Vaccination Produces CD4 T Cells with a Novel CD154−CD40-Dependent Cytolytic Mechanism This information is current as Rhea N. Coler, Thomas Hudson, Sean Hughes, Po-wei D. of October 1, 2021. Huang, Elyse A. Beebe and Mark T. Orr J Immunol published online 21 August 2015 http://www.jimmunol.org/content/early/2015/08/20/jimmun ol.1501118 Downloaded from Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision http://www.jimmunol.org/ • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: by guest on October 1, 2021 http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 21, 2015, doi:10.4049/jimmunol.1501118 The Journal of Immunology Vaccination Produces CD4 T Cells with a Novel CD154–CD40-Dependent Cytolytic Mechanism Rhea N. Coler,*,†,‡ Thomas Hudson,* Sean Hughes,* Po-wei D. Huang,* Elyse A. Beebe,* and Mark T. Orr*,† The discovery of new vaccines against infectious diseases and cancer requires the development of novel adjuvants with well-defined activities. The TLR4 agonist adjuvant GLA-SE elicits robust Th1 responses to a variety of vaccine Ags and is in clinical devel- opment for both infectious diseases and cancer. We demonstrate that immunization with a recombinant protein Ag and GLA-SE also induces granzyme A expression in CD4 T cells and produces cytolytic cells that can be detected in vivo. Surprisingly, these in vivo CTLs were CD4 T cells, not CD8 T cells, and this cytolytic activity was not dependent on granzyme A/B or perforin. Unlike previously reported CD4 CTLs, the transcription factors Tbet and Eomes were not necessary for their development. CTL activity was also independent of the Fas ligand–Fas, TRAIL–DR5, and canonical death pathways, indicating a novel mechanism of Downloaded from CTL activity. Rather, the in vivo CD4 CTL activity induced by vaccination required T cell expression of CD154 (CD40L) and target cell expression of CD40. Thus, vaccination with a TLR4 agonist adjuvant induces CD4 CTLs, which kill through a previ- ously unknown CD154-dependent mechanism. The Journal of Immunology, 2015, 195: 000–000. ajor histocompatibility complex class II–restricted CD4 directly, ex vivo work has described CD4 CTLs that express T cells have traditionally been characterized as Th perforin and the most well characterized cytolytic granzyme, M cells based on their ability to modify or enhance the granzyme B (reviewed in Refs. 3 and 4). These CD4 CTLs have http://www.jimmunol.org/ immune response mediated by CD8 T cells, B cells, and innate been implicated in the control of a number of viral infections, immune cells. Help is mediated by both cell–cell interactions, including those caused by lymphocytic choriomeningitis virus, such as CD154–CD40 cross talk with B cells, and secretion of influenza virus, mousepox virus, and West Nile virus in mice (5– cytokines, including TNF and IFN-g, which cause maturation of 8). Human CD4 CTLs expressing lytic granules have also been phagocytic cells such as macrophages. CD8 T cells also produce described for infections caused by HIV, human CMV, and EBV some of these same cytokines but can also directly kill target cells as well as for those caused by mycobacteria, including bacillus presenting a cognate MHC class I:peptide complex. CD8 CTLs Calmette-Gue´rin and Mycobacterium tuberculosis (9–16). Hu- use two primary mechanisms of cytolysis: exocytosis of lytic gran- man and mouse CD4 CTLs can also kill via cell–cell contact by by guest on October 1, 2021 ules containing perforin and granzymes and cell surface recep- expressing FasL or the related surface protein TRAIL, which tors, including Fas ligand (FasL), that bind receptors on the target binds Fas or death receptor 5 (DR5), respectively, on target cells cell that initiate a cell death pathway. Death of the target cell can to induce death (9, 17, 18). Of note, Woodworth et al. (19) found proceed via several different signaling pathways, including a cas- that M. tuberculosis––specific CD4 CTLs were induced in mice pase 3– or caspase 7–dependent pathway and Bad/Bax pathway of infected with M. tuberculosis, but unlike those produced by viral mitochondria cytochrome c release (1). infection, these CD4 CTLs killed via an undefined mechanism CD4 T cells with lytic activity have also been described; how- that was independent of perforin, Fas–FasL, and TNFR1. ever, early work was based on long-term cultured CD4 T clones, The major lineages of CD4 T cell differentiation, including Th1, suggesting this may be an in vitro artifact resulting from chronic Th2, Th17, regulatory T, and T follicular helper cells, have been Ag stimulation and IL-2 signaling (2). More recent in vivo and, linked to expression of a fate-determining transcription factor, Tbet, GATA3, RORgt, Foxp3, and Bcl-6, respectively. CTL ac- tivity was originally ascribed to a subset of Th1 cells, although *Infectious Disease Research Institute, Seattle, WA 98102; †Department of Global other groups found that nonpolarized CD4 T cells could also me- ‡ Health, University of Washington, Seattle, WA 98105; and PAI Life Sciences, Seat- diate CTL activity. More recently, the T-box transcription factor tle, WA 98102 Eomes was found to be necessary for the expression of granzyme Received for publication May 15, 2015. Accepted for publication July 22, 2015. B in mouse CD4 T cells stimulated via CD134 and CD137, a reg- This work was supported in whole or in part by federal funds from the National imen sufficient to produce CD4 CTLs (20). Similarly, ectopic ex- Institute of Allergy and Infectious Diseases, National Institutes of Health, Depart- ment of Health and Human Services, under Award AI101488 to M.T.O. and Contract pression of Eomes drove perforin and FasL expression in mouse HHSN272200800045C to R.N.C. Th2 cells, converting them to CD4 CTLs (21). The exact con- The content of this publication is solely the responsibility of the authors and does not ditions necessary to induce CD4 CTLs in vitro and in vivo are necessarily reflect the official views of the National Institutes of Health or policies of still being established, but it seems clear that both Ag concentra- the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. tion and IL-2 availability can affect CD4 CTL programming (22). Address correspondence and reprint requests to Dr. Mark T. Orr, Infectious Disease Given the contribution of CD4 CTLs to the immune response Research Institute, 1616 Eastlake Avenue E, Suite 400, Seattle, WA 98102. E-mail to a number of bacterial and viral infections, it would be useful address: [email protected] to develop a vaccination scheme that can intentionally elicit these Abbreviations used in this article: DR5, death receptor 5; FasL, Fas ligand; WT, cells. We have developed a number of adjuvants that preferentially wild-type. augment Th1 or Th2 responses or boost Ab responses to protein Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 Ags indicating the induction of T follicular helper cells (23–26). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501118 2 CD4 CTL KILL VIA CD154–CD40 Using the recombinant M. tuberculosis protein Ag ID93, we have recipients. Target cells (#107) were i.v. injected into immunized animals found that the synthetic TLR4 agonist GLA augments IFN-g and and matched unimmunized controls 1 wk after immunization. One day TNF CD4 T cell responses when formulated in an oil-in-water later, splenocytes were harvested from recipients and RBCs were lysed. Depending on the experiment, cells were stained for CD45.1 (clone A20), stable emulsion (24, 26). We now report that this vaccination CD45.2 (clone 104), and I-Ab (eBioscience and BioLegend). Cells were scheme also elicits CD4 T cells that express granzyme A and are gated as singlets . lymphocytes . CFSE+ donors . CD45.1+ CD45.22 or 2 2 lytic in vivo. CD45.1 CD45.2+ . I-Ab+ or I-Ab . CFSEhi or CFSElo. Specific hi lo lysis was calculated as [1 – (CFSE immunized/CFSE immunized)/average hi lo (CFSE unimmunized/CFSE unimmunized)]*100, as described previously (5). Materials and Methods In some experiments, immunized mice were depleted of CD4+ cells (clone Mice and immunizations GK1.5) or CD8+ cells and NK1.1+ cells (clones 53-6.7 and PK136, re- spectively) 2 d prior to transfer of target cells by i.p. injection of 0.25 mg a b Wild-type (WT) C57BL/6, B6.SJL-Ptprc Pepc /BoyJ (CD45.1), 129X1/ of each depleting Ab. SvJ-Gzmatm1Ley Gzmbtm2.1Ley/J (Gzm A/B2/2, B6.Cg-Tg(Cd4-cre) 1Cwi/BfluJ (CD4-Cre+), B6.129S1(cg)-Eomestm1.1Bflu/J (Eomes fl/fl), Statistics Tbet2/2, B6Smn.C3-Faslgld/J (FasL2/2), B6.MRL-Faslpr/J (Fas2/2), C57BL/6-Pfr1tm1Sdz/J (Pfr2/2), B6N.129S1-Casp3tm1Flv/J (Casp32/2), The Student t test was used to analyze data sets consisting of only two B6.129S6-Casp7tm1Flv/J (Casp72/2), B6.129X1-Baxtm1Sjk/J (Bax2/2), groups. ANOVA analysis with the Bonferroni correction for multiple com- B6;129S-Tnfrsf1atm1Imx Tnfrsf1btm1Imx/J (TNFR1/22/2), B6.129P2- parisons was used when more than two groups were analyzed.