figura 1"/> Figura 1. Gráfico Representante fluxo de expansão vivo baseado ex AAPC de CTL humanos para imunoterapia adotiva em alogênico HSCT <img src="/files/ftp_upload/2801/2801fig2.jpg" alt="Figura 2"/> Figura 2. Representante resultado coloração tetrâmero de CMV CTL específicos gerados por AAPC após uma semana de cultura <img src="/files/ftp_upload/2801/2801fig3.jpg" alt="Figura 3"/> Figura 3. Resultado coloração intracelular de citocinas Representante da CMV CTL específicos gerados pela AAPC (CMV especificidade foi de 61%) <img src="/files/ftp_upload/2801/2801fig4.jpg" alt="Figura 4"/> Figura 4. Coloração resultado Representante do M-450 contas Epoxy após conjugação de proteínas marcadas com anti-camundongo IgG1-PE e anti-mouse IgG2-FITC

<ol>
	<li>Walter, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+cellular+immunity+against+cytomegalovirus+in+recipients+of+allogeneic+bone+marrow+by+transfer+of+T-cell+clones+from+the+donor.">Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.</a> N Engl J Med. 333, 1038-1038 (1995).</li><li>Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+cell+transfer:+a+clinical+path+to+effective+cancer+immunotherapy.">Adoptive cell transfer: a clinical path to effective cancer immunotherapy.</a> Nat Rev Cancer. 8, 3669-3669 (2008).</li><li>Oelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+induction+and+expansion+of+antigen-specific+cytotoxic+T+cells+by+HLA-Ig-coated+artificial+antigen-presenting+cells.">Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells.</a> Nat Med. 9, 619-619 (2003).</li><li>Oelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+antigen-presenting+cells:+artificial+solution+for+real+diseases.">Artificial antigen-presenting cells: artificial solution for real diseases.</a> Trends Mol Med. 11, 412-412 (2005).</li></ol>

Não há conflitos de interesse declarados.

O sistema AAPC descrevemos aqui é um sistema eficiente para a expansão ex vivo de CTL humana contra uma variedade de antígenos. Cuidados especiais devem ser tomados com relação à qualidade da conjugação de proteínas e distribuição uniforme da AAPC e CTL na cultura placa de 96 poços. Usando essa abordagem, temos sido capazes de expandir CTL por mais de oito semanas, durante a qual expandiu-antígeno específico CTL até um milhão de vezes 4. Houve vários sistemas APC artificial utilizando linhas celulares ou outras plataformas acelular 5, no entanto, de acordo com os dados publicados a cada sistema tem seu perfil único no que diz respeito à expansão e especificidade suporte a aplicações diferentes. Importante, uma vez que a qualidade do CTL é tão importante como a quantidade, o polyfunctionality do CTL CMV-específico gerado pelo nosso sistema são esperadas para conferir eficiência anti-viral superior.

<table border="1"><tbody><tr><th> Reagente </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> Vacutainer tubo (contém heparina) </td><td> Becton Dickinson </td><td> 367874 </td></tr><tr><td> Humana CD8 + T kit de isolamento de células </td><td> Miltenyi </td><td> 130-094-156 </td></tr><tr><td> Dynabeads Epoxy M-450 </td><td> Invitrogen </td><td> 140,11 </td></tr><tr><td> Dynal Magnet MPC-1 </td><td> Invitrogen </td><td> 120-01D </td></tr><tr><td> Ficoll-Paque Plus </td><td> GE Healthcare </td><td> 17-1440-03 </td></tr><tr><td> Meio RPMI 1640 </td><td> Gibco </td><td> 11875 </td></tr><tr><td> HLA-A2-Ig dímero X </td><td> Becton Dickinson </td><td> 551263 </td></tr><tr><td> iTAgMHC tetrâmero (HLA-A2-CMV)-PE </td><td> Beckman Coulter </td><td> T20099 </td></tr><tr><td> Falcon clara de 96 poços da placa de microtitulação </td><td> Becton Dickinson </td><td> 353077 </td></tr><tr><td> Rato anti-rato IgG2a-FITC </td><td> Becton Dickinson </td><td> 553390 </td></tr><tr><td> Cabra anti-rato IgG1-PE </td><td> Invitrogen </td><td> P21129 </td></tr><tr><td> Soro humano tipo AB </td><td> Atlanta biológicos </td><td> S40110 </td></tr><tr><td> Camundongo anti-humano-FITC CD8a </td><td> Sigma-Aldrich </td><td> F0772 </td></tr><tr><td> Camundongo anti-humano CD8a-APC </td><td> Becton Dickinson </td><td> 340684 </td></tr><tr><td> Camundongo anti-humano-FITC IFNγ </td><td> Becton Dickinson </td><td> 340449 </td></tr><tr><td> Camundongo anti-humano TNFa-PE </td><td> Becton Dickinson </td><td> 340512 </td></tr></tbody></table>

hla-ig based artificial antigen presenting cells for efficient ex vivo expansion of human ctl

CTL com função efetora ideal desempenham papéis críticos na mediação proteção contra diversas infecções intracelulares e câncer. No entanto, os indivíduos podem apresentar microambiente imune supressivo e, em contraste com a ativação CTL, as suas células autólogas apresentando antígeno pode tender a tolerize ou anergize CTL antígeno específico. Como resultado, embora ainda em fase experimental, baseada em CTL imunoterapia adotiva evoluiu para se tornar um tratamento promissor para diversas doenças, como câncer e infecções por vírus. Em experimentos ex vivo inicial expandiu CMV (citomegalovírus) CTL específicos têm sido utilizados para o tratamento de infecção por CMV em pacientes imunocomprometidos óssea alogênico transplante de medula. Embora seja comum ter a vida em risco viremia CMV nesses pacientes, nenhum dos pacientes que receberam expandiu CTL CMV desenvolver doenças relacionadas, implicando a imunidade anti-CMV é estabelecido pela CTL adotivamente transferidos 1. Resultados promissores também têm sido observados para melanoma e pode ser estendido para outros tipos de câncer 2. Embora existam muitas maneiras de ex vivo estimular e expandir CTL humanos, abordagens atuais são restritas pelas limitações de custo e técnicas. Por exemplo, o padrão ouro atual é baseado no uso de DC autólogo. Isso exige de cada paciente para doar um número significativo de leucócitos e também é muito caro e trabalhoso. Além disso, detalhada caracterização in vitro da DC expandiu CTL revelou que estes só têm função efetora suboptimal 3. Aqui apresentamos um sistema altamente eficiente baseada AAPC para a expansão ex vivo de CTL CMV humano específico para a imunoterapia adotiva (Figura 1). A AAPC foram feitas pelo acoplamento de células de tamanho esferas magnéticas com humano HLA-A2-Ig dímero e anti-CD28mAb 4. Uma vez AAPC são feitos, eles podem ser carregados com vários peptídeos de interesse, e permanecer funcional por meses. Neste relatório, AAPC foram carregados com um peptídeo dominante da CMV, pp65 (NLVPMVATV). Após o cultivo humano purificado CD8 + CTL de um doador saudável com AAPC por uma semana, CMV CTL específica pode ser aumentado dramaticamente na especificidade até 98% (Figura 2) e ampliado mais de 10.000 vezes. Se mais CTL CMV-específicos são necessários, uma maior expansão pode ser facilmente alcançado por estimulação repetitiva com AAPC. Caracterização fenotípica e funcional mostra estas células expandidas têm fenótipo efetor uma memória e fazer uma quantidade significativa de ambos os TNFa e IFNγ (Figura 3).

Watch this Scientific Journal Video about HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL at JoVE.com

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

Um método novo CD independente para indução e expansão de células T antígeno-específicos é descrito. HLA A2-Ig baseada Células Antigen Presenting artificial (AAPC) são carregados com HLA-A2 peptídeos restrito a expandir de forma eficiente CTL de especificidade do antígeno diversas. Esta tecnologia tem um grande potencial para CTL baseada imunoterapia adotiva.

HLA-Ig Baseado Artificial células apresentadoras de antígenos para a eficiente Ex vivo Ampliação da CTL Humanos

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

hla-ig-based-artificial-antigen-presenting-cells-for-efficient-ex

Research

JoVE Journal

Immunology and Infection

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

15.3K visualizações.  Johns Hopkins University. Um método novo CD independente para indução e expansão de células T antígeno-específicos é descrito. HLA A2-Ig baseada Células Antigen Presenting artificial (AAPC) são carregados com HLA-A2 peptídeos restrito a expandir de forma eficiente CTL de especificidade do antígeno diversas. Esta tecnologia tem um grande potencial para CTL baseada imunoterapia adotiva.

Vídeo: HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Specific Antigen In Vivo Ensaio Killing usando células CFSE rotuladas Alvo

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

Imunologia e Infecção

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

Expansão ex vivo de células tumorais T-reativa por Meios de 1/Ionomycin Bryostatin eo Common Gamma Cadeia Formulação Citocinas

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Expansão, Purificação e Avaliação Funcional de Células NK do sangue periférico

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Expansão do sangue periférico humano γδ células T usando zoledronato

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Antígeno Artificial célula apresentadora de ativação mediada (AAPC) e expansão de células T Natural Killer

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

Aplicação Clínica de<em&gt; A Bela Adormecida</em&gt; Artificial e Antigen Presenting Cells para modificar geneticamente células T de Sangue do Cordão Umbilical e periférica

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

Avaliando a inata Sensing de HIV-1 CD4 Infected<sup&gt; +</sup&gt; As células T por células dendríticas plasmocitóides Usando um<em&gt; Ex vivo</em&gt; Co-cultura do sistema.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Assassino Artificial células apresentadoras de antígenos (KaAPC) para a eficiente<em&gt; In Vitro</em&gt; Depleção de células T humanas antígeno específico da

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Isolamento de linfócitos da pele humana para análise fenotípica e<em&gt; Ex Vivo</em&gt; Cultura de Células

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Desenvolvimento de uma colite Modelo Antigen-driven para o Estudo de apresentação de antígenos por células apresentadoras de antígenos às células T

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...