Geometria e silêncio das mulheres nos tapetes berberes do médio atlas

A investigação desenvolvida neste estudo pretende conduzir a uma reflexão sobre a relação que se estabelece entre a geometria e o pensamento abstracto de mulheres berberes, maioritariamente analfabetas e que, até há bem pouco tempo (talvez ainda em algumas regiões rurais, mais isoladas, de Marrocos), viviam segregadas. Falamos, sim, desses berberes que chegaram e se instalaram nas montanhas de Marrocos, muito antes da chegada dos árabes e que, ainda hoje continuam a fazer história, deixando que o seu quotidiano, o seu modo de vida seja ditado pelo enquadramento numa paisagem magnífica, que dá azo a um tempo mais lento, mais contemplativo, mais devoto. Reflectir-se-á sobre a originalidade dos tapetes berberes, cujo produto final não está delineado, à partida, mas que tem a ver com uma concepção interior da obra, que promove a liberdade de criação destas mulheres. Procurar-se-á compreender o tapete como uma forma de arte doméstica, conceptual, que emerge de um pensamento que, sendo geométrico (universal), se torna abstracto ou só compreensível por um grupo restrito de iniciados.1 O tapete tornou-se o segredo destas mulheres. E só o é, porque nós (todos, à excepção das mulheres berberes) não o conseguimos entender. Falamos de um “código” que só é perceptível quando é conhecida a matriz do pensamento. Pode até conhecer-se a técnica e a estética dos tapetes, identificar-se as diferentes tipologias e os materiais utilizados, mas tornar-se-á impossível criá-los, como estas mulheres o fazem, sem planos pré-estabelecidos. É essa maneira de produzir, quase inata para estas mulheres, que está fora do nosso alcance lógico

Impact of peptide:HLA complex stability for the identification of SARS-CoV-2-specific CD8⁺T cells.

Induction of a lasting protective immune response is dependent on presentation of epitopes to patrolling T cells through the HLA complex. While peptide:HLA (pHLA) complex affinity alone is widely exploited for epitope selection, we demonstrate that including the pHLA complex stability as a selection parameter can significantly reduce the high false discovery rate observed with predicted affinity. In this study, pHLA complex stability was measured on three common class I alleles and 1286 overlapping 9-mer peptides derived from the SARS-CoV-2 Spike protein. Peptides were pooled based on measured stability and predicted affinity. Strikingly, stability of the pHLA complex was shown to strongly select for immunogenic epitopes able to activate functional CD8+T cells. This result was observed across the three studied alleles and in both vaccinated and convalescent COVID-19 donors. Deconvolution of peptide pools showed that specific CD8+T cells recognized one or two dominant epitopes. Moreover, SARS-CoV-2 specific CD8+T cells were detected by tetramer-staining across multiple donors. In conclusion, we show that stability analysis of pHLA is a key factor for identifying immunogenic epitopes

Highly Active Microbial Phosphoantigen Induces Rapid yet Sustained MEK/Erk- and PI-3K/Akt-Mediated Signal Transduction in Anti-Tumor Human γδ T-Cells

BACKGROUND: The unique responsiveness of Vgamma9Vdelta2 T-cells, the major gammadelta subset of human peripheral blood, to non-peptidic prenyl pyrophosphate antigens constitutes the basis of current gammadelta T-cell-based cancer immunotherapy strategies. However, the molecular mechanisms responsible for phosphoantigen-mediated activation of human gammadelta T-cells remain unclear. In particular, previous reports have described a very slow kinetics of activation of T-cell receptor (TCR)-associated signal transduction pathways by isopentenyl pyrophosphate and bromohydrin pyrophosphate, seemingly incompatible with direct binding of these antigens to the Vgamma9Vdelta2 TCR. Here we have studied the most potent natural phosphoantigen yet identified, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), produced by Eubacteria and Protozoa, and examined its gammadelta T-cell activation and anti-tumor properties. METHODOLOGY/PRINCIPAL FINDINGS: We have performed a comparative study between HMB-PP and the anti-CD3epsilon monoclonal antibody OKT3, used as a reference inducer of bona fide TCR signaling, and followed multiple cellular and molecular gammadelta T-cell activation events. We show that HMB-PP activates MEK/Erk and PI-3K/Akt pathways as rapidly as OKT3, and induces an almost identical transcriptional profile in Vgamma9(+) T-cells. Moreover, MEK/Erk and PI-3K/Akt activities are indispensable for the cellular effects of HMB-PP, including gammadelta T-cell activation, proliferation and anti-tumor cytotoxicity, which are also abolished upon antibody blockade of the Vgamma9(+) TCR Surprisingly, HMB-PP treatment does not induce down-modulation of surface TCR levels, and thereby sustains gammadelta T-cell activation upon re-stimulation. This ultimately translates in potent human gammadelta T-cell anti-tumor function both in vitro and in vivo upon transplantation of human leukemia cells into lymphopenic mice, CONCLUSIONS/SIGNIFICANCE: The development of efficient cancer immunotherapy strategies critically depends on our capacity to maximize anti-tumor effector T-cell responses. By characterizing the intracellular mechanisms of HMB-PP-mediated activation of the highly cytotoxic Vgamma9(+) T-cell subset, our data strongly support the usage of this microbial antigen in novel cancer clinical trials

ɣδ T cell responses to tumours : recruitment, recognition and functions

Tese de doutoramento, Ciências Biomédicas (Imunologia), Universidade de Lisboa, Faculdade de Medicina, 2013Tumour immunosurveillance postulates that immune cells are able to eliminate transformed cells, as much as they eliminate pathogens or pathogeninfected cells. This theory constitutes the basis of cancer immunotherapy which explores anti-tumour properties of the immune system (both innate and adaptive) for the treatment of human malignancies. γδ T cells are innate-like lymphocytes that account for a small percentage (1- 10%) of human peripheral blood lymphocytes, but represent the majority of T cells in epithelial tissues. Several properties make γδ T cells attractive targets for cancer immunotherapy, namely their MHC-unrestricted cytotoxicity and unique responsiveness to clinical grade available (phospho) agonists. Despite the promise of γδ T cells in cancer immunotherapy, the clinical success has been limited by low percentages of objective responses. This urges more research on the mechanisms that govern the interactions between γδ T cells and tumours. One of the major gaps in the γδ T cell field is the lack of mechanistic knowledge on tumour cell recognition. We decided to analyze which molecules determine haematological tumour recognition by Vγ9Vδ2 T cells, the major γδ T cell subsets in human peripheral blood. We observed widely variable susceptibility of leukaemia and lymphoma cell lines to Vγ9Vδ2 T cell-mediated cytotoxicity. For those tumours that were efficiently targeted by Vγ9Vδ2 T cells, we found that this required the NK receptor NKG2D, but not the signature T cell receptor (TCR). We then analyzed the expression of NKG2D ligands in susceptible or resistant tumour cells lines, and observed that ULBP1 expression (both at the mRNA level and protein level) segregated with susceptible targets. Through a series of loss- and gain-offunction assays, we demonstrated that ULBP1 constitutes a non-redundant determinant of leukaemia and lymphoma cell recognition by human γδ T cells. Importantly, we observed a dramatic heterogeneity of ULBP1 expression in primary samples obtained from leukaemia and lymphoma patients, which can thus contribute to the highly variable outcomes of γδ T cell-based clinical trials in haematological tumours. Another important limitation to the modulation of γδ T cells in cancer immunotherapy is the lack of molecular cues that direct γδ T cell migration to tumours. Given the pivotal role played by chemokines and their receptors in leukocyte migration, we investigated which chemokines could determine γδ T cell recruitment to tumours. We used the “gold standard” pre-clinical transplantable B16 melanoma model and compared chemokine composition of tumour extracts from WT and TCRδ-deficient mice, and observed an accumulation of the CCR2 ligands, CCL2 and CCL12, in tumour extracts from TCRδ-deficient animals. Interestingly, the comparison of WT and CCR2-deficient hosts revealed increased tumour growth in CCR2-/- mice. Critically, we showed that tumours from CCR2-/- (as well as CCL2-/-) mice contained significantly less infiltrating γδ T cells compared to WT tumours, whereas other T cell populations, particularly CD4+ and CD8+ T cells, were not affected. We also analysed myeloid populations, namely macrophages, neutrophils and myeloid-derived suppressor cells (MDSCs), and observed that, as expected from previous literature, these leukocyte populations were reduced in tumours from CCR2-/- mice. Considering that myeloid cell infiltration into tumours is usually associated with poor prognosis, it is noticeable that CCR2-/- mice displayed increased tumour growth when compared to WT mice. Furthermore, as TCRδ-deficient mice also showed increased tumour burden (compared to WT mice), our data suggests a novel protective role for the CCR2/ CCL2 chemokine pathway through the recruitment of cytotoxic γδ T cells. Considering the significant differences between murine and human γδ T cells, and trying to translate these findings into the human setting, we also investigated if human γδ T cells relied on CCR2 for migration. We observed that the Vδ1 subset expresses CCR2 and migrates towards CCL2 in vitro; by contrast with the Vδ2 population that lacks CCR2 expression, even after activation. Moreover, we observed a dramatic variety of CCL2 levels in several cancer types, with some overexpressing and other downmodulating CCL2 when compared to healthy tissue controls. These data collectively highlight the importance of correlating Vδ1 T cell infiltration with CCL2 expression in situ in future cancer clinical studies. While the potent anti-tumour properties of γδ T cells have been widely demonstrated, some recent reports have implied a pro-tumour role for γδ T cells. We therefore set out to investigate which conditions determine the anti- versus protumour properties of γδ T cells. By comparing two different murine tumour models, B16 melanoma and ID8 ovarian carcinoma, we proposed that the pro-tumour role of γδ T cells may be determined by IL-17 production. TCRδ-deficient mice showed reduced survival (compared to WT) when challenged with B16 tumours, but enhanced survival when challenged with ID8 tumours. We demonstrated an accumulation of IL-17-producing γδ T cells in the peritoneal cavity of ID8 tumour bearing mice that seemed to dominate over anti-tumour IFN-γ production. Importantly, the major source of IL-17 in the ID8 tumour model were γδ T cells, and these expressed higher levels of IL-17 (on a per cell basis) than their CD4+T cell counterparts. In summary, in this thesis we have characterized some key features of γδ T cells in tumour immunosurveillance, namely leukaemia/ lymphoma cell recognition; migration and tumour infiltration; and putative pro-tumour properties. We believe these findings make an important contribution to our understanding of γδ T cell function, and may help to improve current cancer immunotherapy protocols based on γδ T cell activation.A teoria da imunovigilância do cancro postula que as células do sistema imunitário são capazes de eliminar células transformadas, da mesma forma que combatem patogénios e células infectadas por patogénios. Esta teoria constitui a base da imunoterapia do cancro, a qual explora as propriedades anti-tumorais do sistema imunitário (tanto do sistema inato como do adaptativo) no tratamento de doenças malignas. As células T γδ são linfócitos que constitutem uma pequena percentagem (1-10%) dos linfócitos periféricos do sangue humano, mas que representam a maioria das células T em tecidos epiteliais. Estas células apresentam algumas propriedades que as tornam uma boa aposta para protocolos de imunoterapia para o cancro, como por exemplo, citotoxicidade independente da apresentação de antigénios por moléculas do complexo major de histocompatibilidade (MHC) e reactividade selectiva a fosfoantigénios que podem ser sintetizados em larga escala. Contudo, apesar do entusiasmo inicial, o sucesso clínico do uso de células T γδ tem sido limitado dadas as baixas percentagens de resposta clínica objectiva obtidas. Estes resultados revelam a necessidade de mais investigação acerca dos mecanismos que determinam uma interação produtiva entre células T γδ e células tumorais. Uma das maiores lacunas na biologia das células T γδ são os mecanismos que controlam o reconhecimento de células tumorais. Por esse motivo decidimos analisar quais as moléculas envolvidas na detecção de tumores hematológicos por células Vγ9Vδ2, as quais constituem a maior fracção das células T γδ no sangue periférico humano. Observámos uma grande variabilidade de susceptibilidade de linhas celulares de leucemia e linfoma à citotoxicidade mediada por células Vγ9Vδ2. Verificámos que o receptor NKG2D é necessário, enquanto que o receptor de células T (TCR) é dispensável, para a eliminação de linhas tumorais susceptíveis. Analisámos a expressão de ligandos de NKG2D nas linhas celulares tumorais resistentes e susceptíveis, e observámos que a expressão de ULBP1 (tanto ao nível do mRNA como ao nível da proteína) está associada a tumores susceptíveis. Realizámos ensaios de perda e ganho de função in vitro e concluímos que o ULBP1 é um determinante não redundante do reconhecimento de leucemias e linfomas por parte das células T γδ humanas. De realçar que observámos uma grande heterogeneidade de expressão de ULBP1 em amostras primárias de doentes de leucemia e linfoma, o que poderá contribuir para a variabilidade de respostas observadas em ensaios clínicos hematológicos baseados em células T γδ. Outro aspeto limitante para o sucesso da utilização de células T γδ na imunoterapia do cancro, é a falta de conhecimento dos fatores que controlam a migração e infiltração tumoral das células T γδ . Dado o papel fulcral das quimiocinas e dos seus receptores na migração de leucócitos, investigámos o seu envolvimento o recrutamento tumoral das células T γδ. Usámos o modelo murino pré-clinico de melanoma, baseado na injecção da linha tumoral B16, e comparámos a composição de quimiocinas em extratos proteicos de tumores provenientes de murganhos suficientes (“selvagens”) ou deficientes em linfócitos T γδ (Tcrd-/-), tendo observado uma acumulação de ligandos de CCR2 (CCL2 e CCL12) em extratos tumorais de animais Tcrd−/−. Curiosamente, verificámos também que os murganhos Ccr2-/-apresentavam tumores maiores do que os murganhos selvagens. Analisámos a composição leucocitária e concluímos que os tumores de murganhos Ccr2-/- e também de murganhos Ccl2-/- continham significativamente menos células T γδ infiltrantes do que os murganhos selvagens. Verificámos também que a migração de outras populações de linfócitos T, nomeadamente células CD4+ ou CD8+, não foram afetadas. Analisámos as populações mielóides, incluindo macrófagos, neutrófilos e “myeloid-derived suppressor cells” (MDSCs), e observámos (como previsto na literatura) que estas populações leucocitárias se encontravam reduzidas em tumores originários de murganhos Ccr2-/- . Considerando que a infiltração de tumores por células mielóides está geralmente associada com mau prognóstico, é notável que os murganhos Ccr2-/- apresentem tumores maiores em comparação com murganhos selvagens. Adicionalmente, como os murganhos Tcrd-/- também apresentaram tumores aumentados (em comparação com murganhos selvagens), os nossos dados sugerem uma nova função protetora da via inflamatória CCR2/CCL2 através do recrutamento de células T γδ citototóxicas. Dadas as diferenças significativas entre células T γδ de murganho e humanas, e no sentido de aplicar estas descobertas à medicina, investigámos se as células T γδ humanas dependiam de CCR2/CCL2 para a sua migração. Verificámos que a subpopulação Vδ1 expressa CCR2 e migra para CCL2 in vitro; pelo contrário, a subpopulação Vδ2 não expressa CCR2 (mesmo após ativação)e não responde a CCL2. Adicionalmente, observámos uma grande variabilidade de níveis de expressão de CCL2 em vários tipos de tumores humanos; alguns tipos sobrexpressam, enquanto que outros subexpressam CCL2 comparativamente com os respetivos tecidos saudáveis. Estes dados salientam a importância de correlacionar a infiltração de células T Vδ1 com a expressão de CCL2 in situ em ensaios clínicos futuros. Apesar das potentes propriedades anti-tumourais das células T γδ estarem bem estabelecidas, alguns estudos recentes reportaram uma atividade pro-tumoral das células T γδ. Consequentemente, proposemo-nos investigar as condições que determinam as propriedades anti- ou pro-tumorais das células T γδ. Através da comparação de dois modelos murinos de melanoma (linha B16) e de carcinoma do ovário (linha ID8), mostrámos que o efeito pro-tumoral das células T γδ se associa a elevada produção de produção de interleucina-17 (IL-17). Verificámos que murganhos Tcrd-/- apresentam sobrevivência reduzida (comparado com murganhos selvagens) após transplante do tumor B16, mas sobrevivência aumentada após transplante do tumor ID8. A acumulação de células T γδ produtoras de IL-17 exclusivamente na cavidade peritoneal de murganhos transplantados com tumores ID8, levou-nos a propôr um efeito pro-tumoral dependente de IL-17. É importante notar que a maior fonte de IL-17 nos tumores ID8 foram as células T γδ, e estas que estas expressaram níveis mais elevados de IL-17 (ao nível de cada célula) em comparação com as células T CD4+ . Resumindo, nesta tese caracterizámos vários aspetos-chave da resposta das células T γδ a tumores , nomeadamente o reconhecimento molecular de linfomas e leucemias; os mecanismos de migração e infiltração tumoral; e possíveis propriedades pro-tumorais. Acreditamos que estas descobertas contribuem significativamente para o conhecimento da biologia das células T γδ, e esperamos que possam melhorar os protocolos atuais de imunoterapia do cancro baseados da ativação de células T γδ.Fundação para a Ciência e Tecnologia (FCT, SFRH/BD47342/2008); Molecular Biology Organization- Young Investigator Programme; European Research Council (StG_26052) e Fundação Calouste Gulbenkia

Identification of a panel of ten cell surface protein antigens associated with immunotargeting of leukemias and lymphomas by peripheral blood γδ T cells

©2010 Ferrata Storti Foundation. This is an open-access paperBackground Vγ9Vδ2 T lymphocytes are regarded as promising mediators of cancer immunotherapy due to their capacity to eliminate multiple experimental tumors, particularly within those of hematopoietic origin. However, Vγ9Vδ2 T-cell based lymphoma clinical trials have suffered from the lack of biomarkers that can be used as prognostic of therapeutic success. Design and Methods We have conducted a comprehensive study of gene expression in acute lymphoblastic leukemias and non-Hodgkin’s lymphomas, aimed at identifying markers of susceptibility versus resistance to Vγ9Vδ2 T cell-mediated cytotoxicity. We employed cDNA microarrays and quantitative real-time PCR to screen 20 leukemia and lymphoma cell lines, and 23 primary hematopoietic tumor samples. These data were analyzed using state-of-the-art bioinformatics, and gene expression patterns were correlated with susceptibility to Vγ9Vδ2 T cell mediated cytolysis in vitro. Results We identified a panel of 10 genes encoding cell surface proteins that were statistically differentially expressed between “γδ-susceptible” and “γδ-resistant” hematopoietic tumors. Within this panel, 3 genes (ULBP1, TFR2 and IFITM1) were associated with increased susceptibility to Vγ9Vδ2 T-cell cytotoxicity, whereas the other 7 (CLEC2D, NRP2, SELL, PKD2, KCNK12, ITGA6 and SLAMF1) were enriched in resistant tumors. Furthermore, some of these candidates displayed a striking variance of expression among primary follicular lymphomas and T-cell acute lymphoblastic leukemias. Conclusions Our results suggest that hematopoietic tumors display a highly variable repertoire of surface proteins that can impact on Vγ9Vδ2 cell-mediated immunotargeting. The prognostic value of the proposed markers can now be evaluated in upcoming Vγ9Vδ2 T cell-based lymphoma/leukemia clinical trials.This work was supported by the European Molecular Biology Organization (YIP Installation Grant to BS-S),Fundação Calouste Gulbenkian (SDH Oncologia 2008 - Projecto 99293) and Fundação para a Ciência e Tecnologia/FCT (PTDC/BIA-BCM/71663/2006)

IRF8 deficiency induces the transcriptional, functional, and epigenetic reprogramming of cDC1 into the cDC2 lineage

Conventional dendritic cells (cDCs) consist of two major functionally and phenotypically distinct subsets, cDC1 and cDC2, whose development is dependent on distinct sets of transcription factors. Interferon regulatory factor 8 (IRF8) is required at multiple stages of cDC1 development, but its role in committed cDC1 remains unclear. Here, we used Xcr1-cre to delete Irf8 in committed cDC1 and demonstrate that Irf8 is required for maintaining the identity of cDC1. In the absence of Irf8, committed cDC1 acquired the transcriptional, functional, and chromatin accessibility properties of cDC2. This conversion was independent of Irf4 and was associated with the decreased accessibility of putative IRF8, Batf3, and composite AP-1-IRF (AICE)-binding elements, together with increased accessibility of cDC2-associated transcription-factor-binding elements. Thus, IRF8 expression by committed cDC1 is required for preventing their conversion into cDC2-like cells

Tumor-associated neutrophils suppress pro-tumoral IL-17+ γδ T cells through induction of oxidative stress

<div><p>Interleukin 17 (IL-17)–producing γδ T cells (γδ17 T cells) have been recently found to promote tumor growth and metastasis formation. How such γδ17 T-cell responses may be regulated in the tumor microenvironment remains, however, largely unknown. Here, we report that tumor-associated neutrophils can display an overt antitumor role by strongly suppressing γδ17 T cells. Tumor-associated neutrophils inhibited the proliferation of murine CD27⁻ Vγ6⁺ γδ17 T cells via induction of oxidative stress, thereby preventing them from constituting the major source of pro-tumoral IL-17 in the tumor microenvironment. Mechanistically, we found that low expression of the antioxidant glutathione in CD27⁻ γδ17 T cells renders them particularly susceptible to neutrophil-derived reactive oxygen species (ROS). Consistently, superoxide deficiency, or the administration of a glutathione precursor, rescued CD27⁻ Vγ6⁺ γδ17 T-cell proliferation in vivo. Moreover, human Vδ1⁺ γδ T cells, which contain most γδ17 T cells found in cancer patients, also displayed low glutathione levels and were potently inhibited by ROS. This work thus identifies an unanticipated, immunosuppressive yet antitumoral, neutrophil/ROS/γδ17 T-cell axis in the tumor microenvironment.</p></div

Tumor-associated neutrophils suppress γδ 17 T-cell responses.

<p>Frequency of <b>(A)</b> total and IL-17⁺ γδT cells and <b>(B)</b> CD8⁺ and CD4⁺ T cells in the PEC of tumor-free and B16-F0 tumor-bearing mice. Data were pooled from four different experiments. <b>(C)</b> Representative FACS plots and summary of neutrophil, monocyte, and Treg cell frequency in the PEC of tumor-free and B16 tumor-bearing mice. Data were pooled from four independent experiments. <b>(D)</b> Frequency of IL-17⁺ γδ T cells in B16 tumor–bearing mice injected with vehicle (PBS) or mAb αGr-1, αLy6G, αCD115 + clodronate liposomes, and αCD25. <b>(E)</b> Representative FACS plots and frequency of neutrophils in tumor-free liver and within Hepa 1–6 intrahepatic tumor developed in C57BL/6 mice and <b>(F)</b> frequency of IL-17⁺ γδ T cells within Hepa 1–6 tumors developed in mice deficient/depleted for neutrophils (Neu −) or respective controls (Neu +). Red and blue circles represent αGr-1 mAb-treated or PBS-treated C57BL/6 mice, respectively, whereas red and blue triangles represent <i>Genista</i> homozygous or littermate controls, respectively. <b>(G)</b> Left: intrahepatic Hepa 1–6 tumor growth in mice with (heterozygous littermate control, <i>n</i> = 10) and without (<i>Genista</i> homozygous, <i>n</i> = 4) mature neutrophils. Data were pooled from two independent experiments. Right: intrahepatic Hepa 1–6 tumor growth in C57Bl/6J WT (<i>n</i> = 5) and <i>Il17</i>^−/− (<i>n</i> = 5) mice treated with αGr-1. Data presented as mean ± SEM. Statistical analysis was performed using Student <i>t</i> test or Mann-Whitney test. Data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004990#pbio.2004990.s001" target="_blank">S1 Data</a>. Lipo, clodronate liposome; mAb, monoclonal antibody; PEC, peritoneal exudate cell; RLU, relative luminescence units; Treg, regulatory T; γδ17, IL-17–producing γδ T cell.</p

Neutrophils selectively inhibit the proliferation of Vγ6+ γδ T cells.

<p>Representative FACS plots and/or frequency (gated on CD45⁺ lymphocytes) of <b>(A)</b> γδ T cells, <b>(B)</b> CD4⁺ and CD8⁺ T cells, and <b>(C)</b> Vγ1⁻Vγ4⁻ γδ T cells (gated on γδ T cells) in intraperitoneal B16 or intrahepatic Hepa 1–6 tumors, developed in mice deficient/depleted for neutrophils (Neu −) or respective controls (Neu +). Red and blue circles represent αGr-1 mAb-treated or PBS-treated C57BL/6 mice, respectively, whereas red and blue triangles represent <i>Genista</i> homozygous or littermate controls, respectively. Data were pooled from two (Hepa 2–6) and three to five (B16) independent experiments. <b>(D)</b> Representative FACS plots of γδ T-cell phenotype in PBS− (Neu +) or αGr-1 (Neu −) mAb-treated B16 tumor–bearing mice. <b>(E)</b> Frequency of BrdU⁺ Vγ6⁺ T cells in B16 tumor–bearing mice and of Ki67⁺ Vγ6⁺ T cells in Hepa 1–6 tumor–bearing mice at days 9 and 21 post–tumor inoculation, respectively. Statistical analysis was performed using Student <i>t</i> test or Mann-Whitney test. Data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004990#pbio.2004990.s001" target="_blank">S1 Data</a>. BrdU, bromodeoxyuridine; mAb, monoclonal antibody; TCR, T-cell receptor.</p