PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Vaccine. Author manuscript; available in PMC 2014 January 2.
Published in final edited form as:
PMCID: PMC3529748
NIHMSID: NIHMS419379

T cell Vaccinology: Exploring the known unknowns

Abstract

The objective of modern vaccine development is the safe generation of protective long-term immune memory, both prophylactic and therapeutic. Live attenuated vaccines generate potent cellular and humoral immunity [13], but numerous problems exist with these vaccines, ranging from production and storage issues to adverse reactions and reversion to virulence. Subunit vaccines are safer, more stable, and more amenable to mass production. However the protection they produce is frequently inferior to live attenuated vaccines and is typically confined to humoral, and not cellular immunity. Unfortunately, there are presently no subunit vaccines available clinically that are effective at eliciting cellular responses let alone cellular memory [4]. This article will provide and overview of areas of investigation that we see as important for the development of vaccines with the capacity to induce robust and enduring cellular immune responses.

Introduction

The importance of T cell immunity to vaccination is at least 2 fold. First, though neutralizing antibody is admittedly the reason for the success of the majority of vaccines thus far, this parameter is far less relevant (if not irrelevant) when considering therapeutic vaccination against established diseases such as HIV, Hepatitis C, Mycobacterium tuberculosis (mTB), or cancer. In these situations of chronic infection/disease, it is clear that potent cellular immunity is a prerequisite for vaccine efficacy [5]. Second, even in situations where neutralizing antibody is protective, the biology of the infectious agent may make the generation of cellular immunity far more desirable than just antibody. A good example is influenza where a cellular response against antigens conserved across multiple strains would be expected to protect the host more broadly that the typical yearly vaccine-elicited neutralizing antibody responses against the highly variable HA and NA proteins. Therefore, there is a significant need for the development and implementation of new vaccine paradigms capable of priming robust CD4 and CD8+ T cell immunity.

The production of such a vaccine will require the identification not only of the appropriate antigens to serve as immunologic targets, but also the discovery of robust adjuvants, capable of performing the tasks listed above [69]. Following the conceptual leadership of Charlie Janeway [10], all those interested in the generation of adaptive immune responses from vaccination have spent the last 3 decades searching for entities that elicit innate immunity for use as vaccine adjuvants. Numerous advances have been made in the last 10 years regarding the identification of new innate signaling pathways as well as the identification of the agonists/ligands that instigate their stimulation [1113]. These discoveries have presented the vaccine world with a veritable plethora of agonists and ligands to incorporate into rationally designed vaccine adjuants in the hopes that better days are ahead for the generation of enduring, vaccine elicited cellular responses.

Unfortunately, these advances in innate immunity have not necessarily translated into significant clinical advances in vaccines and/or vaccine adjuvants. Years of exploration have revealed that the mere generation of innate immunity does not guarantee that adaptive immunity will follow. That is, there is a distinction between innate immunity that simply elicits inflammation and innate immunity that effectively transitions into robust adaptive immunity, specifically T cell immunity. The failure to produce advances in vaccine biology commensurate with the explosion of information on innate activating pathways is due, at least in part, to a failure in addressing a number of “known unknowns” (with apologies to Donald Rumsfeld) in vaccine biology. In calling them as such, we recognize that not all of these known unknowns are universally agreed upon as known, unknown or even as unknowns worthy of knowing. Be that as it may, this review will present a short list of vaccinology considerations and the rationale behind why these are important parameters to explore for the purposes of ongoing and future vaccine design and development.

Will the real signal 3 please stand up?

It has long been appreciated that additional signals outside of the trimolecular complex (MHC-peptide-antigen) are required for fulminate T cell activation [14]. In the mid 1980’s, CD28 was identified, and has since been well characterized, as a required costimulatory molecule for functional T cell responses [15, 16]. Through associated kinase activity, ligation of CD28 with either CD80 or CD86 (B71/2) on antigen presenting cells results in upregulated and stabilized IL-2 mRNA expression, IL-2 production, mTOR activation, and subsequent upregulation of the pro-survival factor Bcl-xL. [1719]. Collectively CD28 provided the necessary signal(s) along side of TCR activation for robust T cell proliferation and survival, the so-called 2 signal hypothesis of T cell activation [20].

However, T cells do more than just proliferate, being called upon to express a variety of effector functions ranging from the production of pro- and anti-inflammatory cytokines to lysis of antigen bearing target cells [21, 22]. In addition, after pathogen/target clearance, T cells must contract to a memory pool that will remain poised for rapid helper/effector functions upon reintroduction of cognate antigen [23]. The two signal model of T cell activation is not sufficient for providing information that can direct either the effector or memory arms of the response, and thus a minimal “3 signal” model was proposed. By way of analogy, in the dance that is a developing T cell response, signal 1 (MHC-peptide-TCR) serves to promote selection of a capable dance partner, signal 2 (CD28) enhances this recognition and promotes a more firm embrace between the dancers, while signal 3 serves as the music telling the dancers whether they are in a tango, foxtrot, or waltz.

Immunologically, the concept of signal 3 was coined and formalized for CD8+ T cells when Mescher and colleagues proposed that cytokines of the immunological milieu played a deterministic role in the fate of CD8 T cell responses to in vitro activation cultures and adjuvant antigen priming [22]. They and others would go on to show that type I IFN and IL-12 acting through their receptors on the surface of T cells could program both the acute T cell response as well as the kind and quality of the T cell memory response. [2430]. In the realm of CD4 helper T cell biology this paradigm fits with the ever expanding world of helper T cell differentiation in which specific cytokines direct CD4+ T cell fate into various cytokine producing Th1, Th2, Th17, etc. T cell subsets [31]. Molecularly speaking, the factors capable of providing “signal 3” support were universally inflammatory cytokines, typically involving the induction of STAT1 or STAT4 signaling into the responding T cells [28]. Activation of these signaling modalities eventually culminates in regulating the expression of transcription factors (Tbet, Eomes, RORγT, GATA3) that ultimately shape the fate of the cell [2527, 29, 30]. This has been confirmed in numerous model systems where T cells deficient in various cytokine receptors show impairments in primary and secondary responses to certain pathogen challenge or vaccination [23, 32].

While the capacity for IL-12 or type 1 I IFN to serve as signal 3 mediators is undisputed, a growing number of observations indicate that these signaling pathways can at times be a liability for the generation of immune memory [26]. Recent studies in both mouse and human indicate a central role for STAT3-inducing cytokines in the development of T cell memory, primarily due to the capacity of STAT3, and subsequent induction of SOCS3, to limit STAT4 activation and downstream Tbet expression [33, 34]. Thus, T cell memory is achieved only through the limitation of STAT4/Tbet. Indeed, unrestricted Tbet expression, which is downstream of both STAT1 and STAT4 “signal 3” mediators, has gained a reputation for being too much of a good thing, serving to severely blunt the development of long term memory [35, 36]. As immune memory is the object of vaccination, these data question what the optimal signal 3 mediators are in a vaccine setting.

In the search for non-cytokine signaling pathways, various TNF Receptor family members appear to support signal 3 functions in responding T cells [37], specifically under conditions of vaccination in which the generation of memory T cells is preferred over the generation of primary effectors. Recently we published that the generation of immune memory in response to vaccination using a combined TLR/CD40 adjuvant [38, 39] is independent of all the usual cytokine signal 3 suspects, instead relying upon the TNFR family members CD27 and OX40 [40]. In these experiments both the primary and memory CD8+ response is critically dependent upon signals through CD27 principally and residually through OX40. In contrast, the CD4 response to this vaccination is more dependent upon OX40 with a residual dependence on CD70 [41]. TNFR signaling promotes T cell survival through NFkB activation of the pro-survival member Bcl-xL [37, 4245]. Besides contributing to T cell survival and memory formation, van Lier and colleagues showed that CD27 was specifically required for the survival of lower affinity T cells into the memory pool [46]. When present, these lower affinity cells resulted in T cell pool with greater cross reactivity and better immune protection to challenge with a disparate, but related pathogen. Thus TNF receptors, with their capacity to promote the survival of immunity against a more broad diversity of pathogens, may represent a form of “signal 3” that might be specifically preferred during vaccination.

Collectively, the data suggest that the notion of a “signal 3” may have a good deal of flexibility, and it is even likely that signals beyond cytokines or TNF receptors may be competent for supporting complete T cell activation and programming of memory. A true reductionist approach suggests perhaps a 4 signal model consisting of peptide/MHC and CD28 to support initial activation (signals 1 and 2), cytokine mediated expansion and differentiation matched to the inflammatory milieu (Signal 3a), and TNFR-mediated survival (signal 3b) of both effectors and long lived memory. A major area of focus for the future should be the exploration and identification of the spectrum of pathways capable of contributing to signals 3a/b and how to best target these for the purposes of vaccine development.

DC activation profile… is more simply better?

Given that the Dendritic Cell (DC) is saddled with the responsibility of providing the majority, if not all, of signals 1–3b described above, it is somewhat surprising that we have relatively little quantitative understanding of what the optimal antigen-presenting DC looks like. Since its discovery in the mid to late 1970s by Steinmann and Cohen [4749], numerous studies have confirmed the critical function of DC in bridging the innate and adaptive immune system[50]. Various subsets of DCs have been identified in both rodents and humans, each with common and unique T cell stimulatory capacities. The central position of DCs in eliciting T cell responses made it obvious that the creation of effective vaccines required harnessing the capability of DCs to prime an effective adaptive immune response. Therefore, in order to develop vaccines that produce T cell responses, it became imperative that an adjuvant must induce the maturation and activation of the appropriate DC subset.

The question remains as to which DC subset is best suited to prime a given T cell response [51]. Numerous lines of evidence indicate that specific DC subsets in both mouse and human specialize in cross presenting exogenously provided antigens in the class I pathway [50, 5258]. Targeting these DC subsets, either by the form of antigen or nature of the adjuvant, could optimize a vaccine toward eliciting particular kinds of T cell expansion. For example, we recently demonstrated that Langerhans cells (LC) in the skin can be induced to cross-present antigen to CD8 T cells using a TLR7 agonist-conjugate vaccination platform [59]. In contrast, Heath and colleagues have shown that dermal CD103+ DCs, not LCs, are the dominant cross presenting DC subset during HSV challenge [6062]. In further contrast, both CD103+ and CD103− DCs in the lung have been variably implicated as the major cross presenting DC in models as diverse as influenza challenge and apoptotic cell encounter [52, 6366]. Therefore, the data suggest that the apparent specialization of specific DC subsets for different modes of antigen presentation may have more to do with the nature of the challenge and/or form of the antigen/adjuvant than the functional capacity of the DC.

In addition, we have little to no quantitative appreciation as to what DC “maturation” and “activation” mean at the molecular level. In the tradition of the model described above, the simplest definition of DC “activation” would be the production by the DC of all the necessary factors (antigen/MHC, CD80/86 expression, IL-12/IFN production, CD70/OX40L expression) that can deliver all 4 signals. This however does not answer the question of how much of each of these signals can be considered enough. Is a 2-fold elevation of CD86 expression above background sufficient? Or does it need to be 5 fold? Can 4 fold more signal 3 compensate for 2 fold less signal 2? And how much of, and which, signal 3 is preferred? We, and many others, have identified the expression of the TNF ligands CD70 and OX40L on the DCs as potent initiators of CD8 and CD4 T cell expansion, respectively [3941, 52, 58, 6776]. Given the potency of the receptors for these ligands to serve as signal 3a/b mediators in supporting T cell expansion, they are attractive targets for priming an effective adaptive immune response following vaccination. Indeed, the targeting of CD27 in the context of infection or dendritic cell immunization can even promote CD8 T cell memory in the absence of CD4 T cells [77]. This CD8 memory phenotype/functionality would be preferred in individuals with diminished CD4 T cell counts, such as HIV+ or HepC+ individuals. While these data seem to suggest that CD27 stimulation is always a boon to the development of T cell memory, we (RMK, unpublished data) and others [46, 7882] have observed a deleterious effect of over exuberant CD27 stimulation on the development of competent CD8 T cell memory. Substantial quantitative analysis will need to be done on the effect of specific DC activation/maturation profiles on subsequent development of immunity before we will have a clear picture of what, when, and for how long which activation markers/costimulatory molecules are required to prime protective CD4 and CD8 T cell memory.

Antigen Dose… the antigen hungry T cell response

The appropriate dose of antigen in a vaccine study is a crucial determinant of vaccine efficacy. Ours and others [38, 8387] data reveal that small changes in the dose of antigen produces a corresponding increase in the magnitude of the T cell response as well as the level of protective immunity generated. Some older data might seem to suggest that high doses of antigen can result in the phenomenon known as high zone tolerance. However, these studies were largely focused on the effect of high levels of antigen stimulation on either B cells [88] or on T cell clones in vitro [8993], and no in vivo equivalent for T cells has been observed with the exception of clonal exhaustion which is discussed in further detail below. Simply put, all recent in vivo data indicate that T cell responses are highly dose responsive… the more antigen you put in, the more T cells you get out (Figure 1). With this in mind, it comes as considerable surprise that the mechanism underlying this direct relationship between antigen dose and T cell magnitude is unclear. Indeed, if anything, the literature is somewhat contradictory to these empirical findings. CD8+ T cells, upon encounter with sufficient antigen and costimulatory signals, undergo preprogrammed rounds of proliferation regardless of the ongoing presence or absence of the antigen [84, 9497]. If the presence or absence of antigen does not effect the extent of proliferation for an antigen specific T cell, this questions how the amount of antigen can affect the magnitude of the response? One answer to this question is that antigen dose must affect the degree to which T cells are recruited into the response. However recent bar coding studies [98, 99] have confirmed what most of us suspected from older experiments using adoptive transfer or more recent experiments using tetramer enrichment of endogenous repertoires; namely that recruitment of antigen specific T cells into most responses is close to 100%. While the solution to this apparent conundrum almost certainly lies in how increasing antigen produces subtle, but critical, variations in T cell survival, recruitment and/or proliferation, the increase in magnitude of a T cell response to larger antigen doses is still an unspoken mystery of T cell biology.

Figure 1
Increases in antigen input produce a corresponding increase in the magnitude of the T cell response

Be that as it may, the data in experimental models regarding the direct relationship between antigen dose and T cell magnitude is unyielding. Despite this, the antigen doses used in clinical vaccines are surprisingly low. The antigen doses used in most animal mouse vaccine studies are generally in the range of 1–10mg/kg. In a 20g mouse, this comes to somewhere between 20–200μg of antigen. Curiously, the same μg amounts of antigen are used in the majority of clinical vaccines (e.g. HepB vaccines use 5–20ug HepBsAg), despite the fact that humans represent a 3000 fold increase in mass and at least a 200 fold increase in surface area! [100] Given the degree to which we experimentally appreciate the importance of antigen dose to successful T cell responses, this raises the question as to why the antigen doses are not scaled up in the clinic to meet the size of a larger host? As far back as the late 1800s, it was observed that various metabolic parameters were similar between the same or even different species when using surface area, not mass, for scaling purposes [101]. Therefore drugs are often dosed as a function of mg/m2 rather than mg/kg. A mathematical correction is made for the fact that these parameters do not change in straight-line proportion to surface area[100], but in the end, using this type of calculation for vaccine purposes serves to effectively reduce the recommended antigen dosing for higher animals. For example, the recommended calculation for an antigen with an optimal dose of a 4 mg/kg (12.06 mg/m2) in a mouse produces a scaled and corrected dose in the human of 0.32mg/kg.

Three points are noteworthy. First, the calculation above is used predominantly for more traditional drugs and therapeutics and is actually not followed at all when identifying a dose of antigen appropriate for a vaccine. If it were, then the 4mg/kg dose mentioned above would translate into injecting over 19mgs of protein into a 60kg human patient! Clearly this is ridiculously high for the purposes of generating either antibody or T cell responses. However, it may well be closer to appropriate than the ~20ug used in many current vaccines in the clinic, a dose that translates to 0.0003 mg/kg (< 0.01mg/m2) which is well below anything even remotely successful for generating T cell responses in mouse or man. Second, since the biology of secondary lymphoid tissue into which the vaccine drains is arguably more related to surface area than mass, there is a reasonable argument that antigen doses should indeed be scaled to surface area. However, not only is the average volume of an individual human lymph node larger than that of a mouse, but the local draining lymphoid tissue in a human is far more diffuse than in the mouse. Any 1cm of mouse surface area might have access to one or two lymph nodes. In contrast, the same 1cm in the human might drain to 5–20 lymph nodes, depending on the precise location. To make matters even worse for T cell vaccine enthusiasts, antigen processing and presentation are woefully inefficient in response to non-live (subunit) vectors, far less efficient than the direct recognition of antigen by specific B cells. Collectively, this means that an antigen will be heavily diluted by the larger volume of human draining lymphoid tissue as compared to the mouse, resulting in the greater likelihood that the local concentration of protein might slip below the threshold necessary for effective presentation to T cells but not below the threshold for the induction of antibody responses. Third, the majority of vaccines use seroconversion (antibody) as a primary readout of vaccine efficacy. As antibody responses are far less responsive to, and dependent upon, increases in antigen dose, the lowest dose of antigen capable of inducing antibody is utilized. Thus, while it is little wonder that smaller amounts of antigen have been deemed acceptable in the clinic, it is less well appreciated that this dose is chosen to the complete detriment of T cell responses.

Ultimately this suggest that any attempts to determine the appropriate antigen dose for eliciting T cell responses in humans should take these factors into account. While the 19mgs/dose in the example above is surely overkill, it may be an order of magnitude closer to the right dose compared to the many-orders-of-magnitude-too-low that is currently in clinical practice. If preclinical animal models have any significance, the vast majority of our clinical vaccines may be starving the potential for antigen-hungry T cell responses right out of existence.

Antigen:Adjuvant… what is a vaccine’s Golden Ratio?

While antigen dose has both an intuitive and historical connection to vaccine efficacy, the influence of the adjuvant dose is far less appreciated. Innate cytokines have a substantial impact on the development of adaptive immunity, in particular with respect to the differentiation of responding cells into effectors. Indeed, the 3-signal model described above incorporates the necessary function of these cytokines, originally IL-1, IL-12 and type I IFN [21, 22, 102104]. However, an excess of a cytokine such as IL-12 has the capacity to drive responding cells into terminal differentiation, providing a robust primary effector response but a dramatically reduced magnitude of long lived immune memory [33]. Regulators of metabolism such as mTOR control not only the source of energy within the responding T cells but are now seen to regulate the formation of immune memory in a fashion analogous to excess IL-12 [105]. Indeed, the deleterious effects of both mTOR and IL-12 on immune memory have a common root as both promote over expression of the differentiation factor Tbet which, when left unchecked, enacts a transcriptional program that suppresses the ability of another transcription factor, Eomesodermin, to promote long lived memory [35, 36].

The upshot is that these cytokines and transcription factors operate on a goldilocks principle in which the contribution of each must be “just right” in order for the appropriate development of a long lived memory pool. As it is the responsibility of the adjuvant to induce the production and release of these inflammatory cytokines and factors, this implies that the dose of adjuvant could have important consequences on the induction, or lack thereof, of immune memory. This prediction bears out as we have observed the dose of adjuvant to have an impact on the phenotype, though not the magnitude, of the antigen specific T cell response, with the cells becoming more effector-like (hi KLRG1 expression [106]) as the dose of adjuvant increases (Figure 2). This suggests that the wrong dose of adjuvant might create levels of inflammation that begin to impinge on the goldilocks threshold. Thus there is reason to be mindful of the dose of adjuvant in our vaccines to be sure they are optimal for the generation of cellular immune memory. While more may be better for antigen doses (see above) the same is unlikely to be true for the adjuvant.

Figure 2
Adjuvant dose influences the phenotype, not necessarily the magnitude, of the T cell response

Emulsions… murky waters for T cell responses

It has been said that there are only two things one needs to know about physics; F=ma and don’t spit into the wind. Examining the last 100 years of vaccine development reveals an equivalent two-rule mantra for vaccinology; always use an adjuvant, and always have the adjuvant form an antigen depot. While the need to incorporate the adjuvant is indisputable, the requirement for an antigen depot is highly suspect. The history behind the use of an antigen depot, originally through the use of precipitates and later though the use of various kinds of emulsions, can be traced back to the simple fact that they worked. In the early 1900’s, Alexander Glenny postulated that his precipitation of antigen using aluminum potassium sulfate resulted in better immune protection due to the precipitate forming a slowly dissipating antigen depot, thereby allowing the host an extended period of time for antigen recognition and response [107110]. This hypothesis regarding the importance of the antigen depot largely persists to this day despite evidence to the contrary. For example, while the antigen in the precipitate does indeed persist for many weeks in vivo [111], the removal of antigen precipitate depots/nodules two weeks after initial vaccination has no impact on the magnitude of the antibody response[112, 113]. These and other data have served to fuel the understanding that the affect of the precipitate/emulsion on the efficacy of the vaccine has far more to do with its activation of innate immunity than any requirement for the formation of an antigen depot. However, this knowledge has not necessarily translated upwards and changed the formulation of adjuvants in the clinic. This is again mostly due to that fact that an antigen depot does not seem to have any deleterious effect on the magnitude of antibody elicited. Since the majority of vaccines in the clinic are geared toward the production of humoral immunity, this has led to the perception that, though an antigen depot may not be necessary for vaccine efficacy, it probably can’t hurt.

Recent data indicates that at least for the purposes of cellular immunity, the last point above is likely to be wrong. While T cell responses are certainly observable following vaccination with emulsion-based adjuvants [114116], recent data indicate that the magnitude of the T cell response generated is a fraction of what it could be in the absence of the emulsion. Estaban Celis at the Lee Moffit Cancer Center and Willem Overwijk at MD Anderson have shown convincingly that the success of a soluble adjuvant formulation in promoting robust CD8+ T cell expansion and immune protection against cancer growth can be substantially reversed by incorporating the use of an emulsion ([117] and WO personal communication). Similar data in our hands is consistent with their observations (Figure 3). The emulsion appears to compromise the T cell response as a result of the very antigen depot that was ironically once viewed as so critical to the vaccine success. T cells generated by the vaccine recirculate and become trapped at the site of the antigen depot [118]. In the situation of a tumor bearing host, the injection site can become the chief competitor to the tumor site in attracting tumor specific T cells, distracting the cells away from the intended target and effectively sabotaging the entire purpose of the vaccination (WO, personal communication). The solution to this problem (pun intended) may be as simple as just removing the emulsion. For saponin/MF59/QS21-based adjuvants this will be problematic as the emulsion is the adjuvant. However, TLR-based adjuvants (Oncovir, MPL, CpG) can easily be formulated without the use of an emulsion.

Figure 3
The capacity of a vaccine to induce cellular immunity is severely blunted by its formulation as an emulsion

Antigen persistence… different forms, different outcomes

It is worth clarifying at this point that the deleterious effects of antigen trapped in a depot are almost certainly due to the form and site in which the antigen persists and not due to the simple persistence of antigen alone. Antigen persists long after the clearance of a number of infectious agents [119125] and this persistence can be beneficial to the immune protection of the host. A good example is influenza where flu antigen persists in the lung draining lymphoid tissue well beyond the sterile clearance of the virus [119121, 123]. In these circumstances, stimulation of flu-specific memory T cells by the persisting antigen within the lymphoid tissue can effect their phenotype, function [119, 123]. The subsequent trafficking of these cells into the lung parenchyma provides an enhanced degree of immune protection against reinfection, an enhancement that dissipates along with the loss of the persisting antigen over time. This type of antigen persistence must also be distinguished from the excessive antigen load observed after challenge with certain chronic viral infections such as LCMV clone 13, HepC or HIV [126131]. It is probably not coincidence that the immune exhaustion seen in the host after these chronic infections bears striking resemblance to the loss of T cell function in a host containing an emulsion-based vaccine adjuvant (Figure 3). Collectively the data suggest the tentative conclusion that long term, lymphoid based antigen persistence has the potential for substantial immune benefit while chronic antigen depots within peripheral compartments (from either chronic viral replication or emulsion based adjuvants) is highly detrimental.

The logical questions to follow such a conclusion are i) what is the half life of antigen following subunit vaccination, ii) what is the effect of a (non-emulsion) adjuvant on that persistence, and iii) would increasing the persistence of antigen within lymphoid tissue after vaccination have the same beneficial effect as does the persistence of antigen after infectious challenge? While it is generally assumed that soluble whole antigen has a relatively short half life within the host, recent data from our lab has shown a surprising longevity for whole antigen (3+ weeks) when in a soluble formulated adjuvant and at a dose that produces a robust primary T cell response (BAT and RMK, manuscript in preparation). Our data seem to suggest that long term antigen persistence is not only inevitable after an appropriately adjuvanted, formulated and dosed subunit vaccination, but is probably beneficial as well. While a more complete understanding of the impact/necessity of antigen persistence will be tied closely to the issues of both antigen dose and adjuvant:antigen ratio, the data indicate the need to more closely monitor this “unknown” as a possible predictor of overall vaccine efficacy with respect to the generation of T cell responses.

Phenotypic correlates of vaccine efficacy… what are we actually shooting for?

The major contributions of vaccination to global public health have taken place in the last 50 years[132] including the eradication of Smallpox in 1980 and the near-elimination of Polio today. There are now estimated to be over 120 vaccines in routine use worldwide, which annually save about 2.5–3 million lives, and could, with optimal use, save almost twice as many [132134]. It is not an overstatement to say that vaccination is the most successful medical intervention known to mankind. In light of these achievements, it is humbling to acknowledge that, though our capacity to quantitate an immune response has dramatically elevated in recent years, our ability to interpret these data in a way that predicts immune protection in humans is still lacking. For example, live attenuated infectious agents (ie. Vaccinia virus, MMR) produce both humoral and cellular immunity lasting for many decades [135138]. However, we still know relatively little about the phenotypic correlates that characterize this T cell memory. To complicate matters further, the vast majority of basic immunology data is derived from animal models, and we have yet to fully ascertain how well vaccine principles from these models (in particular the ones described in detail above) are directly applicable to the human. A good case study on this point is TGN1412, an anti-CD28 superagonist antibody, which induced robust Treg responses in mice yet led to cytokine storm and shock in humans in Phase I trials[139]. These results underscore the fact that we do not always know how well animal model data, even sometimes in higher primates, apply to the human response and immunologic protection.

In recent years much work has been done to describe phenotypic and functional differences between T cells that persist past the primary immune response [106, 140143]. These different memory cell subsets have been characterized phenotypically by their cell surface expression of markers, such as KLRG1, CD127, CCR7 and CD62L, into either central or effector memory subsets. Due to the self-renewing nature of central memory T cells (Tcm) and their preferential localization to lymphoid organs, as compared to the shorter life span of effector memory T cells (Tem) that preferentially localize in the peripheral tissues, the field has largely considered the generation of Tcm’s as the primary goal of vaccination [144]. But is this phenotype universally preferred or is it specific to the infectious challenge being protected against? Several studies have suggested a more critical role for Tem [121, 123, 145, 146], or the more recently identified tissue resident memory (Trm) subset [147150], in T cell mediated immune protection, especially with regards to infections that are largely restricted to peripheral tissues. While Tcm CD8 T cells have proliferative capacity upon reinfection [144, 151], Tem and Trm cells have the capacity to immediately produce perforin and granzyme in a lytic response [148,152154]. This functional distinction may be responsible for the recent observations that T cell mediated immune protection against influenza may be more dependent on T cell effector/effector memory cells than Tcm [155]. In contrast, protection against a systemic infection, such as LCMV in the mouse or Listeria monocytogenes in mouse or human, may best be mediated by Tcm cells [144].

Even less is known about whether T cell memory can be generated that will preferentially result in different effector responses after rechallenge. In CD4 T cell biology, both Th1-like memory and Th0-like memory cells have been identified, the former giving rise to only Th1 effectors and the latter capable of giving rise to a diversity of T cell phenotypes and functionalities [156158]. Just how broadly this applies to other subset phenotypes is yet unknown, as is any information on the potential for pre-committed CD8+ memory T cell populations. For example, what might a memory precursor CD8+ T cell that is pre-committed to IL-17 production (Tc17) look like and what benefits might it have? The tissue specific inflammatory properties of IL-17 may well produce an environment that is optimal for fighting diseases such as cancer or other chronic infections. Indeed, recent data has shown successful tumor regression following recruitment of CD8 T cells which produce IL-17[159, 160]. If this degree of diversity can be achieved in the pool of memory cells, then perhaps we may need to tailor our vaccines to generate different memory T cell subsets depending on the infection and/or diseases against which they might protect.

The situation is further complicated by the fact that the majority of observations regarding T cell memory phenotype and immunologic protection have been made in animal models, raising the additional question as to whether any of these correlates truly apply in a human clinical setting. It is noteworthy that the original distinction of memory T cell subsets into Tem and Tcm was made for human T cells [154, 161], indicating at least some degree of connection between mouse and human data. Further, there appears to be a good correlation between the phenotype of T cell exhaustion observed in animal models with that seen in the clinic in situations of chronic viral infection and cancer [162165]. A few inroads have been made in attempting to prospectively define immunologic correlates of protection predicated on vaccines that are already known to work effectively in the clinical setting [162, 166168]. One in particular found that robust humoral responses to the yellow fever vaccine correlated with expression of the B cell growth factor TNFRSF17, and robust cellular responses correlated with C1qB and eukaryotic translation initiation factor 2 alpha kinase 4 expression [167]. While the importance of these genes is intuitive once identified, it is informative that no preceding mouse model or study predicted that these genes would be the major harbingers of immune protection. Correlates of protection even more unexpected than these may well be lurking just below the surface, and a comprehensive “systems biology” analysis of both live and attenuated vaccines will be necessary before these correlates are fully appreciated.

Conclusions

Historically, the vaccinologist can be compared to one who looses his keys in a large parking lot but spends his time only able to look in one small corner of it for the simple reason that that is the location of the lampost. This is not meant as a metaphor for an effort in futility, but as a reminder that we can only effectively study that which we can see. More broadly put, our conclusions are only as comprehensive as our tools of analysis are at distinguishing between the knowns, the unknowns and the known unknowns. Thankfully, a number of technological developments have given us the capacity to measure T cells and antibody producing cells down to vanishingly small numbers, as well as to measure many qualitative and quantitative aspects of innate immune activation. It remains to be determined just how much of the parking lot these tools will allow us to see, or how predictive those observations will be, but the unknowns described above are good places to start shining the light.

Highlights

  1. Subunit vaccines are safer than live attenuated vaccines but poorly generate cellular immunity.
  2. A Subunit vaccine that can elicit potent cellular immunity is an unmet need in vaccine development.
  3. We describe six areas of inquiry in which further investigation is needed for vaccine development.

Acknowledgments

The authors thank Catherine Haluszczak for expert lab assistance. R.M.K. is a founder of ImmuRx Inc., a vaccine company whose intellectual property is based on the combined TLR agonist/anti-CD40 immunization platform. R.M.K. is an inventor on patent applications filed by the University of Colorado and licensed by ImmuRx Inc. MAB was supported Irvington Institute-Cancer Research Institute/Eugene V. Weissman Memorial Fellowship. Remaining authors were supported by grants from the NIH (AI06877, AI066121) and the Department of Defense (W81XWH-07-1-0550). DoD support was associated with funding for the Center for Respiratory Biodefense at National Jewish Health.

Footnotes

The remaining authors have no conflicts to report.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

1. Cox RJ, Brokstad KA, Ogra P. Influenza virus: immunity and vaccination strategies. Comparison of the immune response to inactivated and live, attenuated influenza vaccines. Scand J Immunol. 2004 Jan;59(1):1–15. [PubMed]
2. Hobson P, Barnfield C, Barnes A, Klavinskis LS. Mucosal immunization with DNA vaccines. Methods. 2003 Nov;31(3):217–24. [PubMed]
3. Polo JM, Dubensky TW., Jr Virus-based vectors for human vaccine applications. Drug Discov Today. 2002 Jul 1;7(13):719–27. [PubMed]
4. Coffman RL, Sher A, Seder RA. Vaccine adjuvants: putting innate immunity to work. Immunity. 2010 Oct 29;33(4):492–503. [PMC free article] [PubMed]
5. Trumpfheller C, Longhi MP, Caskey M, Idoyaga J, Bozzacco L, Keler T, et al. Dendritic cell-targeted protein vaccines: a novel approach to induce T-cell immunity. J Intern Med. 2012 Feb;271(2):183–92. [PMC free article] [PubMed]
6. Mondino A, Khoruts A, Jenkins MK. The anatomy of T-cell activation and tolerance. Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2245–52. [PubMed]
7. Pulendran B, Smith JL, Jenkins M, Schoenborn M, Maraskovsky E, Maliszewski CR. Prevention of peripheral tolerance by a dendritic cell growth factor: flt3 ligand as an adjuvant. J Exp Med. 1998 Dec 7;188(11):2075–82. [PMC free article] [PubMed]
8. Jenkins MK. The ups and downs of T cell costimulation. Immunity. 1994 Sep;1(6):443–6. [PubMed]
9. Kearney ER, Pape KA, Loh DY, Jenkins MK. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo. Immunity. 1994 Jul;1(4):327–39. [PubMed]
10. Janeway CA., Jr Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harbor symposia on quantitative biology. 1989;54(Pt 1):1–13. [PubMed]
11. Barber GN. Cytoplasmic DNA innate immune pathways. Immunol Rev. 2011 Sep;243(1):99–108. [PubMed]
12. McGettrick AF, O’Neill LA. Regulators of TLR4 signaling by endotoxins. Sub-cellular biochemistry. 2010;53:153–71. [PubMed]
13. Saleh M. The machinery of Nod-like receptors: refining the paths to immunity and cell death. Immunol Rev. 2011 Sep;243(1):235–46. [PubMed]
14. Prlic M, Hernandez-Hoyos G, Bevan MJ. Duration of the initial TCR stimulus controls the magnitude but not functionality of the CD8+ T cell response. J Exp Med. 2006 Sep 4;203(9):2135–43. [PMC free article] [PubMed]
15. Harding FA, McArthur JG, Gross JA, Raulet DH, Allison JP. CD28-mediated signalling co-stimulates murine T cells and prevents induction of anergy in T-cell clones. Nature. 1992 Apr 16;356(6370):607–9. [PubMed]
16. Ledbetter JA, Martin PJ, Spooner CE, Wofsy D, Tsu TT, Beatty PG, et al. Antibodies to Tp67 and Tp44 augment and sustain proliferative responses of activated T cells. J Immunol. 1985 Oct;135(4):2331–6. [PubMed]
17. Linsley PS, Brady W, Grosmaire L, Aruffo A, Damle NK, Ledbetter JA. Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation. J Exp Med. 1991 Mar 1;173(3):721–30. [PMC free article] [PubMed]
18. Nunes JA, Truneh A, Olive D, Cantrell DA. Signal transduction by CD28 costimulatory receptor on T cells. B7-1 and B7-2 regulation of tyrosine kinase adaptor molecules. The Journal of biological chemistry. 1996 Jan 19;271(3):1591–8. [PubMed]
19. Pages F, Ragueneau M, Klasen S, Battifora M, Couez D, Sweet R, et al. Two distinct intracytoplasmic regions of the T-cell adhesion molecule CD28 participate in phosphatidylinositol 3-kinase association. The Journal of biological chemistry. 1996 Apr 19;271(16):9403–9. [PubMed]
20. Mueller DL, Jenkins MK, Schwartz RH. Clonal expansion versus functional clonal inactivation: a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Annu Rev Immunol. 1989;7:445–80. [PubMed]
21. Curtsinger JM, Lins DC, Mescher MF. Signal 3 determines tolerance versus full activation of naive CD8 T cells: dissociating proliferation and development of effector function. J Exp Med. 2003 May 5;197(9):1141–51. [PMC free article] [PubMed]
22. Curtsinger JM, Schmidt CS, Mondino A, Lins DC, Kedl RM, Jenkins MK, et al. Inflammatory cytokines provide a third signal for activation of naive CD4+ and CD8+ T cells. J Immunol. 1999 Mar 15;162(6):3256–62. [PubMed]
23. Masopust D, Kaech SM, Wherry EJ, Ahmed R. The role of programming in memory T-cell development. Curr Opin Immunol. 2004 Apr;16(2):217–25. [PubMed]
24. Agarwal P, Raghavan A, Nandiwada SL, Curtsinger JM, Bohjanen PR, Mueller DL, et al. Gene regulation and chromatin remodeling by IL-12 and type I IFN in programming for CD8 T cell effector function and memory. J Immunol. 2009 Aug 1;183(3):1695–704. [PMC free article] [PubMed]
25. Filatenkov AA, Jacovetty EL, Fischer UB, Curtsinger JM, Mescher MF, Ingulli E. CD4 T cell-dependent conditioning of dendritic cells to produce IL-12 results in CD8-mediated graft rejection and avoidance of tolerance. J Immunol. 2005 Jun 1;174(11):6909–17. [PubMed]
26. Keppler SJ, Aichele P. Signal 3 requirement for memory CD8+ T-cell activation is determined by the infectious pathogen. Eur J Immunol. 2011 Nov;41(11):3176–86. [PubMed]
27. Keppler SJ, Theil K, Vucikuja S, Aichele P. Effector T-cell differentiation during viral and bacterial infections: Role of direct IL-12 signals for cell fate decision of CD8(+) T cells. Eur J Immunol. 2009 Jul;39(7):1774–83. [PubMed]
28. Kolumam GA, Thomas S, Thompson LJ, Sprent J, Murali-Krishna K. Type I interferons act directly on CD8 T cells to allow clonal expansion and memory formation in response to viral infection. J Exp Med. 2005 Sep 5;202(5):637–50. [PMC free article] [PubMed]
29. Mescher MF, Curtsinger JM, Agarwal P, Casey KA, Gerner M, Hammerbeck CD, et al. Signals required for programming effector and memory development by CD8+ T cells. Immunol Rev. 2006 Jun;211:81–92. [PubMed]
30. Pearce EL, Shen H. Generation of CD8 T cell memory is regulated by IL-12. J Immunol. 2007 Aug 15;179(4):2074–81. [PubMed]
31. Zhu J, Yamane H, Paul WE. Differentiation of effector CD4 T cell populations (*) Annu Rev Immunol. 2010;28:445–89. [PMC free article] [PubMed]
32. Kaech SM, Wherry EJ. Heterogeneity and cell-fate decisions in effector and memory CD8+ T cell differentiation during viral infection. Immunity. 2007 Sep;27(3):393–405. [PMC free article] [PubMed]
33. Cui W, Liu Y, Weinstein JS, Craft J, Kaech SM. An interleukin-21-interleukin-10-STAT3 pathway is critical for functional maturation of memory CD8+ T cells. Immunity. 2011 Nov 23;35(5):792–805. [PMC free article] [PubMed]
34. Siegel AM, Heimall J, Freeman AF, Hsu AP, Brittain E, Brenchley JM, et al. A critical role for STAT3 transcription factor signaling in the development and maintenance of human T cell memory. Immunity. 2011 Nov 23;35(5):806–18. [PMC free article] [PubMed]
35. Li Q, Rao RR, Araki K, Pollizzi K, Odunsi K, Powell JD, et al. A central role for mTOR kinase in homeostatic proliferation induced CD8+ T cell memory and tumor immunity. Immunity. 2011 Apr 22;34(4):541–53. [PMC free article] [PubMed]
36. Rao RR, Li Q, Odunsi K, Shrikant PA. The mTOR kinase determines effector versus memory CD8+ T cell fate by regulating the expression of transcription factors T-bet and Eomesodermin. Immunity. 2010 Jan 29;32(1):67–78. [PubMed]
37. Watts TH. Tnf/Tnfr family members in costimulation of T cell responses. Annu Rev Immunol. 2005;23:23–68. [PubMed]
38. Ahonen CL, Doxsee CL, McGurran SM, Riter TR, Wade WF, Barth RJ, et al. Combined TLR and CD40 triggering induces potent CD8+ T cell expansion with variable dependence on type I IFN. J Exp Med. 2004 Mar 15;199(6):775–84. [PMC free article] [PubMed]
39. Sanchez PJ, McWilliams JA, Haluszczak C, Yagita H, Kedl RM. Combined TLR/CD40 stimulation mediates potent cellular immunity by regulating dendritic cell expression of CD70 in vivo. J Immunol. 2007 Feb 1;178(3):1564–72. [PubMed]
40. Sanchez PJ, Kedl RM. An alternative signal 3: CD8(+) T cell memory independent of IL-12 and type I IFN is dependent on CD27/OX40 signaling. Vaccine. 2011 Feb 1;30(6):1154–61. [PMC free article] [PubMed]
41. Kurche JS, Burchill MA, Sanchez PJ, Haluszczak C, Kedl RM. Comparison of OX40 ligand and CD70 in the promotion of CD4+ T cell responses. J Immunol. 2010 Aug 15;185(4):2106–15. [PMC free article] [PubMed]
42. DeBenedette MA, Shahinian A, Mak TW, Watts TH. Costimulation of CD28− T lymphocytes by 4-1BB ligand. J Immunol. 1997 Jan 15;158(2):551–9. [PubMed]
43. Shuford WW, Klussman K, Tritchler DD, Loo DT, Chalupny J, Siadak AW, et al. 4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses. J Exp Med. 1997 Jul 7;186(1):47–55. [PMC free article] [PubMed]
44. Takahashi C, Mittler RS, Vella AT. Cutting edge: 4-1BB is a bona fide CD8 T cell survival signal. J Immunol. 1999 May 1;162(9):5037–40. [PubMed]
45. Vaitaitis GM, Wagner DH., Jr High distribution of CD40 and TRAF2 in Th40 T cell rafts leads to preferential survival of this auto-aggressive population in autoimmunity. PloS one. 2008;3(4):e2076. [PMC free article] [PubMed]
46. van Gisbergen KP, Klarenbeek PL, Kragten NA, Unger PP, Nieuwenhuis MB, Wensveen FM, et al. The costimulatory molecule CD27 maintains clonally diverse CD8(+) T cell responses of low antigen affinity to protect against viral variants. Immunity. 2011 Jul 22;35(1):97–108. [PubMed]
47. Steinman RM, Adams JC, Cohn ZA. Identification of a novel cell type in peripheral lymphoid organs of mice. IV. Identification and distribution in mouse spleen. J Exp Med. 1975 Apr 1;141(4):804–20. [PMC free article] [PubMed]
48. Steinman RM, Cohn ZA. Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution. J Exp Med. 1973 May 1;137(5):1142–62. [PMC free article] [PubMed]
49. Steinman RM, Cohn ZA. Identification of a novel cell type in peripheral lymphoid organs of mice. II. Functional properties in vitro. J Exp Med. 1974 Feb 1;139(2):380–97. [PMC free article] [PubMed]
50. Steinman RM. Decisions about dendritic cells: past, present, and future. Annu Rev Immunol. 2011 Apr 23;30:1–22. [PubMed]
51. Nussenzweig MC, Steinman RM, Gutchinov B, Cohn ZA. Dendritic cells are accessory cells for the development of anti-trinitrophenyl cytotoxic T lymphocytes. J Exp Med. 1980 Oct 1;152(4):1070–84. [PMC free article] [PubMed]
52. Ballesteros-Tato A, Leon B, Lund FE, Randall TD. Temporal changes in dendritic cell subsets, cross-priming and costimulation via CD70 control CD8(+) T cell responses to influenza. Nat Immunol. 2010 Mar;11(3):216–24. [PMC free article] [PubMed]
53. den Haan JMM, Lehar SM, Bevan MJ. CD8+ but Not CD8− Dendritic Cells Cross-prime Cytotoxic T Cells In Vivo. J Exp Med. 2000 Dec 11;192(12):1685–96. [PMC free article] [PubMed]
54. Desch AN, Randolph GJ, Murphy K, Gautier EL, Kedl RM, Lahoud MH, et al. CD103+ pulmonary dendritic cells preferentially acquire and present apoptotic cell-associated antigen. J Exp Med. 2011 Aug 29;208(9):1789–97. [PMC free article] [PubMed]
55. Dudziak D, Kamphorst AO, Heidkamp GF, Buchholz VR, Trumpfheller C, Yamazaki S, et al. Differential antigen processing by dendritic cell subsets in vivo. Science. 2007 Jan 5;315(5808):107–11. [PubMed]
56. Hildner K, Edelson BT, Purtha WE, Diamond M, Matsushita H, Kohyama M, et al. Batf3 deficiency reveals a critical role for CD8alpha+ dendritic cells in cytotoxic T cell immunity. Science. 2008 Nov 14;322(5904):1097–100. [PMC free article] [PubMed]
57. Jongbloed SL, Kassianos AJ, McDonald KJ, Clark GJ, Ju X, Angel CE, et al. Human CD141+ (BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens. J Exp Med. 2010 Jun 7;207(6):1247–60. [PMC free article] [PubMed]
58. Soares H, Waechter H, Glaichenhaus N, Mougneau E, Yagita H, Mizenina O, et al. A subset of dendritic cells induces CD4+ T cells to produce IFN-gamma by an IL-12-independent but CD70-dependent mechanism in vivo. J Exp Med. 2007 May 14;204(5):1095–106. [PMC free article] [PubMed]
59. Oh JZ, Kurche JS, Burchill MA, Kedl RM. TLR7 enables cross-presentation by multiple dendritic cell subsets through a Type I IFN-dependent pathway. Blood. 2011 in press. [PubMed]
60. Allan RS, Smith CM, Belz GT, van Lint AL, Wakim LM, Heath WR, et al. Epidermal viral immunity induced by CD8alpha+ dendritic cells but not by Langerhans cells. Science. 2003 Sep 26;301(5641):1925–8. [PubMed]
61. Allan RS, Waithman J, Bedoui S, Jones CM, Villadangos JA, Zhan Y, et al. Migratory dendritic cells transfer antigen to a lymph node-resident dendritic cell population for efficient CTL priming. Immunity. 2006 Jul;25(1):153–62. [PubMed]
62. Bedoui S, Whitney PG, Waithman J, Eidsmo L, Wakim L, Caminschi I, et al. Cross-presentation of viral and self antigens by skin-derived CD103+ dendritic cells. Nat Immunol. 2009 May;10(5):488–95. [PubMed]
63. Hao X, Kim TS, Braciale TJ. Differential response of respiratory dendritic cell subsets to influenza virus infection. J Virol. 2008 May;82(10):4908–19. [PMC free article] [PubMed]
64. Ho AW, Prabhu N, Betts RJ, Ge MQ, Dai X, Hutchinson PE, et al. Lung CD103+ dendritic cells efficiently transport influenza virus to the lymph node and load viral antigen onto MHC class I for presentation to CD8 T cells. J Immunol. 2011 Dec 1;187(11):6011–21. [PubMed]
65. Kim TS, Braciale TJ. Respiratory dendritic cell subsets differ in their capacity to support the induction of virus-specific cytotoxic CD8+ T cell responses. PloS one. 2009;4(1):e4204. [PMC free article] [PubMed]
66. Smed-Sorensen A, Chalouni C, Chatterjee B, Cohn L, Blattmann P, Nakamura N, et al. Influenza a virus infection of human primary dendritic cells impairs their ability to cross-present antigen to CD8 T cells. PLoS pathogens. 2011 Mar;8(3):e1002572. [PMC free article] [PubMed]
67. Akiba H, Oshima H, Takeda K, Atsuta M, Nakano H, Nakajima A, et al. CD28-independent costimulation of T cells by OX40 ligand and CD70 on activated B cells. J Immunol. 1999 Jun 15;162(12):7058–66. [PubMed]
68. Arens R, Schepers K, Nolte MA, van Oosterwijk MF, van Lier RA, Schumacher TN, et al. Tumor rejection induced by CD70-mediated quantitative and qualitative effects on effector CD8+ T cell formation. J Exp Med. 2004 Jun 7;199(11):1595–605. [PMC free article] [PubMed]
69. Borst J, Hendriks J, Xiao Y. CD27 and CD70 in T cell and B cell activation. Curr Opin Immunol. 2005 Jun;17(3):275–81. [PubMed]
70. Bullock TN, Yagita H. Induction of CD70 on dendritic cells through CD40 or TLR stimulation contributes to the development of CD8+ T cell responses in the absence of CD4+ T cells. J Immunol. 2005 Jan 15;174(2):710–7. [PubMed]
71. Croft M. The role of TNF superfamily members in T-cell function and diseases. Nat Rev Immunol. 2009 Apr;9(4):271–85. [PMC free article] [PubMed]
72. Laouar A, Haridas V, Vargas D, Zhinan X, Chaplin D, van Lier RA, et al. CD70+ antigen-presenting cells control the proliferation and differentiation of T cells in the intestinal mucosa. Nat Immunol. 2005 Jul;6(7):698–706. [PMC free article] [PubMed]
73. McWilliams JA, Sanchez PJ, Haluszczak C, Gapin L, Kedl RM. Multiple innate signaling pathways cooperate with CD40 to induce potent, CD70-dependent cellular immunity. Vaccine. 2010 Feb 10;28(6):1468–76. [PMC free article] [PubMed]
74. Rowley TF, Al-Shamkhani A. Stimulation by soluble CD70 promotes strong primary and secondary CD8+ cytotoxic T cell responses in vivo. J Immunol. 2004 May 15;172(10):6039–46. [PubMed]
75. Taraban VY, Rowley TF, Al-Shamkhani A. Cutting edge: a critical role for CD70 in CD8 T cell priming by CD40-licensed APCs. J Immunol. 2004 Dec 1;173(11):6542–6. [PubMed]
76. Taraban VY, Rowley TF, Tough DF, Al-Shamkhani A. Requirement for CD70 in CD4+ Th Cell-Dependent and Innate Receptor-Mediated CD8+ T Cell Priming. J Immunol. 2006 Sep 1;177(5):2969–75. [PubMed]
77. Dong H, Franklin NA, Roberts DJ, Yagita H, Glennie MJ, Bullock TN. CD27 Stimulation Promotes the Frequency of IL-7 Receptor-Expressing Memory Precursors and Prevents IL-12-Mediated Loss of CD8+ T Cell Memory in the Absence of CD4+ T Cell Help. J Immunol. Mar 14; [PMC free article] [PubMed]
78. Penaloza-MacMaster P, Ur Rasheed A, Iyer SS, Yagita H, Blazar BR, Ahmed R. Opposing effects of CD70 costimulation during acute and chronic lymphocytic choriomeningitis virus infection of mice. J Virol. 2011 Jul;85(13):6168–74. [PMC free article] [PubMed]
79. Beishuizen CR, Kragten NA, Boon L, Nolte MA, van Lier RA, van Gisbergen KP. Chronic CD70-driven costimulation impairs IgG responses by instructing T cells to inhibit germinal center B cell formation through FasL-Fas interactions. J Immunol. 2009 Nov 15;183(10):6442–51. [PubMed]
80. Nolte MA, van Olffen RW, van Gisbergen KP, van Lier RA. Timing and tuning of CD27-CD70 interactions: the impact of signal strength in setting the balance between adaptive responses and immunopathology. Immunol Rev. 2009 May;229(1):216–31. [PubMed]
81. Wensveen FM, Unger PP, Kragten NA, Derks IA, ten Brinke A, Arens R, et al. CD70-driven costimulation induces survival or Fas-mediated apoptosis of T cells depending on antigenic load. J Immunol. 2012 May 1;188(9):4256–67. [PubMed]
82. Arens R, Tesselaar K, Baars PA, van Schijndel GM, Hendriks J, Pals ST, et al. Constitutive CD27/CD70 interaction induces expansion of effector-type T cells and results in IFNgamma-mediated B cell depletion. Immunity. 2001 Nov;15(5):801–12. [PubMed]
83. Badovinac VP, Haring JS, Harty JT. Initial T cell receptor transgenic cell precursor frequency dictates critical aspects of the CD8(+) T cell response to infection. Immunity. 2007 Jun;26(6):827–41. [PMC free article] [PubMed]
84. Badovinac VP, Porter BB, Harty JT. Programmed contraction of CD8(+) T cells after infection. Nat Immunol. 2002 Jul;3(7):619–26. [PubMed]
85. Hamilton SE, Harty JT. Quantitation of CD8+ T cell expansion, memory, and protective immunity after immunization with peptide-coated dendritic cells. J Immunol. 2002 Nov 1;169(9):4936–44. [PubMed]
86. Kaech SM, Ahmed R. Memory CD8+ T cell differentiation: initial antigen encounter triggers a developmental program in naive cells. Nat Immunol. 2001 May;2(5):415–22. [PMC free article] [PubMed]
87. Kedl RM, Rees WA, Hildeman DA, Schaefer B, Mitchell T, Kappler J, et al. T Cells Compete for Access to Antigen-bearing Antigen-presenting Cells. J Exp Med. 2000 Oct 9;192(8):1105–14. [PMC free article] [PubMed]
88. Defrance T, Casamayor-Palleja M, Krammer PH. The life and death of a B cell. Advances in cancer research. 2002;86:195–225. [PubMed]
89. Diener E, Feldmann M. Mechanisms at the cellular level during induction of high zone tolerance in vitro. Cell Immunol. 1972 Sep;5(1):130–6. [PubMed]
90. Swinton J, Schweitzer AN, Anderson RM. Two signal activation as an explanation of high zone tolerance: a mathematical exploration of the nature of the second signal. Journal of theoretical biology. 1994 Jul 7;169(1):23–30. [PubMed]
91. Lamb JR, Skidmore BJ, Green N, Chiller JM, Feldmann M. Induction of tolerance in influenza virus-immune T lymphocyte clones with synthetic peptides of influenza hemagglutinin. J Exp Med. 1983 May 1;157(5):1434–47. [PMC free article] [PubMed]
92. Matis LA, Glimcher LH, Paul WE, Schwartz RH. Magnitude of response of histocompatibility-restricted T-cell clones is a function of the product of the concentrations of antigen and Ia molecules. Proc Natl Acad Sci U S A. 1983 Oct;80(19):6019–23. [PubMed]
93. Suzuki G, Kawase Y, Koyasu S, Yahara I, Kobayashi Y, Schwartz RH. Antigen-induced suppression of the proliferative response of T cell clones. J Immunol. 1988 Mar 1;140(5):1359–65. [PubMed]
94. Kadowaki N, Ho S, Antonenko S, de Waal Malefyt R, Kastelein RA, Bazan F, et al. Subsets of Human Dendritic Cell Precursors Express Different Toll-like Receptors and Respond to Different Microbial Antigens. J Exp Med. 2001 Sep 17;194(6):863–70. [PMC free article] [PubMed]
95. Mercado R, Vijh S, Allen SE, Kerksiek K, Pilip IM, Pamer EG. Early programming of T cell populations responding to bacterial infection. J Immunol. 2000 Dec 15;165(12):6833–9. [PubMed]
96. van Stipdonk MJ, Lemmens EE, Schoenberger SP. Naive CTLs require a single brief period of antigenic stimulation for clonal expansion and differentiation. Nat Immunol. 2001 May;2(5):423–9. [PubMed]
97. Corbin GA, Harty JT. Duration of infection and antigen display have minimal influence on the kinetics of the CD4+ T cell response to Listeria monocytogenes infection. J Immunol. 2004 Nov 1;173(9):5679–87. [PubMed]
98. Gerlach C, van Heijst JW, Schumacher TN. The descent of memory T cells. Ann N Y Acad Sci. 2011 Jan;1217:139–53. [PubMed]
99. van Heijst JW, Gerlach C, Swart E, Sie D, Nunes-Alves C, Kerkhoven RM, et al. Recruitment of antigen-specific CD8+ T cells in response to infection is markedly efficient. Science. 2009 Sep 4;325(5945):1265–9. [PubMed]
100. Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human studies revisited. Faseb J. 2008 Mar;22(3):659–61. [PubMed]
101. Rubner M. Ueber den einfluss der korpergrosse auf stoff- und kraftwechsel. Z Biol. 1883;19:535–62.
102. Curtsinger J, Valenzuela J, Agarwal P, Lins D, Mescher M. Cutting Edge: Type I IFNs provide a third signal to CD8 T cells to stimulate clonal expansion and differentition. Journal of Immunology. 2005;174:4465–9. [PubMed]
103. Curtsinger JM, Valenzuela JO, Agarwal P, Lins D, Mescher MF. Type I IFNs provide a third signal to CD8 T cells to stimulate clonal expansion and differentiation. J Immunol. 2005 Apr 15;174(8):4465–9. [PubMed]
104. Valenzuela J, Schmidt C, Mescher M. The roles of IL-12 in providing a third signal for clonal expansion of naive CD8 T cells. J Immunol. 2002 Dec 15;169(12):6842–9. [PubMed]
105. Araki K, Youngblood B, Ahmed R. The role of mTOR in memory CD8 T-cell differentiation. Immunol Rev. 2011 May;235(1):234–43. [PMC free article] [PubMed]
106. Joshi NS, Cui W, Chandele A, Lee HK, Urso DR, Hagman J, et al. Inflammation directs memory precursor and short-lived effector CD8(+) T cell fates via the graded expression of T-bet transcription factor. Immunity. 2007 Aug;27(2):281–95. [PMC free article] [PubMed]
107. Glenny AT. The Principles of Immunity applied to Protective Inoculation against Diphtheria. The Journal of hygiene. 1925 Dec;24(3–4):301–20. [PMC free article] [PubMed]
108. Glenny AT. Insoluble Precipitates in Diphtheria and Tetanus Immunization. British medical journal. 1930 Aug 16;2(3632):244–5. [PMC free article] [PubMed]
109. Glenny AT, Barr M. The precipitation of diphtheria toxoid by potash alum. The Journal of Pathology and Bacteriology. 1931;34(2):131–8.
110. Glenny AT, Sudmersen HJ. Notes on the Production of Immunity to Diphtheria Toxin. The Journal of hygiene. 1921 Oct;20(2):176–220. [PMC free article] [PubMed]
111. White RG, Coons AH, Connolly JM. Studies on antibody production. III. The alum granuloma. J Exp Med. 1955 Jul 1;102(1):73–82. [PMC free article] [PubMed]
112. Holt LB. Quantitative studies in diphtheria prophylaxis; the primary response. British journal of experimental pathology. 1949 Aug;30(4):289–97. pl. [PubMed]
113. Holt LB. Quantitative studies in diphtheria prophylaxis: the second response. British journal of experimental pathology. 1950 Apr;31(2):233–41. [PubMed]
114. Rosenberg SA, Yang JC, Kammula US, Hughes MS, Restifo NP, Schwarz SL, et al. Different adjuvanticity of incomplete freund’s adjuvant derived from beef or vegetable components in melanoma patients immunized with a peptide vaccine. J Immunother. 2010 Jul-Aug;33(6):626–9. [PMC free article] [PubMed]
115. Rosenberg SA, Yang JC, Restifo NP. Cancer immunotherapy: moving beyond current vaccines. Nat Med. 2004 Sep;10(9):909–15. [PMC free article] [PubMed]
116. Speiser DE, Schwarz K, Baumgaertner P, Manolova V, Devevre E, Sterry W, et al. Memory and effector CD8 T-cell responses after nanoparticle vaccination of melanoma patients. J Immunother. 2010 Oct;33(8):848–58. [PubMed]
117. Assudani D, Cho HI, DeVito N, Bradley N, Celis E. In vivo expansion, persistence, and function of peptide vaccine-induced CD8 T cells occur independently of CD4 T cells. Cancer Res. 2008 Dec 1;68(23):9892–9. [PMC free article] [PubMed]
118. Reinhardt RL, Bullard DC, Weaver CT, Jenkins MK. Preferential accumulation of antigen-specific effector CD4 T cells at an antigen injection site involves CD62E-dependent migration but not local proliferation. J Exp Med. 2003 Mar 17;197(6):751–62. [PMC free article] [PubMed]
119. Jelley-Gibbs DM, Brown DM, Dibble JP, Haynes L, Eaton SM, Swain SL. Unexpected prolonged presentation of influenza antigens promotes CD4 T cell memory generation. J Exp Med. 2005 Sep 5;202(5):697–706. [PMC free article] [PubMed]
120. Kim TS, Hufford MM, Sun J, Fu YX, Braciale TJ. Antigen persistence and the control of local T cell memory by migrant respiratory dendritic cells after acute virus infection. J Exp Med. 2010 Jun 7;207(6):1161–72. [PMC free article] [PubMed]
121. Takamura S, Roberts AD, Jelley-Gibbs DM, Wittmer ST, Kohlmeier JE, Woodland DL. The route of priming influences the ability of respiratory virus-specific memory CD8+ T cells to be activated by residual antigen. J Exp Med. 2010 Jun 7;207(6):1153–60. [PMC free article] [PubMed]
122. Turner DL, Cauley LS, Khanna KM, Lefrancois L. Persistent antigen presentation after acute vesicular stomatitis virus infection. J Virol. 2007 Feb;81(4):2039–46. [PMC free article] [PubMed]
123. Zammit DJ, Turner DL, Klonowski KD, Lefrancois L, Cauley LS. Residual antigen presentation after influenza virus infection affects CD8 T cell activation and migration. Immunity. 2006 Apr;24(4):439–49. [PMC free article] [PubMed]
124. Mori I, Komatsu T, Takeuchi K, Nakakuki K, Sudo M, Kimura Y. Parainfluenza virus type 1 infects olfactory neurons and establishes long-term persistence in the nerve tissue. The Journal of general virology. 1995 May;76( Pt 5):1251–4. [PubMed]
125. Schwarze J, O’Donnell DR, Rohwedder A, Openshaw PJ. Latency and persistence of respiratory syncytial virus despite T cell immunity. American journal of respiratory and critical care medicine. 2004 Apr 1;169(7):801–5. [PubMed]
126. Kaufmann DE, Walker BD. Programmed death-1 as a factor in immune exhaustion and activation in HIV infection. Current opinion in HIV and AIDS. 2008 May;3(3):362–7. [PubMed]
127. Moskophidis D, Lechner F, Pircher H, Zinkernagel RM. Virus persistence in acutely infected immunocompetent mice by exhaustion of antiviral cytotoxic effector T cells. Nature. 1993 Apr 22;362(6422):758–61. [PubMed]
128. Nolz JC, Harty JT. Protective capacity of memory CD8+ T cells is dictated by antigen exposure history and nature of the infection. Immunity. 2011 May 27;34(5):781–93. [PMC free article] [PubMed]
129. Recher M, Lang KS, Navarini A, Hunziker L, Lang PA, Fink K, et al. Extralymphatic virus sanctuaries as a consequence of potent T-cell activation. Nat Med. 2007 Nov;13(11):1316–23. [PubMed]
130. Shin H, Blackburn SD, Intlekofer AM, Kao C, Angelosanto JM, Reiner SL, et al. A role for the transcriptional repressor Blimp-1 in CD8(+) T cell exhaustion during chronic viral infection. Immunity. 2009 Aug 21;31(2):309–20. [PMC free article] [PubMed]
131. Shin H, Wherry EJ. CD8 T cell dysfunction during chronic viral infection. Curr Opin Immunol. 2007 Aug;19(4):408–15. [PubMed]
132. Fauci AS, Morens DM. The perpetual challenge of infectious diseases. The New England journal of medicine. Feb 2;366(5):454–61. [PubMed]
133. Maurice JM, Davey S. World Health Organization., UNICEF., World Bank., ebrary Inc. State of the world’s vaccines and immunization. 3. xxxiii. Geneva: World Health Organization; 2009. p. 169.
134. Andre FE. Vaccinology: past achievements, present roadblocks and future promises. Vaccine. 2003 Jan 30;21(7–8):593–5. [PubMed]
135. Amanna IJ, Carlson NE, Slifka MK. Duration of humoral immunity to common viral and vaccine antigens. The New England journal of medicine. 2007 Nov 8;357(19):1903–15. [PubMed]
136. Amanna IJ, Slifka MK, Crotty S. Immunity and immunological memory following smallpox vaccination. Immunol Rev. 2006 Jun;211:320–37. [PubMed]
137. Crotty S, Felgner P, Davies H, Glidewell J, Villarreal L, Ahmed R. Cutting edge: long-term B cell memory in humans after smallpox vaccination. J Immunol. 2003 Nov 15;171(10):4969–73. [PubMed]
138. Hammarlund E, Lewis MW, Hansen SG, Strelow LI, Nelson JA, Sexton GJ, et al. Duration of antiviral immunity after smallpox vaccination. Nat Med. 2003 Sep;9(9):1131–7. [PubMed]
139. Suntharalingam G, Perry MR, Ward S, Brett SJ, Castello-Cortes A, Brunner MD, et al. Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. The New England journal of medicine. 2006 Sep 7;355(10):1018–28. [PubMed]
140. Intlekofer AM, Banerjee A, Takemoto N, Gordon SM, Dejong CS, Shin H, et al. Anomalous type 17 response to viral infection by CD8+ T cells lacking T-bet and eomesodermin. Science. 2008 Jul 18;321(5887):408–11. [PMC free article] [PubMed]
141. Parish IA, Kaech SM. Diversity in CD8(+) T cell differentiation. Curr Opin Immunol. 2009 Jun;21(3):291–7. [PubMed]
142. Sullivan BM, Juedes A, Szabo SJ, von Herrath M, Glimcher LH. Antigen-driven effector CD8 T cell function regulated by T-bet. Proc Natl Acad Sci U S A. 2003 Dec 23;100(26):15818–23. [PubMed]
143. Takemoto N, Intlekofer AM, Northrup JT, Wherry EJ, Reiner SL. Cutting Edge: IL-12 inversely regulates T-bet and eomesodermin expression during pathogen-induced CD8+ T cell differentiation. J Immunol. 2006 Dec 1;177(11):7515–9. [PubMed]
144. Wherry EJ, Teichgraber V, Becker TC, Masopust D, Kaech SM, Antia R, et al. Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat Immunol. 2003 Mar;4(3):225–34. [PubMed]
145. Ely KH, Cookenham T, Roberts AD, Woodland DL. Memory T cell populations in the lung airways are maintained by continual recruitment. J Immunol. 2006 Jan 1;176(1):537–43. [PubMed]
146. Hikono H, Kohlmeier JE, Takamura S, Wittmer ST, Roberts AD, Woodland DL. Activation phenotype, rather than central- or effector-memory phenotype, predicts the recall efficacy of memory CD8+ T cells. J Exp Med. 2007 Jul 9;204(7):1625–36. [PMC free article] [PubMed]
147. Belz GT, Bedoui S, Kupresanin F, Carbone FR, Heath WR. Minimal activation of memory CD8+ T cell by tissue-derived dendritic cells favors the stimulation of naive CD8+ T cells. Nat Immunol. 2007 Oct;8(10):1060–6. [PubMed]
148. Gebhardt T, Mueller SN, Heath WR, Carbone FR. Peripheral tissue surveillance and residency by memory T cells. Trends Immunol. 2012 Oct 1; [PubMed]
149. Mackay LK, Stock AT, Ma JZ, Jones CM, Kent SJ, Mueller SN, et al. Long-lived epithelial immunity by tissue-resident memory T (TRM) cells in the absence of persisting local antigen presentation. Proc Natl Acad Sci U S A. 2012 May 1;109(18):7037–42. [PubMed]
150. Shin H, Iwasaki A. A vaccine strategy that protects against genital herpes by establishing local memory T cells. Nature. 2012 Oct 17; [PMC free article] [PubMed]
151. Marzo AL, Klonowski KD, Le Bon A, Borrow P, Tough DF, Lefrancois L. Initial T cell frequency dictates memory CD8+ T cell lineage commitment. Nat Immunol. 2005 Aug;6(8):793–9. [PMC free article] [PubMed]
152. Hamann D, Baars PA, Rep MH, Hooibrink B, Kerkhof-Garde SR, Klein MR, et al. Phenotypic and functional separation of memory and effector human CD8+ T cells. J Exp Med. 1997 Nov 3;186(9):1407–18. [PMC free article] [PubMed]
153. Masopust D, Vezys V, Marzo AL, Lefrancois L. Preferential localization of effector memory cells in nonlymphoid tissue. Science. 2001 Mar 23;291(5512):2413–7. [PubMed]
154. Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature. 1999 Oct 14;401(6754):708–12. [PubMed]
155. Marzo AL, Yagita H, Lefrancois L. Cutting edge: migration to nonlymphoid tissues results in functional conversion of central to effector memory CD8 T cells. J Immunol. 2007 Jul 1;179(1):36–40. [PMC free article] [PubMed]
156. Pepper M, Jenkins MK. Origins of CD4(+) effector and central memory T cells. Nat Immunol. 2011 Jun;12(6):467–71. [PubMed]
157. Pepper M, Linehan JL, Pagan AJ, Zell T, Dileepan T, Cleary PP, et al. Different routes of bacterial infection induce long-lived TH1 memory cells and short-lived TH17 cells. Nat Immunol. 2010 Jan;11(1):83–9. [PMC free article] [PubMed]
158. Pepper M, Pagan AJ, Igyarto BZ, Taylor JJ, Jenkins MK. Opposing signals from the Bcl6 transcription factor and the interleukin-2 receptor generate T helper 1 central and effector memory cells. Immunity. 2011 Oct 28;35(4):583–95. [PMC free article] [PubMed]
159. Garcia-Hernandez Mde L, Hamada H, Reome JB, Misra SK, Tighe MP, Dutton RW. Adoptive transfer of tumor-specific Tc17 effector T cells controls the growth of B16 melanoma in mice. J Immunol. Apr 15;184(8):4215–27. [PMC free article] [PubMed]
160. Hinrichs CS, Kaiser A, Paulos CM, Cassard L, Sanchez-Perez L, Heemskerk B, et al. Type 17 CD8+ T cells display enhanced antitumor immunity. Blood. 2009 Jul 16;114(3):596–9. [PubMed]
161. Reiner SL, Sallusto F, Lanzavecchia A. Division of labor with a workforce of one: challenges in specifying effector and memory T cell fate. Science. 2007 Aug 3;317(5838):622–5. [PubMed]
162. Baitsch L, Baumgaertner P, Devevre E, Raghav SK, Legat A, Barba L, et al. Exhaustion of tumor-specific CD8(+) T cells in metastases from melanoma patients. J Clin Invest. 2011 Jun;121(6):2350–60. [PMC free article] [PubMed]
163. Rehermann B. Chronic infections with hepatotropic viruses: mechanisms of impairment of cellular immune responses. Seminars in liver disease. 2007 May;27(2):152–60. [PubMed]
164. Wherry EJ. T cell exhaustion. Nat Immunol. 2011 Jun;12(6):492–9. [PubMed]
165. Youngblood B, Wherry EJ, Ahmed R. Acquired transcriptional programming in functional and exhausted virus-specific CD8 T cells. Current opinion in HIV and AIDS. 2012 Jan;7(1):50–7. [PMC free article] [PubMed]
166. Miller JD, van der Most RG, Akondy RS, Glidewell JT, Albott S, Masopust D, et al. Human effector and memory CD8+ T cell responses to smallpox and yellow fever vaccines. Immunity. 2008 May;28(5):710–22. [PubMed]
167. Querec TD, Akondy RS, Lee EK, Cao W, Nakaya HI, Teuwen D, et al. Systems biology approach predicts immunogenicity of the yellow fever vaccine in humans. Nat Immunol. 2009 Jan;10(1):116–25. [PubMed]
168. Welters MJ, Kenter GG, de Vos van Steenwijk PJ, Lowik MJ, Berends-van der Meer DM, Essahsah F, et al. Success or failure of vaccination for HPV16-positive vulvar lesions correlates with kinetics and phenotype of induced T-cell responses. Proc Natl Acad Sci U S A. 2010 Jun 29;107(26):11895–9. [PubMed]