Cross-presenting human ␥␦ T cells induce robust CD8؉ ␣␤ T cell responses Marle` ne Brandesa,1, Katharina Willimanna, Gilles Bioleyb,2, Nicole Le´ vyc, Matthias Eberla,3, Ming Luod, Robert Tampe´ e, Fre´ de´ ric Le´ vyc,4, Pedro Romerob, and Bernhard Mosera,3,5 aInstitute of Cell Biology, University of Bern, CH-3000 Bern 9, Switzerland; bDivision of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne Branch, University Hospital (Centre Hospitalier Universitare Vaudois), CH-1005 Lausanne, Switzerland; cLudwig Institute for Cancer Research, Lausanne Branch, CH-1066 Epalinges, Switzerland; dDepartment of Microbiology, University of Alabama, Birmingham, AL 35294; and eInstitute of Biochemistry, Biocenter, Johann Wolfgang Goethe-University, D-60438 Frankfurt am Main, Germany Edited by Peter Cresswell, Yale University School of Medicine, New Haven, CT, and approved December 18, 2008 (received for review October 7, 2008) ␥␦ T cells are implicated in host defense against microbes and receptors and the transformation of these cells into professional tumors but their mode of function remains largely unresolved. APCs, termed ␥␦ T-APCs, capable of inducing CD4ϩ T cell Here, we have investigated the ability of activated human responses (9, 10). Of note, antigen-presenting ␥␦ T cells have also .(V␥9V␦2؉ T cells (termed ␥␦ T-APCs) to cross-present microbial and been reported in cows (11); pigs (12); and, most recently, mice (13 tumor antigens to CD8؉ ␣␤ T cells. Although this process is thought Reactivity to HMB-PP-expressing microbes and certain tumors to be mediated best by DCs, adoptive transfer of ex vivo antigen- suggested to us a role for human ␥␦ T-APCs in the induction of ϩ loaded, human DCs during immunotherapy of cancer patients has pathogen/tumor-specific CD8 T effector cells. Rapid and uniform shown limited success. We report that ␥␦ T-APCs take up and activation in response to a single stimulus of IPP or HMB-PP process soluble proteins and induce proliferation, target cell killing represents a highly useful tool for investigating ␥␦ T cell functions and cytokine production responses in antigen-experienced and and allowed us to examine the ability of ␥␦ T-APCs to cross-present ϩ .naïve CD8؉ ␣␤ T cells. Induction of APC functions in V␥9V␦2؉ T cells soluble microbial and tumor antigens to CD8 responder cells was accompanied by the up-regulation of costimulatory and MHC class I molecules. In contrast, the functional predominance of the Results ؉ immunoproteasome was a characteristic of ␥␦ T cells irrespective of Human ␥␦ T-APCs Efficiently Cross-Present Soluble Proteins to CD8 IMMUNOLOGY their state of activation. ␥␦ T-APCs were more efficient in antigen ␣␤ T Cells. First, we examined the ability of ␥␦ T-APCs to induce cross-presentation than monocyte-derived DCs, which is in con- ␣␤ T cell proliferation in response to the complex protein trast to the strong induction of CD4؉ ␣␤ T cell responses by both mixture Mycobacterium tuberculosis purified protein derivative types of APCs. Our study reveals unexpected properties of human (PPD). ␥␦ T-APCs or monocyte-derived DCs were loaded with (T-APCs in the induction of CD8؉ ␣␤ T effector cells, and justifies PPD, washed and then cocultured with autologous, 5- (and 6- ␦␥ their further exploration in immunotherapy research. carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled responder cells. Using bulk CD3ϩ T cells as responder cells, both ϩ ϩ anti-microbial immunity ͉ antigen cross-presentation CD8 T cells and CD4 T cells showed clear proliferation responses, as assessed by reduction in CFSE signals (Fig. 1A). Similar antigen-dependent responses were obtained with puri- mmunity to many pathogens and tumors involves major his- fied naïve CD8ϩ ␣␤ T cells as responder cells. Itocompatibility complex class I (MHC I) restricted, cytotoxic ϩ ␣␤ To confirm these initial findings in support of cross- CD8 T cells, which kill affected leukocytes and nonhema- presentation by ␥␦ T-APCs, we turned to an experimental model topoietic tissue cells. Microbes and tumors frequently interfere that allowed more detailed investigations. This model included with antigen processing or presentation and thus inhibit appro- the well defined influenza virus-encoded matrix protein M1 that priate antigen-presenting cell (APC) function; also, many mi- induces strong CD8ϩ ␣␤ T cell responses to M1p58–66, the crobes do not infect APCs. However, dendritic cells (DCs), the immunodominant peptide contained within M1, in HLA A*0201 prototype professional APCs (1), can take up exogenous mate- (HLA-A2)-positive individuals (14). First, cross-presentation rial derived from infected cells and tumors and direct these to was studied in a HLA-A2-restricted CD8ϩ ␣␤ T cell clone, which intracellular compartments with access to the MHC I pathway, produces IFN-␥ in response to M1p58–66-presenting, HLA- a process known as antigen ‘‘cross-presentation’’ (2, 3). Such A2ϩ APCs (labeling and gating strategy of the IFN-␥ assay is DCs can trigger expansion and differentiation of microbe/tumor- specific CD8ϩ ␣␤ T cells. Natural DC subsets in humans that are specialized in antigen cross-presentation are not well defined. Author contributions: M.B., K.W., G.B., N.L., M.E., F.L., P.R., and B.M. designed research; ␥␦ T cells are essential constituents of innate anti-microbial M.B., K.W., G.B., N.L., M.E., M.L., R.T., and F.L. performed research; M.L., R.T., and P.R. contributed new reagents/analytic tools; M.B., K.W., G.B., N.L., M.E., F.L., P.R., and B.M. and anti-tumor defense, yet their role in adaptive immunity is analyzed data; and M.B. and B.M. wrote the paper. less clear (4–6). ␥␦ T cells are a distinct subset of CD3ϩ T cells The authors declare no conflict of interest. featuring T cell receptors (TCRs) that are encoded by V␥- and V␦-gene segments (4, 5). In peripheral blood of healthy indi- This article is a PNAS Direct Submission. ␥␦ 1Present address: Lymphocyte Biology Section, Laboratory of Immunology, National Insti- viduals T cells make up 2–10% of total T cells, and of these tute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892. the majority (typically Ͼ80%) express V␥9V␦2-TCRs. A distin- 2Present address: Laboratory of Cancer Vaccinotherapy, Institut National de la Sante´etde guishing feature, their TCRs are selective for conserved non- la Recherche Me´dicale U601, Centre de Lutte Contre le Cancer Rene´Gauducheau, 44800 peptide compounds of microbial or tumor cell origin, including Saint Herblain Nantes, France. the isoprenoid metabolites isopentenyl pyrophosphate (IPP) and 3Present address: Department of Medical Biochemistry and Immunology, School of Med- (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), icine, Cardiff University, Cardiff CF14 4XN, United Kingdom. which are recognized in a MHC-independent fashion (7, 8). In 4Present address: Debiopharm SA, Case Postale 5911, CH-1002 Lausanne, Switzerland. ϩ agreement, V␥9V␦2 T cells are highly expanded in patients 5To whom correspondence should be addressed. E-mail: [email protected]. suffering from microbial infections. This article contains supporting information online at www.pnas.org/cgi/content/full/ We have recently reported that IPP-stimulation of human blood 0810059106/DCSupplemental. ϩ V␥9V␦2 T cells leads to the expression of lymph node migration © 2009 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810059106 PNAS ͉ February 17, 2009 ͉ vol. 106 ͉ no. 7 ͉ 2307–2312 Downloaded by guest on September 27, 2021 T+PPD TT- - PPD DC + PPD of MHC I molecules. HLA-mismatched B cells used as feeder cells A during in vitro activation of V␥9V␦2ϩ T cells failed to cross-present 21.2 0.4 10.6 M1 (Fig. 1B). Of note, ␥␦ T-APCs from different donors gave Bulk reproducible results, which is in contrast to the strikingly variable CD3+ ␥␦ 4D responses obtained with DCs (Fig. 1B). Of interest, T-APCs 2.7 0.2 5.8 C were able to take up and process M1 protein over a wide range of 2.7 0.2 5.8 culture time and still showed antigen presentation function after prolonged culture in the absence of antigen (Figs. S3 and S4). 0.6 Naive 2.4 0.7 In the next step, we tested M1p58–66-pulsed ␥␦ T-APCs for CD8+ ϩ ␣␤ OR54DC their ability to induce proliferation in blood CD8 T cells. M1p58–66-specific cells (0.01–0.5%), assessed by M1p58–66- tetramer staining, are primarily found in the memory T cell compartment of healthy HLA-A2ϩ individuals (14). Responses obtained with M1p58–66-pulsed ␥␦ T-APCs were unmatched in 2.1 94.9 0.7 94.4 0.6 97.3 terms of potency and efficacy, as compared with DCs, monocytes and B cells (Fig. S5). Moreover, ␥␦ T-APCs were also very adept in cross-presentation of M1, involving the uptake and intracel- 8DC lular processing of exogenous protein, to this polyclonal M1p58– 0.5 <0.1 0.1 66-reactive CD8ϩ ␣␤ T cell compartment (Fig. 1C). Striking CFSE variation in responses to DCs prompted us to evaluate different strategies for DC generation, including substituting IL-15 for P =0.017 B ) 100 100 IL-4 during monocyte differentiation (data not shown), and %(s applying CD40-signaling as opposed to shear force in combina- l l e tion with LPS to induce DC maturation. None of these treat- c+ 50 50 ments led to substantial improvements (Fig. 1D), and in all - N subsequent experiments shear force/LPS-treated DCs were used. F I 0 0 0 0.04 0.4 4 T DC DC ␥␦ M1 ( M) Antigen Cross-Presentation by T-APCs Involves Proteasome Activ- M1 p58-66 ity and de Novo Synthesized MHC I Molecules. The route(s) of antigen processing leading to peptide loading onto MHC I within ␥␦ C D P = 0.016 ) T-APCs are not known.