Exploiting cross-priming to generate protective CD8 T-cell immunity rapidly Nhat-Long L. Phama, Lecia L. Peweb, Courtney J. Fleenorb, Ryan A. Langloisa, Kevin L. Leggea,b,c, Vladimir P. Badovinaca,c, and John T. Hartya,b,c,1 aInterdisciplinary Graduate Program in Immunology and Departments of bMicrobiology, and cPathology, University of Iowa, Iowa City, IA 52242 Edited by Michael J Bevan, University of Washington, Seattle, WA, and approved May 28, 2010 (received for review April 6, 2010) The number of memory CD8 T cells generated by infection or vac- of systemic inflammation is key to evoking a response that can cination correlates strongly with the degree of protection ob- respond rapidly to booster immunization. However, the labori- served in infection and tumor models. Therefore, rapid induction ous and personalized nature of DC immunization is a major of protective numbers of effector and memory CD8 T cells may be hurdle for translating this approach to large-scale vaccination of outbred humans. Overcoming this limitation requires an “off- crucial in the case of malignancy, pandemic infection, or bioterror- ” ism. Many studies have shown that amplifying T-cell numbers by the-shelf approach to immunization that induces little systemic inflammation but still results in maturation of DC capable of prime-boost vaccination is most effective with a substantial time stimulating CD8 T-cell responses. Here, we demonstrate an al- interval between immunizations. In contrast, immunization with ternative vaccination strategy that exploits the cross-priming peptide-coated mature dendritic cells (DCs) results in a CD8 T-cell pathway in the absence of adjuvants to generate rapidly pro- response exhibiting accelerated acquisition of memory character- tective CD8 T-cell immunity against multiple pathogens. istics, including the ability to respond to booster immunization within days of initial priming. However, personalized DC immuni- Results zation is too costly, labor intensive, and time-consuming for large- Cross-Priming with Cell-Associated Antigen Accelerates CD8 T-Cell scale vaccination. Here, we demonstrate that in vivo cross-priming Memory. Disposal of apoptotic cells by DC limits inflammation with cell-associated antigens or antigen-coated, biodegradable and provides a mechanism for cross-priming CD8 T cells against microspheres in the absence of adjuvant quickly generates CD8 T cell-associated antigens (13). Immunization of naïve C57BL/6 − − IMMUNOLOGY cells that display the phenotype and function of long-term (B6) mice with irradiated Act-mOva.Kb / splenocytes, which memory populations. Importantly, cross-primed CD8 T cells can cannot directly present the ovalbumin (Ova) epitope (14), cross- b respond to booster immunization within days of the initial immu- primed functional H-2K –restricted Ova257-specific CD8 T cells nization to generate rapidly large numbers of effector and memory that are detectable in both peripheral blood and spleen (Fig. 1 A and B and Fig. S1A) in the absence of overt systemic inflamma- T cells that can protect against bacterial, viral, and parasitic in- fi fections, including lethal influenza and malaria-causing Plasmo- tion (Fig. S1B). More importantly, Ova257-speci c CD8 T cells that were primed with either DC-Ova or irradiated Act-mOva. dium infection. Thus, accelerated CD8 T-cell memory after in vivo − − 257 Kb / splenocytes acquired phenotypic (CD127hi, KLRG-1lo) and cross-priming in the absence of adjuvant is generalizable and can functional (∼35–40% produced IL-2 after antigen stimulation be exploited to generate protective immunity rapidly. and exhibited low granzyme B) memory characteristics at day 7 after immunization (Fig. 1C and Fig. S1C) (3, 12). This result protective immunity | vaccination contrasts sharply with the effector phenotype (CD127lo,KLRG-1hi) and function (reduced IL-2, increased frequency of granzyme B- D8 T cells are critical in protecting the host from infection expressing cells) of Ova-specific CD8 T cells stimulated by in- Cby intracellular pathogens. During infection, antigen-specific fection with Listeria monocytogenes expressing Ova (LM-Ova). CD8 T cells undergo proliferative expansion to increase in Thus, similar to DC immunization, cross-priming CD8 T cells number, followed by contraction and generation of a stable pool with cell-associated antigen results in accelerated acquisition of of long-lived memory cells that provide enhanced resistance to memory phenotype and function. reinfection (1). The number of memory CD8 T cells correlates strongly with the level of protection in experimental models of Cross-Primed CD8 T Cells Respond Vigorously to Short-Interval infection (2–5). To date, prime-boost immunization remains the Boosting. A cardinal feature of memory CD8 T cells is their most successful approach to generate high numbers of memory robust proliferative response upon reexposure to antigen (1). T cells and enhanced resistance (6, 7). However, most current Consistent with their accelerated memory phenotype, Ova257- prime-boost strategies, which are based on the use of adjuvants specific CD8 T cells in cross-primed mice underwent vigorous to amplify initial T-cell responses, require several months be- secondary expansion in response to three different booster tween each immunization to achieve the greatest amplification of regimens: virulent Listeria monocytogenes expressing Ova immunological memory. Clearly, reducing the time interval be- (virLM-Ova), attenuated actA-deficient Listeria monocytogenes tween priming and boosting would be beneficial in the case of expressing Ova (attLM-Ova), and Vaccinia virus expressing the pandemic outbreaks or in immunotherapy of cancer, when time Ova257–264 epitope (VacV-Ova) delivered at day 7 after initial is of the essence. immunization (short-interval booster immunization) (Fig. 1D). Infection of mice with intracellular pathogens stimulates ro- Listeria boosting induced an enormous response in cross- ∼ bust CD8 T-cell responses that initially exhibit an “effector” primed mice: 60% of circulating CD8 T cells in peripheral phenotype and acquire memory phenotype and function rela- tively slowly after the infection is cleared (5, 8). Similarly, subunit vaccines that use adjuvants to mimic the inflammatory conditions Author contributions: N.-L.L.P., V.P.B., and J.T.H. designed research; N.-L.L.P., L.L.P., C.J.F.., of infection also induce T-cell responses that are slow to acquire and R.A.L. performed research; K.L.L. contributed new reagents/analytic tools; N.-L.L.P., memory function (9–11). In contrast, immunization with peptide- L.L.P., C.J.F., R.A.L., K.L.L., V.P.B., and J.T.H. analyzed data; and N.-L.L.P. and J.T.H. wrote coated mature dendritic cells (DC) in the absence of additional the paper. adjuvant evokes CD8 T cells that display memory characteristics The authors declare no conflict of interest. within days of the initial priming (12). Importantly, systemic in- This article is a PNAS Direct Submission. flammatory cytokines induced by infection or adjuvant prevent 1To whom correspondence should be addressed. E-mail: [email protected]. accelerated memory differentiation by DC-primed CD8 T cells. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Thus, priming of naïve CD8 T cells by mature DC in the absence 1073/pnas.1004661107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1004661107 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 A B C D Fig. 1. Cross-priming with cell-associated anti- gen followed by short-interval booster immuni- zation rapidly generates protective CD8 T-cell immunity. Naïve C57BL/6 (B6) mice received ∼107 irradiated WT or Kb−/−mOva splenocytes (i.v.). (A) b Detection by K /Ova257 tetramer staining. (B)Ki- netics of Ova257-specific CD8 T-cell response (mean frequency ± SEM, n = 3) in PBL. (C) Phe- E notypic and functional status of Ova257-specific CD8 T cells at day 7 after DC immunization, cross- priming, or virLM-Ova infection (mean ± SEM, n = 3). (D) Kinetics of Ova257-specific CD8 T-cell re- sponse (mean frequency ± SEM, n = 3) in PBL with different booster immunizations as indicated. Numbers indicate fold difference at day 54. (E) Bacteria count (mean ± SEM, n = 3) in spleen and liver ∼65 h after a lethal dose of virLM-Ova. LOD, limit of detection. *Statistical analysis was per- formed using an unpaired, two-tailed t test. blood were specificfortheOva257 epitope within 1 wk after Cross-Priming with Autologous Peripheral Blood Mononuclear Cells. boosting. Importantly, this enormous CD8 T-cell response was To avoid alloreactivity, cross-priming CD8 T-cell responses in not observed in mice that received the booster immunizations humans would require reinfusion of syngeneic antigen-coated PBMC. To address the feasibility of this approach, we first de- after initial priming with irradiated WT splenocytes without − − Ova (Fig. 1D) and thus is a function of the presence of initially termined that 106 irradiated Act-mOva.Kb / splenocytes (Fig. cross-primed CD8 T cells. As observed in our DC immuniza- S3 A and B)or106 irradiated Ova-coated syngeneic splenocytes tion model, inducing systemic inflammation with CpG oligo- (Fig. S4 A–C) primed CD8 T cells capable of responding to deoxynucleotide, a Toll-like receptor 9 agonist, prevented rapid short-interval boosting. We were able to obtain 106 peripheral acquisition of memory characteristics by cross-primed CD8 blood mononuclear cells (PBMC) from ∼150 μL of mouse blood. Tcells(Fig. S2 A–C). Thus, cross-priming