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 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 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 . 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 –Fas, –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 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/ 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– , including TNF and IFN-g, which cause maturation of 8). Human CD4 CTLs expressing lytic granules have also been phagocytic cells such as . 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 (FasL), that bind receptors on the target binds Fas or death 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 7–dependent pathway and Bad/Bax pathway of infected with M. tuberculosis, but unlike those produced by viral mitochondria 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 , 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, , WA 98102; †Department of Global other groups found that nonpolarized CD4 T cells could also me- ‡ Health, University of , 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, ; 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 . . 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. Statistical Cd40tm1Kik/J (CD402/2), and B6.129S2-Cd40lgtm1Imx/J (CD1542/2) analyses were performed using Prism software (GraphPad Software, La mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Jolla, CA). Splenocytes from DR52/2 mice on the C57BL/6 background (27) were a kind gift from Stephen Schoenberger (La Jolla Institute for Allergy and Results Immunology, La Jolla, CA). CD4-Cre+ and Eomesfl/fl mice were inter- Expression of cytolytic markers in CD4 T cells after Downloaded from + 0/0 2 fl/fl bred to establish CD4-Cre Eomesfl/fl (Eomes ) and CD4-Cre Eomes immunization (Eomesfl/fl) lines. Mice were immunized by i.m. injection with the re- combinant protein ID93 unadjuvanted or formulated with the adjuvant Parenteral immunization with ID93 + GLA-SE produces ID93- GLA-SE to provide a final vaccine dose of 0.5 mgAgand5mgGLA-SE specific CD4 T cells that produce a number of Th1-associated (28). All mice were maintained in specific pathogen–free conditions. All procedures were approved by the Infectious Disease Research Institute cytokines upon stimulation, including IFN-g,TNF,IL-2,and institutional animal care and use committee. GM-CSF, as well as expression of CD154 (CD40L) (24). How- ever, -producing CD8 T cells are not evident (29). To http://www.jimmunol.org/ Intracellular cytokine and tetramer staining determine whether immunization produced cytolytic CD8 T cells At 1 wk after immunization, splenocytes were plated at 2 3 106 cells per that were not detectable by cytokine secretion, we used an in vivo well and stimulated for 2 h at 37˚C with ID93 (10 mg/ml) or unstimulated. killing assay. CD45.1 congenic splenocytes were differentially GolgiPlug (BD Biosciences) was added, and the cells were incubated labeled with high or low concentrations of CFSE ex vivo and foranadditional8hat37˚C.Cellswerewashedandsurfacestained pulsed with ID93 or left unpulsed. Labeled cells were mixed 1:1 with fluorochrome-labeled Abs to CD4 (clone RM4-5) and CD8 (clone 53-6.7) (BioLegend and eBioscience) in the presence of anti-CD16/32 and i.v. injected into immunized or unimmunized animals. Eigh- (clone 2.4G2) for 20 min. Cells were washed and permeabilized with teen hours later there was a substantial reduction in the frequency Cytofix/Cytoperm (BD Biosciences) for 20 min. Cells were washed of ID93-pulsed target cells in the immunized recipients compared by guest on October 1, 2021 with Perm/Wash (BD Biosciences) and stained intracellularly with with the unimmunized controls, indicating the presence of ID93- fluorochrome-labeled Abs to CD154 (clone MR1), IFN-g (clone XMG- 1.2), IL-2 (clone JES6-5H4), and TNF (clone MP6-XT22) (BioLegend and specific CTLs (Fig. 1A, 1B). Surprisingly, CD8 T cells from these eBioscience) for 20 min. Cells were washed and resuspended in PBS. Then same animals did not degranulate upon in vitro stimulation with #106 events were collected on an LSRFortessa flow cytometer (BD ID93, as measured by surface capture of CD107a during stimulation Biosciences). Data were analyzed with FlowJo v9. Cells were gated as (data not shown). Further, immunization did not induce the ex- . . + 2 2 + . singlets lymphocytes CD4 CD8 or CD4 CD8 cytokine pos- pression of granzyme A, granzyme B, or perforin in CD44hi CD8 itive. ID93-specific response frequencies were determined by subtract- ing the frequency of response positives of unstimulated cells from ID93- T cells (Fig. 1C). Unexpectedly, we found that a significant number hi stimulated cells in matched samples. of CD44 CD4 T cells contained granzyme A, but not granzyme B Alternatively, splenocytes were stained for 90 min at 37˚C with an I-Ab or perforin (Fig. 2A–D). Granzyme A was further enriched in ID93- tetramer presenting the dominant epitope of Rv3619 (VIYEQANAHGQ), specific CD4 T cells identified with an I-Ab tetramer loaded with the one of the components of ID93 (24). APC-labeled tetramers were provided by the National Institutes of Health Tetramer Core Facility. dominant epitope from Rv3619, one of the component of Cells were then surface stained with fluorochrome-labeled Abs to CD4, ID93. In addition, these cells had an increased level of expression of CD8, CD44 (clone IM7), and lineage (CD19 [clone 1D3], CD11b [clone the transcription factor Eomes, which has been linked to the devel- b M1/70], and I-A [clone AF6-120.1]) in the presence of anti-CD16/32 for opment of cytolytic CD4 T cells (Fig. 2E) (20, 21). 20 min. Cells were washed and permeabilized with Cytofix/Cytoperm (BD Biosciences) for 20 min. Cells were washed with Perm/Wash (BD CD4 cells mediate MHCII-restricted cytotoxicity Biosciences) and stained intracellularly with fluorochrome-labeled Abs to granzyme A (clone GzA-3G8.5), granzyme B (clone GB11), perforin (clone To determine whether MHCII-restricted CD4 T cells were medi- eBioOMAK-D), and Eomes (clone Dan11mag) (BioLegend, eBioscience, ating the in vivo cytotoxicity we observed after immunization, we andBDBiosciences).CellswerewashedandresuspendedinPBS.Then assessed the killing of MHCII+ and MHCII2 target cells pulsed 6 #10 events were collected on an LSRFortessa flow cytometer (BD with ID93. ID93-pulsed MHCII+ cells were almost completely Biosciences). Data were analyzed with FlowJo v9. Cells were gated as . . 2 . + 2. hi + . ablated in vaccinated recipients, whereas there was minimal kill- singlets lymphocytes lin CD4 CD8 CD44 tetramer 2 intracellular marker. ing of MHCII ID93-pulsed cells (Fig. 3A, 3B). Induction of these MHCII-restricted CTLs largely depended on the GLA-SE In vivo killing adjuvant, as immunization with ID93 alone elicited very little Target CD45.1 splenocytes were isolated and RBCs were lysed. Cells were specific in vivo killing, in line with the paucity of ID93-specific split equally and labeled with 4 mM or 0.4 mM CFSE for 10 min in PBS at CD4 T cells generated by immunization with ID93 alone (Fig. 3B) hi 37˚C. Cells were washed twice with RPMI 1640 + 20% FCS. CFSE cells (24). Similar results of adjuvant-dependent induction of MHCII- were then incubated for 1 h at 37˚C with ID93 (10 mg/ml) and washed. CFSEhi and CFSElo cells were combined 1:1 in PBS. In some experiments restricted in vivo killing were obtained with a second fusion CD45.1 and CD402/2, Fas2/2, or DR52/2 (all CD45.2) target cells were protein, F3, consisting of two Leishmania proteins, adjuvanted mixed 1:1 prior to CFSE and ID93 labeling and were analyzed in the same with GLA-SE (data not shown). The Journal of Immunology 3

FIGURE 1. ID93 + GLA-SE immunization produces cytolytic cells. (A) ID93 + GLA-SE (black line) and unimmunized (gray fill) mice received a 1:1 mix of ID93-pulsed (CFSEhi) and -unpulsed (CFSElo) CD45.1 cells. At 18 h later, CFSEhi cells were specifically re- duced in the ID93 + GLA-SE–immunized recipients. (B) The frequency of in vivo killing of CFSEhi ID93- pulsed cells was determined for immunized animals relative to the unimmunized controls. (C) The fre- quency of granzyme A, granzyme B, and perforin was determined for naive (CD44lo) and memory (CD44hi) CD8 T cells from unimmunized (black bars) and im- munized (white bars) animals; n = 3–5 animals per group. Data are representative of eight experiments with similar results. Bars represent mean + S.D. Downloaded from

To confirm that this was mediated by CD4 T cells and not ca- in vivo killing of MHCII+ cells in vaccinated animals, whereas nonical cytolytic cell populations such as CD8 T cells or NK CD8 T cell and NK cell depletion had no effect (Fig. 3C). With cells, we depleted CD4 T cells or CD8 T cells and NK cells after the data taken together, we find that ID93 + GLA-SE produces http://www.jimmunol.org/ vaccination. Depletion of CD4 T cells completely abolished the a novel population of CD4 T cells that expresses granzyme A by guest on October 1, 2021

FIGURE 2. ID93 + GLA-SE immunization elicits granzyme A–expressing CD4 T cells. (A) ID93-spe- cific CD4 T cells were identified by tetramer staining after immunization. The frequency of (B) granzyme A, (C) granzyme B, and (D) perforin or (E) mean fluo- rescence intensity (MFI) of Eomes was determined for naive (CD44lo), memory (CD44hi), or Rv3619 tetramer– specific CD4 T cells from unimmunized (black bars) and immunized (white bars) animals; n = 5 animals. Data are representative of three experiments with sim- ilar results. Bars represent mean + SD. 4 CD4 CTL KILL VIA CD154–CD40

FIGURE 3. ID93 + GLA-SE immunization produces cytolytic CD4 T cells. (A) ID93 + GLA- SE (black line) and unimmunized (gray fill) mice received a 1:1 mix of ID93-pulsed (CFSEhi) and -unpulsed (CFSElo) cells. At 18 h later, MHCII+ CFSEhi cells were specifically eliminated in the ID93 + GLA-SE–immunized recipients. (B) The frequency of in vivo killing of MHCII+ and MHCII2 cells was determined for animals im- munized with ID93 alone or ID93 + GLA-SE. (C) Depletion of CD4 T cells, but not CD8 T cells and NK cells, following vaccination with ID93 + GLA-SE was sufficient to ablate the in vivo killing of ID93-pulsed MHCII+ cells; n =5 Downloaded from animals per group. Data are representative of three experiments with similar results. Bars rep- resent mean + SD. http://www.jimmunol.org/

and Eomes and kills ID93-pulsed cells in vivo in an MHCII- ID93-specific cells remaining in the vaccinated Tbet2/2 mice, restricted manner. as determined by CD154 expression upon ID93 stimulation (Fig. 5C). Tbet deficiency resulted in a modest, but significant by guest on October 1, 2021 CD4 cytotoxicity is independent of granzyme, perforin, Eomes, (p , 0.01), reduction in specific lysis of MHCII+ cells in vivo FasL, TRAIL, and canonical death signaling pathways (Fig. 5D). However, there was substantial residual in vivo cyto- ThelackofperforinexpressionbyID93-specificCD4Tcells toxicity activity, indicating that neither transcription factor was suggested that the in vivo cytolytic activity may not depend on essential for the development of CD4 CTLs following vaccination the expression of granzyme A. To test this hypothesis, we im- and that CD4 CTL activity did not depend on Th1 differentiation munized WT and granzyme A/B–deficient mice. Granzyme A controlled by Tbet. staining in memory and ID93-specific CD4 T cells was abolished In addition to producing perforin and granzyme-containing lytic in the granzyme A/B–deficient animals, confirming the speci- granules, CTLs can kill target cells via cell–cell interactions, pri- ficity of the staining (Fig. 4A). Despite the loss of granzyme A marily FasL on the CTL binding to Fas on the target cell. To test expression, the in vivo cytolytic capacity was not diminished in this possibility we immunized WT and FasL2/2 mice with ID93 + the immunized GzmA/B2/2 mice(Fig.4B).Further,theinvivo GLA-SE. At 1 wk after immunization, WT and FasL2/2 cohorts killing was not dependent on perforin expression, as killing was were able to kill MHCII+ target cells with similar efficiency not ablated in vaccinated Pfr2/2 animals (Fig. 4C). Thus the (Fig. 6A). To determine whether this killing was mediated by re- in vivo CD4 T cell–mediated cytotoxicity does not depend on dundant lytic granule and Fas–FasL pathways we examined the exocytosis of lytic granules containing granzymes or perforin. killing of a mixture of CD45.1 and Fas2/2 target cells in WT and The T-box transcription factor Eomes is critical for the devel- perforin-deficient recipients. Immunized perforin-deficient recip- opment of cytolytic CD4 T cells by CD134 and CD137 stimulation ients killed Fas2/2 targets more efficiently than CD45.1 targets, (20) and is upregulated along with Tbet in ID93-specific CD4 indicating that perforin-dependent cytolytic granules and the Fas– T cells following vaccination with ID93 + GLA-SE (Ref. 24 and FasL pathway are dispensable for CD4 CTL activity following Fig. 2E). As global Eomes deficiency is embryonic lethal, we vaccination (Fig. 6B). bred Eomesfl/fl and CD4-Cre mice to generate either CD4-Cre2 The TNF receptor superfamily member DR5 can also initiate Eomesfl/fl (Eomesfl/fl) or CD4-Cre+ Eomesfl/fl (Eomes0/0), which cell death on target cells when engaged by its ligand TRAIL on lack Eomes expression in all T cells (30). Eomes deficiency re- cognate CTLs (31). To test this possibility, we transferred a 1:1 mix duced the frequency of ID93-specific Th1 cells by ∼50% fol- of CD45.1 and DR52/2 target cells into immunized WT and Pfr2/2 lowing vaccination (Fig. 5A). To our surprise, Eomes was not recipients. As with Fas deficiency, we found that neither DR5 nor necessary for the killing of MHCII+ target cells in vivo (Fig. 5B). perforin was necessary for in vivo cytotoxicity (Fig. 6C). On the To determine whether the residual Th1 cells were sufficient to me- basis of these results, none of the canonical CTL effector pathways diate in vivo cytotoxicity, we immunized WT and Tbet2/2 mice. are necessary for CD4 CTL activity after vaccination. As expected, Tbet was necessary for the generation of ID93- To determine whether these cells kill via one of the canonical specific Th1 cells; however, there was a modest frequency of cell death signaling pathways or a unique pathway, we immunized The Journal of Immunology 5

CD4 cytotoxicity depends on CD40 and CD154 In the absence of Tbet, there was a partial loss of in vivo cyto- toxicity, along with a complete loss of Th1 cytokine production (Fig. 5C, 5D). However, even in these animals substantial CTL activity remained, along with some ID93-specific CD154 ex- pression by CD4 T cells. B cells are the most prevalent MHCII- expressing splenocytes in our target cell population, and they constitutively express CD40. To determine whether CD154 was necessary for in vivo killing of MHCII+ cells, we immunized WT and CD1542/2 animals and transferred in a 1:1 mix of CD45.1 and CD402/2 target cells. Immunization of CD1542/2 mice gen- erated a greater frequency of ID93-specific CD4 T cells, as de- termined by the frequency of CD4 T cells producing TNF, or IL-2 upon ID93 stimulation or by I-Ab tetramer labeling (Fig. 7A, 7B). Unlike the other knockouts tested, CD1542/2 mice failed to kill WT MHCII+ target cells. Similarly, WT mice were unable to kill CD402/2 MHCII+ target cells (Fig. 7C). Thus ID93 + GLA- SE immunization produces CD4 CTLs that express CD154 that + binds CD40 on MHCII cells presenting the cognate Ags, re- Downloaded from sulting in the death of the target cell. FIGURE 4. In vivo cytotoxicity does not require granzyme A/B or perforin. (A and B)WTandGzmA/B2/2 mice were immunized with ID93 + GLA-SE or left unimmunized. (A) The frequency of granzyme A ex- Discussion pression by memory (CD44hi) and Rv3619 tetramer–specific CD4 T cells The TLR4 agonist adjuvant GLA-SE augments a robust Th1 and 2 2 was increased in vaccinated WT, but not GzmA/B / immunized, animals. Ab response when paired with recombinant protein vaccine Ags + In vivo cytotoxicity of MHCII cells was not impaired by (B) granzyme (24, 26). We now find that this combination also induces CD4 http://www.jimmunol.org/ C A/B or ( ) perforin deficiency relative to WT; n = 5 animals per group. T cells with the ability to kill cells bearing cognate MHCII:pep- Data are representative of (A and B) two or (C) four experiments with tide complexes. Although CD4 CTLs have been noted for more similar results. Bars represent mean + SD. than three decades, to our knowledge reports of vaccine-elicited CD4 CTLs have been rare. Unlike in previous reports, we find a cohort of WT mice and transferred in either WT targets or tar- that this CD4 CTL activity does not rely on any of the canonical gets lacking a canonical cell death signaling component—caspase CTL mechanisms, including perforin, granzymes, Fas–FasL, or 3, caspase 7, Bax, or TNFR1/2 (1, 32). Surprisingly, none of these + TRAIL–DR5; rather, the CD4 CTLs induce cell death by ligating signaling components were necessary for the killing of MHCII CD40 on target cells via expression of CD154. Unlike other by guest on October 1, 2021 targets in vaccinated recipients (Fig. 6D). These data suggested CD4 CTLs, these cells do not require the expression of the tran- a novel killing pathway that was independent of the known ef- scription factor Eomes for their cytolytic activity. fector and signaling pathways associated with CTL activity. CD4 T cells have traditionally been characterized by their ability to provide help to a number of different immune responses, in- cluding augmenting production of Abs and formation of memory cells, modifying APCs to more efficiently prime CD8 T cells, and activating macrophages to resist intracellular infec- tions. In addition, direct cytolytic activity of CD4 T cells was noted in cell lines but largely dismissed as an artifact of the long-term stimulation and culturing needed to detect the cytolytic activity (2). More recently, CD4 CTLs specific for a number of viral in- fections have been isolated directly ex vivo. These include CD4 CTLs that recognize HIV, influenza, EBV, and many other pathogens (reviewed in Refs. 4 and 33). By adapting an in vivo killing assay, Jellison et al. (5) demonstrated that CD4 CTLs arise during murine lymphocytic choriomeningitis virus infection. No- tably, these CD4 CTLs are almost as efficient at killing target cells in vivo as canonical CD8 CTLs, although the kinetics of killing is slower for CD4 CTLs (34). CD4 CTLs likely play an important role in controlling pathogens that infect macrophages and other APCs as well as other cell types that express MHC class II either FIGURE 5. Development of cytolytic CD4 T cells by vaccination is constitutively or upon infection. In mice perforin expression by independent of the T-box transcription factors Eomes and Tbet. (A)The CD4 T cells is important for the control of both influenza and B frequency of ID93-specific Th1 cells and ( ) in vivo cytotoxicity against ectromelia virus (6, 8). In humans the frequency of malaria- MHCII+ ID93-pulsed target cells were determined in ID93 + GLA-SE– 2 2 specific CD4 T cells that degranulate upon stimulation corre- immunized Eomesfl/fl and Eomes0/0 mice. (C and D)WTandTbet / mice were immunized with ID93 + GLA-SE. (C) Only the WT animals devel- lates with protection against experimental malaria challenge, oped ID93-specific Th1 cells, (D) but both populations killed MHCII+ whereas the frequency of malaria-specific IFN-g–producing CD4 ID93-pulsed target cells in vivo; n = 5 animals per group . Data are rep- or CD8 T cells does not correlate with protection, suggesting resentative of (A and B) four or (C and D) two experiments with similar a role for CD4 CTLs in controlling malaria infection (35). In HIV results. Bars represent mean + SD. the frequency of Gag-specific CD4 T cells that express granzyme 6 CD4 CTL KILL VIA CD154–CD40

FIGURE 6. In vivo CD4 T cell cytotoxicity is inde- pendent of canonical cytolytic pathways. (A) WT and FasL2/2 immunized mice both kill MHCII+ CD45.1 cells. (B) WT and Pfr2/2 immunized mice both kill CD45.1 and Fas2/2 MHCII+ WT cells. (C) WT and Pfr2/2 immunized mice kill CD45.1 and DR52/2 MHCII+ target cells. (D) WT immunized mice kill WT, Casp32/2, Casp72/2, Bax2/2, and TNFR1/22/2 MHCII+ targets; n = 5 animals per group . Data are representative of two to five experiments with similar results. Bars represent mean + SD. Downloaded from

A and other cytolytic markers correlates with suppression of or Fas depends on the identity of the pathogen driving CTL de- viral load and prevention of disease progression in patients not velopment as well as Ag abundance and concentrations of IL-2 http://www.jimmunol.org/ undergoing antiretroviral therapy (11). Although the granzyme (4, 16, 22, 33). However, these pathways are not sufficient to ex- A expression by CD4 T cells elicited by immunization that plain the activity of some CD4 CTLs. Using an in vivo killing we observed following vaccination was not necessary for CD4 assay similar to the one we use in this study, Woodworth et al. (19) CTL activity in this study, it is intriguing to speculate about its found that M. tuberculosis–specific CD4 CTLs arise during infec- potential benefits. Induction of granzyme A–expressing CD4 tion of mice. This activity was independent on perforin, Fas–FasL, T cells via the GLA-SE adjuvant may be a beneficial compo- and TNF, leading the authors to conclude there was a novel, but nent of an efficacious vaccine. undefined, mechanism of CD4 CTL. Similarly, perforin- and HumanandmurineCD4CTLshavebeenshowntokillvia Fas-independent CD4 CTLs have been observed in murine g- by guest on October 1, 2021 perforin- or Fas-dependent pathways, or both. The use of perforin herpesvirus 68 infection (36). On the basis of our observations in this article, we speculate that these CD4 CTL activities may also depend on CD154 engagement of CD40 on target cells. Granzyme/perforin- and Fas-dependent cell death requires the activation of executioner caspase pathways, including 3 and 7, or mitochondrial cytochrome c effluxmediatedbyBad/ Bax (1). We found that these pathways are dispensable for CD4 CTL activity in this system. CD40 signals through a variety of TRAF molecules to engage a number of downstream signaling pathways (reviewed in Ref. 37). It is possible that one or more of these TRAF pathways may be engaged to drive target cell death in this system. CD4CTLactivityhasbeenassociatedwithIFN-g–producing CD4 T cells, suggesting that they are not a unique lineage, but rather a subset of Th1 cells. However, recent work by Brown and Swain (22) demonstrated that nonpolarizing conditions (i.e., Th0) are more effective for producing CD4 CTLs than Th1 or Th2 polarizing conditions. In addition, expression of the T-box transcription factor Eomes is essential for the expression of granzyme B by CD4 T cells stimulated via CD134 and CD137 (20). Ectopic expression of Eomes was sufficient to convert non- cytolytic Th2 cells into CTLs, suggesting that Eomes-dependent FIGURE 7. In vivo CD4 T cell cytotoxicity depends on CD40–CD154 CD4 CTLs represent a unique differentiation pathway (21). Type I interactions. Immunization with ID93 + GLA-SE produced similar fre- IFNs may also play a role in some CD4 CTL development by quencies of ID93-specific CD4 T cells, as determined by the frequency of acting on the transcription factors Blimp-1 and Tbet (38). The (A) IFN-g–, TNF-, or IL-2–producing cells upon ID93 stimulation or (B) staining with an I-Ab tetramer. (C) CD1542/2 immunized mice do not de- CD154-dependent CD4 CTLs we report in this article do not velop cytolytic CD4 T cells upon vaccination. Similarly, immunized WT require Tbet expression, strongly suggesting that they are not a mice develop cytolytic CD4 T cells that kill WT, but not CD402/2 MHCII+, subset of Th1 cells. To our surprise they also did not require target cells; n = 5 animals per group. Data are representative of four Eomes expression, indicating that they develop via a unique dif- experiments with similar results. Bars represent mean + SD. ***p , 0.001. ferentiation mechanism. CD154 expression upon Ag stimulation The Journal of Immunology 7 has been proposed as an indicator of CD4 T cell specificity re- 7. Brien, J. D., J. L. Uhrlaub, and J. Nikolich-Zugich. 2008. West Nile virus- specific CD4 T cells exhibit direct antiviral cytokine secretion and cytotoxicity gardless of Th subset differentiation; thus CD4 CTL activity via this and are sufficient for antiviral protection. J. Immunol. 181: 8568–8575. pathway may be common to all Ag-experienced CD4 T cells (39). 8. Fang, M., N. A. Siciliano, A. R. Hersperger, F. Roscoe, A. Hu, X. Ma, CD4 T cell–expressed CD154 ligation of CD40 on B cells and A. R. Shamsedeen, L. C. Eisenlohr, and L. J. Sigal. 2012. Perforin-dependent CD4+ T-cell cytotoxicity contributes to control a murine poxvirus infection. APCs plays a central role in many adaptive immune responses. Proc. Natl. Acad. Sci. USA 109: 9983–9988. CD40 is constitutively expressed on B cells throughout devel- 9. Appay, V., J. J. Zaunders, L. Papagno, J. Sutton, A. Jaramillo, A. Waters, opment. CD40 signaling is essential for the development of P. Easterbrook, P. Grey, D. Smith, A. J. McMichael, et al. 2002. Characterization of CD4(+) CTLs ex vivo. J. Immunol. 168: 5954–5958. T cell–dependent Ag responses, including forma- 10. Zheng, N., M. Fujiwara, T. Ueno, S. Oka, and M. Takiguchi. 2009. Strong ability tion, somatic hypermutation, isotype switching, dif- of Nef-specific CD4+ cytotoxic T cells to suppress human immunodeficiency ferentiation, and generation of plasma cells (reviewed in Ref. 37). virus type 1 (HIV-1) replication in HIV-1-infected CD4+ T cells and macro- phages. J. Virol. 83: 7668–7677. Defects in CD154–CD40 result in one of several types of hyper- 11. Soghoian, D. Z., H. Jessen, M. Flanders, K. Sierra-Davidson, S. Cutler, T. Pertel, IgM syndrome (X linked in the cases of CD154 deficiency) S. Ranasinghe, M. Lindqvist, I. Davis, K. Lane, et al. 2012. HIV-specific cyto- marked by a lack of class switching and somatic hypermutation lytic CD4 T cell responses during acute HIV infection predict disease outcome. Sci. Transl. Med. 4: 123ra25. (40). CD40 engagement on APCs upregulates MHC and costimu- 12. Long, H. M., A. M. Leese, O. L. Chagoury, S. R. Connerty, J. Quarcoopome, latory molecules necessary for T cell priming, augments IL-12 L. L. Quinn, C. Shannon-Lowe, and A. B. Rickinson. 2011. Cytotoxic CD4+ T cell responses to EBV contrast with CD8 responses in breadth of lytic cycle secretion, and promotes cross presentation. CD40 engagement choice and in lytic cycle recognition. J. Immunol. 187: 92–101. on both B cells and APCs can promote survival by upregulating 13. Nikiforow, S., K. Bottomly, G. Miller, and C. Munz.€ 2003. Cytolytic CD4(+)-T- the prosurvival genes Bcl-XL and c-FLIP (41–43). Considering cell clones reactive to EBNA1 inhibit Epstein-Barr virus-induced B-cell prolif- eration. J. Virol. 77: 12088–12104. this prosurvival effect of CD154–CD40 signaling, we were sur- 14. van Leeuwen, E. M., E. B. Remmerswaal, M. H. Heemskerk, I. J. ten Berge, and Downloaded from prised to find that CD154 and CD40 were essential for the in vivo R. A. van Lier. 2006. Strong selection of virus-specific cytotoxic CD4+ T-cell CTL activity observed after vaccination. However, CD40 has also clones during primary human cytomegalovirus infection. Blood 108: 3121–3127. 15. Canaday, D. H., R. J. Wilkinson, Q. Li, C. V. Harding, R. F. Silver, and been implicated in cell death under certain circumstances. For ex- W. H. Boom. 2001. CD4(+) and CD8(+) T cells kill intracellular Mycobacterium ample, CD40 signaling can increase expression of Fas on tumor tuberculosis by a perforin and Fas/Fas ligand-independent mechanism. J. cells, increasing susceptibility to FasL-mediated killing (44–46). Immunol. 167: 2734–2742. 16. Lewinsohn, D. M., T. T. Bement, J. Xu, D. H. Lynch, K. H. Grabstein, Engagement of CD40 on tumor cells can inhibit proliferation S. G. Reed, and M. R. Alderson. 1998. Human purified protein derivative- via an undefined mechanism (47, 48). More directly, human tri- specific CD4+ T cells use both CD95-dependent and CD95-independent cyto- http://www.jimmunol.org/ lytic mechanisms. J. Immunol. 160: 2374–2379. meric CD154 can induce of CD40-expressing solid 17. Green, D. R., and T. A. Ferguson. 2001. The role of Fas ligand in immune tumors (46). Finally, the CD40 agonist Ab dacetuzumab is used privilege. Nat. Rev. Mol. Cell Biol. 2: 917–924. to treat metastatic melanomas and B cell lymphomas by inducing 18. Kayagaki, N., N. Yamaguchi, M. Nakayama, A. Kawasaki, H. Akiba, K. Okumura, and H. Yagita. 1999. Involvement of TNF-related apoptosis- apoptosis (49, 50). Thus, the ability to induce CD154-dependent inducing ligand in human CD4+ T cell-mediated cytotoxicity. J. Immunol. CD4 CTLs by vaccination with an adjuvanted protein may be of 162: 2639–2647. particular benefit for therapeutic cancer vaccines. 19. Woodworth, J. S., Y. Wu, and S. M. Behar. 2008. Mycobacterium tuberculosis- specific CD8+ T cells require perforin to kill target cells and provide protection In summary, we find that the TLR4 agonist adjuvant GLA-SE in vivo. J. Immunol. 181: 8595–8603. induces CD4 CTLs with the ability to kill autologous B cells that 20. Qui, H. Z., A. T. Hagymasi, S. Bandyopadhyay, M. C. St Rose, by guest on October 1, 2021 present a cognate MHCII:peptide complex. This activity is in- R. Ramanarasimhaiah, A. Me´noret, R. S. Mittler, S. M. Gordon, S. L. Reiner, A. T. Vella, and A. J. Adler. 2011. CD134 plus CD137 dual costimulation dependent of all previously defined CTL mechanisms and in- induces Eomesodermin in CD4 T cells to program cytotoxic Th1 differentiation. stead depends on CD154 engagement of CD40 on target cells. This J. Immunol. 187: 3555–3564. 21. Eshima, K., S. Chiba, H. Suzuki, K. Kokubo, H. Kobayashi, M. Iizuka, finding represents a newly defined function for this receptor– K. Iwabuchi, and N. Shinohara. 2012. Ectopic expression of a T-box transcrip- ligand pair and may be related to the observations of antitumor tion factor, eomesodermin, renders CD4(+) Th cells cytotoxic by activating both activity of CD40 engagement. Future efforts will be dedicated to perforin- and FasL-pathways. Immunol. Lett. 144: 7–15. 22. Brown, D. M., C. Kamperschroer, A. M. Dilzer, D. M. Roberts, and S. L. Swain. defining the signaling mechanisms necessary for CTL activity 2009. IL-2 and antigen dose differentially regulate perforin- and FasL-mediated and application of this to the development of effective CTL cytolytic activity in antigen specific CD4+ T cells. Cell. Immunol. 257: 69–79. vaccines. 23. Orr, M. T., E. A. Beebe, T. E. Hudson, J. J. Moon, C. B. Fox, S. G. Reed, and R. N. Coler. 2014. A dual TLR agonist adjuvant enhances the immunogenicity and protective efficacy of the tuberculosis vaccine antigen ID93. PLoS One 9: Acknowledgments e83884. We thank Stephen Schoenberger for provision of DR52/2 splenocytes, 24. Orr, M. T., C. B. Fox, S. L. Baldwin, S. J. Sivananthan, E. Lucas, S. Lin, T. Phan, J. J. Moon, T. S. Vedvick, S. G. Reed, and R. N. Coler. 2013. Adjuvant for- Chris Fox for provision of GLA-SE, Chris Wilson for helpful discussions, mulation structure and composition are critical for the development of an ef- and the National Institutes of Health Tetramer Core Facility (Contract fective vaccine against tuberculosis. J. Control. Release 172: 190–200. HHSN272201300006C) for provision of MHC class II tetramers. 25. Coler, R. N., S. L. Baldwin, N. Shaverdian, S. Bertholet, S. J. Reed, V. S. Raman, X. Lu, J. DeVos, K. Hancock, J. M. Katz, et al. 2010. A synthetic adjuvant to enhance and expand immune responses to influenza vaccines. PLoS One 5: Disclosures e13677. 26. Coler, R. N., S. Bertholet, M. Moutaftsi, J. A. Guderian, H. P. Windish, The authors have no financial conflicts of interest. S. L. Baldwin, E. M. Laughlin, M. S. Duthie, C. B. Fox, D. Carter, et al. 2011. Development and characterization of synthetic glucopyranosyl lipid adjuvant system as a vaccine adjuvant. PLoS One 6: e16333. References 27. Diehl, G. E., H. H. Yue, K. Hsieh, A. A. Kuang, M. Ho, L. A. Morici, L. L. Lenz, 1. Lieberman, J. 2003. The ABCs of granule-mediated cytotoxicity: new weapons D. Cado, L. W. Riley, and A. Winoto. 2004. TRAIL-R as a negative regulator of in the arsenal. Nat. Rev. Immunol. 3: 361–370. innate immune cell responses. Immunity 21: 877–889. 2. Fleischer, B. 1984. Acquisition of specific cytotoxic activity by human T4+ 28. Bertholet, S., G. C. Ireton, D. J. Ordway, H. P. Windish, S. O. Pine, M. Kahn, T lymphocytes in culture. Nature 308: 365–367. T. Phan, I. M. Orme, T. S. Vedvick, S. L. Baldwin, et al. 2010. A defined tu- 3. Marshall, N. B., and S. L. Swain. 2011. Cytotoxic CD4 T cells in antiviral berculosis vaccine candidate boosts BCG and protects against multidrug- immunity. J. Biomed. Biotechnol. 2011: 954602. resistant Mycobacterium tuberculosis. Sci. Transl. Med. 2: 53ra74. 4. Soghoian, D. Z., and H. Streeck. 2010. Cytolytic CD4(+) T cells in viral im- 29. Baldwin, S. L., L. K. Ching, S. O. Pine, M. Moutaftsi, E. Lucas, A. Vallur, munity. Expert Rev. Vaccines 9: 1453–1463. M. T. Orr, S. Bertholet, S. G. Reed, and R. N. Coler. 2013. Protection against 5. Jellison, E. R., S. K. Kim, and R. M. Welsh. 2005. Cutting edge: MHC class II- tuberculosis with homologous or heterologous protein/vector vaccine approaches restricted killing in vivo during viral infection. J. Immunol. 174: 614–618. is not dependent on CD8+ T cells. J. Immunol. 191: 2514–2525. 6. Brown, D. M., A. M. Dilzer, D. L. Meents, and S. L. Swain. 2006. CD4 T cell- 30. Zhu, Y., S. Ju, E. Chen, S. Dai, C. Li, P. Morel, L. Liu, X. Zhang, and B. Lu. mediated protection from lethal influenza: perforin and -mediated 2010. T-bet and eomesodermin are required for T cell-mediated antitumor im- mechanisms give a one-two punch. J. Immunol. 177: 2888–2898. mune responses. J. Immunol. 185: 3174–3183. 8 CD4 CTL KILL VIA CD154–CD40

31. Dorothe´e, G., I. Vergnon, J. Menez, H. Echchakir, D. Grunenwald, M. Kubin 43. Wong, B. R., R. Josien, S. Y. Lee, B. Sauter, H. L. Li, R. M. Steinman, and S. Chouaib, and F. Mami-Chouaib. 2002. Tumor-infiltrating CD4+ T lym- Y. Choi. 1997. TRANCE ( [TNF]-related activation- phocytes express APO2 ligand (APO2L)/TRAIL upon specific stimulation with induced cytokine), a new TNF family member predominantly expressed in autologous lung carcinoma cells: role of IFN-alpha on APO2L/TRAIL expression T cells, is a dendritic cell-specific survival factor. J. Exp. Med. 186: 2075–2080. and -mediated cytotoxicity. J. Immunol. 169: 809–817. 44. Eliopoulos, A. G., C. Davies, P. G. Knox, N. J. Gallagher, S. C. Afford, 32. Tite, J. P. 1990. Evidence of a role for TNF-alpha in cytolysis by CD4+, class II D. H. Adams, and L. S. Young. 2000. CD40 induces apoptosis in carcinoma cells MHC-restricted cytotoxic T cells. Immunology 71: 208–212. 33. Brown, D. M. 2010. Cytolytic CD4 cells: direct mediators in infectious disease through activation of cytotoxic ligands of the tumor necrosis factor superfamily. and malignancy. Cell. Immunol. 262: 89–95. Mol. Cell. Biol. 20: 5503–5515. 34. Hildemann, S. K., J. Eberlein, B. Davenport, T. T. Nguyen, F. Victorino, and 45. Lee, J. K., N. Seki, T. J. Sayers, J. Subleski, E. M. Gruys, W. J. Murphy, and D. Homann. 2013. High efficiency of antiviral CD4(+) killer T cells. PLoS One R. H. Wiltrout. 2005. Constitutive expression of functional CD40 on mouse renal 8: e60420. cancer cells: induction of Fas and Fas-mediated killing by CD40L. Cell. 35. Bijker, E. M., A. C. Teirlinck, R. Schats, G. J. van Gemert, M. van de Vegte- Immunol. 235: 145–152. Bolmer, L. van Lieshout, J. IntHout, C. C. Hermsen, A. Scholzen, L. G. Visser, 46. Alexandroff, A. B., A. M. Jackson, T. Paterson, J. L. Haley, J. A. Ross, and R. W. Sauerwein. 2014. Cytotoxic markers associate with protection against D. L. Longo, W. J. Murphy, K. James, and D. D. Taub. 2000. Role for CD40- malaria in human volunteers immunized with Plasmodium falciparum spor- CD40 ligand interactions in the immune response to solid tumours. Mol. ozoites. J. Infect. Dis. 210: 1605–1615. Immunol. 37: 515–526. 36. Stuller, K. A., and E. Flan˜o. 2009. CD4 T cells mediate killing during persistent 47. Tong, A. W., M. H. Papayoti, G. Netto, D. T. Armstrong, G. Ordonez, gammaherpesvirus 68 infection. J. Virol. 83: 4700–4703. J. M. Lawson, and M. J. Stone. 2001. Growth-inhibitory effects of CD40 ligand 37. Elgueta, R., M. J. Benson, V. C. de Vries, A. Wasiuk, Y. Guo, and R. J. Noelle. 2009. Molecular mechanism and function of CD40/CD40L engagement in the (CD154) and its endogenous expression in human breast cancer. Clin. Cancer . Immunol. Rev. 229: 152–172. Res. 7: 691–703. 38. Hua, L., S. Yao, D. Pham, L. Jiang, J. Wright, D. Sawant, A. L. Dent, 48. von Leoprechting, A., P. van der Bruggen, H. L. Pahl, A. Aruffo, and T. J. Braciale, M. H. Kaplan, and J. Sun. 2013. Cytokine-dependent induction of J. C. Simon. 1999. Stimulation of CD40 on immunogenic human malignant CD4+ T cells with cytotoxic potential during influenza virus infection. J. Virol. melanomas augments their cytotoxic T -mediated lysis and induces 87: 11884–11893. apoptosis. Cancer Res. 59: 1287–1294. 39. Frentsch, M., O. Arbach, D. Kirchhoff, B. Moewes, M. Worm, M. Rothe, 49. Lewis, T. S., R. S. McCormick, I. J. Stone, K. Emmerton, B. Mbow, Downloaded from A. Scheffold, and A. Thiel. 2005. Direct access to CD4+ T cells specific for J. Miyamoto, J. G. Drachman, I. S. Grewal, and C. L. Law. 2011. Proapoptotic defined according to CD154 expression. Nat. Med. 11: 1118–1124. signaling activity of the anti-CD40 monoclonal antibody dacetuzumab circum- 40. Bhushan, A., and L. R. Covey. 2001. CD40:CD40L interactions in X-linked and vents multiple oncogenic transformation events and chemosensitizes NHL cells. non-X-linked hyper-IgM syndromes. Immunol. Res. 24: 311–324. Leukemia 25: 1007–1016. 41. Hennino, A., M. Be´rard, P. H. Krammer, and T. Defrance. 2001. FLICE- 50. Lewis, T. S., R. S. McCormick, K. Emmerton, J. T. Lau, S. F. Yu, inhibitory protein is a key regulator of germinal center B cell apoptosis. J. Exp. Med. 193: 447–458. J. A. McEarchern, I. S. Grewal, and C. L. Law. 2011. Distinct apoptotic signaling characteristics of the anti-CD40 monoclonal antibody dacetuzumab and ritux- 42. Tuscano, J. M., K. M. Druey, A. Riva, J. Pena, C. B. Thompson, and J. H. Kehrl. http://www.jimmunol.org/ 1996. Bcl-x rather than Bcl-2 mediates CD40-dependent survival in imab produce enhanced antitumor activity in non-Hodgkin lymphoma. Clin. the germinal center. Blood 88: 1359–1364. Cancer Res. 17: 4672–4681. by guest on October 1, 2021