Direct in vivo evidence of CD4 T cell requirement for CTL response and memory via pmhc-i targeting and CD40L signaling

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1 Article Direct in vivo evidence of CD4 T cell requirement for CTL response and memory via pmhc-i targeting and CD40L signaling Khawaja Ashfaque Ahmed,*,,1 Lu Wang,*,,1 Manjunatha Ankathatti Munegowda,*, Sean J. Mulligan, John R. Gordon, Philip Griebel, and Jim Xiang*,,2 *Research Unit, Saskatchewan Cancer Agency, Departments of Oncology and Physiology and Division of Respirology, Critical Care and Sleep Medicine, Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan Canada RECEIVED DECEMBER 19, 2011; REVISED FEBRUARY 21, 2012; ACCEPTED MARCH 10, DOI: /jlb Abbreviations: 4D four-dimensional, B6 C57BL/6, CD40L CD40 ligand, CMFDA 5-chloromethylfluorescein diacetate, CMTMR chloromethyltetramethylrhodamine, DTR diphtheria toxin receptor, FITC- CD8 FITC-anti-CD8 antibody, ILN inguinal LN, KO knockout, LCMV lymphocytic choriomeningitis virus, OT-I/II MHC class I/II-restricted, OVAspecific, CD8 T cell, OVAI OVA , OVAII OVA , OVA- EL4 OVA-loaded EL4, pdc plasmacytoid DC, PE-tetramer PE-H-2K b / OVAI tetramer, pmhc-i peptide/mhc complex-i The online version of this paper, found at includes supplemental information. ABSTRACT CD4 T cell help contributes critically to DC-induced CD8 CTL immunity. However, precisely how these three cell populations interact and how CD4 T cell signals are delivered to CD8 T cells in vivo have been unclear. In this study, we developed a novel, two-step approach, wherein CD4 T cells and antigen-presenting DCs productively engaged one another in vivo in the absence of cognate CD8 T cells, after which, we selectively depleted the previously engaged CD4 T cells or DCs before allowing interactions of either population alone with naïve CD8 T cells. This protocol thus allows us to clearly document the importance of CD4 T-licensed DCs and DC-primed CD4 T cells in CTL immunity. Here, we provide direct in vivo evidence that primed CD4 T cells or licensed DCs can stimulate CTL response and memory, independent of DC-CD4 T cell clusters. Our results suggest that primed CD4 T cells with acquired pmhc-i from DCs represent crucial immune intermediates for rapid induction of CTL responses and for functional memory via CD40L signaling. Importantly, intravital, two-photon microscopy elegantly provide unequivocal in vivo evidence for direct CD4-CD8 T cell interactions via pmhc-i engagement. This study corroborates the coexistence of direct and indirect mechanisms of T cell help for a CTL response in noninflammatory situations. These data suggest a new dynamic model of three-cell interactions for CTL immunity derived from stimulation by dissociated, licensed DCs, primed CD4 T cells, and DC-CD4 T cell clusters and may have significant implications for autoimmunity and vaccine design. J. Leukoc. Biol. 92: ; Introduction CD8 CTLs recognize and kill malignant and pathogen-infected cells. Gaining a better understanding of the mechanisms guiding the generation of protective memory CTLs remains a top priority for health researchers for devising ways to develop effective vaccines and immunotherapies [1]. Several studies reported that the generation of primary CTL responses to noninflammatory antigens [1 3] and certain viral pathogens [4] largely depend on CD4 T cell help. Although primary CTL responses to acute infections can occur in the absence of CD4 T help, there is general agreement that all CTL responses depend on CD4 T cell help for the generation of efficient memory (that is, the ability to respond robustly on re-encounter with the same antigen) [1, 5, 6]. Since the discovery of a critical requirement for CD4 T cell help in the orchestration of robust CTL immunity [7], numerous studies sought to define the mechanisms for this interesting phenomenon. However, exactly how CD4 T cell helper signals are delivered to CD8 T cells in noninflammatory situations remains a mystery. Based on the observation that antigen ligands for the CD4 and CD8 T cells must be carried by the same antigen-presenting DCs for effective helper-dependent CTL responses, several distinct models have been proposed. Three-cell interaction model suggests that naive CD4 and CD8 T cells must interact simultaneously with a common DC, forming a ternary cluster (DC-CD4 -CD8 ), wherein the CD4 T cell provides help through local secretion of IL-2 [7]. However, several imaging studies subsequently demonstrated that CD4 and CD8 T cells interact only in a random manner with antigen-bearing DCs within LNs [8, 9], which brought into question how a rare antigen-specific CD4 1. These authors contributed equally to this study. 2. Correspondence: Saskatoon Cancer Center, 20 Campus Dr., Saskatoon, Saskatchewan S7N 4H4, Canada. jim.xiang@saskcancer.ca /12/ Society for Leukocyte Biology Volume 92, August 2012 Journal of Leukocyte Biology 289

2 T cell and an equally rare antigen-specific CD8 T cell might simultaneously find the same antigen peptide-carrying DC [10]. Thus, an alternative model of sequential two-cell interactions by APCs or DC-licensing model [3] was proposed, wherein DC first stimulates the CD4 T cell, which reciprocally offers CD40L-dependent licensing signals to DCs, enabling licensed DCs to activate CTL responses [2]. However, the DC-licensing model was first challenged by the observation that efficient memory CTL responses required CD40 expression on CD8 T cells but not on DCs. This experiment led to a model of CD4 -CD8 T cell interactions, wherein direct CD40L-CD40 interactions between CD4 and CD8 T cells deliver helper signals [11]. Recently, we demonstrated that on in vitro activation by DCs, CD4 T cells acquired membrane patches (including antigen-presenting machinery) from DCs and used these to directly target helper effects onto cognate CD8 T cells [12], suggesting an alternate mechanisms for direct CD4 -CD8 T cell interactions (i.e., the model of sequential two-cell interactions by CD4 T-APCs or primed CD4 Th-APC model). This was also supported by further data from others [13 15] and our own laboratory [16, 17]. Although one recent study provided evidence for cognate CD4 T cell-mediated DC licensing but lacked any direct in vivo evidence for CTL memory programming, the authors relied exclusively on in vitro activation of monoclonal CD8 T cells by sorted, licensed DCs [18]. The physiological relevance of the DC-licensing model has again been challenged by recent two-photon imaging data of lymphocyte-dc interactions within intact LNs [19]. Germain and colleagues [19] suggested that despite their rarity, naive antigen-specific CD8 T cells receive helper signals from equally rare DC-CD4 T clusters under the guidance of CCR5-specific chemokines (CCL3 and CCL4). In spite of several developments in the field, the issue of the mechanisms for functional delivery of CD4 T cell help, whether CD8 T cells receive helper signals indirectly through licensed DCs, directly from primed CD4 T cells, or by forming a three-cell cluster, remains unresolved [19]. It is important to point out that previous studies used diverse experimental systems and proposed disparate mechanisms for CD4 T help in CTL immunity. Unfortunately, the controversy grew, as they did not rule out other possibilities with the same experimental system. Recently, with the advent of more sophisticated imaging technologies, it became possible to actually see DCs and CD4 T cells in all of their dynamic DC-CD4 T cell interactions. These imaging studies provided in vivo evidence for the existence of short (during the first 2 8 h and h after immunization)- and long-lasting interactions, with the latter occurring between 8 and 24 h. Thus, in a physiological condition, antigen-specific interactions between CD4 T cells and antigen-presenting DCs could occur in at least two different scenarios: first DC-CD4 T cell interactions without cognate CD8 T cells on the clusters and second with cognate-cd8 T cells forming ternary clusters with the DC-CD4 T cells. In light of the latest imaging data, which suggested that CD8 T cells receive help from DC-CD4 T cell clusters [19], many obvious questions arise, such as: whether DC licensing by CD4 T cells truly exists in vivo and has any physiological relevance; what role, if any, primed CD4 T cells could play in CTL immunity after their dissociation from DC-CD4 T cell clusters; and whether the formation of ternary DC-CD4 -CD8 T cell clusters is absolutely essential for the delivery of helper signals to CD8 T cells. These important issues remain to be investigated in vivo, and their resolution is crucial for designing better strategies for vaccination and immunotherapies. In this study, we developed a new immunization model system, where the three central players in CTL induction (i.e., the DCs and CD4 and CD8 T cells) can be manipulated independently in vivo. Using this model, we analyzed the importance of licensed DCs and primed CD4 T cells in the induction of CTL responses, as well as some of the mechanisms regulating primed CD4 T cell activation of naive CD8 T cells in vivo. MATERIALS AND METHODS Mice Female B6 (CD45.2 ), B6.1 (CD45.1 ), KO, transgenic OVAI-TCR OT-I, OVAII-TCR OT-II, and CD11c-DTR mice (8 12 weeks of age) were all obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and housed in specific pathogen-free conditions in the animal facilities at the University of Saskatchewan (Canada). CD11c-DTR/H-2K b / mice were generated by backcrossing CD11c-DTR mice with H-2K b / mice. All animal studies were performed in compliance with the regulations of the University of Saskatchewan Institutional Animal Care and Use Committee. Reagents and tumor cell lines DT was purchased from Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada). The rgm-csf and ril-4 were obtained from R&D Systems (Minneapolis, MN, USA). The OVAI (SIINFEKL) [17], OVAII (ISQAVHAA- HAEINEAGR), irrelevant LCMV gp (GLNGPDIYKGVYQFKSVEFD), and Mut1 (FEQNTAQP) [17] peptides were synthesized by Multiple Peptide Systems (San Diego, CA, USA). The biotin-labeled and fluorescent dye (FITC and PE)-labeled antibodies, specific for CD4 (GK1.5), CD11c (HL3), and CD45.1 (A20), were obtained from BD PharMingen Canada (Missisauga, Ontario, Canada). The depleting anti-cd8 and anti-cd4 antibodies were purified from ascites of hybridoma cell lines and GK1.5, respectively. The anti-cd8 (53-5.8), anti-cd20 (AISB12), PE-anti-CD4 (RM 4-4), and anti-pdc (120G8) antibodies were obtained from BD PharMingen Canada, ebioscience (San Diego, CA, USA), and Dendritics (Lyon, France), respectively. The PE-tetramer and FITC-CD8 (YTS and ) were obtained from Beckman Coulter (Miami, FL, USA) and Caltag (Burlingame, CA, USA), respectively. The mouse thymoma cell line EL4 and OVA-expressing B16 melanoma cell line BL6-10ova were available in our laboratory [12]. DC preparation Bone marrow-derived DCs from CD11c-DTR and CD11c-DTR/H-2K b / mice were generated in culture medium containing GM-CSF (20 ng/ml) and IL-4 (20 ng/ml), pulsed with 0.3 mg/ml OVA (Sigma-Aldrich Canada Ltd.) [12], and referred to as DTR-DC OVA and DTR-(K b / )DC OVA, respectively. The potentially contaminating CD3 and CD19 cells in DC OVA preparation were depleted with anti-cd3 and anti-cd19 magnetic beads (Miltenyi Biotech, Auburn, CA, USA). CD11c high GFP DTR-DC OVA was purified further by sorting on a Beckman Coulter EPICS Elite for immunization of mice. Preparation of OVA-EL4 thymoma cells EL4 thymoma cells were loaded with OVA protein by osmotic shock, as described previously with a slight modification [20]. The amount of OVA as- 290 Journal of Leukocyte Biology Volume 92, August

3 Ahmed et al. CD4 T cell help in CTL immunity sociated with OVA-EL4 cells was quantitated by an OVA ELISA kit (Alpha Diagnostic International, San Antonio, TX, USA). OVA-EL4 ( ) cells was estimated to contain ng cellular OVA using OVA-EL4 cell lysates and an OVA detection kit (Alpha Diagnostic International) by ELISA analysis. Preparation of T cells The naive polyclonal CD8 T cells (purity, 90%) were prepared from splenocytes of B6.1 or B6 mice, as described earlier [12]. OT-I CD8 and OT-II CD4 T cells (purity, 96%) were purified from mouse splenocytes using CD4 and CD8 T cell enrichment kits (Stemcell Technologies, Vancouver, British Columbia, Canada), respectively. Novel immunization protocols We developed three novel immunization protocols. In Protocol I, B6 mice (six/group) were first depleted of endogenous CD8 T cells by i.p. injection with anti-cd8 antibody (3.155; 500, 200, and 200 g/mouse at Days 5, 3, and 1) [21]. The efficiency of antibody-mediated depletion of endogenous CD8 (CD8 ) T cells [22] was determined by flow cytometry analysis of FITC-CD8 (YTS 169.4)-stained mouse spleen/blood samples, 12 h after the last treatment with depleting antibody. At Day 0, the mice were i.v.-immunized with OVA-pulsed, FACS-sorted CD11c-DTR mouse DCs (DTR-DC OVA ; cells/mouse) for facilitating the DC- CD4 T cell interactions in the absence of CD8 T cells. As DC/CD4 T cell interactions (multiple short-lasting and then long-lasting contacts) last for the first 24 h after immunization [9, 23], we allowed the DC-CD4 T cell interactions in vivo to progress for 24 h and then depleted CD4 T cell-licensed DTR-DCs and/or DC-primed CD4 T cells by injection of DT (300 ng/mouse, i.p.) [24] and/or anti-cd4 antibody (GK1.5; 500 g/ mouse, i.p.) [5] for generation of different groups of mice, which possessed the primed CD4 T cells, the licensed DCs, both, or neither populations. Then, 12 h later, mice were reconstituted with naive polyclonal CD8 T cells ( cells/mouse) of B6 mice for induction of OVA-specific CTL responses, as detected by tetramer analysis, 6 days post-dtr- DCova immunization. To assess the role for CD40L signaling of CD4 T cells at the CD4-CD8 T interface, we used naïve polyclonal CD8 T cells from CD40 / mice for reconstitution in this protocol. To determine whether antibody treatment affects the subsequent transfer of naive CD8 T cells in CD8 T cell-depleted mice, we examined the amount of transferred CD8 T cells in mice, 1 day after T cell ( cells/mouse) transfer by flow cytometry. To assess the efficiency of GK1.5 antibody-mediated depletion of DC-primed CD4 T cells, B6 mice were first i.v.-transferred with 90% pure CFSE-labeled, naïve OT-II CD4 T cells ( cells/mouse). Then, mice were i.v.-immunized with DC OVA ( cells/ mouse) and 24 h later, i.p.-treated with GK1.5 antibody (500 g/mouse). After 12 h, mouse spleen samples were stained with noncompeting PE-anti- CD4 (RM4-4) antibody and analyzed by flow cytometry. Protocol II was designed using physiologically generated OVA-cross-presenting DCs by cell-associated OVA (OVA-EL4 cells) immunization. In this protocol, CD11c-DTR mice (six mice/group) were first depleted of CD8 T cells by treatment of anti-cd8 antibody (53-5.8; 1, 0.5, and 0.2 mg/ mouse at Days 5, 3, and 1, respectively) [25] and then i.v.-immunized with irradiated (20,000 rads) OVA-EL4 cells ( cells/mouse) to generate cross-priming DC OVA in vivo. Mice were subjected to various treatments, similarly as mentioned in Protocol I. OVA-specific CTL responses were detected by flow cytometry, 7 days post-ova-el4 immunization. To ascertain that anti-cd8 antibody treatment, which depletes CD8 T cells, will not deplete CD8 (CD8 ) DCs [26], the spelnocytes of antibody-treated CD11c-DTR mice were stained with PE-anti-CD11c (PE- CD11c) and FITC-CD8, 1 day after the last injection of antibody and analyzed by flow cytometry. To ascertain DT-mediated depletion of endogenous DCs in CD11c-DTR mice, CD11c-DTR mice were i.p.-injected with DT (4 ng/g mouse body weight) twice (36 h and 3 days after OVA- EL4 injection). Twelve hours after the first DT treatment, mouse spleen/ blood samples were stained with PE-anti-CD11c antibody alone or PE-anti- CD11c and FITC-anti-MHC-II antibodies and analyzed by flow cytometry. To eliminate the potential CTL responses derived from phagocytosis of irradiated OVA-EL4 cells by the other host APCs, such as CD11c low pdcs and B cells, which will not be depleted by DT treatment, we also treated CD11c-DTR mice with anti-cd20 (AISB12) and anti-pdc (120G8) antibodies (300 g/mouse) to deplete endogenous B cells and pdcs. One day after the treatment, mouse splenocytes were stained with FITC-anti-B220 antibody (as B cells and pdcs express B220) and analyzed by flow cytometry. Protocol III, similar to Protocol I, was designed using peptide(s)-pulsed DTR-DCs for assessment of the critical role of acquired pmhc-i on primed CD4 T cells [17]. In this protocol, B6 mice (six/group) were first depleted of CD8 T cells with antibody treatment, and 200 OT-II CD4 T cells were adoptively transferred into each mice for enhancement of OVAII-specific CD4 T cell responses. The next day, mice were i.v.-immunized with DTR-DCs ( cells/mouse), pulsed with peptides (1 g/ ml, 37 C for 1 h) in various combinations. These include DC pmhc-ii pmhc-i (OVAII OVAI), DC pmhc-i noncognate LCMV-II (OVAI gp ), DC pmhc-i (OVAI) or DC pmhc-ii (OVAII) only, and a mixture of DC pmhc-i (OVAI) and DC pmhc-ii (OVAII). In another set of experiments, FACS-sorted DTR-(K b / ) DC OVA ( cells/mouse), derived from CD11c-DTR/H-2K b / mice, was i.v.-injected. Twenty-four hours after the immunization, mice were treated with DT (300 ng/mouse, i.p.) to deplete injected DCs. Then, 12 h later, mice were i.v.-transferred with polyclonal B6.1 (CD45.1 ), naïve CD8 T cells ( cells/mouse). OVA-specific CTL responses were detected by flow cytometry, 6 days post- OVA, peptide-pulsed DC or DC OVA immunization. MHC-I tetramer staining In the above three protocols, the primary CD8 T cell responses were assessed 6 and 7 days subsequent to DTR-DC OVA or peptide-pulsed DTR-DCs and OVA-EL4 immunizations, respectively. Mouse peripheral blood samples were stained with PE-tetramer and FITC-CD8 or FITC-CD45.1 (FITC- CD45.1) antibody and analyzed to enumerate OVA-specific CD8 or CD45.1 T cells by flow cytometry [17]. The recall CD8 T cell responses were assessed using FITC-CD8 antibody and PE-tetramer staining by flow cytometry, 4 days after DC OVA (i.v., cells/mouse) boost at Day 40 or 120, subsequent to the primary immunization in Protocol I. Cytotoxicity assay An in vivo cytotoxicity assay was performed, as described previously [16]. Splenocytes were harvested from naive B6 mouse spleens and incubated with high (3.0 M, CFSE high ) or low (0.6 M, CFSE low ) concentrations of CFSE to generate differentially labeled target cells. The CFSE high cells were pulsed with OVAI (SIINFEKL), whereas the CFSE low cells were pulsed with irrelevant Mut1 (FEQNTAQP) and served as an internal control [16]. These peptide-pulsed target cells were i.v.-coinjected at a 1:1 ratio into the above-immunized mice, 6 days after immunization in Protocol I. Sixteen hours later, the residual, antigen-specific CFSE high and control CFSE low target cells remaining in the recipients spleens were analyzed by flow cytometry. Intravital two-photon imaging CMTMR-labeled in vitro DC OVA -or(k b / )DC OVA -activated OT-II CD4 T cells ( cells) were i.v.-injected into B6 recipient mouse. Twentyfour hours later, an equal amount of the CMFDA-labeled, OVA-specific OT-I CD8 T cells was injected into the same recipient. ILNs, collected 3 h later and immobilized in an imaging chamber, were perfused continuously with RPMI-1640 medium, bubbled with 95% O 2 and 5% CO 2 at 37 C, and imaged in the T-zone region. Measurements were made in at least five independent experiments. Imaging was performed with a two-photon, laserscanning microscope (Olympus BX51WIF, fitted with a LUMPLFL/IR 40 W/0.8 NA Olympus objective lens). Samples were excited with a MaiTai Ti-Saphire laser (Spectra-Physics), tuned to a wavelength of 850 nm, so as to excite both fluorophors equally. Emission of the relevant wavelengths was acquired simultaneously with the use of selective emission-bandpass Volume 92, August 2012 Journal of Leukocyte Biology 291

4 nm (50 nm bandpass) and 605 nm (70 nm bandpass) filters. Images were collected with a typical voxel size of m, and volume dimension varied in each case. Imaris (Bitplane, Zurich, Switzerland) was used for 4D image analysis and automated tracking of cells. The confinement ratio, corresponding to the ratio of the distance between the initial and a final position of each cell to the total distance, was covered by the same cell. The duration of individual, new contacts, formed during the imaging session, was quantitated by individual inspection of each time slice at each z-slice level in the 4D imaging data set. Statistical analysis was performed with GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). All significant values were determined using the unpaired two-tailed t test. Animal studies To assess protective immunity, the primed CD4 T cell intact group of mice was i.v.-challenged with BL6-10ova tumor cells ( cells/mouse) at Day 120 after the primary immunization with DTR-DC OVA. Three weeks after tumor cell challenge, black melanoma colonies in mouse lungs were counted. Metastatic foci, too numerous to count, were assigned an arbitrary value of 300 [5, 12]. Data analysis Unless stated otherwise, data are expressed as mean (sd) and evaluated using two-tailed unpaired t test using Prism software. Probability values of P 0.05 and P 0.01 are considered statistically not significant and very significant, respectively. RESULTS CTL induction following DC-CD4 T cell interactions To study the abilities of antigen-presenting DCs and cognate CD4 T cells, which had primed and licensed one another previously to subsequently activate cognate CTL responses, we developed a novel immunization protocol (Protocol I). Accordingly, we first depleted B6 mice of their CD8 T cells using antibody and then immunized them with FACSsorted OVA-pulsed CD11c-DTR mouse CD11c high GFP DCs (DTR-DC OVA )(Fig. 1A), which expressed CD11c, Ia b, CD40, CD80, and OVAI pmhc-i, but not CD4 and CD8, and stimulated the host CD4 T cell-dependent CTL responses (Supplemental Fig. 1A and B). One day after immunization, we selectively depleted the immunizing DTR-DC OVA (by DT treatment) or the host s CD4 T cells (with GK1.5 antibody treatment) to allow these DCs to interact for 24 h with recipients CD4 T cells for DC licensing and CD4 T cell priming. Twelve hours later, we then reconstituted the animals with naïve B6 CD8 T cells to assess the ability of the residual, licensed DCs or primed CD4 T cells to drive CTL responses. As shown in Fig. 1B and C, a single-dose treatment of antibody and GK1.5 was able to deplete almost completely endogenous, naïve CD8 (CD8 ) and primed (active) CD4 T cells [5], respectively. CD4 T depletion lasted for at least another 4 days after treatment (data not shown). Licensed DCs (Fig. 1D, a) and primed CD4 T cells (Fig. 1D, c) were able to stimulate CFSE-CD8 T cell divisions. In addition, a singledose treatment of DT abrogated FACS-sorted, DTR-DC OVA - stimulated CTL responses completely (Fig. 1D, b) [20]. Furthermore, as shown, 77% (2.45%/3.17%) of reconstituted, polyclonal, naïve CD8 T cells still survived in antibody treated B6 mice (Fig. 1E), indicating that the residual antibody in antibody-treated mice could deplete only partly the subsequently transferred polyclonal CD8 T cells, which will not affect our experiments. Therefore, our data indicate that this protocol is feasible for dissecting the role of DCs and CD4 and CD8 T cells in immune responses, leading to independently manipulating in vivo-licensed DC- and primed CD4 T cell-stimulated CTL responses. Following the above protocol, we demonstrated that mice that possessed licensed DC OVA and primed CD4 T cells stimulated OVA-specific CTL responses efficiently ( % OVA-specific CTLs; Fig. 1F, b). Interestingly, mice that possessed the primed CD4 T cells, but no longer had any OVApresenting, licensed DCs (Fig. 1F, d), were able to induce stronger cognate CTL priming ( % OVA-specific CTLs) than mice that possessed the licensed DC OVA ( % OVA-specific CTLs; Fig. 1F, c; P 0.01 vs. licensed DC group). The possibility of primary CTL responses, derived from the B6 host DCs through cross-priming of dead DTR-DC OVA after DT treatment, is negligible, as the primary CTL response was found to be abrogated completely in mice with B6 host DCs but depleted in primed CD4 T cells and licensed DTR-DC OVA (Fig. 1F, e). It was demonstrated previously that DCs with acquired pmhc complexes from dead donor cells, which synthesized the complexes, became crossdressed DCs, capable of stimulating T cell responses [27, 28]. Although in vitro cross-dressed APCs with acquisition of pmhc-i complexes from the surface of other infected APCs have been found to stimulate CTL responses [28, 29], some recent in vivo evidence demonstrated that in vivo cross-dressed DCs can only drive the memory CD8 T cell but not the primary CD8 T cell responses [30], indicating that the above 1.10% OVA-specific, primary CTL responses should be derived from the primed CD4 T cell stimulation but not the potentially cross-dressed host DCs. In addition, we demonstrated that the primary, OVA-specific CTL responses, stimulated by in vitro-generated DC OVA in CD11c-DTR mice, were similar in the presence and absence of the host DCs (Supplemental Fig. 1C), confirming that the in vivo cross-dressed DCs do not contribute to the primary CTL responses. Therefore, our above results provide in vivo evidence that CD4 T cell-licensed DCs alone can stimulate CTL responses [18]. The more-efficient CTL responses, stimulated by primed CD4 T cells rather than by licensed DCs (Fig. 1F), may be a result of CD4 T cell replication [9, 23] in the primed CD4 T-intact group, leading to increased CD8 T cell expansion [17]. Rapid generation of licensed DCs and primed CD4 T cells in vivo We next asked how long antigen-presenting DCs and cognate CD4 T cells need to interact to induce optimal DC licensing and CD4 T cell priming. To do this, we immunized CD8 T cell-depleted B6 mice with DTR-DC OVA, as described in Fig. 1F, but depleted the immunizing DTR-DC OVA or primed CD4 T cells at various times (i.e., 0, 6, 12, 24, or 36 h) thereafter. Twelve hours after the depletion, we reconstituted the mice with polyclonal CD8 T cells to assess the extent to which the residual CD4 T cells or DC OVA had become primed or licensed. In agreement with a previous report of rapid DC licensing [18], we also found that DCova had rapidly 292 Journal of Leukocyte Biology Volume 92, August

5 V Ahmed et al. CD4 T cell help in CTL immunity A Pre-sort Post-sort B C CD11c-DTR DCova CD11c-DTR DCova Isotype Ab anti-cd8 Ab (3.155) 68.2% 96.2% a b PE-CD11c GFP PE- CD4 FITC- CD8 99% depleted PE-CD4 Isotype Ab No depletion Naive Active CFSE anti-cd4 Ab >99% depletion Naive Active D F H PBS anti-cd4 Ab DT anti-cd4 Ab Licensed-DCs intact Licensed-DCs killed DTR -DCova PBS anti-cd4 Ab or immunization DT anti-cd4 Ab or a b CD8 -/- DT isotype Ab injection CFSE-labeled OT-I transfer 0 h 24 h 36 h CFSE profile in spleen 3 days post OT- I transfer CFSE PE- tetramer Relative cell number Deplete: - CD8 T cells (Ab 3.155) Days DTR-DCova immunization C57BL/6 Day 0 Deplete: - none - CD4 CD8 T (B6) cell T cells reconstitution - DTR-DCova - CD4 T cell DTR DCova 24 h 36 h Day 6 FITC-CD8 Assess CTL induction (FACS) Unimmunized control PBS Isotypic Ab anti-cd4 Ab Diphtheria toxin (DT) anti-cd4 Ab DT a 0.02 (0.01) b 1.52 (0.12) c 0.56 (0.07) d 1.10 (0.14)* e 0.02 (0.02) Unimmunized control a Mut I 50.7% 0% L H Ova I 49.3% PBS Isotypic Ab Mut I 81.2% 63(5)% L H Ova I 18.8% anti-cd4 Ab Mut I 62.6% L H CFSE Ova I 37.4% PE - CD8 Mut I 71.1% 25(4)% 42(6)% * Diphtheria toxin (DT) b c d e L H Ova I 28.9% anti-cd4 Ab DT Mut I 49.7% 0% L H Ova I 50.3% DT isotype Ab Primed-CD4 T intact c G CD8 tetramer T cells (%) E FITC-CD45.1 a Isotype Ab anti-cd8 Ab 3.17% b 2.45% CD8-PE anti-cd4 Ab injected Diphtheria toxin (DT) injected ** 1.2 * * * 1 * * h 6h 12h 24h 36h Time point of injection with DT and anti-cd4 Ab Figure 1. CTL induction following DTR-DC-CD4 T interactions in B6 mice in Protocol I. (A) To check the purity of sorted DTR-DC OVA, OVA-pulsed, CD11c-DTR mouse CD11c high GFP DTR-DC OVA, sorted by FACS, was analyzed by flow cytometry. (B) To assess CD8 T cell depletion, splenocytes of mice with CD8 T cell depletion by anti-cd8 antibody treatment were stained with PE-CD4 and FITC- CD8 antibody and analyzed by flow cytometry. (C) To assess primed CD4 T cell depletion, splenocytes of mice transferred with CFSE-labeled OT-II CD4 T cells and then immunized with DC OVA, followed by anti-cd4 antibody treatment, 1 day after immunization, were stained with PE-CD4 antibody and analyzed by flow cytometry. (D) To assess the depletion of DTR-DC stimulation, splenocytes of CD8 gene KO mice immunized with DTR- DC OVA and then treated with anti-cd4 antibody (a) or DT (c) and anti-cd4 antibody and DT (b), 1 day after immunization, followed by transfer of CFSE-labeled OT-I CD8 T cells, 12 h after treatment, were stained with PE-CD8 antibody and analyzed by flow cytometry, 3 days post-t cell transfer, as described in the T cell proliferation assay in Materials and Methods. (E) To assess transferred CD8 T cell survival in anti-cd8 antibody-treated mice, splenocytes of mice treated with anti-cd8 antibody and then transferred CD45.1 mouse CD8 T cells, 2 days after treatment, were stained with FITC-anti-45.1 and PE-anti-CD8 antibodies and analyzed by flow cytometry, 12 h post-t cell transfer. (F) A schematic protocol using OVA protein-pulsed DTR-DCs to study the direct role of DC-primed CD4 T cells in induction of CTL responses. B6 mice were depleted of CD8 T cells; some were kept unimmunized, whereas others were immunized with DTR-DC OVA, and then, 1 day later, the mice were untreated (a) or treated with PBS isotype antibody (b), anti-cd4 antibody to deplete primed CD4 T cells (c), DT to deplete licensed DCs (d), and both anti-cd4 antibody and DT (e) to deplete licensed DCs and primed CD4 T cells. All animals were reconstituted with B6 polyclonal CD8 T cells for induction of CTL responses. (G) B6 mice were treated essentially as above, but the times for DC OVA and CD4 T cell depletion and therefore, DC OVA licensing and CD4 T cell priming were designed for 0, 6, 12, 24, or 36 h after DC OVA immunization. Six days after DTR-DC OVA immunization (F and G), tail blood samples were stained with PE-tetramer and FITC-CD8 and analyzed by flow cytometry. (F) Scatter plots of blood cells from the different groups of mice, gating on PE-tetramer/CD8 double-positive cells (boxes). The numbers in the upper right section of each panel represent the mean percentage of CD8 T cells, which were double-positive ( sd). (H) In vivo killing of OVAI-pulsed splenocytes, labeled with CFSE high (H) in licensed DC OVA and primed CD4 T cell groups at Day 7 post-dc OVA immunization. The value in the left bottom panel corner represents the percentage of OVA-specific killing compared with Mut1-pulsed and CFSE low -labeled control cells (L). *P 0.01 versus cohorts of licensed DC OVA intact group with anti-cd4 antibody treatment; **P 0.01 versus cohorts of primed CD4 T cell intact group with DT treatment at two different times. One representative experiment of two is displayed. Volume 92, August 2012 Journal of Leukocyte Biology 293

6 become licensed to drive CTL responses efficiently, after 6 h of interacting with cognate CD4 T cells (Fig. 1G). Similarly, CD4 T cell priming also occurred quickly (i.e., within 6 h), although CD4 T cells that interacted with DC OVA for 24 h were more efficient in driving CTL responses. At all timepoints studied, primed CD4 T cells induced clonal expansion of CD8 T cells more efficiently than did the licensed DC OVA (P 0.01). In vivo-licensed DC- and primed CD4 T-stimulated CTLs are functional effectors To assess the functional effect of licensed DC- and primed CD4 T-stimulated CTLs, we performed an in vivo cytotoxicity assay by adoptively transferring OVA-specific, OVAI-pulsed/ CFSE high -labeled splenocytes and irrelevant Mut1-pulsed/ CFSE low -labeled splenocytes (internal control) into the immunized mice with the primed CD4 T cells, the licensed DCs, both, or neither populations, as shown in Fig. 1F. After 16 h of the transfer, the loss of OVAI-pulsed/CFSE high -labeled target cells, which represents OVA-specific killing activity in the mice, was quantitatively detected by flow cytometric analysis of mouse splenocytes. We found that there was substantial (25% and 42%) loss of OVAI-pulsed/CFSE high -labeled target cells in mice with licensed DCs and primed CD4 T cells (Fig. 1H, c and d), respectively, indicating that in vivo-licensed DC- and primed CD4 T-stimulated CTLs are functional effectors, and the latter is more efficient than the former (P 0.01). CTL induction by physiologically DC-primed CD4 T cells We next assessed whether DCs, which acquired cellular OVA antigen under physiological conditions, would become licensed to drive CTL responses themselves and prime cognate CD4 T cells for stimulation of CD8 T cell responses. To address this question, we designed Protocol II, wherein we loaded EL4 thymoma cells with OVA (OVA-EL4) using osmotic shock [20] and then induced cell apoptosis by irradiation, such that they would be scavenged by the host s DCs, following injection of irradiated OVA-EL4 cells into transgenic CD11c-DTR mice. DCs, which acquire such cell-associated antigens, have been reported to cross-present them efficiently and thereby, induce antigen-specific CTL responses [20, 31]. As noted above, we also confirmed that OVA-EL4 immunization induced the host DC- and CD4 T cell-dependent CTL responses (Fig. 2A). To ascertain DT-mediated depletion of endogenous DTR-DCs, CD11c-DTR mice were i.p.-injected with DT (4 ng/g mouse body weight). We found that a single dose of DT treatment deleted CD11c high GFP and CD11c high MHC- II DTR-DCs completely (Fig. 2B, c and f) [20]. It has been demonstrated that DT-depleted, endogenous DCs recovered 2 days after DT treatment [32]. Therefore, CTL responses in a primed CD4 T cell group of mice with a second DT treatment will not be interfered by recovered DCs via phagocytosis of irradiated OVA-EL4 cells. As there are some other APC subsets, such as B cells and pdcs, in addition to CD11c DCs, in CD11c-DTR mice, we also treated mice with depleting antibodies AISB12 and 120G8, to deplete B220 B cells and B220 pdcs. We found a complete depletion ( 99%) of B220 cells in AISB12 antibody- 120G8 antibody-injected (Fig. 2C, black) compared with isotype control antibody-injected (Fig. 2C, gray) mice, which is consistent with previous reports [33, 34]. Therefore, DT plus AISB12 and 120G8 antibody treatment of CD11c-DTR mice will deplete CD11c-DTR DCs and macrophages completely [24], as well as pdcs and B cells. To assess whether the anti-cd8 antibody (53-5.8) treatment depletes CD8 (CD8 ) T cells and CD8 (CD8 ) DCs, we i.p.-injected CD11c-DTR mice with anti-cd8 antibody (53-5.8). One day after the last antibody injection, mouse splenocytes were analyzed by flow cytometry. We found that the anti- CD8 antibody (53-5.8) treatment depleted CD8 (CD8 ) T cells completely (Fig. 2D), which is consistent with a previous report [25]. However, the anti-cd8 antibody (53-5.8) did not deplete CD8 CD11c DCs, including CD8 high ones (Fig. 2E). Therefore, the antibody treatment to deplete CD8 T cells in this protocol will not affect CD8 DC subsets to cross-prime the OVA-EL4 antigen to T cells. Our data thus indicate that this protocol is also feasible for independently manipulating physiologically licensed DC- and primed CD4 T cell-stimulated CTL responses. Accordingly, we first depleted endogenous CD8 T cells with antibody treatment and generated mice with intact licensed DC OVA, primed CD4 T cells, neither population, or both populations, followed by reconstitution of polyclonal B6.1 CD8 T cells. We found that the outcomes in this protocol (Fig. 2F), which showed that more efficient CTL responses were found in the primed CD4 intact group ( %; Fig. 2F, c) than in licensed DC intact group ( %; Fig. 2F, b; P 0.01), mirrored the above findings in Fig. 1F. To rule out the potential involvement of host pdcs and B cells in primed CD4 T cell-stimulated CTL responses, we repeated the experiment using DT treatment to deplete CD11c-DTR macrophages/myeloid DCs [32] and coadministration of neutralization antibodies AISB12 and 120G8 to deplete B220 B cells and B220 pdcs [34]. We found that the primed CD4 T cells in the absence of endogenous pdcs and B cells stimulated CTL ( %) responses (Fig. 2F, d) similar to those ( %) in the presence of pdcs and B cells (Fig. 2F, c; P 0.05), confirming that CTL ( %) responses in the DT-treated group are mostly derived from primed CD4 T cell stimulation but not from pdc or B cell stimulation. The more efficient CTL responses stimulated by primed CD4 T cells than by licensed DCs (Fig. 2F) may be a result of a combination of CD4 T cell replication [9, 23] in the primed CD4 T-intact group, leading to increased CD8 T cell expansion [17], and CD4 DC depletion after GK1.5 antibody treatment in the licensed DC-intact group, leading to decreased CD8 T cell expansion. Our results thus provide physiologically relevant in vivo evidence that following DC- CD4 T interactions, the dissociated, primed CD4 T cells can directly orchestrate efficient CD8 CTL responses. pmhc-i plays a critical role in direct induction of CTL responses by primed CD4 T cells We further investigated the molecular mechanisms by which DC-primed CD4 T cells were able to stimulate CTLs directly, 294 Journal of Leukocyte Biology Volume 92, August

7 V PE-CD11c Ahmed et al. CD4 T cell help in CTL immunity A PE- tetramer B OVA-EL-4 injected PBS Diphtheria toxin (DT) anti-cd4 Ab 0.75(0.06) 0.02(0.02) 0.01(0.01) B6 FITC-CD8 CD11c-DTR CD11c-DTR Diphtheria toxin (DT) PBS Diphtheria toxin (DT) a 0.0 b 1.52 c 0.03 (0.0) (0.12) (0.01) C Relative cell number anti-banti-pdc Abs Abs-treated F Normal FITC-B220 Deplete: - CD8β T cells (Ab ) D PE- CD4 a Osmotically OVA -loaded EL4 cells CD11c-DTR Isotype Ab anti-cd8β Αb (53 5.8) b FITC- CD8 99% depleted E Deplete: - none - CD4 CD8 T (B6.1) cell T cells reconstitution - DTR-DCova - host s APCs - CD4 T cell DTR DCova Isotype Ab anti-cd8b Ab CD8 - CD8 high CD8 - CD8 high CD8 T FITC-CD8 Assess CTL induction (FACS) 0.13 CD8 T PE - CD11c GFP d 1.48 e 1.56 f 0.02 PBS (0.14) injected (0.16) (0.01) PE- tetramer Days Day 0 36 h 48 h Day 7 PBS Isotypic Ab anti-cd4 Ab Diphtheria toxin (DT) anti-banti-pdc AbsDT anti-cd4 Ab DT a 0.84 (0.07) b 0.23(0.03) c 0.55(0.05) * d 0.58 (0.06)** e 0.02 (0.01) FITC-MHC Class II FITC-CD45.1 Figure 2. CTL induction by stimulation of physiologically generated DC OVA and primed CD4 T cells in CD11c-DTR mice in Protocol II. (A) DT or anti-cd4 antibody-treated CD11c-DTR mice were i.v.-immunized with irradiated OVA-EL4 cells. Mouse bloods were stained with FITC-CD8 and PE-tetramer staining and analyzed by flow cytometry, 7 days postimmunization. (B) To assess CD11c MHC-II GFP DC depletion, splenocytes of CD11c-DTR mice, i.p.-treated with DT, were stained with PE-CD11c antibody or PE-CD11c and FITC-MHC-II antibody and analyzed by flow cytometry, 1 day post-dt treatment. Panel represents the mean percentage of GFP CD11c high or CD11c high MHC-II DCs in the total cell population assessed, and values in parentheses represent the sd. (C) Histograms demonstrate depletion ( 99%) of B220 cells in mice with treatment of anti-cd20 and anti-pdc antibodies (black) for depletion of B220 B cells and pdcs compared with treatment of isotype control antibodies (gray). (D) To assess CD8 T cell depletion, splenocytes of mice with CD8 T cell depletion by anti-cd8 antibody treatment were stained with PE-CD4 and FITC-CD8 antibodies and analyzed by flow cytometry. (E) To assess the depletion of CD8 DCs by treatment of anti-cd8 antibody, splenocytes of CD11c-DTR mice treated with anti-cd8 antibody were stained with PE-CD11c and FITC-CD8 and analyzed by flow cytometry, 1 day post-dt treatment. Panel represents the mean percentage of CD11c CD8 high and CD11c CD8 DCs in the total cell population. (F) OVA-EL4 cells were irradiated prior to i.v. injection into CD8 T cell-depleted CD11c-DTR mice, as a means of delivering the cellular OVA in a physiologically relevant way to the animal s APCs for subsequent cross-presentation. Thirty-six hours later, the recipients were treated with (a) PBS and isotype control antibody, (b) anti-cd4 antibody, (c) DT, and (d) a cocktail designed to deplete all host APCs or (e) with both DT and anti-cd4 antibody, and 12 h later, they were reconstituted with CD45.1 CD8 T cells. Seven days after OVA-EL4 immunization, OVA-specific CD8 T cell responses were assessed by flow cytometry by gating on PE-tetramer/CD8 double-positive cells (boxes). The number in the upper right section of each panel represents the mean percentage of CD8 T cells, which were double-positive ( sd). *P 0.01 versus cohorts of anti- CD4 antibody-injected group (b), and **P 0.05 versus cohorts of toxin (no anti-cd4 Ab) injected group (c). One representative experiment of two is displayed. despite the absence of cognate antigen-carrying DCs at the time of CTL induction. To address this question, we investigated the roles of MHC-I and -II molecules on our immunizing populations of DTR-DCs, pulsing these cells with MHC-Ispecific OVAI peptide alone, MHC-II-specific OVAII peptide alone, or both or with irrelevant LCMV MHC-II-specific peptide (gp ), prior to using them to immunize mice (endogenous CD8 T cells depleted), as described in Protocol III. After 24 h of DC immunization, we depleted peptide-pulsed DTR-DCs by injecting DT and then transferred polyclonal B6.1 CD8 T cells into these mice. On the 6th day of immunization, the CTL immune response was assessed using tetramer staining and flow cytometry. As a control for whether the OVA-specific pmhc-i and pmhc-ii had to be presented on the same DC, we included a group of mice that was immunized with a mixture of DCs carrying OVA-specific pmhc-i or pmhc-ii but not both. Following the protocol, we found that only the primed CD4 T cells, which had interacted with DCs, simultaneously presenting cognate MHC-II (OVAII) and MHC-I peptide (OVAI; DC pmhc-i pmhc-ii), stimulated CTL ( %) responses (Fig. 3A, a). Simultaneous presentations of both of these epitopes, but by separate DCs (DC pmhc-i DC pmhc-ii), did not lead to cognate CTL induction (Fig. 3A, e), and presentation of only OVA-specific pmhc-i (Fig. 3A, c) or pmhc-ii epitope (Fig. 3A, d) or irrelevant MHC-II epitope gp plus pmhc-i (DC pmhc- I MHC-II; Fig. 3A, b) did not enable CTL responses. As further confirmation of the critical nature of the priming DC OVA, presenting pmhc-i and pmhc-ii, while interacting with CD4 T cells, we included a group of mice, which were immunized with DTR-(K b / )DC OVA (which would be fully competent to present pmhc-ii but not pmhc-i). As expected, CD4 T cells without acquired pmhc-i from mice immunized with DTR- Volume 92, August 2012 Journal of Leukocyte Biology 295

8 A PE- tetramer Deplete: - CD8 T cells (Ab 3.155) Days DC pmhci pmhcii Ova Ova Peptide(s) pulsed DTR-DC DC pmhci MHCII Ova gp II C57BL/6 Day 0 24 h 36 h Day 6 Primed CD4 T intact B6 mice DC pmhci Ova I ( ) Deplete: - DTR-DC CD8 T (B6.1) cell reconstitution DC pmhcii Ova II ( ) Assess CTL induction (FACS) DC pmhci / DC pmhcii Ova or Ova a 1.15 (0.12) b 0.05 (0.02) c 0.04 (0.02) d 0.02 (0.01) e 0.03 (0.02) B PE- tetramer Primed CD4 T intact B6 mice a DTR-DCova 1.09 (0.16) DTR-(K b-/- ) DCova b 0.02 (0.01) FITC-CD45.1 Figure 3. pmhc-i complexes of primed CD4 T cells play a pivotal role in CTL induction in Protocol III. (A) CD8 T cell-depleted B6 mice were adoptively transferred with 200 OT-II CD4 T cells, followed by immunization of mice with DTR-DCs, pulsed with MHC-I- or MHC- II-OVA peptides (OVAI or OVAII, re- FITC-CD45.1 spectively) or both (DC pmhc-i pmhc-ii) or with OVAI and irrelevant LCMV MHC-II peptide (gp ; DC pmhc-i MHC-II). Control mice were given a mixture of two DC populations, which had been pulsed independently with OVAI or OVAII (DC pmhc-i/dc pmhc-ii). The mice were then treated with DT prior to B6.1 CD8 T cell reconstitution to generate mice with intact, primed CD4 T cells. (B) CD8 T celldepleted B6 mice were immunized with OVA protein-pulsed DTR-DC OVA and DTR-(K b / )DC OVA lacking pmhc-i, followed by DT treatment and B6.1 CD8 T cell reconstitution to generate mice with intact, primed CD4 T cells. Six days after DC immunization, OVA-specific CD8 T cell responses were assessed by gating on PE-tetramer/CD45.1 double-positive cells (boxes), largely as in Fig. 1F. One representative experiment of two is displayed. (K b / )DC OVA, bearing only pmhc-ii but not pmhc-i, did not induce any CTL responses (Fig. 3B). Our data indicate that DCs must express cognate pmhc-i and -II simultaneously to enable otherwise primed CD4 T cells to stimulate CTL responses directly. Our data, using various models, strongly suggest that primed CD4 T cells (with acquired pmhc-i) are able to stimulate naïve CD8 T cells. A recent study on trogocytosis has elegantly shown that cross-dressed DCs cannot stimulate a primary CTL response [30] and thus, support our conclusion that a primed CD4 T cell with acquired pmhc-i could play an important role in CTL immunity. Intravital two-photon microscopy confirms the role of pmhc-i in direct CD4 -CD8 T cell cooperation To confirm the above targeting role of pmhc-i, we performed a two-photon imaging study in ILNs of mice, allowing transfer of in vitro DC OVA -or(k b / )DC OVA -activated, CMTMR-labeled (primed) OT-II CD4 T cells and CMFDA-labeled, naïve OT-I CD8 T cells. In the presence of DC OVA -activated (primed) OT-II CD4 T cells, antigen-specific OT-I CD8 T cells (Fig. 4A, left, green) formed persistent contacts with primed OT-II CD4 T cells (Fig. 4A, left, red) and moved if necessary to maintain the contacts (Supplemental Movie 1). On the other hand, using (K b / )DC OVA -activated (primed) OT-II CD4 T cells, no stable interactions were found between primed OT-II CD4 T cells without acquired pmhc-i (Fig. 4A, right, red) and CD8 T cells (Fig. 4A, right, green; Supplemental Movie 2). In the presence of pmhc-i-acquired OT-II CD4 T cells, OT-I CD8 T cells showed a slower (mean velocity vs m/min; P ) and more confined (confinement ratio vs ; P ) movement (Fig. 4B), indicating that the primed CD4 T cell pmhc-i controls the motile behavior and the ability to form stable conjugates between primed CD4 T cells and CD8 T cells. Interactions between primed CD4 T cells without acquired pmhc-i with CD8 T cells were only intermittent (90% of contacts lasted 5 min), whereas primed CD4 T cells with acquired pmhc-i established long-lasting contacts with CD8 T cells (95% of contacts lasted 5 min; Fig. 4C), confirming the important role of pmhc-i in targeting primed CD4 T cells to CD8 T cells in vivo. The duration of interactions between primed CD4 T cells and naïve OT-I CD8 T cells was probably underestimated in these experiments, as highly motile conjugates could exit the imaging volume during a 40- to 60-min imaging period. Therefore, our two-photon imaging data clearly confirm the critical role of pmhc-i in targeting primed CD4 T cells to cognate CD8 T cells in vivo. CD4 T cell CD40L signaling programs CD8 T cell memory development A general requirement for CD40-CD40L signaling in the induction of CTL response and memory has been reported previously [12, 16, 17], but we wished to investigate these issues in-depth, specifically exploring the role of CD4 T cells in these processes. According to Protocol I, we immunized CD8 T-depleted B6 mice with DTR-DC OVA to facilitate DC-CD4 T interactions. One day later, we depleted the immunizing DC OVA and primed CD4 T cells by DT and anti-cd4 antibody treatment, leaving the mice with cognate-primed CD4 T cells and licensed DCs, respectively. Thirty-six hours later, we reconstituted these mice with naïve, polyclonal CD8 T cells, derived from WT B6 mice. In addition, we reconstituted other groups of mice with naïve polyclonal CD8 T cells, derived 296 Journal of Leukocyte Biology Volume 92, August

9 Ahmed et al. CD4 T cell help in CTL immunity A DCova-activated OTII Naive OTI (Kb-/-) DCova-activated OTII Naive OTI B V elocity of OTI CD8 T cells (μm/min ) *** 0.5 *** *** C onfinement Ratio of OTI CD8 T cell s C Contact Duration (s) DCova-activated OTII (Kb-/-) DCova-activated OTII DCova-activated OTII (Kb-/-) DCova-activated OTII OTI pathway OTI pathway Figure 4. Two-photon in vivo imaging confirms the role of pmhc-i-acquired CD4 T cells in targeting naive CD8 T cells. (A) Time-lapse images of DC OVA -or(k b / )DC OVA -activated (primed) OT-II CD4 T cells interacting with OVA-specific OT-I CD8 T cells (shown with blue arrows; see also Supplemental Movie 1). Automated tracking of CD8 T cell migration was measured by velocity in the T cell zone. Individual cells are displayed as color-coded tracks to represent increasing time from start of imaging (blue) to end of imaging (yellow). Tracks were overlaid after normalizing their starting coordinates (lower panel; see also Supplemental Movies 1 and 2). (B) Left, mean velocity; right, confinement ratio; corresponded to the ratio of the distance between the initial and final positions of each cell to the total distance covered by the same cell. (C) Contact durations between DC OVA (blue label)- or (K b / )DC OVA (yellow label)-activated (primed) OT-II CD4 T cells and OT-I CD8 T cells. Data were pooled from five independent experiments. ***P from CD40 / mice, such that the cognate CD8 T cells would be fully immunocompetent and incapable of receiving a CD40L signal, respectively, from the fully primed CD4 T cells and licensed DC OVA. This approach allows us to interrogate the long-standing issue of the role(s) played by cognate CD4 T helper signals, delivered directly to the CD8 T cells they are activating. We found that in the primed CD4 T cell group, WT CD8 CTLs, activated by primed CD4 T cells providing CD40L signaling, had regular primary CTL responses ( % OVAspecific CTLs; Fig. 5A, a) and recall responses at Day 120 after the primary DTR-DC OVA immunization ( % OVAspecific CTLs; Fig. 5A, b). However, in the primed CD4 T cell group, CD8 CTLs with CD40 deficiency (unable to receive the primed CD4 T cell CD40L signaling), activated by primed CD4 T cells, had regular primary CTL responses ( % OVA-specific CTLs; Fig. 5A, c) but defect in their capacity to mount memory responses ( % OVAspecific CTLs; Fig. 5A, d; P 0.01), indicating that CD40L signaling by the primed CD4 T cell is essential for programming CD8 T cell memory development. In contrast, the reconstitution of naïve polyclonal CD8 T cells, derived from CD40 / mice, did not affect primary CTL responses (Fig. 5B, a and c; P 0.05) and CD8 memory T recall responses in the licensed DC OVA intact group of mice (Fig. 5B, b and d; P 0.05), indicating that the licensed DC CD40L signal is not involved in programming CD8 T cell memory development. The above findings in the primed CD4 T cell intact mouse group were confirmed further in our animal studies by challenging the mice with BL6-10ova tumor cells, 120 days after immunization. We found that mice with reconstitution of WT B6 CD8 T cells were all protected against tumor cell challenge, whereas mice reconstituted with CD8 (CD40 / )T cells had numerous tumor lung colonies (Fig. 5C). Taken together, we provide direct in vivo evidence that CD40L from in vivo-primed CD4 T cells, but not licensed DCs, contributed critical signals for programming of CTL memory, although they did not affect CTL priming. DISCUSSION Trogocytosis, the intercellular membrane molecule transfer between immune cells, plays an important role in immune regulation [35]. Several reports in the literature [13 15, 36, 37] and our earlier findings [12, 16, 17] provided some in vitro evidence that CD4 T cells can capture cell surface proteins, including pmhc-i and costimulatory molecules from professional APCs, and use these acquired molecules to modulate CTL responses. Some recent studies further supported the acquisition of pmhc complexes by CD4 T cells via in vivo trogocytosis [15, 38, 39]. We also demonstrated that CD4 T cells harvested from DC OVA -immunized mice expressed pmhc-i complexes, possibly via in vivo trogocytosis [12, 16, 17]. In this study, our novel approach herein allowed us to study CD4 - CD8 T cell interactions in the absence of antigen-presenting Volume 92, August 2012 Journal of Leukocyte Biology 297

10 A PE-tetramer CD8 T (B6) CD8 T (CD40 -/- ) Primed-CD4 T Intact Groups 120 days post-primary Primary day 6 Boost day 4 a 1.12(0.15) * 1.75(0.25) ** b 1.01(0.21) 0.04(0.02) B PE-tetramer CD8 T (B6) CD8 T (CD40 -/- ) Licensed-DCova Intact Groups 120 days post primary Primary day 6 Boost day 4 a 0.50(0.06) * 1.02(0.16) ** b 0.48(0.07) 0.98(0.17) C Tumor incidence (%) Mean of LMC Primed-CD4 T Intact (DT injected) Groups PBS CD8 T(B6) CD8 T(CD40 -/- ) 6/6 (100%) 0/6 (0%) 6/6 (100%) >300 0 >300 Figure 5. Primed CD4 T cell CD40L signaling for CTL memory development. B6 mice were treated as in Fig. 1F, with the exception that the polyclonal CD8 T cells used FITC-CD8 FITC-CD8 for reconstitution were also derived from CD40 / mice. Six days after primary DTR-DC OVA immunization or 4 days after DC OVA boost at Day 120, subsequent to the primary DTR-DC OVA immunization, blood samples of (A) primed CD4 T cell intact and (B) licensed DC intact mouse groups were stained with PE-tetramer and FITC-CD8 and analyzed by flow cytometry. Scatter plots of blood cells from the different groups of mice, gating on PE-tetramer/CD8 double-positive cells (boxes). The numbers in the upper right section of each panel represent the mean percentage of CD8 T cells that were double-positive ( sd). (A) *P 0.05, and **P 0.01 versus cohorts of CD8 (CD40 / ) T cells, respectively; (B) *P 0.05, and **P 0.01 versus cohorts of CD8 (CD40 / ) T cells, respectively. (C) At Day 120 after DTR- DC OVA immunization, immunized mice with the primed CD4 T cell intact group were i.v.-challenged with BL6-10ova tumor cells. Mouse lungs were removed 3 weeks after tumor cell challenge, and black melanoma foci were counted. One representative experiment of two is displayed. LMC, Lung metastatic colony. DCs and thus, provided an opportunity to assess the role of pmhc-i on primed CD4 T cells in CTL induction in vivo. Accordingly, we demonstrated that DCs must simultaneously express cognate pmhc-i and pmhc-ii to enable otherwise primed CD4 T cells to stimulate CTL responses directly. The pathways of acquisition of pmhc-i complexes by CD4 T cells include internalization and recycling of synapse-composed molecules [37, 40] and/or an association-dissociated process [35]. The acquisition of pmhc-i complexes by CD4 T cells further facilitates a direct targeting of CD4 T cell helper signals to CD8 T cells via pmhc-i/tcr interactions, even in the absence of antigen-carrying DCs. Importantly, our intravital two-photon imaging studies unequivocally confirm the critical role of acquired pmhc-i in targeting primed CD4 T cells to cognate CD8 T cells. Moreover, the ability of primed CD4 T cells to secrete chemokines CCL3 and CCL4 following activation [19] could also help direct cooperation of CD4 - CD8 T cells in vivo. The targeting role of acquired pmhc-i on primed CD4 T cells may also be applied to interpret the generation of antigen-specific regulatory T and double-negative regulatory T cells in vivo after an encounter with the antigen-presenting DCs [16, 41 43]. However, it has also been demonstrated that the primed CD4 T cells with acquired pmhc-i could become susceptible to CTL killing in an antigen-specific manner [44] as a type of negative immune modulation. CD40L signaling has been found to be associated with CTL immunity. For example, Bourgeois et al. [11] reported that CD40 deficiency had a major impact on CTL memory responses and suggested that the T cell help of CD4 to the CD8 T cell involves direct CD4 -CD8 T interactions by forming DC-CD4 -CD8 T cell clusters. However, in addition to CD4 T cells, DCs express CD40L [45]. Janssen et al. [5] demonstrated that the presence of CD4 T cells during CTL priming is an absolute requirement for memory CTLs, and CD4 T cells deliver IL-2 signals by forming DC-CD4 -CD8 T clusters. Therefore, the direct in vivo evidence for the cellular source of CD40L in CTL memory development is still unclear. Our novel approach provides an opportunity to independently assess the role of CD40L signaling derived from primed CD4 T cells or licensed DCs in CTL memory development. Our data show that CD40L of primed CD4 T cells provided critical signals for programming of CTL memory in DC OVA -stimulated CD4 T cell-dependent CTL responses, although they did not affect CTL priming. It has been demonstrated that long-lived memory CTLs could be generated in the absence of CD40 expression in inflammatory experimental models using recombinant Listeria monocytogenes as stimulatory pathogen [46 48]. In these studies, recombinant L. monocytogenes stimulated CD4 T cell-independent CTL responses, in which DCs licensed by pathogenic danger signals were able to stimulate CD8 T cells directly. In this study, our data also demonstrate that CD40L of licensed DCs is not involved in CTL priming or programming of CTL memory, indicating that similarly to danger signal-licensed DCs, CD4 T cell-licensed DCs can stimulate CTL responses in the absence of CD40 expression on CD8 T cells. Our findings in this study provide us with new knowledge of CD4 T cells in in vivo CTL immunity. Our findings also reconcile previous, somewhat concerning observations vis-à-vis the passive three-cell interaction model (Fig. 6A), the licensed DC model (Fig. 6B), and the primed CD4 T-APC model (Fig. 6C) and propose a new dynamic model of three-cell interactions for CTL immunity derived from stimulation of dissociated licensed DCs, primed CD4 T cells, and DC-CD4 T clusters (Fig. 6D). Such a dynamic three-cell interaction model would dramatically reduce the dependence on an antigen-presenting DCs, simultaneously engaging cognate, naïve CD4 298 Journal of Leukocyte Biology Volume 92, August