The Journal of Immunology

Alterations in Antigen-Specific Naive CD4 T CellPrecursors after Sepsis Impairs Their Responsiveness toPathogen Challenge

Javier Cabrera-Perez,*,† Stephanie A. Condotta,‡ Britnie R. James,x Sakeen W. Kashem,*,†

Erik L. Brincks,x Deepa Rai,‡ Tamara A. Kucaba,x Vladimir P. Badovinac,‡,{ and

Thomas S. Griffith*,x,‖,#,**

Patients surviving the acute stages of sepsis develop compromised T cell immunity and increased susceptibility to infection. Little is

known about the decreased CD4 T cell function after sepsis. We tracked the loss and recovery of endogenous Ag-specific CD4 T cell

populations after cecal ligation and puncture–induced sepsis and analyzed the CD4 T cell response to heterologous infection during

or after recovery. We observed that the sepsis-induced early loss of CD4 T cells was followed by thymic-independent numerical

recovery in the total CD4 T cell compartment. Despite this numerical recovery, we detected alterations in the composition of naive

CD4 T cell precursor pools, with sustained quantitative reductions in some populations. Mice that had experienced sepsis and

were then challenged with epitope-bearing, heterologous pathogens demonstrated significantly reduced priming of recovery-

impaired Ag-specific CD4 T cell responses, with regard to both magnitude of expansion and functional capacity on a per-cell

basis, which also correlated with intrinsic changes in Vb clonotype heterogeneity. Our results demonstrate that the recovery of

CD4 T cells from sepsis-induced lymphopenia is accompanied by alterations to the composition and function of the Ag-specific

CD4 T cell repertoire. The Journal of Immunology, 2015, 194: 000–000.

CD4 Th cells influence the function of a variety of innateand adaptive immune cells critical for the successfulgeneration of a productive and protective immune re-

sponse (1). For example, effective primary CD8 T cell responses(2, 3), the formation of functional CD8 T cell memory (4–7),efficient isotype switching in primary and memory B cellresponses (8, 9), and the effector function of macrophages (10)all develop with the “help” of CD4 T cells. CD4 T cells are able tofunction in such an array of immunological settings because ef-fector CD4 T cells can take on different phenotypes [i.e., Th1,Th2, Th9, Th17, follicular Th (1)], based on the cytokines and

costimulatory molecules present at the time of Ag recognition. Inturn, this plasticity enables CD4 T cells to drive a response that isbest suited for the situation. Because of their importance ina broad variety of immune responses, perturbations in the CD4T cell compartment can have dramatic consequences on theoverall fitness of the immune system.Sepsis strikes 750,000 Americans every year (11), with

∼210,000 of these patients dying (12). Although sepsis has beendefined as a systemic inflammatory response syndrome in thepresence of a disseminated infection (13–15), it has become clearin the past decade that sepsis is not just the symptoms of a com-plicated infection. Instead, sepsis is now viewed as a syndromestemming from the dysregulation of immune responses due toan invasive pathogen, a phenomenon that results in system-widecollateral damage (16). Sepsis-induced immune suppression isintricately related to the process of lymphocyte apoptosis thatoccurs after a septic event (17, 18). Sepsis-induced lymphopeniatransiently creates a reduction in the numbers of immune cells,including T cells. Although the total T cell compartment recoversnumerically after a septic event, it is unknown whether differentAg-specific T cell subpopulations can revert back to the antigenicdiversity seen before sepsis and whether changes in populationdiversity can affect the functionality of the immune system. Grossquantitation of CD4 T cells reveals that they are severely depletedduring the acute stage of sepsis but gradually recover throughoutits immunosuppressive phase (19). However, there are knowledgegaps regarding the mechanism(s) driving this CD4 T cell recovery,the quality/functionality of the “recovered” CD4 T cell compart-ment, and the extent to which sepsis impairs Ag-specific CD4T cell function in surviving animals.In this study, we used peptide:MHC II (p:I-Ab) tetramer-

enrichment technology (20) to examine quantitative shifts withinthe endogenous naive Ag-specific CD4 T cell repertoire at dif-ferent time points after sepsis. Our findings suggest that the nu-

*Microbiology, Immunology, and Cancer Biology Graduate Program, University ofMinnesota Medical School, Minneapolis, MN 55455; †Medical Scientist TrainingProgram, University of Minnesota Medical School, Minneapolis, MN 55455;‡Department of Pathology, University of Iowa Carver College of Medicine, IowaCity, IA 52242; xDepartment of Urology, University of Minnesota Medical School,Minneapolis, MN 55455; {Interdisciplinary Program in Immunology, University ofIowa Carver College of Medicine, Iowa City, IA 52242; ‖Masonic Cancer Center,University of Minnesota Medical School, Minneapolis, MN 55455; #Center for Im-munology, University of Minnesota Medical School, Minneapolis, MN 55455; and**Minneapolis VA Health Care System, Minneapolis, MN 55417

Received for publication July 7, 2014. Accepted for publication December 12, 2014.

This work was supported by a U.S. Department of Veterans Affairs Merit ReviewAward (to T.S.G.), an award from the American Heart Association (to S.A.C.), andNational Institutes of Health Grants 2T32AI007313 (to J.C.-P.) and AI83286 andAI114543 (to V.P.B.).

Address correspondence to Dr. Thomas S. Griffith, Department of Urology, Univer-sity of Minnesota, MMC Mayo, 420 Delaware Street SE, Minneapolis, MN 55455.E-mail address: tgriffit@umn.edu

Abbreviations used in this article: B6, C57BL/6; BCS, bovine calf serum; BV, Bril-liant Violet; Ca-2W1S, 2W1S-expressing Candida albicans; CLP, cecal ligation andpuncture; gD, gp D; IAV, influenza A virus; i.n., intranasal(ly); LCMV, lymphocyticchoriomeningitis virus; Lm-OVA, OVA323–339-expressing ActAD L. monocytogenes;Lm-2W1S, 2W1S-expressing ActAD Listeria monocytogenes; LN, lymph node; NP,nucleoprotein; p:I-Ab, peptide:MHC II; tg, transgenic; Treg, regulatory T cell; WT,wild-type.

Copyright� 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401711

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merical restoration of the CD4 T cell repertoire after sepsis occursvia a peripherally driven mechanism that is, in part, independentof Ag availability. Although the total CD4 T cell populationrecovers numerically, examination of individual Ag-specific pop-ulations revealed an asymmetric recovery in different Ag-specificprecursor populations. Our results also suggest that, if inade-quately recovered, Ag-specific CD4 T cell populations show im-pairments in expansion and function in response to pathogenchallenge after sepsis. The implications of these findings withinthe context of long-term increased susceptibility to secondaryinfections (and the associated increased risk for mortality) arediscussed.

Materials and MethodsMice

Euthymic and thymectomized C57BL/6 (B6) mice were purchased fromThe National Cancer Institute. Thy1.1/1.1 TCR-transgenic (tg) SMARTA(lymphocytic choriomeningitis virus [LCMV] gp61–77-specific) and SM1(Staphylococcus typhimurium FliC447–460-specific) B6 mice were obtainedfrom Drs. David Masopust and Marc Jenkins (University of Minnesota),respectively. All mice were housed in the same facilities for $4 wk, re-gardless of their source. Animal procedures were performed according toNational Institutes of Health guidelines and were approved by the Uni-versity of Minnesota Institutional Animal Care and Use Committee. In allin vivo experiments, groups consisted of four or more animals, andexperiments were repeated at least two times with similar results beforereporting.

Cecal ligation and puncture

Septic injury was induced by cecal ligation and puncture (CLP) (21).Briefly, mice were anesthetized, the abdomen was shaved and disinfected,and a midline abdominal incision was made. The distal third of the cecumwas ligated with 4-0 silk suture and punctured once using a 25-g needle toextrude a small amount of cecal content. The cecum was returned to theabdomen, the peritoneum was closed via continuous suture, and the skinwas sealed using surgical glue (Vetbond; 3M, St. Paul, MN). Saline (1 ml)was provided s.c. following the procedure for resuscitation, and bupiva-caine was administered at the incision site for postoperative analgesia. Thislevel of injury was used to create a chronic septic state characterized by theloss of appetite and body weight, ruffled hair, shivering, diarrhea, and/orperiorbital exudates and with a 5–10% mortality rate. Sham-treated miceunderwent the same procedure, excluding CLP.

BrdU incorporation and detection

To assess CD4 T cell proliferation, sham- and CLP-treated mice were givena BrdU pulse (2mg in 0.2 ml/mouse i.p.; Sigma, St. Louis, MO) on day 6 aftersurgery. Blood was collected at the indicted times, followed by RBC lysis inACK buffer. Cells were surface stained with PE CD4 (clone GK1.5; Bio-Legend, San Diego, CA), after which they were fixed with Cytofix/Cytopermsolution (BD Biosciences, San Diego, CA) and treated with DNase I (300mg/ml in PBS; Sigma) for 1 h. BrdU was detected by intracellular stainingwith FITC-conjugated anti-BrdU (clone BU20A) or an IgG1 isotype (cloneP3.6.2.8.1) mAb (both from eBioscience, San Diego, CA).

Adoptive cell transfers

SM1 or SMARTA TCR-tg CD4 T cells were obtained from the spleens ofnaive SMARTA or SM1 mice. Contaminating memory phenotype (CD44hi

CD11ahiCD49hi) TCR-tg cells were consistently ,5%. The purified cellswere transferred to naive B6 mice 1 d before sham or CLP surgery.

Experimental pathogens and infections

2W1S (EAWGALANWAVDSA)-expressing ActAD Listeria mono-cytogenes (attenuated Lm-2W1S; 107 PFU/mouse) or OVA323–339

(ISQAVHAAHAEINEAGR)-expressing ActAD L. monocytogenes (atten-uated Lm-OVA; 107 PFU/mouse) was grown and injected i.v., as previ-ously described (22). 2W1S-expressing Candida albicans (Ca-2W1S) wasderived from clinical isolate SC5314 (23). Ca-2W1S was grown to logphase (OD600 of 1.5) in YDAP medium, washed, and counted by hemo-cytometer before being resuspended at a concentration of 5 3 104 yeasts/i.v. challenge. Recombinant Lm-2W1S and Ca-2W1S were obtained fromDrs. Marc Jenkins and Daniel Kaplan (University of Minnesota), respec-tively. For HSV-1 (KOS strain) and influenza A virus (IAV; strain X31)

inocula, frozen viral stocks were thawed, and 2.5 3 104 PFU and 3000EID50 units, respectively, was administered per mouse. Viral stocks wereobtained from culture conditions that were described previously (24–26).Intravenous challenges using 0.1-ml injection volumes were used for all ofthe inoculants described, with the exception of IAV X31, which was givenintranasally (i.n.) using 0.02-ml aliquots/nostril. Infected mice were housedunder the appropriate biosafety level.

Tetramers and peptides

I-Ab–specific tetramers containing 2W1S (EAWGALANWAVDSA),LCMVgp66–77 (DIYKGVYQFKSV), LLO190-201 (NEKYAQAYPNVS)protein of L. monocytogenes, or OVA323–339 (ISQAVHAAHAEINEAGR)peptides were obtained from Dr. Marc Jenkins. Biotinylated soluble I-Ab

molecules containing HSVgp D (gD)290–305 (IPPNWHIPSIQDA) or IAVnucleoprotein (NP)311–325 (QVYSLIRPNENPAHK) peptides (27, 28) co-valently attached to the I-Ab b-chain were produced with the I-Ab

a-chain in Drosophila melanogaster S2 cells, purified, and made intotetramers with streptavidin-PE (eBioscience) or streptavidin-allophycocyanin(BioLegend), as previously described (20, 29). Peptides used to elicit cyto-kine production or expand endogenous Vb repertoire for quantification weresynthesized by Bio-Synthesis (Lewisville, TX).

Quantitation of endogenous Ag-specific CD4 T cellpopulations using p:I-Ab tetramer-based enrichment

To quantify the number of Ag-specific CD4 T cells within the spleens ofsham- or CLP-treated mice, a tetramer-based enrichment protocol (29)using p:I-Ab tetramers was used. Briefly, spleens were harvested for eachmouse analyzed, a single-cell suspension was prepared, and allophyco-cyanin- and PE-conjugated tetramers were added at a 1:400 dilution intetramer staining buffer (PBS containing 5% bovine calf serum [BCS],2 mM EDTA, and 50 mM dasatinib, 1:50 normal mouse serum, and 1:100anti-CD16/32 mAb). The cells were incubated in the dark at room tem-perature for 1 h, followed by a wash in 10 ml cold FACS Buffer. Thetetramer-stained cells were resuspended in 0.2 ml FACS Buffer, mixedwith 0.05 ml each anti-allophycocyanin and anti-PE mAb-conjugatedmagnetic MicroBeads (Miltenyi Biotec), and incubated in the dark onice for 30 min. The cells were washed and resuspended in 3 ml cold FACSBuffer and passed over a MACS separation column (Miltenyi Biotec)to enrich for the tetramer-specific cells. Columns were washed threetimes with 3 ml cold FACS Buffer before eluting the bound fractionwith 5 ml cold FACS Buffer. The resulting enriched fractions werestained with a mixture of fluorochrome-labeled mAb (see later dis-cussion). Cell numbers for each sample were determined using Accu-Check Counting Beads (Invitrogen). Samples were analyzed using anLSR II flow cytometer (BD) and FlowJo software (TreeStar, Ashland,OR). The percentage of tetramer+ events was multiplied by the totalnumber of cells in the enriched fraction to calculate the total number ofAg-specific CD4 T cells in the spleen.

CD4 T cell assays

In vivo peptide stimulation was used to determine Ag-specific CD4 T cellfunction by intracellular cytokine production, as previously described (22,30, 31). Briefly, infected mice were injected i.v. with 100 mg of the ap-propriate peptide. After 2 h, spleens were harvested in media containing10 mg/ml brefeldin A. The resulting cell suspensions were fixed, per-meabilized, and stained with anti–IFN-g and anti-TNF mAbs. To specificallyexamine the function of Ag-specific Th17 cells, sham and CLP-treated micewere infected 30 d after surgery with Ca-2W1S epicutaneously (23). On day7 postinfection, the spleen and skin-draining (inguinal, brachial, axillary, andcervical) lymph nodes (LNs) were harvested and dissociated into a single-cell suspension. The resultant cells were stimulated for 4 h with PMA(50 ng/ml) and ionomycin (1.5 mM) in complete RPMI 1640 media sup-plemented with monensin (1 mM). After stimulation, cell debris was fil-tered, and the samples underwent tetramer enrichment, as described. Aftertetramer enrichment and subsequent staining for cell surface markers,0.1-ml aliquots from the enriched and flow-through fractions weresuspended in fixation/permeabilization buffer for 20 min at 4˚C, andthen stained for intracellular IFN-g and IL-17 accumulation overnightin permeabilization buffer (both from eBioscience). After staining, cellswere resuspended in FACS buffer, and 0.02 ml counting beads (eBioscience)was added to each sample immediately before acquisition.

Flow cytometry

To assess the expression of cell surface proteins, cells were incubated withfluorochrome-conjugated mAb at 4˚C for 30 min. The cells were thenwashed with FACS buffer (PBS containing 2% BCS and 0.2% NaN3). For

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some experiments, the cells were fixed with PBS containing 2% parafor-maldehyde. In procedures requiring intracellular staining, cells werepermeabilized following surface staining using the transcription factorstaining kit (eBioscience), stained for 1 h at 4˚C with a second set offluorochrome-conjugated mAbs, and suspended in FACS buffer for ac-quisition. The following fluorochrome-conjugated mAbs were used forboth surface and intracellular staining: Horizon V500 Thy1.2 (clone53-2.1; BD Biosciences), Brilliant Violet (BV) 510 and FITC CD3 (clone17A2; BioLegend), BV421 and BV605 CD4 (clone GK1.5; BioLegend),BV650 CD8 (clone 53-6.7; BioLegend), Alexa Fluor 700 CD44 (cloneIM7; BioLegend), allophycocyanin and BV421 IL-17A (clone TC11-18H10.1; BioLegend), allophycocyanin and BV650 IFN-g (cloneXMG1.2; BioLegend), PE-Cy7 IL-2 (clone JES6-5H4; BioLegend), AlexaFluor 647 CD49d (clone R1-2; BioLegend), PerCP-Cy5.5 B220 (cloneRA3-6B2; eBioscience), PerCP-Cy5.5CD11b (clone M1/70; eBioscience),PerCP-Cy5.5CD11c (clone N418; eBioscience), PerCP-Cy5.5 F4/80(clone BM8; eBioscience), FITC Foxp3 (clone FJK-15S; eBioscience),FITC CD11a (clone M17/4; eBioscience), FITC TNF-a (clone MP6-XT22; eBioscience), and PE-Cy7 CD11a (clone M17/4; eBioscience).FlowJo software (TreeStar) was used for analysis of samples acquired onan LSR II flow cytometer (BD).

Ag-specific TCR Vb repertoire flow cytometry assay

Assessment of TCR Vb repertoire diversity was performed using a modi-fication of a previously reported method (29, 32). Briefly, sham- or CLP-treated mice were injected with 50 mg 2W1S peptide and 5 mg LPS on day30 postsurgery. Four days later, splenic T cells were enriched for allo-phycocyanin–2W1S:I-Ab tetramer-binding cells. The enriched populationwas subsequently divided into two equal aliquots and stained for surfacemarkers along with 13 available TCR Vb mAbs multiplexed onto fourseparate flow cytometry detection channels, using directly conjugated Absand/or biotinylated mAbs detected afterward with Streptavidin-BV421(BioLegend). The TCR Vb mAbs used were FITC-conjugated mAbagainst mouse Vb2 (clone B20.6), Vb4 (clone KT4), Vb6 (clone RR4-7),Vb7 (clone TR310), Vb8.1/8.2 (clone KJ16-133.18), and Vb8.3 (clone8C1); PE-conjugated mAb against Vb5.1/5.2 (clone MR9-4), Vb8.1/8.2(clone MR5-2), Vb8.3 (clone 1B3.3), Vb9 (clone MR10-2), and Vb10(clone B21.5); PerCP–eFluor 710–conjugated Vb13 (clone MR12-3); and

biotinylated mAb against Vb3 (clone KJ25), Vb4, Vb5.1/5.2, Vb6, Vb10,and Vb14 (clone 14-2). Unless otherwise specified, mAbs that detected thesame TCR Vb clonotype were from the same clone and vendor. All mAbsused to detect Vb clonotype distribution were purchased from BD Bio-sciences, BioLegend, or eBioscience.

Statistical analyses

Data were analyzed using GraphPad Prism (La Jolla, CA). Specific tests todetermine statistical significance are indicated in the figure legends. Datascatter plots are presented as mean values 6 SEM, and data shown as bargraphs are presented as mean + SEM.

ResultsNumerical recovery of CD4 T cells after sepsis occurs bya thymic-independent mechanism

Sepsis can be investigated experimentally using the CLP model(21), which is used frequently to assess the acute complicationsand mortality associated with severe septic events. The CLPmodel used in our studies induces a mild septic state resulting in∼10% acute mortality (Fig. 1A). This degree of injury createsimmune defects similar to more severe models and permits thelong-term study of immune system responses in septic mice (33–35). In addition, mice that experience this milder sepsis demon-strate the same symptoms that are characteristic of severe exper-imental peritonitis, including cachexia, weight loss, piloerection,and lethargy. Consistent with previous data (19), we found a sig-nificant decrease in the total number of CD4 T cells in the spleen,inguinal LNs, and blood 2 d after septic injury, and a numericalrecovery was apparent by day 30 (Fig. 1B, 1C). These results ledus to conclude that the attenuated CLP procedure that producesa mild septic insult can be used to interrogate the CD4 T cell lossand recovery in the context of sepsis.

FIGURE 1. Numerical recovery of CD4 T cells after CLP-induced sepsis occurs by a thymus-independent mechanism. (A) Kaplan–Meier survival curve

of experimental cohorts after undergoing sham or CLP surgery. (B) Number of CD4 T cells in spleen and inguinal LNs (pLN) on days 2 and 30 after sham

or CLP surgery. (C) Thymectomized and euthymic mice underwent CLP surgery, and the number of CD4 T cells in the peripheral blood was measured over

time. (D) Thymectomized and euthymic mice underwent sham or CLP surgery. BrdU was injected i.p. 6 d later, and the frequency of peripheral blood CD4

T cells incorporating BrdU was determined 24 h later. Data shown are representative of at least two independent experiments, with four or five mice/group

in each experiment. *p, 0.05, ***p, 0.005, ****p, 0.001, Mann-Whitney U test (A) or one-way ANOVA (B–D), with multiple-testing correction using

the Holm–Sidak method, and a = 0.05, when deemed appropriate. ns, not significant.

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CD4 T cell recovery is not usually dependent on thymic-derivedT cells in models of experimentally induced lymphopenia, becausethe export rate of naive T cells from the thymus is not modulated byperturbations in the periphery (36). In contrast, work by Unsingeret al. (17) suggested that CD4 T cells did not homeostaticallyproliferate when transferred into CLP-treated recipients. Thesecontradictory findings led us to examine the number of CD4 Tcells in the blood of sham- and CLP-treated euthymic (wild-type[WT]) and thymectomized mice. CD4 T cell loss and recoverywere similar in WT and thymectomized mice, with no statisticallysignificant difference at any of the time points analyzed (Fig. 1C).Next, sham- and CLP-treated WT and thymectomized mice weregiven BrdU on day 6 after surgery to measure proliferation of theperipheral blood CD4 T cells. We found statistically higher fre-quencies of BrdU+ CD4 T cells in CLP-treated mice versus shams,regardless of thymic presence or absence (Fig. 1D). Together,these results suggest that CD4 T cell recovery after sepsis occursby a thymus-independent mechanism.

CD4 T cells that numerically recover after septic insult acquirean “Ag-experienced” phenotype

As naive T cells homeostatically proliferate to fill lymphopenicniches, their surface phenotype changes to resemble T cells that haveencountered their cognate Ag (37, 38). Importantly, this change inphenotype can be independent of cognate Ag recognition (39). Werecently reported that the numerical recovery of CD8 T cells afterseptic injury is driven by homeostatic proliferation, as indicated byan increased frequency of CD11ahiCD44hi CD8 T cells comparedwith sham mice (34), even when cognate Ag was not encountered.Currently, there is no canonical phenotype characterizing CD4T cells that have undergone similar processes, but several pheno-types have been suggested. For example, CD11a and CD49dcoexpression indicate an “Ag-experienced” CD4 T cell phenotype(40, 41). We noted that CD4 T cell recovery after sepsis was con-comitant with an increased frequency of CD11ahiCD49dhi CD4T cells among the total CD4 T compartment (Fig. 2A, 2B). CD44and CD127 coexpression also was suggested to be a phenotype forCD4 T cells after Ag-independent expansion (42), and we foundthat CLP-treated mice had an increased frequency of CD44hi

CD127hi CD4 T cells (Fig. 2A, 2B). To determine the extent towhich these changes were dependent on TCR interaction withcognate Ag, we transferred TCR-tg Sm1 [tg epitope: FliC427–441

from Staphylococcus enterica ser. typhimurium flagellin (43)] andSMARTA [tg epitope: gp61–77 from LCMV (44)] CD4 T cells intoB6 mice prior to sham or CLP surgery. These are two disparatelydifferent pathogens, and neither is normally found in specificpathogen–free mice. We observed significantly increased frequen-cies of CD11ahiCD49dhi Sm1 and SMARTA CD4 T cells in thespleens of CLP-treated mice compared with sham-treated mice after30 d (Fig. 2C). Although Ag cross-reactivity cannot be excluded,these phenotypic changes in TCR-tg CD4 T cells led us to concludethat the acquisition of an “Ag-experienced” phenotype was occur-ring without cognate Ag present in the septic host. Together, thedata in Figs. 1 and 2 suggest that lymphopenia-induced homeostaticproliferation plays a major role in the numerical recovery of theCD4 T cell compartment after sepsis.

Asymmetric recovery of Ag-specific naive CD4 T cells aftersepsis

The massive attrition of peripheral CD4 T cells and evidencesupporting a peripheral mechanism of CD4 T cell recovery aftersepsis led us to question the possibility of discrete changes withinindividual Ag-specific CD4 T cell populations. We used p:I-Ab

tetramer-based enrichment (20, 29) (Fig. 3A) to quantify six

endogenous Ag-specific CD4 T cell populations of various size,clonotype composition, and immunodominance. CLP-treated miceshowed acute reductions in all populations examined on day 2(consistent with the global lymphopenia) compared with sham-treated mice, but the numerical recovery of the different Ag-specific CD4 T cell populations 30 d post-CLP was asymmetric(Fig. 3B). Quantitatively, the 2W1S- and Listeria LLO190–201-specific CD4 T cell populations were reduced in CLP-treatedversus sham-treated mice, whereas the number of IAV NP311–325-specific CD4 T cells increased in CLP-treated mice over shammice. In contrast, LCMVgp66–77-specific, HSV-1gD290–305-spe-cific, and OVA323–339-specific CD4 T cell populations recovered

FIGURE 2. Numerical recovery of CD4 T cells after sepsis is accom-

panied by the acquisition of an “Ag-experienced” phenotype in an Ag-

independent manner. (A) The phenotype of CD4 T cells in the spleen was

assessed 30 d after sham or CLP surgery. Representative flow cytometry

plots depicting CD11a/CD49d and CD44/CD127 expression in CD4 T

cells 30 d after sham or CLP surgery. (B) Frequency of CD11ahiCD49dhi

and CD44hiCD127hi CD4 T cells 2 and 30 d after sham or CLP surgery. (C)

Salmonella FliC447–458-specific Sm1 (CD90.1/CD90.1; 5 3 105/mouse)

and LCMVgp61–77-specific SMARTA (CD90.1/CD90.2; 106/mouse) CD4

T cells were adoptively transferred together into naive CD90.2 B6 mice 1 d

before sham or CLP surgery. After 30 d, the frequency of CD11ahiCD49dhi

endogenous CD90.2/CD90.2 CD4 T cells and adoptively transferred

TCR-tg CD4 T cells was determined. Data shown are representative of at least

two independent experiments with four or five mice/group in each ex-

periment. *p , 0.05, **p , 0.01, ****p , 0.001, one-way ANOVA with

multiple-testing correction using the Holm–Sidak method, and a = 0.05,

when deemed appropriate. ns, not significant.

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(i.e., no statistical difference between sham and day-30 CLPmice). It is important to emphasize that the changes in individualAg-specific CD4 T cell populations were not evident when ex-amining bulk CD4 T cells in an Ag-independent manner (Fig. 1).These findings suggest that the loss of peripheral CD4 T cells aftersepsis-induced lymphopenia is asymmetric among the individualAg-specific CD4 T cell populations examined and that recoveryresults in the overall changes in the composition of the naive CD4T cell pool in sepsis survivors.

Incomplete naive precursor recovery after sepsis correlateswith reduced proliferative capacity and cytokine productionduring Ag-specific CD4 T cell responses

The magnitude of an Ag-specific CD4 T cell response after primingdirectly correlates with the size of the precursor pool (29, 45).Seeing the numerical changes in Ag-specific CD4 T cell pop-ulations in CLP-treated mice, we examined the impact of septicinjury on Ag-specific CD4 T cell responses after secondary het-erologous pathogen challenge (Fig. 4A). To test this, we used anAg-specific approach to track the CD4 T cell response to themodel Ag (Ca-2W1S) (23). This design allowed us to model anopportunistic superinfection that is common in sepsis survivorsduring convalescence (46). When CLP-treated mice were infected

with Ca-2W1S on day 2 after surgery, CD4 T cell responses werereduced significantly compared with sham mice (data not shown),which was not surprising given the dramatic reduction in CD4T cell numbers at this time point. When mice were inoculated withCa-2W1S 30 d after surgery, we still saw a significant reductionin the peak 2W1S-specific CD4 T cell–proliferative response inCLP-treated mice compared with sham-treated mice given thesame infection (Fig. 4B, 4C). To determine whether the pathogenused influenced the 2W1S-specific CD4 T cell response, sham-and CLP-treated mice were infected with Lm-2W1S 30 d aftersurgery. Assessing the CD4 T cell response to 2W1S or the en-dogenous LLO190–201 epitope of Listeria listeriolysin-O revealeda significantly reduced expansion for both Ag-specific CD4 Tcell populations in CLP-treated mice compared with sham mice(Fig. 4D, 4E). We next examined the response in Ag-specificCD4 T cell populations that numerically recovered by 30 d afterCLP surgery. Postinfection with recombinant attenuated Lm-OVA(Fig. 4F, 4G) or HSV-1 (Fig. 4H, 4I), we found that the quanti-tative expansion of OVA323–339-specific or HSVgD290–305-specific(47, 48) CD4 T cells was similar, regardless of sham or CLPsurgery. Additionally, we did not see any statistically significantdifference in the proliferative capacity of the NP311-specific CD4T cell population in sham- and CLP-treated mice after i.n. IAV

FIGURE 3. Numerical recovery of CD4 T cells after

sepsis is accompanied by asymmetric changes in Ag-spe-

cific repertoire heterogeneity. Mice underwent sham or CLP

surgery, and the number of Ag-specific CD4 T cells specific

for 2W1S, L. monocytogenes LLO190–201, IAV NP311–325,

LCMVgp66–77, HSVgD290–302, and OVA323–339 was deter-

mined 2 and 30 d later using p:I-Ab tetramer enrichment. (A)

Representative flow plots showing gating strategy used in

tetramer-enriched cell fractions to detect the frequency of

Ag-specific CD4 T cell populations. Shown is an example

used to detect 2W1S:I-Ab–specific CD4+ T cells. Gating for

p:I-Ab–specific cells was determined using CD8+ T cells as

an internal negative control for tetramer binding. (B)

Number of Ag-specific, naive CD4 T cell precursors across

the six epitopes in sham- and CLP-treated mice 2 or 30 d

after surgery. Data shown are the combined results from two

to four independent experiments/population analyzed, with

three to five mice/group in each experiment. ****p, 0.001,

group-wise, one-way ANOVA with multiple-testing correc-

tion using the Holm–Sidak method, and a = 0.05. ns, not

significant.

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infection (Fig. 4J, 4K), which was interesting because this Ag-specific CD4 T cell population increased numerically after sepsis-induced lymphopenia (Fig. 3). However, the majority of theNP311-specific CD4 T cells in uninfected CLP-treated miceadopted a memory phenotype (i.e., CD44hi; data not shown) at day30 postsurgery. Recent data suggest that naive CD8 T cells(cognate Ag inexperienced) give rise to more effector CD8T cells than primary memory CD8 T cells when analyzed on aper-cell basis after cognate infection (49). Although it remains tobe tested whether the same phenomenon is true for naive andmemory CD4 T cell responses, these results suggest that thephenotype of the CD4 T cells (naive versus “memory”-like), inaddition to the numbers of cells present, might contribute to thein vivo response to cognate Ag recognition. In summary, the datain Fig. 4 show that the proliferative capacity of an Ag-specificCD4 T cell population after sepsis correlates with the degree(reduced versus complete or increased) of numerical recovery ofits naive precursor population, and the differences seen are intrinsic

to the Ag-specific CD4 T cell populations examined and are not dueto the pathogen used.We next examined the function of Ag-specific CD4 T cell

populations that underwent a sepsis-induced numerical reduction(2W1S specific) or not (OVA323 specific) in CLP-treated miceinfected with Lm-2W1S or Lm-OVA 2 or 30 d after surgery viain vivo peptide restimulation (31) (Fig. 5A), which permits eval-uation of cytokine production with almost no background staining(Fig. 5B). There was a persistent reduction in the frequency andnumber of IFNg+ (Fig. 5C) or TNFa+IFNg+ (Fig. 5D) 2W1S-specific CD4 T cells in CLP-treated mice compared with shamcontrols. In contrast, there was no difference in the frequencyor number of IFNg+ or TNFa+IFNg+ OVA323–339-specific CD4T cells (Fig. 5E–G). Because CD4 T cells can adopt differenteffector phenotypes based on the pathogen encountered, we ex-amined the function of total and 2W1S-specific CD4 T cells afteran epicutaneous Ca-2W1S infection that primes for a Th17 re-sponse (Fig. 6A) (23, 50, 51). There was no significant difference

FIGURE 4. Expansion of epitope-specific populations correlates with precursor pool recovery after septic injury. (A) Experimental design. Mice were

infected with Ca-2W1S (53 104 yeasts in 0.1 ml i.v.), Lm-2W1S or Lm-OVA (107 CFU in 0.1 ml i.v.), HSV-1 (2.53 104 PFU in 0.1 ml i.v.), or IAV (X31;

3000 EID50 in 0.02 ml i.n.) 30 d after sham or CLP surgery. After another 7–12 d, the frequency and number of Ag-specific CD4 T cells were determined in

the spleen. Representative flow plots showing the frequency (B) and number (C) of 2W1S-specific CD4 T cells in the spleens from sham- and CLP-treated

mice 7 d after i.v. infection with Ca-2W1S. Representative flow plots showing the frequency (D) and number (E) of LLO190-specific and 2W1S-specific

CD4 T cells in the spleens from sham- and CLP-treated mice 7 d after i.v. infection with Lm-2W1S. Representative flow plots showing the frequency (F)

and number (G) of OVA323-specific CD4 T cells in the spleens from sham- and CLP-treated mice 7 d after i.v. infection with Lm-OVA. Representative flow

plots showing the frequency (H) and number (I) of gD290-specific CD4 T cells in the spleens from sham- and CLP-treated mice 9 d after i.v. infection with

HSV-1. Representative flow plots showing the frequency (J) and number (K) of NP311-specific CD4 T cells in the lungs and spleens from sham- and CLP-

treated mice 12 d after i.n. infection with X31. Data shown are the combined results from two to four independent experiments/pathogen tested, with three

to five mice/group in each experiment. *p , 0.05, **p , 0.01, group-wise, one-way ANOVA, followed by multiple-testing correction using the Holm–

Sidak method, with a = 0.05. ns, not significant.

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in the frequency and number of IL-17A+ CD4 T cells from shamand CLP-treated mice after PMA/ionomycin stimulation (Fig. 6B,6C). However, after stimulation, significant reductions were seenin the frequency and number of 2W1S-specific CD4 T cells fromCLP-treated mice making IL-17A (Fig. 6B, 6D). Together, thesedata demonstrate that (at least) for 2W1S-specific CD4 T cells,sepsis leads to fewer naive precursors with a reduced capacity toproliferate and make effector cytokines (on a per-cell basis) fol-lowing antigenic stimulation.

Sepsis alters TCR clonotype composition

Data in Figs. 1 and 2 suggest that homeostatic proliferation is onemeans by which CD4 T cells recover from sepsis-induced lym-phopenia. However, homeostatic proliferation is limited in that itcan only recreate the peripheral T cell pool from the availablediversity. Given that changes have been detected in T cell reper-toire diversity in septic patients (52), we analyzed TCRb variablechain isotype (TCR Vb) frequencies within the total and 2W1S-specific CD4 T cell populations to determine the extent to whichsepsis affects clonotype diversity. Due to the technical limitationof analyzing naive Ag-specific CD4 T cell populations # 200cells, we used a method to approximate clonotype diversity of anAg-specific CD4 T cell population whereby excess peptide Ag

was injected i.v. (29), and the resultant expansion of Ag-specificCD4 T cells was examined 4 d later via p:I-Ab tetramer enrich-ment. To maximize Vb identification within the Ag-specificpopulation, we used TCR Vb-specific mAb multiplexed into twoflow cytometry panels (Fig. 7A). Similar to previous data (19), wedid not detect significant changes in the TCRVb clonotypes whenexamined at the total CD4 T cell population level (Fig. 7B, 7C).Although Vb distribution of 2W1S-specific CD4 T cells in sham-treated mice was consistent with previous data (29), a skewing inthe number of 2W1S-specific CD4 T clonotypes (specificallyVb2, Vb5.1/5.2, Vb6, Vb8.1/8.2, and Vb10b) from CLP-treatedmice was seen (Fig. 7B, 7D). These data show that an Ag-specificCD4 T cell population that is numerically truncated in a postsepsishost after recovery also has altered clonal diversity (based on TCRVb usage), which could contribute to the subsequent reduction infunction of Ag-specific CD4 T cells.

DiscussionSepsis represents an unmet challenge in medicine. Despite modernintensive care practices, mortality from sepsis holds at 30–50%(53). Patients surviving a septic event often have suppressedimmune function, a state that is thought to contribute to the in-creased susceptibility to (and mortality from) secondary nosocomial

FIGURE 5. Inadequate recovery of precursor population correlates with inadequate CD4 T cell function after pathogen challenge. (A) Experimental

design. Mice were infected with attenuated Lm-2W1S or Lm-OVA (107 CFU in 100 ml i.v.) 2 or 30 d after sham or CLP surgery. After another 7 d, the mice

were injected i.v. with 100 mg 2W1S56–68 or OVA323–339 peptide. Spleens were harvested 2 h later, and the frequency and number of IFNg+ or TNFa+IFNg+

CD44+2W:I-Ab+ or OVA323:I-Ab+ CD4 T cells were determined. (B) Representative flow plots of intracellular IFN-g and TNF-a detection in CD44+2W:I-Ab+

CD4 T cells after in vivo peptide restimulation. Plots show cells gated from internal control populations (CD44lo2W:I-Ab2 CD4 T cells) denoted as

background (“BKG”) or 2W:I-Ab–enriched CD4 T cells from sham- or CLP-treated mice. Frequency and number of CD44+2W:I-Ab+–specific CD4 T cells

in the spleen producing IFN-g (C) or TNF-a and IFN-g (D). (E) Representative flow plots of intracellular IFN-g and TNF-a detection in the CD44+OVA323:I-Ab+

CD4 T cells after in vivo peptide restimulation. Plots show cells gated from internal control populations (CD44loOVA323:I-Ab2 CD4 T cells) denoted as

background (“BKG”) or OVA323:I-Ab–enriched CD4 T cells from sham- or CLP-treated mice. Frequency and number of CD44+OVA323:I-A

b+–specific CD4

T cells in the spleen producing IFN-g (F) or TNF-a and IFN-g (G). Data shown are the combined results of two independent experiments, with three to five

mice/group in each experiment. **p, 0.01, ***p, 0.005, ****p, 0.001, group-wise, one-way ANOVA, followed by Holm–Sidak correction with a = 0.05.

ns, not significant.

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infections. A number of studies examined the numerical andfunctional changes in various immune cell subsets after sepsis, butthey did so at the total population level. The goal of this study wasto analyze the quantitative and qualitative changes in CD4 T cells,but at the level of Ag-specific populations, which permits a more

rigorous and sensitive analysis of how these cells perform undervarious immunological settings. With this in mind, we performed,for the first time to our knowledge, quantitative and qualitativeanalyses of multiple Ag-specific CD4 T cell populations in septicmice, before and after secondary heterologous infections. Our datademonstrate that a septic event induces deletion within each en-dogenous Ag-specific CD4 T cell population examined and thatthis event is followed by a recovery of the Ag-specific repertoire inan irregular, or asymmetric, fashion. Moreover, our results showthat sepsis-induced numerical changes to certain Ag-specific CD4T cell populations can affect the function of the cells in questionduring the subsequent response to a pathogenic challenge.Clearly defining the mechanism by which lymphocyte apop-

tosis occurs after sepsis is difficult, because no single intrinsic orextrinsic pathway dominates (54, 55). Similarly, there has beenlimited investigation into the mechanism(s) behind lymphocyterecovery after a septic event. The sole publication examiningthe process of CD4 T cell recovery following septic injury sug-gested that CD4 T cells did not undergo homeostatic proliferationduring recovery from sepsis (19). This conclusion was largelyreached by adoptively transferring a large number of TCR-tg CD4(OT-II) T cells into septic mice 7 d after surgery. Although thesecells were introduced into a lymphopenic environment, it could beargued that, because these CD4 T cells did not “experience” theseptic event, any T cell–intrinsic changes that occur during sepsiswould not be present in these cells. Furthermore, it is clear thatadoptive-transfer experiments that use nonphysiologically largeinput numbers of TCR-tg T cells do not accurately recapitulatethe endogenous Ag-specific T cell response (56). In contrast, thesimilar recovery of CD4 T cell numbers in thymectomized andeuthymic mice, along with the similar rates of BrdU incorporationin CD4 T cells of CLP-treated euthymic and thymectomized mice,suggests that CD4 T cells do indeed undergo homeostatic prolif-eration after septic injury. Unsinger et al. (19) then went on toshow increased frequencies of CD4 T cells in septic mice with“activated” (CD69+) and “memory” (CD44hiCD62Llo) pheno-types, leading them to suggest that the majority of activated andmemory CD4 T cells arise from endogenous sources. Our data inFig. 3 are consistent with these findings by Unsigner et al. (19),but extend them to identify cells with phenotypes consistent with“homeostatic proliferation” for both endogenous CD4 T cells andadoptively transferred TCR-tg CD4 T cells (37, 42). It remains tobe determined what the driving factor(s) is for the numerical re-covery of CD4 T cells after sepsis. In addition to recovery byhomeostatic proliferation, it is likely that some CD4 T cell pop-ulations respond directly to antigenic epitopes present in theproteins expressed by the various commensal bacterial specieswithin the gut or to self-Ag, leading to a difference in functionalpotential compared with those CD4 T cells truly undergoing Ag-independent homeostatic proliferation. The cecum contains a highconcentration of microbes that are a combination of Gram-positiveand Gram-negative bacterial species, and the Ag expressed bythese bacteria can be recognized by T cells and can drive effectorresponses, despite being commensal bacteria (57). It is tempting tospeculate that the above-normal numerical recovery in the NP311-specific CD4 T cell population in CLP-treated mice is due to di-rect antigenic stimulation as a result of cross-reactivity with someyet-to-be defined epitope expressed by the gut commensal bacte-ria, especially because the majority of NP311-specific CD4 T cellsin CLP-treated mice were also CD44hi (data not show). As a re-sult, this population of cells could be considered a “memory”population with different functional characteristic, such as pro-ducing fewer effectors after influenza infection (49), over true“naıve” cells. The bacterial constituents of the gut microbiome are

FIGURE 6. Ag-specific CD4 T cells have functional deficits in Th17-

polarized responses. (A) Experimental design. On day 30 after sham or

CLP surgery, mice were infected epicutaneously with Ca-2W1S (108

yeasts in 0.05 ml). After 7 d, lymphocytes obtained from the skin-draining

(inguinal, brachial, axillary, and cervical) LNs of infected mice were

stimulated for 4 h with PMA/ionomycin. The stimulated samples were

enriched for 2W1S-specific CD4 T cells, and production of IFNg and IL-

17Awas assayed by flow cytometry. (B) Representative flow plots showing

the gating strategy to identify IL-17A+IFNg2 cells within bulk and 2W:I-Ab–

specific CD4 T cells. Frequency and number of IL-17A+ in bulk (C) and

2W1S-specific (D) CD4 T cells in infected sham- or CLP-treated mice.

Data shown are representative results from two independent experiments,

with five mice/group in each experiment. *p , 0.05, **p , 0.01, Welch t

test. ns, not significant.

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unique to each individual (especially humans), and they can bestrongly influenced by a variety of factors (58). Consequently, theextent of recovery and function of a particular Ag-specific T cellpopulation after a septic event can easily be different as a resultof the intestinal “health” of the individual, regardless of possiblegenetic similarities (e.g., same mouse strain from different ven-dors). As reagents become available to track CD4 T cell pop-ulations specific to Ag expressed by specific gut commensalbacteria, it will be interesting to investigate the potential impactof this component of polymicrobial sepsis on the recovery andfunction of such Ag-specific CD4 T cell populations in septicmice.When examined at the bulk CD4 T cell level, our data are

consistent with a number of other studies demonstrating sepsis-induced changes in the basic numerical and functional charac-teristics of CD4 T cells. It is important to emphasize that thesepsis-induced changes in the different Ag-specific CD4 T cellpopulations would not have been identified had we examinedCD4 T cells as a whole. This includes the changes in naive pre-cursor numbers, proliferative capacity, and cytokine production bythe different Ag-specific populations. Although we showed re-cently that sepsis significantly decreases the Ag sensitivity ofmemory CD8 T cells (35), it remains to be determined to what

extent Ag sensitivity is affected in naive or memory Ag-specificCD4 T cell populations. In addition, TCR Vb repertoire usage onCD4 T cells from sham- and CLP-treated mice was investigatedpreviously. The investigators found no skewing of the repertoiretoward one particular Vb subtype (19); however, this conclusionwas based on the analysis of bulk CD4 T cells. Just as we onlyobserved stochastic changes in the number of naive CD4 T cellswhen we examined Ag-specific CD4 T cell populations using p:I-Ab

tetramers, alterations in Vb repertoire usage after sepsis were ob-served when examining the endogenous 2W1S-specific CD4 T cellpopulation. We realize that the method for examining Vb repertoireusage required in vivo 2W1S56–68 peptide immunization to expandthis Ag-specific population of cells, but it is important to emphasizethat this technique results in an expanded T cell population that isreflective of the clonotype diversity of the naive starting population(29). Even after the expansion, 2W1S:I-Ab tetramer enrichment andthe multiplexed flow cytometry panel of Vb-specific mAbs wereneeded to complete the analysis of the endogenous 2W1S-specificCD4 T cell population. These results show the power of using thesereagents and techniques to analyze small numbers of endogenousAg-specific CD4 T cell populations.Pools of Ag-specific CD4 T cell “precursors” are maintained in

the periphery by frequent, low-level signals from self-Ag:MHC II

FIGURE 7. Sepsis alters the TCR clonotype composition of Ag-specific CD4 T cell population. (A) Experimental design. Mice were injected i.v. with 50

mg 2W1S56–68 peptide (along with LPS) 30 d after sham or CLP surgery. Splenocytes were harvested 4 d later and tetramer-enriched, as previously

described. The resultant sample was used to determine the clonotype composition using two multiplexed flow cytometry panels consisting of the indicated

murine TCRVb mAbs. Representative flow plots showing the gating strategy used to identify Vb usage on 2W:I-Ab–specific CD4 T cells using TCR panel

1. (B) Usage profile for TCRVb gene segments in bulk and 2W:I-Ab–specific CD4 T cells from three representative individual sham- or CLP-treated mice.

Averaged frequency of TCRVbof bulk (C) and 2W:I-Ab–specific (D) CD4 T cells in sham- or CLP-treated mice. Data are combined from two independent

experiments, each having 5 mice/group. *p , 0.05, **p , 0.01, group-wise, one-way ANOVA analyses followed by Holm–Sidak correction with a = 0.05.

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and cytokines [most notably, IL-7 for naive CD4 T cells (59) andIL-15 for naive CD8 T cells (60)]. The increased availability ofthese resources turns survival signals into mitogenic stimuli thatrestore T cell numbers through proliferative expansion in sit-uations where T cell numbers drop acutely (61). With this in mind,there is considerable effort being spent to identify therapeuticstrategies designed to enhance T cell recovery and function aftersepsis, and the administration of agents that promote lymphocyteproliferation [e.g., IL-2, IL-7, and IL-15 (62-64)] or block thefunction of inhibitory molecules [e.g., PD-1 (65, 66) and CTLA-4(67)] are producing encouraging results. For example, adminis-tration of IL-7 to septic mice shortly after sepsis induction canprevent T cell apoptosis and restore function (63, 64). Moreover,disruption of the PD-1:PD-L1 signaling pathway improves sur-vival in animal models of sepsis (68, 69) and reverses T cell ex-haustion in sepsis patients (66). Additional work is needed todetermine the impact of such therapies at the level of Ag-specificT cell populations where, even if apparently subtle, physiologi-cally meaningful changes may actually be present.In the current study, we assessed CD4 T cell function after sepsis

primarily within the context of a “Th1” response, but it is importantto emphasize that sepsis likely affects other CD4 T cell subsetsneeded for a variety of immunological responses. For example,Th1 cells also provide necessary signals for B cell isotype switch-ing (70), and IL-4 from Th2 cells also facilitates B cell isotypeswitching to IgG1 and IgE (71). Th17 cells are important in im-munity to extracellular fungal and bacterial pathogens (72), and wesaw a reduction in the Th17 response when using a secondaryepicutaneous C. albicans after sepsis (Fig. 6). Thus, the loss orimproper function of CD4 T cell responses is detrimental for im-munity to a wide range of pathogens, especially those that fre-quently cause secondary infections in septic patients. In addition tothe different “effector” CD4 Th subsets, CD4+CD25+Foxp3+ reg-ulatory T cells (Tregs) have been found at an increased frequencyin septic patients, especially early after diagnosis (73–75). Subse-quently, it was determined that the increased frequency of Tregswas the consequence of decreases in the effector CD4 T cell pop-ulations (76), suggesting that Tregs are more resistant to sepsis-induced apoptosis than are conventional CD4 T cells (16). Re-gardless, the role of Tregs in the immunosuppression after sepsishas not been rigorously investigated and merits study. We also re-alize that the sepsis-induced alterations in the Ag-specific CD4T cell populations that we examined could be due to CD4 T cell–intrinsic and/or -extrinsic factors. Because CD4 T cell stimulationrequires Ag presentation from professional APCs, changes in thenumber and/or function of dendritic cells could contribute to theobserved modulation in CD4 T cell function (77–79).The development and integration of a number of reagents and

techniques over the last decade have permitted the characterizationof Ag-specific endogenous naive and memory T cell responses toa handful of experimental bacterial (e.g., L. monocytogenes) orviral (e.g., LCMV) pathogens in exquisite detail. These fea-tures have given us the ability to track the quantity and quality ofendogenous CD4 T cells reactive to antigenic epitopes withinthese pathogens. Yet, one weakness of these (and other well-characterized) experimental pathogens is that they are typicallynot seen as nosocomial infection threats for septic patients. Mostseptic patients potentially face complications arising from sec-ondary infection by the extracellular pathogens Candida, Pseu-domonas, and Staphylococcus (46, 80, 81), and these pathogenshave been used most often to examine alterations in animal sur-vival in experimental models of sepsis. It is important to keep inmind that our use of Lm-2W1S for most of the studies was to takeadvantage of the wealth of information regarding the infectivity/

pathogenicity of this pathogen, the characteristic CD4 T cell re-sponse that it elicits, and the availability of reagents to criticallyinvestigate different aspects of this response. Moreover, althoughour analysis of sepsis-induced alterations in the responsiveness of2W1S-specific CD4 T cells was largely performed after secondaryinfection with attenuated Lm-2W1S, we found similar reductionsin the proliferative capacity of 2W1S-specific CD4 T cells post-infection with Ca-2W1S (Fig. 4). These data serve as a startingpoint for future studies using yet-to-be-developed reagents fortracking CD4 T cell populations specific for Ag within pathogensthat commonly plague sepsis patients.In summary, the data presented in this article elucidate with

detail how CD4 T cells are affected by sepsis. Our results reveal thatthe recovery of individual Ag-specific CD4 T cell populations aftera septic event is skewed, which is not evident when examining thebulk CD4 T cell pool. In addition, we show that an incompleterecovery of the Ag-specific T cell repertoire after sepsis stems fromthe attrition of Ag-specific TCR diversity and that this phenomenoncorrelates with altered CD4 T cell responses long after sepsis.Ultimately, this study increases our collective understanding ofwhy septic patients more easily acquire secondary infections.

AcknowledgmentsWe thank Dr. Jon Linehan (Jenkins Lab, Center for Immunology,

University of Minnesota) for assistance with the production of the gD290

and NP311:I-Ab tetramers.

DisclosuresThe authors have no financial conflicts of interest.

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