impact of sepsis on immunity

Impact of sepsis on CD4 T cell immunityJavier Cabrera-Perez,*, Stephanie A. Condotta, Vladimir P. Badovinac,,

and Thomas S. Griffith*,,,#,1

*Microbiology, Immunology, and Cancer Biology Graduate Program, Medical Scientist Training Program, Center forImmunology, and Department of Urology, University of Minnesota Medical School, Minneapolis, Minnesota, USA;

#Minneapolis Veterans Administration Health Care System, Minneapolis, Minnesota, USA; and Department of Pathologyand Interdisciplinary Program in Immunology, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA

RECEIVED JANUARY 31, 2014; REVISED MARCH 8, 2014; ACCEPTED MARCH 19, 2014. DOI: 10.1189/jlb.5MR0114-067R

ABSTRACTSepsis remains the primary cause of death from infec-tion in hospital patients, despite improvements in antibi-otics and intensive-care practices. Patients who sur-vive severe sepsis can display suppressed immunefunction, often manifested as an increased susceptibil-ity to (and mortality from) nosocomial infections. Notonly is there a significant reduction in the number ofvarious immune cell populations during sepsis, butthere is also decreased function in the remaining lym-phocytes. Within the immune system, CD4 T cells areimportant players in the proper development of nu-merous cellular and humoral immune responses. De-spite sufficient clinical evidence of CD4 T cell loss inseptic patients of all ages, the impact of sepsis onCD4 T cell responses is not well understood. Recentfindings suggest that CD4 T cell impairment is a multi-pronged problem that results from initial sepsis-in-duced cell loss. However, the subsequent lymphope-nia-induced numerical recovery of the CD4 T cellcompartment leads to intrinsic alterations in pheno-type and effector function, reduced repertoire diver-sity, changes in the composition of naive antigen-specific CD4 T cell pools, and changes in the repre-sentation of different CD4 T cell subpopulations (e.g.,increases in Treg frequency). This review focuses onsepsis-induced alterations within the CD4 T cell com-partment that influence the ability of the immune sys-tem to control secondary heterologous infections.The understanding of how sepsis affects CD4 T cellsthrough their numerical loss and recovery, as well asfunction, is important in the development of futuretreatments designed to restore CD4 T cells to theirpresepsis state. J. Leukoc. Biol. 96: 767777; 2014.

IntroductionHistorical accounts of sepsis help to explain why this syn-dromecurrently defined as a SIRS in the presence of a dis-seminated infectionremains a serious challenge to modernmedicine [1]. The term sepsis () is first found in rela-tion to disease in the writings of the Greek physician Hippo-crates (c. 460370 BC) as the reason behind the odiferousbiological decay of the body and a bad prognosis for thewound-healing process [2]. Galen (Roman gladiatorial sur-geon; 130200 AD) would misinterpret this notion 500 yearslater [3], claiming that sepsis was essentially a good omen ininfections (e.g., pus bonum et laudabile, or part of ahealthyand welcomed suppuration) [4]. Galens humoristic viewsabout the nature of sepsis became medical dogma for morethan 15 centuries, until the germ theory of infection gainedacceptance and shed light on the nature and propagation ofdisseminated infections [5]. To this day, sepsis remains apoorly understood disease process [6]. In spite of the techno-logical leaps in critical care, overall case mortality from septicevents is still high, ranging between 30% and 50% [7]. Septiccauses are responsible for 200,000 deaths/year in the UnitedStates [8], making it a leading cause of death in hospitals ofthe 21st century. The elderly are a patient population with ahigh incidence (accounting for nearly 60% of all septic cases)that is vulnerable to the consequences of sepsis [9], showing100-fold higher mortality rates than the general population[10]. Collectively, the burden of morbidity, mortality, reducedquality of life, and excessive cost of sepsis on the healthcaresystem ($1416 billion/year) [11] are clear indicators of howmuch of an unmet medical challenge this condition truly rep-resents [12].

Within the last 40 years, our collective knowledge regardingthe pathophysiology of sepsis has grown exponentially. Specifi-cally, it has become clear that sepsis is not just the symptomsof a complicated infection; instead, we now know that sepsis ismore like a bad immune response to a complicated infection[6]. In other words, sepsis represents the dysregulation of im-mune responses as a result of an invading pathogen and the

1. Correspondence: University of Minnesota, Dept. of Urology, MMC Mayo, 420Delaware St., SE, Minneapolis, MN 55455, USA. E-mail: [email protected]

Abbreviations: c chain, BTLAB- and T-lymphocyte attenuator,CARScompensatory anti-inflammatory response syndrome, CLPcecalligation and puncture, DTHdelayed-type hypersensitivity,FoxP3forkhead box P3, GITRglucocorticoid-induced TNFR-related pro-tein, HSVherpes simplex virus, LCMVlymphocytic choriomeningitis vi-rus, PD-1programmed cell death protein 1, PD-L1programmed celldeath protein 1 ligand, ROR-tretinoic acid receptor-related orphan re-ceptor , SIRSsystemic inflammatory response syndrome,TregCD4

CD25forkhead box P3 regulatory T cell

Review

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ensuing system-wide collateral damage. The crux of the sepsismystery resides in knowing the parts of the immune systemthat remain defective after sepsis and are ultimately detrimen-tal to patients. In this review, we will dissect how sepsis affectsthe recovery and maintenance of a diverse, functional T cellrepertoire, as well as to investigate potential therapies that im-prove survival and enhance function of T cells early and lateafter a septic event. The understanding of these areas is cru-cial for the development and translation of potential therapiesto restore immune system function in recovering sepsispatients.

SEPSIS-INDUCED IMMUNOPATHOLOGY

The birth of molecular immunology paved the way for the ear-liest interpretations of what happens to the immune systemduring/after a septic event. At first, the reproducible observa-tion of elevated inflammatory markers in the serum of pa-tients, coupled with the high mortality rates, led to the idea thatthe systemic invasion of pathogens was forcing our own bodies touse massive retaliation to regain homeostasis (Fig. 1A) [13], aphenomenon referred to as SIRS.

This theory of hyperinflammation has dictated the directionof basic and translational sepsis research for the last 30 years[14]. This is not surprising, given that SIRS is a key compo-nent of septic pathophysiology. SIRS represents the spillover ofelevated inflammatory mediators into the circulation [15] thatare released during the course of an immune response [16].These mediators locally promote cell death and leukocyte re-cruitment, as well as coagulation events that limit the systemicspread of infection and create an uninviting environment forthe offending pathogen [17]. When amplified systemically, thesame mediators cause localized edema and promote neutro-

phil infiltration that can lead to cardiovascular dysfunction[18], and factors causing local thrombosis can initiate dissemi-nated intravascular coagulation [11]. In accordance with thisexhuberant immune response model of sepsis, the vast ma-jority of therapeutics tested in the 1980s and 1990s was aimedat blocking proinflammatory responses [1922]. Unfortu-nately, strategies that dampen inflammation have been over-whelmingly ineffective in reducing mortality when tested inclinical settings [5, 23, 24].

Perhaps the most important contributor to re-evaluating theimmunopathophysiological mechanisms of sepsis was RogerBone [25], who in light of numerous therapeutic failures,noted several phenomena that were not consistent with thetraditional exuberant inflammation model of sepsis. Bonenoticed that most patients would survive the SIRS phase withadequate supportive care, and the cytokine storm wouldeventually subside, but mortality remained elevated long aftera septic episode resolved. There was also clear evidence of an-ti-inflammatory cytokines circulating during sepsis, includingIL-4, IL-10, TGF-, and CSFs [26]. In addition, he observedthat apoptotic cell death seemed to be present in sepsis in avariety of cell types, including lymphocytes. These largely ig-nored pieces of the sepsis puzzle led to Bones postulate that alarge population of patients surviving the early events of sepsiswould enter into an immunological state characterized by hy-poinflammation and immunosuppression, which he termed aCARS [25]. More recent evidence suggests that SIRS andCARS are interdependent and concurrent during the courseof sepsis [27] (Fig. 1B). Indeed, we now know that a sizeablenumber of patients surviving the early events of sepsis enterinto an immunological state characterized by T cell exhaus-tion, unresolved infection, and defective antigen presentation[28]. It is also becoming clear that apoptosis of lymphoid and

A Exhuberant Responsemodel of sepsis

B Mixed SIRS/CARSmodel of sepsis End-organ damage Hemodynamic instability

Immunosuppression Susceptibility to opportunistic infections

Sepsis

SIRS CARS

IL-4, IL-10,TGF-

IL-1, IL-6,TNF-

Figure 1. Evolving concepts in the etiologicalbasis for sepsis. The conceptual understand-ing of the pathophysiology of sepsis hasevolved over the past 40 years from a simple,linear model of exuberant inflammation toa complicated interplay between opposingfactions within the immune response. (A)The classic theory (and current consensusdefinition) of sepsis was popularized in the1970s and views sepsis as a linear conse-quence of uncontrolled inflammation causedby the innate immune system in response toan invading pathogen. The inflammatory re-sponse is here depicted as a dial or gradientthat encompasses immunological states rang-ing from homeostasis to sepsis. (B) Cur-rently, one of the more widely accepted theo-ries about sepsis is that it stems from the in-terplay between two opposite immunologicalpoles or forces (depicted here as scales). Sev-eral clues point to this as a reasonable alter-native to the classic model. First, clinical studies have found that undesirable concentrations of pro- and anti-inflammatory cytokines can be de-tected in the serum of septic patients. In addition, lymphocytes can be detected as undergoing apoptosis and proliferating simultaneously. Balancebetween immunological extremes varies from patient to patients and influences the outcome of the septic episode: some patients may experiencecardiovascular collapse and organ ischemia, whereas others might recover from hemodynamic instability but end up immunosuppressed and vul-nerable to secondary opportunistic infections.

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nonlymphoid tissue [29, 30] and suppression of lymphocyteresponses after the acute phase events [31] are of paramountimportance to the protracted course and infectious complica-tions often seen in septic patients [32].

APOPTOSIS AND LYMPHOCYTEIMMUNOSUPPRESSION IN SEPSIS

The focus of basic and clinical research regarding lymphocyteapoptosis in sepsis has grown considerably over the past 20years. The studies published have added credibility to the ideathat apoptosis and immune suppression are not only impor-tant players in the pathophysiology of sepsis but are also intri-cately intertwined. Work performed in the 1990s dramaticallyadvanced the understanding of apoptotic cell death throughthe identification of numerous cell death-inducing moleculesand their cognate receptors, as well as the molecular compo-nents of the cell death machinery. Incorporation of the widerange of cell death reagents and genetically modified miceinto the sepsis arena helped to define some of the proteinsimportant in sepsis-induced lymphocyte apoptosis and the im-portance of sepsis-induced apoptosis on the development ofthe subsequent immune suppression. General inhibition ofapoptosis, via overexpression of Bcl-2, increased survival aftersepsis induction [33]. In addition, a variety of caspases is acti-vated during sepsis-induced apoptosis, and the administrationof caspase inhibitors also improves survival [34, 35]. However,the molecular mechanism by which lymphocyte apoptosis oc-curs after sepsis has remained difficult to define, as no singleextrinsic or intrinsic pathway appears to be dominant [36].Interestingly, there are data to suggest that the TRAIL pathwayis important in the establishment of sepsis-induced immunesuppression [37, 38].

In support of the numerous animal-based studies examin-ing the relationship between sepsis-induced lymphocyte apo-ptosis and immune suppression, Hotchkiss and colleagues[39] showed that postmortem tissue samples from septic pa-tients had considerable amounts of apoptotic cell death(specifically, 56.3% of spleens, 47.1% of colons, and 27.7%of ileums sampled). Furthermore, tissue immunohistochem-istry revealed increased caspase-3 activity in septic versusnonseptic patients, with 2550% of cells positive in thesplenic white pulp of six septic but none of the nonsepticpatients, providing evidence that lymphocyte apoptosis wasincreased significantly in septic patients [40]. Several otherstudies have added credibility to the theory that lymphocyteapoptosis plays a role in the immune suppression character-istic of the late events in sepsis. Le Tulzo et al. [41] exam-ined freshly isolated lymphocytes of critically ill septic pa-tients and showed a higher degree of apoptosis in the ear-lier stages of septic shock, as well as delayed T cellreconstitution, compared with nonseptic individuals. Evi-dence for the expression of intracellular proapoptotic mole-cules has also been reported in human lymphocytes fromseptic patients. Weber and colleagues [42] analyzed mRNAexpression of several Bcl-2 family molecules in circulatinglymphocytes, comparing patients with severe sepsis withnonseptic critically ill patients. One interesting finding in

this study was the marked up-regulation of Bim, a proapop-totic molecule whose deletion is associated with completeprotection from apoptosis in animal sepsis in sepsis-derivedlymphocytes. This is a potentially insightful finding into thespecific pathways of apoptotic death that are dominant insepsis, given that Bim is the only component of the apopto-sis cascade whose deletion induces complete protectionfrom apoptotic cell death in septic mice [36].

IMPACT OF SEPSIS ON CD4 T CELLRESPONSES

CD4 Th cells are among the most important peripheral lym-phocyte subsets when it comes to the orchestration of success-ful immune responses, influencing innate and adaptive im-mune cells through cytokine production and cell-to-cell inter-action [43]. CD4 T cells are essential for effective primary CD8T cell responses [44, 45] and the formation of functional CD8T cell memory [4649], as well as efficient isotype switching inprimary and memory B cell responses [50, 51]. A defining fea-ture of antigen-specific CD4 T cells is that upon recognition ofantigenand depending on the cytokines and costimulatorymolecules presentsubsets of effector CD4 T cells take on aspecific phenotype best suited to drive a response against theperceived threat. These differentiation pathways enable theactivated and differentiated CD4 T cell to exert specific effec-tor functions, such as produce cytokines, activate other cells,and change immune cell migration patterns necessary to clearthe pathogen recognized.

Several polarities or effector differentiation pathways arewell-described within CD4 T cells (Fig. 2). For example, Th1cells are induced in response to viral, bacterial, and protozoanintracellular infections [52]. Classically, CD4 T cells from thissubset are induced by available IL-12 and IFN- in the inflam-matory milieu and produce cytokines, such as IL-2 and IFN-,which go on to activate intracellular killing mechanisms inmacrophages [53, 54]. In addition, Th1 cells provide necessarysignals for isotype switching in B cell responses (e.g., IgG2a inmice) [55]. In contrast, Th2 cells (activated in the presence ofIL-4) produce predominantly IL-4, IL-5, and IL-13 and are im-portant for the clearance of helminthic infections [56]. Th2cells also enhance B cell isotype switching (to IgG1 and IgE)via IL-4 secretion, as well as the alternative activation of macro-phages to promote tissue repair [57]. Lastly, Th17 cells, whichare effector CD4 T cells that can produce IL-17, IL-22, andTNF-, are important in immunity to extracellular fungal andbacterial pathogens (especially at mucosal surfaces) [58]through the recruitment and activation of neutrophils [59].Thus, the loss or improper function of CD4 T cell responses isdetrimental for immunity to a wide range of pathogens.

Whether CD4 T cells are involved directly in the early stagesof septic injury is debated. Several animal studies have shownCD4 T cells to mediate directly the host response to sepsis [60,61], whereas others have concluded that CD4 T cells have noimpact in the inflammatory response [62]. Regardless of theirdirect effect on the acute response to septic injury, several ob-servations point to CD4 T cells as a subset of leukocytes thatmight be important to consider when discussing sepsis-induced

Cabrera-Perez et al. Sepsis-related changes to CD4 T cell responses

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immunosuppression. These observations can be grouped intothree general categories: (1) altered effector CD4 T cell phe-notype and/or function, (2) altered peripheral CD4 T cell di-versity, and (3) altered Treg frequency and/or function.

Altered effector CD4 T cell phenotype and/orfunctionDuring initial TCR engagement, costimulatory signals lowerthe threshold for T cell activation [63]. A T cell receiving onlyantigen-specific TCR stimulation in the absence (or inhibition)of costimulation is rendered unresponsive or anergic to subse-quent challenges [64]. The inhibition of costimulation by theimmune system helps to attenuate T cell responses by way ofclonal anergy [65] and reduces responses by way of clonaldeletion [57, 66]. Similarly, T cell exhaustion, as seen in situ-ations of chronic viral infection [67] and cancer [68], as a re-sult of prolonged antigen exposure in the presence of low-grade inflammation, makes use of the same mechanisms toattenuate and reduce T cell responses. Modulation of the over-all strength of signal transmitted to a T cell by inflammationcan potentially give rise to an exhuberant immune response[69], and in such cases, the immune system attempts to regainhomeostasis through the same mechanisms that we have de-scribed [70]. Thus, it is now accepted that an important aspectof postseptic T cell dysfunction is a phenomenon similar toanergy or T cell exhaustion [71]. This state includes decreasesin cytokine production, epigenetic changes to T cell transcrip-tion factors, and the up-regulation of inhibitory cell surfaceproteins, such as TRAIL [37, 38], PD-1 [7274], and BTLA[75, 76].

The available empirical data supporting the idea of alteredeffector CD4 T cell function in critically ill sepsis patients dateback to studies in the 1970s and 1980s, showing impaired

DTH skin reactions [77]. Early studies using peripheral bloodshowed that cytokines produced under Th1 or Th2 conditionswere altered in sepsis [7882]. More recently, Boomer andcolleagues [83] used freshly isolated, postmortem spleen andlung tissue samples from 40 patients who died in intensivecare units as a result of severe sepsis compared with similarsamples from nonseptic, control patients. The authors foundalmost no production of IFN-, TNF-, IL-6, and IL-10 after 5h stimulation with -CD3/-CD28, which strongly suggests of astate of impaired T cell function. Whereas some investigatorsbelieved initially that these findings pointed toward a pheno-typic switch in CD4 T cells from Th1 to Th2 [84], changes incytokine secretion are more likely a result of a global state ofanergy [78]. This fact has been reinforced by the finding thatin human septic lymphocytes, there is decreased expression ofT-bet, GATA3, and ROR-ttranscription factors that regulatethe Th1, Th2, and Th17 effector CD4 T cell phenotypes, respec-tively [85]. Animal studies have also shown that histone methyl-ation and chromatin remodeling can occur within the T-bet andGATA3 promoter regions of lymphocytes after sepsis, therebycontributing to the anergic state of CD4 T cells [86].

Other studies have also pointed to indirect evidence of de-fective CD4 T cell function. For example, effective CD4 T cellimmunity is essential for the decrease in frequency and sever-ity of recrudescence in human herpesviral infections [87],such as CMV [88] or HSV [89, 90], and recent studies haveshown a significantly higher rate of CMV/HSV reactivation inseptic patients [89, 91]. CD4 T cell function is also integral toadequate B cell function, including antibody isotype switchingand maintenance of an effective humoral memory. B cells arediminished severely by sepsis-induced apoptosis [28], andthere exists some evidence of perturbations within peripheralB cell subsets early on after septic injury [92]. Moreover, cer-

Th1

Th2

Th17

Treg

Intracellular infections(e.g. Salmonella spp.,Mycobacteria spp.)

IL-2, IFN-,TNF-

Interleukinreceptor

Growth factorreceptor

Extracellular,Helminthic infections(e.g. Cryptococcus spp.,Leishmania spp.)

Epithelial barrier infections(e.g. Staphylococcus spp.,Candida spp.)

Negative regulationof T cell immunity

IL-4, IL-5,IL-10

IL-17, IL-23

IL-10, TGF-

InducedNatural

p:MHC-II TCR

co-stimsignals

CytokineMilieu

APC

Th0

CD4

Tbet

GATA-3

IL-6

IL-4

IL-12

IL-2

TGF-

RA

RORyt

IL-10 FoxP3Nrp1FoxP3

Figure 2. Plasticity of CD4 T cell phenotype is es-sential for generation of optimal responses to a widevariety of pathogens. A naive CD4 T cell has theability to execute one of several effector programs.During an infection, APCs present antigenicepitopes from invading pathogens to CD4 T cells viaMHC II. Along with TCR stimulation, APCs also pro-vide CD4 T cells with costimulatory (co-stim) ligandsand cytokine signals that are optimized for the anti-gen in question. The ensuing cytokine milieu, cre-ated for a specific infection at a specific infectionsite, will polarize CD4 T cells into an effector pheno-type most suitable for helping the innate and adap-tive components of the immune response. As ourdiscussion has focused on the activity of Th1, Th2,and Th17 CD4 T cells, we have only included thesesubtypes in the figure. Thus, optimal immunity isdependent on the correct polarization of CD4 Tcells, which is driven by the context, as well as thetype of antigen encountered (e.g., type of pathogenin question, innate adjuvant effects, and route ofinfection). Polarization can also induce naive CD4 Tcells to become Treg, which work alongside thymus-derived, natural Treg to suppress excessive inflam-mation and modulate the damage inflicted upon thehost by the immune response generated. p:MHC-II,peptide:MHC II; RA, retinoic acid; Nrp1, neuropilin-1.


tain B cells are thought to play a role in the success of the in-nate immune response during sepsis [93, 94]. Recent animalstudies have shown that administration of Ig fractions modi-fied by mild oxidation with ferrous ions improves survival aftersepsis [95, 96]. Interestingly, several investigators have also ob-served alterations in humoral responses after sepsis, specificallyin terms of antigen-specific immunity (e.g. T cell-dependentantibody responses) [9799].

Altered peripheral CD4 T cell diversityAnother important observation in sepsis patients is the consid-erable reduction in circulating CD4 T cells (along with otherlymphocyte populations), which is documented in patients ofall ages [41, 100107] and occurring at the time of highpathogen burden [108110]. However, very little is knownabout how CD4 T cells recover following septic injury, particu-larly the extent to which thymic function and homeostatic pro-liferation are involved. Naive CD4 T cells are normally main-tained in the periphery after thymic egress by frequent low-level signals from self-peptide:MHC II and cytokine signals(most notably, IL-7 signaling for naive CD4 T cells [111], withIL-15 signaling more important for naive CD8 T cells [112]).In situations where T cell numbers drop acutely (such as dur-ing cytotoxic drug regimens, irradiation, and certain viral in-fections), the increased availability of these resources turnssurvival signals into mitogenic stimuli in a process known ashomeostatic proliferation, which promotes a proliferative ex-pansion to restore T cell numbers [113]. During expansion(and despite the antigen-independent nature of this prolifera-tive mechanism), naive T cells acquire the phenotypic featuresof antigen-experienced, memory T cells [114]. One study, ex-amining recovery of T cells after sepsis-induced lymphopenia,argued that (OT-I) CD8 T cells were able to proliferate whenadoptively transferred into a septic host, but (OT-II) CD4 Tcells could only proliferate if cognate antigen or IL-7 was ad-ministered [115]. The authors accordingly concluded that ho-meostatic proliferation was not the main mechanism for CD4T cell recovery. This conclusion leads to an interesting dilem-ma: what endogenous source is reconstituting CD4 T cellsafter sepsis? It is evident that thymic output cannot explain Tcell reconstitution in elderly human patients, given that theexport rate of naive, thymus-derived cells is not modulated byalterations to the peripheral T cell pool (neither by lymphocy-tosis nor lymphopenia) [116]. Indeed, circulating levels of IL-7in athymic (but not necessarily lymphopenic) and elderly indi-viduals are increased significantly [117], which adds more evi-dence to the fact that peripheral mechanisms play a biggerrole than the thymus in the maintenance of circulating T cellnumbers after puberty. Furthermore, animal studies demon-strate that thymic function is impaired severely by sepsis viamassive apoptosis and thymic involution [115, 118]. It is moreplausible that CD4 T cells rely in peripheral (rather than cen-tral) maintenance mechanisms to recover full numericalstrength after sepsis.

The almost laissez faire maintenance of naive T cell num-bers in the peripheryautoregulation through the availabilityof IL-7 and tonic TCR signaling in the context of availablespace within the T cell compartmenthas one important

compromise. To anticipate an ever-changing world of patho-gens, the immune system has evolved to give T cells impressivediversity [119]. However, homeostatic proliferation does notcreate diversity, so much as it can maintain some of the diver-sity. This diversity begins at the antigen-specific populationlevel, where each CD4 T cell binds a specific complex of pep-tide antigen and MHC II via their TCR [120]. That is, oneseemingly homogenous group of CD4 T cells, recognizing thesame antigen, is formed by a diverse set of clones with diver-gent capabilities to form TCR/peptide:MHC II complexes[121]. Thus, an optimal diversity of the CD4 T cell repertoire(both in terms of breadth of antigen recognition, as well asheterogeneity of clonotypes within each antigen-specific popu-lation) is crucial for effective immune responses against invad-ing pathogens.

As we age, the competition within one population of anti-gen-specific T cells might give rise to a culled repertoire. Inanimal studies of lymphopenia, proliferative expansion of na-ive T cells also becomes more dependent on TCR/self-peptide:MHC II tonic signaling. The resultant environment enforcescompetition between clones and minimizes diversity withinantigen-specific populations. This is akin to a democratiza-tion process of the antigen-specific repertoire, whereby theclonal elite is culled in favor of the mediocre majority[122]. In agreement with this, both mouse and human studiesshow a dramatic, age-related decline in the diversity of anti-gen-specific T cells, preferentially losing reactivity over time toepitopes recognized by T cells with low precursor frequencies[123]. This effort to maintain some recognition of a pathogencan sacrifice clonal and antigenic diversity, plausibly generat-ing gaps in the immunological repertoire. Extrapolatingfrom these observations, we can reasonably argue that clonaldiversity in antigen-specific cells might be reduced to a mini-mum in an effort to maintain naive homeostasis and that inthe aging individual, this eventually results in the selection ofclones with poor affinity. In the context of sepsis, a recentstudy showed drastic reductions in clonotype diversity of septicpatients [124], making it tempting to speculate that sepsiscould effectively age the adaptive immune system by acceler-ating the selection of clones with poor affinity within the resul-tant peripheral repertoire.

Recent findings from our group have also shed light on theimpact of sepsis on the recovery of antigen-specific diversitywithin the peripheral T cell pool. Specifically, we studied theeffect of sepsis on a range of antigen-specific CD8 T cell popu-lations specific for LCMV and found significant changes to theantigen-specific precursor populations after sepsis that corre-late with impaired priming for some epitope-specific responses[125]. The data in this study ultimately hinted at changes tothe immunodominance hierarchy of LCMV-specific responsesin septic animals. In agreement with these results examiningantigen-specific CD8 T cell populations, we have seen that asimilar phenomenon occurs in antigen-specific CD4 T cellpopulations (unpublished data). As the survival of naive andmemory antigen-specific cells correlates inversely with clonalabundance [126], it is plausible that the massive apoptosis ofperipheral T cells in septic patients can drive a recovery of an-tigen-specific populations with diminished repertoire diversity,



whereby surviving clones do not adequately represent the im-munodominance hierarchy against a specific antigen [127]. Inthe context of a pathogen-specific response, this idea impliesthat the recovery of a less-diverse repertoire within antigen-specific populations could lead to aberrant responses as a re-sult of changes in the affinity for dominant antigen peptides.This effect would account (at least in part) for the susceptibil-ity to opportunistic infections and diminished lymphocytefunction seen in sepsis.

Increases in Treg frequency and/or functionAn increased frequency of Treg has been found in the periph-ery of septic patients, particularly in the early stages after diag-nosis [102, 128, 129]. These results were later clarified by astudy showing that the increased frequency of Treg was a resultof decreases in the effector populations of CD4 T cells [81].Thus, one conclusion drawn about Treg in sepsis is that theyare more resistant to apoptosis than conventional CD4 T cells[28]. Despite these findings, the role of Treg in septic injury isstill debated. Excessive Treg formation decreases survival in ani-mal models of sepsis [130], as well as improving outcomes andimmunity [131133]. These contrasting results, particularly inhuman studies, may be related to the sensitivity of analyzingFoxP3 expression via flow cytometry, the timing of analysis,and the ability to discern the methylation status of the FoxP3promoter in circulating septic lymphocytes [134]. In animalmodels, the removal of Treg by anti-CD25 mAb has not led toany improvements in survival [135], but this may be a result ofthe expression of CD25 in activated CD4 T cells (and thus,depletion is not limited to CD4 Treg). More recently, someinvestigators have used GITR agonistic antibodies to block Tregfunction, and results from these studies show improved im-mune function and microbial killing in septic animals [99].

Complement depletion, which often occurs as a result ofdisseminated intravascular coagulation, can also have an effecton the balance between CD4 Treg and effector T cells. Re-cently, several reports showed how signaling via C3aR/C5aRdiminishes the function of Treg [136138]. Along these lines,studies in human septic patients have shown strong correla-tions between complement C3 depletion and an increase inthe frequency of Treg, as well as significantly higher postopera-tive complications and hospital stay [139, 140]. Moreover, theadministration of exogenous C3 inhibited Treg expansion andimproved survival in animal models of sepsis [141, 142].Whereas it is true that C3 might also be improving outcomesby preventing organ dysfunction, it is just as plausible thatchanges in the T cell compartments and the paralysis of im-mune responses can be, in part, a result of systemic cell death.

STRATEGIES TO ENHANCE T CELLRECOVERY AND FUNCTION AFTERSEPSIS

Administration of immune-modulatory therapy is a promisingtreatment approach for treating sepsis survivors. Of particularinterest are two therapeutic approaches: the use of c recep-tor-dependent cytokines, such as IL-2, IL-7, and IL-15 [143

145], and the blockade of certain inhibitory molecules (partic-ularly, PD-1 [146, 147], CTLA-4 [148], and TRAIL [38]). How-ever, the potency and safety of some these therapies have tobe enhanced and toxicity minimized before their efficacy canbe tested in clinical settings. Here, we will review the extent towhich these therapies can improve pathogen clearance, increaseCD4 T cell responsiveness, and promote survival in sepsis.

Cytokines of the common c receptor familyCytokine-based, immune-modulatory strategies have proveneffective in boosting anticancer responses, rejuvenating T cell-mediated immunity in settings of chronic viral infections, aswell as accelerating immune reconstitution after bone marrowtransplantation. It is now apparent that although their rejuve-nating effects apply differentially to T cell subsets, severalmembers in the common c family of cytokines promote ho-meostasis and survival in CD4 and CD8 T cells. For example,IL-2 becomes a master regulator of homeostasis during T cell-mediated responses, as well as during T cell recovery in lym-phopenic hosts (promoting survival, expansion of Treg, or acti-vation-induced death in a context-dependent manner). IL-7and IL-15, conversely, are much more specifically required forthe survival and expansion of CD4 and CD8 memory and Tcells during homeostatic expansion. IL-7 is one of the mostpromising cytokine-based therapies to date, with 13 currentlyongoing or recently closed clinical trials examining the effectof IL-7 adjuvant therapy in anti-tumor responses or as abooster of immunity in chronic viral infections. In the contextof sepsis, studies published within the last 3 years have shownIL-7 to improve survival of murine T cells after sepsis inducedby CLP, to increase pathogen clearance and DTH responses ina mouse model of sepsis with candidiasis, and to promote pro-liferation in hyporesponsive PBMC from septic patients [144,145, 149].

The biggest potential drawbacks of a cytokine-based therapyare potential off-target effects. For example, although IL-7 canenhance immune reconstitution after bone marrow transplan-tation in humans, patients with acute graft-versus-host diseasedisplay significantly higher circulating concentrations of IL-7[150]. Similarly, IL-15 can exacerbate autoimmunity (such asin celiac disease), and side-effects of high-dose IL-2 immuno-therapy stem from its ability to cause vascular leak syndrome,an accumulation of intravascular fluid in organs, causingprominent pulmonary edema and liver cell damage [151].

Immune checkpoint modulationAs mentioned previously, sepsis-induced immune dysfunctionshares many similarities with cancers refractory to standardtherapies. One of the newest strategies for therapeutic inter-vention in cancerimmune checkpoint modulationmightlead to benefits in sepsis as well. Most of these new strategiesare based on blockade of pathways that negatively regulate Tcell survival and/or activation following TCR engagement.Thus, whereas cytokine therapies might prevent or lessen ir-reparable losses in the T cell repertoire, immune checkpointmodulation therapies would serve to reverse sepsis-inducedimmunosuppression.


Perhaps the most popular target for checkpoint modulationin current sepsis research is PD-1. A growing number of bio-logicals targeting the PD-1 pathway have been evaluated as im-munotherapy for several types of solid tumors and have shownimpressive efficacy in clinical trials [152, 153], as well as in theeffective reversal of T cell exhaustion in mycobacterial andchronic CMV infections [147, 154]. Interestingly, PD-1 expres-sion has also correlated with mortality and nosocomial infec-tions in sepsis patients [73]. Correspondingly, the pharmaco-logically relevant ligand for PD-1, PD-L1, is up-regulated oncirculating monocytes in human sepsis patients and mousemacrophages during experimental sepsis [73, 74]. Indeed,blockade of the PD-1:PD-L1 signaling pathway improves sur-vival in animal models of sepsis [155, 156] and reverses T cellexhaustion in patients with sepsis [146].

Another potential target for blockade or inhibition isTRAIL, which has been under investigation in clinical trials inthe cancer and bone marrow transplantation fields [157].TRAIL is up-regulated in situations of immune privilege and Tcell exhaustion to induce apoptotic cell death [48, 158]. Asmentioned, one interesting phenomenological defect in sepsispatients is a loss of CD4 T cell-dependent DTH responses. Inother tolerance models where there is administration of anti-gen-coupled apoptotic cells or generation of a large numberof apoptotic cells in vivo, one mechanism to explain the lack

of CD4 T cell immunity is immune regulation by a TRAIL-ex-pressing CD8 T cell population [66, 159, 160]. In line withthese experimental models of tolerance, a population ofTRAIL-expressing CD8 T cells potently inhibits CD4 T cellfunction in sepsis, and the blockade of TRAIL by mAb treat-ment increases T cell function and decreased heterologouspathogen burden during a secondary infection in CLP-in-duced sepsis [37, 38]. Interestingly, low concentrations of solu-ble TRAIL in the plasma correlated with reduced immunefunction and a higher risk of mortality in patients with septicshock [161]. Clearly, additional research is needed to deter-mine the relationship between the mouse and human data onthe potential function of TRAIL in sepsis.

CONCLUDING REMARKS

Sepsis is a complex medical condition that exerts a variety ofconsequences on the immune system. We have examined theimpact of sepsis specifically on CD4 T cell immunity, as thesecells factor into the development of numerous types of re-sponses by the immune system. Moreover, we have focused ourdiscussion on the impact of sepsis on the quantity and qualityof CD4 T cells, given that these characteristics dictate the mag-nitude and success of any prospective CD4 T cell response to

CD4

DR5

BTLA

PD-1CD28 TCR

IL-7receptor

FoxP3CD4

TCR

GITR

CTLA-4

C3a/C5areceptor

Sepsis

Effector CD4 Phenotype & FunctionUpregulation of co-inhibitory,pro-apoptotic signals

Decreased IL-7R expressionEpigenetic changes

CD4 T cell Repertoire DiversityAltered immunodominancehierarchy

Over-represented

Appropriately recovered

Under-represented

Multifactorial insultMetabolic stress

Apoptosis

Lymphopeniainducedproliferation

Oxidative stressInflammation

EarlyAfter Sepsis

BeforeSepsis

LateAfter Sepsis

CD4 TREG Frequency & FunctionIncreased co-inhibitorymolecule expression

Increased resistance toapoptosis

Increased suppressive activity

XX

Figure 3. Sepsis-associatedlymphocyte apoptosis is fol-lowed by a quantitativelyand qualitatively impairedrecovery of CD4 T cellpathogen-specific responses.The colored cells representthree different antigen-spe-cific populations within animmunodominance hierar-chy. Sepsis causes a stochasticloss of CD4 T cells by apo-ptosis, but the causativeagent(s) responsible for thisdecline are not clear. It isthought that the drop in cir-culating lymphocytes stemsfrom a multifactorial insultthat includes excessive proin-flammatory cytokine levels,metabolic stress, increasedlevels of toxic metabolites,reactive oxygen species. andhypoxia/ischemia. Neverthe-less, the end result for a sig-nificant group of patients is astate of lymphopenia that ismost pronounced for certaincell populations (one such

population includes CD4 T cells) with clear reductions in diversity, as well as the eventual numerical recovery of T cells. However, several changes occurto CD4 T cells in the process of recovery. These include cell-intrinsic changes (anergic and proapoptotic phenotypes, as well as hypermethylation of pro-moter regions for important Th cell transcription factors) and regulatory changes (increased fraction of Treg and/or perhaps increases in the functionalcapacity of Treg). Finally, changes to CD4 T cell repertoire diversity are depicted here by showing how antigen-specific populations may be altered afterlymphopenia and recovery, thereby altering the immunodominance hierarchy of a response: one population has an impaired recovery, whereas anotheris over-represented after recovery, and a third population recovers numerically to its level at homeostasis. This change can be demonstrated at the levelof single antigen-specific populations but is not evident otherwise given the numerical recovery of total CD4 T cells. DR5, death receptor 5.



pathogenic challenge. The apoptotic attrition within the CD4T cell compartment during a septic event stochastically affectsall antigen-specific effector CD4 T cell populations (Fig. 3).Acutely, this sudden loss of CD4 T cells dramatically affects avariety of adaptive-immune functions. As CD4 T cells recovernumerically, multiple mechanisms are potentially involved insepsis-induced immune suppression of CD4 T cell responses.Changes in TCR repertoire use, antigen-binding affinity, prolif-erative capacity, and cytokine production can act collectively toalter qualitative and compositional aspects of postseptic CD4 Tcell responses. Furthermore, sepsis-induced alterations in thecomposition of the antigen-specific CD4 T cell repertoire per-sist and may ultimately account for the increased risk of mor-tality [162, 163] and the more poorly perceived general healththat is documented in survivors for years after resolution ofsepsis [164].

We realize that the sepsis-induced changes within the CD4 Tcell compartment that we have highlighted are just a few ofthe many ways in which sepsis affects the immune system. Asmedical advancements have increased the ability of cliniciansto reduce morbidity to acute sepsis, the continued testing oftherapeutics that prevent CD4 T cell loss, accelerate numericalrecovery, boost cellular function, and/or block immunosup-pressive pathways is needed to decrease mortality rates associ-ated with the increased susceptibility to secondary infection. Aseach of these aspects of CD4 T cell function act at differentpoints, it is likely that targeting multiple points will be neededto restore the CD4 T cell response to (near) normal states.

AUTHORSHIP

J.C-P. designed and wrote the paper. S.A.C., V.P.B., and T.S.G.wrote and reviewed the paper.

ACKNOWLEDGMENTS

This study was supported by the University of Minnesota Cen-ter for Immunology Training Grant T32 AI007313 and Medi-cal Scientist Training Program T32 GM008244 (to J.C-P.), anAmerican Heart Association Postdoctoral Fellowship (toS.A.C.), U.S. National Institutes of Health Grant AI83286 (toV.P.B.), and a U.S. Department of Veterans Affairs Merit Re-view Award (to T.S.G.).

DISCLOSURES

The authors disclose no financial conflicts of interest.

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KEY WORDS:apoptosis lymphopenia homeostatic proliferation immunesuppression



impact of sepsis on immunity

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