Central memory T cells mediate long-term immunity to Leishmania major in the absence of persistent parasites

Transcription

1 Centrl memory T cells medite long-term immunity to Leishmni mjor in the sence of persistent prsites Coly Zph 1,3,Jude Uzonn 1,3,Stephen M Beverley 2 & Phillip Scott 1 Infection with Leishmni mjor induces protective immune response nd long-term resistnce to reinfection, which is thought to depend upon persistent prsites. Here we demonstrte tht lthough effector CD4 + T cells re lost in the sence of prsites, centrl memory CD4 + T cells re mintined. Upon secondry infection, these centrl memory T cells ecome tissue-homing effector T cells nd medite protection. Thus, immunity to L. mjor is medited y t lest two distinct popultions of CD4 + T cells: short-lived pthogen-dependent effector cells nd long-lived pthogen-independent centrl memory cells. These dt suggest tht centrl memory T cells should e the trgets for nonlive vccines ginst infectious diseses requiring cell-medited immunity. Experimentl infections with Leishmni mjor hve helped define the requirements for the development of T helper type 1 (T H 1) cells in vivo 1,2.Yet how Leishmni-specific memory CD4 + T cells develop nd re mintined is not understood. This knowledge is criticl for the development of leishmnil vccines. Indeed, lthough mny studies hve een done with experimentl vccines ginst Leishmni 3 7, currently there is no vccine for humn leishmnisis, disese tht cuses considerle moridity nd mortlity throughout the world 8. Resolution of Leishmni infection results in lifelong immunity in oth mice nd humns 2.Control of primry infection does not eliminte ll prsites, nd the persistent prsites my contriute to the ility of heled nimls to mintin immunity. For exmple, very low doses of L. mjor induce protection in BALB/c mice, ut immunity is lost in nimls tht eliminte ll of the prsites 9. Similrly, L. mjor infected interleukin (IL)-10 deficient mice show sterile cure (no persistence of pthogenic orgnisms) nd lose their resistnce to reinfection 10.These results could suggest tht leishmnil infections nd possily other chronic infectious diseses do not induce true memory response, nd tht resistnce in leishmnisis reflects the continued presence of effector T (T EFF ) cells resulting from the persistent existence of pthogenic orgnisms. If leishmnil infections fil to generte memory T cells, it my e difficult to develop nonlive vccine ginst leishmnisis. This hs prompted us to chrcterize the CD4 + T cells tht provide immunity to reinfection with L. mjor. Recent studies hve shown tht memory T cells re heterogeneous, contining susets tht migrte through lymph nodes termed centrl memory T (T CM ) cells nd others tht migrte to tissues nd mke effector cytokines (effector memory T (T EM ) cells) Experiments with severl pthogens indicte tht CD8 + T CM cells re derived from T EM cell popultion, develop once the pthogen hs een clered, nd medite protective immunity 16,17.Much less is known out CD4 + T-cell memory, nd sufficient differences with memory CD8 + T cells hve een noted to suggest tht CD4 + nd CD8 + T-cell medited immunologic memory my e distinct 15. Here we chrcterize the CD4 + T cells tht medite infectioninduced immunity to L. mjor nd directly ddress the role of prsite persistence in the development nd mintennce of CD4 + T-cell memory. Using doptive trnsfer of polyclonl T cells from immune mice, we demonstrte tht Leishmni-specific T CM cells develop in the presence of prsites nd cn provide protection to chllenge infection. We lso show tht wheres effector T cell responses re lost if prsites re eliminted, T CM cells re mintined nd protect mice ginst chllenge infection. These dt suggest tht expnsion of the T CM cell popultion, rther thn trgeting short-lived effector T cells, should e the gol for vccines ginst leishmnisis nd possily other infectious diseses requiring cell-medited immunity. RESULTS mice mintin lymph node homing CD4 + T cells Resistnce to reinfection is thought to e controlled in prt y the presence of T EFF cells tht migrte to the chllenge site nd medite delyedtype hypersensitivity (DTH). This response does not require homing of T cells to the lymph nodes, s tretment of immune C57BL/6 (B6) mice during secondry infection with locking ntiody ginst CD62L, molecule tht llows T cells to enter lymph nodes from the lood, 18 rogted homing to lymph nodes, ut hd no effect on DTH or immunity (see Supplementry Fig. 1 online). Becuse nother criticl component of memory my e the ility of T cells to expnd upon reinfection, we sked whether second popultion of T cells from immune mice nlogous to T CM cells preferentilly homes to the lymph nodes 1 Deprtment of Pthoiology, University of Pennsylvni, 3800 Spruce Street, Phildelphi, Pennsylvni 19104, USA. 2 Deprtment of Moleculr Microiology, Wshington University, 660 South Euclid Avenue, St. Louis, Missouri 63110, USA. 3 These uthors contriuted eqully to this work. Correspondence should e ddressed to P.S. (pscott@vet.upenn.edu). Pulished online 26 Septemer 2004; doi: /nm VOLUME 10 NUMBER 10 OCTOBER 2004 NATURE MEDICINE

2 Dy 0 uninf inf Dy 0 Dy 3 Dy 7 Figure 1 Centrl memory CD4 + T cells develop during L. mjor infection. () cells proliferte in the dln. Mice (Thy1.2) were chllenged with L. mjor fter receiving CFSE-leled CD4 + T cells (Thy1.1) from nive or immune mice, nd histogrms gted on donor cells from the dlns re shown; numers represent percentge of donor CFSE dim T cells. () T CM cells downregulte CD62L nd migrte to lesions. CD4 + T cells were trnsferred s in. Bold numers represent percentge of donor CFSE dim T cells; numers in corners represent percentge of CD62L high or CD62L low CFSE dim cells. Dt re representtive of three or more experiments. Uninf, uninfected; inf, infected. T CM cells downregulte CD62L nd migrte to lesions The lymph node homing molecule CD62L is downregulted fter ctivtion, nd loss of CD62L expression is coupled to the development of tissue-homing T EFF cells during n immune response Before doptive trnsfer, CD4 + T cells from immune mice contined higher percentge of CD62L low nd CD44 high cells thn those from nive nimls (Supplementry Fig. 3 online). At dy 3 fter chllenge, 71% of the cells in the dln tht responded to infection y proliferting expressed high levels of CD62L, wheres y dy 14 only 26% of the proliferting cells still mintined expression of CD62L (Fig. 1). Moreover, y 2 weeks fter infection, 80% of the donor T cells in the lood were CFSE dim, indicting they hd proliferted, nd most hd downregulted CD62L. We were unle to isolte mesurle numers of donor T cells t the site of infection t 3 or 7 d, ut y 2 weeks donor T cells tht hd divided nd lost expression of CD62L most likely tissuehoming emigrnts from the dln were present in the lesions. Although the mjority of proliferting donor T cells in the dln downregulted expression of CD62L, some mintined high levels of CD62L. Becuse we found popultion of CD62L high CFSE dim CD4 + T cells in the nondrining lymph nodes (ndln) (Fig. 1), we elieve tht fter prolifertion in the dln, the suset of donor T cells tht mintined or re-expressed CD62L migrted to nd through the ndln. This would llow the mintennce of popultion of ntigen-experienced, lymph node homing T cells with migrtory nd phenotypic chrcteristics of T CM cells, even s the mjority of the T CM cells differentite into T EFF cells. drining the site of chllenge nd prolifertes. To ddress this question, we trnsferred croxyfluorescein dicette succinimidyl ester (CFSE)- leled CD4 + T cells from nive or immune B6 mice into nive Thy1- disprte B6 recipients; trnsfer of purified CD4 + T cells from immune mice provides protection in nive recipients (Supplementry Fig. 2 online). These mice were susequently chllenged with L. mjor, nd we ssessed the prolifertive response of the donor T cells in the drining poplitel lymph nodes (dln). We found no evidence of spontneous prolifertion y donor T cells from either immune or nive mice fter trnsfer into mice tht were not chllenged with L. mjor. In contrst, donor T cells from oth nive nd immune mice were found to proliferte in the dln fter chllenge (Fig. 1). Prolifertion y immune T cells ws sustntilly greter thn tht y nive T cells. We oserved prolifertion, which incresed over time, of donor T cells from immune mice in the dln s erly s 3 d fter chllenge infection (Fig. 1). These dt indicte tht n expnded popultion of ntigen-rective CD4 + T cells persists in immune mice tht, s indicted y their lymph node homing cpcity, possess the primry ttriute of T CM cells. CD62L defines distinct Leishmni-specific CD4 + T cells We next sked whether differentil expression of CD62L would define not only the migrtory potentil of Leishmni-rective T cells, ut lso their cytokine profiles. When T cells from immune mice were stimulted in vitro with solule leishmnil ntigen (SLA), two distinct popultions of responding cells could e identified sed on CD62L expression (Fig. 2). We found tht interferon-γ (IFN-γ) production ws restricted primrily to the CD62L low cells, wheres n equivlent percentge of CD62L high nd CD62L low cells produced IL-2. Despite the similr percentges of IL-2 producing cells, the men fluorescence intensity (MFI) ws twofold higher in CD62L low CD4 + T cells. To confirm tht ntigen-specific IFN-γ production ws confined to CD62L low CD4 + T cells, we purified CD62L high nd CD62L low frctions from immune mice nd stimulted them with SLA. Although oth CD62L high nd CD62L low CD4 + T cells responded to SLA y proliferting, the production of IFN-γ ws restricted to the CD62L low popultion (Fig. 2). Consistent with these results, the ccumultion of IFN-γ in the superntnts ws significntly higher in the CD62L low popultion (Supplementry Fig. 4 online). To determine whether CD62L expression defined functionlly distinct T cell susets in vivo,we nlyzed the cytokine responses of trnsferred immune cells fter infection. Only cells tht hd proliferted in response to infection produced sustntil levels of IFN-γ (Fig. 2c). NATURE MEDICINE VOLUME 10 NUMBER 10 OCTOBER

3 A smll popultion of donor T cells from nive mice produced IFN-γ in the dln 2 weeks fter infection. But s erly s 1 week fter infection, lrge percentge of immune donor T cells produced IFN-γ in the dln, which incresed y 2 weeks fter infection. Consistent with our in vitro results, the overwhelming mjority of the IFN-γ producing cells in the dln were contined in the CD62L low popultion (Fig. 2d). Furthermore, the donor T cells present in the lesion expressed low levels of CD62L nd >90% of the cells produced IFN-γ.The higher percentge of IFN-γ pos CD62L low cells in the lesion compred with the dln my reflect n dditionl differentition of the effector T cell popultion, such s the cquisition of P- nd E-selectin lignds 21. Although there ws mesurle popultion of proliferting donor T cells in the ndlns, the cells were predominntly IFN-γ neg nd CD62L high (Fig. 2c,d). These results demonstrte phenotypic nd functionl dichotomy of ntigen-experienced CD4 + T cells tht develop fter infection with L. mjor lymph node homing, CD62L high, IFN-γ neg T CM cells nd tissue-homing, CD62L low, IFN-γ pos T EFF cells. T CM cells medite protective immunity To directly ddress whether the T CM cells present in immune mice could differentite into T EFF cells nd medite protection, we purified CD4 + CD62L high T cells from immune mice nd tested their protective cpcity. Immunity ws evident in mice tht received CD4 + CD62L low T cells 3 weeks fter chllenge, consistent with their ility to rpidly produce IFN-γ (Fig. 3). Despite the lck of ntigenspecific IFN-γ production y CD62L high CD4 + T cells (Fig. 2), y 6 weeks mice tht received either CD62L low or CD62L high CD4 + T cells were protected ginst chllenge infection (Fig. 3). We hypothesized tht the CD62L high CD4 + T cells provided protection to L. mjor chllenge y differentiting into tissue-homing T EFF cells. To test this, we trnsferred purified CFSE-leled immune CD62L high CD4 + T cells into nive mice nd chllenged with L. mjor.by 2 weeks fter chllenge, there ws equivlent prolifertion in the dln of mice tht received totl CD4 + T cells nd purified CD62L high cells (Fig. 3c). Criticlly, in mice tht received purified CD62L high T cells, popultion of donor CD4 + T cells ws isolted from the site of infection t 2 weeks tht ws uniformly CFSE dim nd expressed low levels of CD62L (Fig. 3c). These results provide direct evidence tht lymph node homing T CM cells cn medite protective immunity ginst L. mjor infection y differentiting into tissue-homing CD62L low T EFF cells fter chllenge. T CM cells in the ndlns medite protective immunity If T CM cells represent n expnded popultion of ntigen-specific T cells migrting though lymph nodes, then ndln from immune mice should contin T CM cells tht could medite protection. To test this, we c Dy 7 Figure 2 CD62L defines functionlly distinct popultions of CD4 + T cells. (,) Proliferting CD62L high CD4 + T cells do not produce IFN-γ. Totl () or sorted () immune cells were stimulted in vitro with SLA. Numers represent percentge of IFN-γ pos or IL-2 pos CFSE dim T cells or (MFI). (c,d) Production of IFN-γ y immune CD4 + T cells in vivo. Mice were chllenged with L. mjor fter receiving CFSE-leled CD4 + T cells from nive or immune mice. (c) Bold numers represent percentge of donor CFSE dim T cells; numers in the upper left corner represent percentge of IFN-γ pos CFSE dim cells. (d) Numers represent percentge of donor T cells in ech qudrnt. Dt re representtive of two or more experiments. ssessed the ility of cells from the ndln of immune mice to provide protection fter doptive trnsfer. Cells isolted from ndln of immune B6 mice responded to leishmnil ntigen in vitro y proliferting (Fig. 4), nd secreting IL-2 ut not IFN-γ (Fig. 4), demonstrting tht popultion of ntigen-rective T cells, ut not T EFF cells, were present in the ndln. In contrst, cells from the dln of immune mice proliferted nd produced oth IL-2 nd IFN-γ in response to ntigen stimultion. Nevertheless, despite the lck of n effector response y the cells isolted from ndln, these cells were le to confer considerle protection to chllenge infection t levels tht were comprle to cells from the dln (Fig. 4c). These dt demonstrte tht the T CM cells found in the ndln provide pool of T cells tht cn medite protective immunity. T CM cells persist in the sence of prsites One of the most importnt questions regrding immunologic memory is the role of ntigen persistence in the mintennce of functionl memory T cells. To directly ddress whether T CM cells were mintined if prsites were eliminted, we used thymidine 1106 VOLUME 10 NUMBER 10 OCTOBER 2004 NATURE MEDICINE

4 uxotrophic mutnt of L. mjor (dhfr-ts ; prsites designted s such lck the gene for dihydrofolte reductse-thymidylte synthse) tht is unle to cuse pthology or persist in vivo 22.We infected B6 mice with either wild-type L. mjor or dhfr-ts ; t 4 weeks fter infection, when prsites were detected in oth groups of mice, the immune response s mesured y ntigenspecific IFN-γ production nd DTH ws equivlent (Fig. 5,). As expected, y 15 weeks fter infection, mice infected with wildtype L. mjor contined low levels of prsites ( prsites) in the lesion nd the dln, wheres mice infected with dhfr-ts hd no detectle prsites (dt not shown) 22,23.It hs een reported tht immune responses to L. mjor re not mintined in the sence of persistent prsites 9,10.Similrly, we found tht mice infected with wild-type L. mjor mice hd DTH nd ntigen-specific IFN-γ recll responses, wheres mice infected for 25 weeks with dhfr-ts did not show Leishmni-specific DTH or IFN-γ responses (Fig. 5c,d), indicting tht Leishmni-specific T EFF cells were lost in the sence of persistent prsites. To determine whether T CM cells re present in nimls infected for 25 weeks with dhfr-ts, CFSE-leled CD4 + T cells from these mice were trnsferred into nive Thy1-disprte B6 hosts tht were susequently chllenged with L. mjor. Infection-induced prolifertion of CD4 + T cells from dhfr-ts -infected mice ws evident in the dln y 2 weeks fter chllenge, with much smller response y donor T cells from nive mice (Fig. 5e). Thus, T CM cells cn e mintined once prsites re no longer present. Persistent prsites re not required to mintin immunity To test whether the presence of T CM cells in mice tht no longer contin prsites is ssocited with long-term immunity nd resistnce to reinfection, we chllenged the mice with virulent L. mjor 25 weeks fter they hd resolved primry infection with wild-type L. mjor or dhfr-ts prsites. As mentioned ove, t this time no prsites could e detected in the dhfr-ts infected mice. Although the protection evident in mice tht hd resolved primry infection with wild-type L. mjor ws greter, we oserved significnt (P < 0.01) protection in mice tht we hd infected with dhfr-ts prsites (Fig. 5f). These results indicte tht in the sence of n effector response or persistent prsites, sustntil protective immunity is mintined. Our dt show tht T CM cells ut not T EFF cells persist in the sence of prsites, nd thus tht the protective cpcity of CD4 + T cells isolted from mice tht hd resolved infection with dhfr-ts should e found exclusively in the CD62L high comprtment. To directly ddress this, we sorted CD4 + T cells sed upon CD62L expression from mice tht hd resolved infection with dhfr-ts prsites. CD62L high, ut not CD62L low, CD4 + T cells medited protection fter trnsfer into nive hosts (Fig. 5g), further demonstrting tht in the sence of persistent prsites, T EFF cells re lost while T CM cells re mintined. c Figure 3 T CM cells medite protective immunity. (,) Protection y immune T cell popultions. Mice were chllenged with L. mjor fter receiving immune CD62L high or CD62L low cells, nd prsite urdens in lesions ssessed fter 3 () or 6 weeks (). (c) CD62L high CD4 + T cells trnsferred s in cquire the ility to home to infected tissues. Bold numers represent percentge of donor CFSE dim T cells. Numers in dot plots represent percentge of CD62L high or CD62L low CFSE dim cells. Dt re representtive of two or more experiments. *P < 0.01 compred to controls. DISCUSSION We hve found tht two CD4 + T cell popultions prticipte in mintining immunity to L. mjor, only one of which requires persistent prsites. The CD4 + T cells with the chrcteristics of effector T cells tissue homing, CD62L low, IFN-γ pos medite DTH responses, re protective nd depend on the continued presence of prsites. By doptively trnsferring CFSE-leled CD4 + T cells from Thy1-disprte immune mice, we were le to chrcterize nother polyclonl CD4 + T cell popultion ssocited with immunity. These cells hve the chrcteristics of T CM cells: they home to lymph nodes nd do not produce IFN-γ, ut upon chllenge they c Figure 4 Protective T CM cells re present in ndlns. () Prolifertive response y CD4 + T cells from lymph nodes fter stimultion in vitro. Histogrms re gted on CD4 + T cells. () Leishmni-specific IFN-γ nd IL-2 production y lymph node cells stimulted in vitro. (c) Mice were chllenged with L. mjor fter receiving immune cells, nd prsite urdens in lesions ssessed fter 6 weeks. Dt re representtive of two or more experiments. *P < 0.01 compred to controls. NATURE MEDICINE VOLUME 10 NUMBER 10 OCTOBER

5 c e Dy 0 f d WT Lm Lm g Figure 5 Prsite persistence is required for the mintennce of T EFF cells, ut not T CM cells nd protective immunity. ( d) Equivlent immune responses in mice infected for 4 weeks with wild-type L. mjor or dhfr-ts, ut loss of effector responses in the sence of persistent prsites. (,c) Leishmnispecific IFN-γ responses y dln cells from infected mice t 4 () or 25 weeks (c). (,d) DTH in infected mice t 4 () or 25 weeks (d). (e) T CM cells re mintined in the sence of persistent prsites. Mice were chllenged with wild-type L. mjor fter receiving CFSE-leled CD4 + T cells from 25-week infected mice. Donor T cells from the dln re shown; old numers represent percentge of donor CFSE dim T cells. (f)protective immunity in the sence of persistent prsites. Infected mice (25 weeks) were chllenged with wild-type L. mjor nd prsites in lesions ssessed fter 6 weeks. (g) CD62L high, ut not CD62L low, CD4 + T cells from dhfr-ts -infected B6 mice re protective. Mice were chllenged with L. mjor fter receiving sorted cells from dhfr-ts - infected mice (25 weeks), nd prsite urdens ssessed fter 6 weeks. Dt re representtive of two or more experiments. *P < 0.01 compred to controls. WT Lm, wild-type Leishmni mjor. proliferte in the dln, dopt n effector phenotype, migrte to the site of infection nd medite protective immunity. Criticlly, they do not require persistent prsites to e mintined. Becuse Leishmni-specific T CM cells survive in the sence of prsites nd medite protective immunity, expnsion of this T-cell popultion is n pproprite gol for vccines ginst pthogens tht require cellmedited immunity. How T CM cells develop is not understood, lthough previous studies with virl nd cteril infections indicte tht CD8 + T CM cells develop only fter clernce of pthogens 16,17.In contrst, our dt show tht CD4 + T CM cells develop in the presence of persistent prsites; this oservtion is consistent with studies demonstrting tht mice infected with L. mjor contin n expnded popultion of ntigen-rective CD62L high T cells tht cn ecome T H 1 or T H 2 cells 24,25. The discrepncy my reflect criticl intrinsic differences etween memory CD4 nd CD8 T cells 15.It hs een suggested tht CD4 + T CM cells re derived from T cells tht fil to differentite into T EFF cells 26 28, lthough how smll percentge of cells mintin this phenotype is unknown. Cesstion of prolifertion due to CTLA-4 expression might e involved 29 ;however, in our studies, prolifertion y itself does not seem to dictte whether cells ecome T CM cells or effector T cells. Some hve suggested tht the mintennce of cell-medited immunity requires the presence of ntigen 9,10,30,31,ut others hve shown tht memory T cells persist in the sence of ntigenic stimultion Our results demonstrte tht there re t lest two distinct popultions of CD4 + T cells tht develop fter resolution of n infection with L. mjor,only one of which requires the presence of persistent prsites. Although we show tht the pthogenindependent T CM cells medite protective immunity, optiml protection my require oth susets. Previous studies, in which mice were infected with low numers of prsites nd immunity ws lost when prsites were eliminted, my indicte tht sufficient expnsion of the T CM cell pool to provide protection depends on the initil overll T-cell response 9,10.How prsite dose influences the development nd efficcy of T CM cells will e n importnt re for future study. The findings presented here hve relevnce to the development of vccines for diseses tht require cell-medited immunity, such s leishmnisis, tuerculosis nd AIDS 38,39.Vccines tht expnd the effector T cell pool my not led to long-term protection, ecuse these cells re short-lived in the sence of persistent ntigen. However, the pproprite T cells to expnd my e T CM cells. The moleculr mechnisms involved in mintining T CM cells in vivo, s well s the protocols necessry to optimlly induce these cells, re still unknown; defining oth will e criticl for etter understnding CD4 + T cell memory nd for the rtionl development of vccines ginst rod rnge of pthogens. METHODS Animls. C57BL/6J (B6) nd B6.PL-Thy1 /Cy (B6 Thy1.1) mice were otined from the Jckson Lortories. Animls were mintined in specific pthogen free environment nd tested negtive for pthogens in routine screening. All experiments were crried out following the guidelines of the University of Pennsylvni Institutionl Animl Cre nd Use Committee. Prsites nd ntigens. We used wild-type L. mjor (WHO/MHOM/IL/ 80/Friedlin, wild-type L. mjor) nd thymidine uxotrophic L. mjor (E10-5A3, dhfr-ts ) 22 for these studies. We grew prsites to sttionry phse in Grce s insect cell culture medium (Life Technologies) with 20% fetl ovine serum (Hyclone; EU/ml), 100 U/ml of penicillin, 100 µg/ml of streptomycin nd 2 mm glutmine (Sigm). We dded thymidine (10 µg/ml, Sigm) to cultures of dhfr-ts prsites. We hrvested sttionry-phse promstigotes nd wshed them three times in PBS. SLA ws prepred s descried previously VOLUME 10 NUMBER 10 OCTOBER 2004 NATURE MEDICINE

6 Infections. For primry infections, we injected the hind footpds of mice with sttionry-phse wild-type L. mjor prsites or sttionry-phse dhfr-ts prsites nd then llowed the mice to completely resolve the inflmmtory lesion. In some infections, single dose of IL-12 (0.5 µg) ws dded to the dhfr-ts prsites to ensure the induction of strong primry T H 1 response. For secondry infections, immune mice (>12 weeks fter primry infection) were rechllenged in the contrlterl footpd with sttionry phse wild-type L. mjor prsites. Secondry lesion size ws determined y sutrcting the size of the footpd efore infection from the size of the secondry lesion using digitl clipers (Mitutoyo). To quntify prsites in lesions, single-cell suspensions were prepred nd plted in twofold seril dilutions (initil dilution of 1:40) in Grce s insect culture medium. Ech smple ws plted in qudruplicte nd the men of the negtive log prsite titer ws determined fter 7 d of culture t 26 C. CD4 + T cell purifiction nd doptive trnsfer. or immune B6 Thy1.1 mice were depleted of CD8 + T cells y injection with 250 µg of H35 (Rt IgG2) 1 nd 3 d efore they were killed (>95% effective). For in vivo studies, cells were isolted from drining poplitel lymph nodes, nondrining lymph nodes (contrlterl poplitel nd superficil inguinl, xil, rchil nd cervicl lymph nodes) nd spleens, red lood cells were lysed in hypotonic solution, nd in some cses lymphocytes were pooled. CD4 + T cells were purified using T-cell purifiction column (R&D Systems) ccording to the mnufcturer s recommendtions. In some experiments, CD4 + T cells were further seprted sed on expression of CD62L y MACS columns (Miltenyi Biotec) with 95 98% purity of CD62L high nd CD62L low frctions. CD4 + T cells were stined with 5- nd 6-croxyfluorescein dicette succinimidyl ester (CFSE) s previously descried 21,41. Five to ten million CFSE-leled CD4 + T cells were trnsferred vi the retro-oritl plexus into nive Thy1.2 B6 recipients. Mice were chllenged 24 h lter with L. mjor s descried ove. Protection ws ssessed t 3 or 6 weeks fter chllenge infection, nd dt shown re from 3 4 mice per group. In vitro cell culture nd cytokine nlysis. Lymph nodes nd spleens were hrvested from nive or immune B6 mice nd single-cell suspensions prepred in complete tissue culture medium (DMEM supplemented with 10% het-inctivted FBS, 2 mm glutmine, 100 U/ml penicillin, 100 µg/ml streptomycin, 25 mm HEPES, nd M β-mercptoethnol). Lymphocytes were isolted from lesions s previously descried 21.In some experiments drining nd nondrining lymph nodes were not pooled. Cells were leled with CFSE nd plted t /well in round-ottom 96-well culture pltes in medium lone or in the presence of SLA (50 µg/ml). Purified CD4 + T cells were stimulted in the presence of irrdited syngeneic splenocytes ( /well). Superntnts were nlyzed for cytokine production y sndwich ELISA using pired monoclonl ntiodies to detect IL-2 (JES6-1A12 nd JES6-5H4) nd IFN-γ (R46A2 nd polyclonl rit ntiodies to IFN-γ) 21. Flow cytometry nd intrcellulr cytokine stining. Cells were stined with fluorochrome-conjugted monoclonl ntiodies ginst CD4 (RM4-5), CD44 (IM7), CD62L (MEL14), Thy1.1 (OX-7), IL-2 (JES6-5H4) nd IFN-γ (XMG1.2) or isotype-specific control ntiodies (BD Phrmingen) efore dt cquisition on FACSCliur flow cytometer (BD Phrmingen). Anlysis ws crried out using CellQuest Pro softwre (Version 5.1, BD Phrmingen). Intrcellulr cytokine stining ws performed s previously descried 21.To exmine coexpression of CD62L nd cytokines, the metlloproteinse inhiitor TAPI-2 (35 µg/ml; Cliochem) ws included for the finl 4 h of stimultion to prevent ctivtion-induced clevge of CD62L. The concentrtion of TAPI-2 used neither ctivted T cells nonspecificlly nor induced cell deth. All plots re gted on CD4 + T cells; in trnsfers, ll plots re gted on donor (Thy1.1) CD4 + T cells. Sttistics. Results represent the men ± s.d.. Sttisticl significnce ws determined y Student s t-test nd results were considered significnt with P vlue <0.01. Note: Supplementry informtion is ville on the Nture Medicine wesite. ACKNOWLEDGMENTS We thnk K. Joyce for excellent technicl ssistnce, memers of the Deprtment of Pthoiology for constructive discussions nd D. Artis, C.G. Feng, C.A. Hunter, D. Jnkovic, E.J. Perce, S.L. Reiner, H. Shen nd A. Sher for criticl reding of the mnuscript. This work ws supported y US Ntionl Institutes of Helth grnt AI35914 (to P. S.). COMPETING INTERESTS STATEMENT The uthors declre tht they hve no competing finncil interests. Received 1 April; ccepted 30 July 2004 Pulished online t 1. Reiner, S.L. & Locksley, R.M. The regultion of immunity to Leishmni mjor. Annu. Rev. Immunol. 13, (1995). 2. Scks, D.L. & Noen-Truth, N. The immunology of susceptiility nd resistnce to Leishmni mjor in mice. Nt. Rev. Immunol. 2, (2002). 3. Afonso, L.C. et l. The djuvnt effect of interleukin-12 in vccine ginst Leishmni mjor. Science 263, (1994). 4. Gurunthn, S., Prussin, C., Scks, D.L. & Seder, R.A. Vccine requirements for sustined cellulr immunity to n intrcellulr prsitic infection. Nt. Med. 4, (1998). 5. Gurunthn, S., Klinmn, D.M. & Seder, R.A. DNA vccines: immunology, ppliction, nd optimiztion. Annu. Rev. Immunol. 18, (2000). 6. Gicheru, M.M. et l. Vervet monkeys vccinted with killed Leishmni mjor prsites nd interleukin-12 develop type 1 immune response ut re not protected ginst chllenge infection. Infect. Immun. 69, Rhee, E.G. et l. Vccintion with het-killed Leishmni ntigen or recominnt leishmnil protein nd CpG oligodeoxynucleotides induces long-term memory CD4 + nd CD8 + T cell responses nd protection ginst Leishmni mjor infection. J. Exp. Med. 195, (2002). 8. Desjeux, P. Leishmnisis. Pulic helth spects nd control. Clin. Dermtol. 14, (1996). 9. Uzonn, J.E., Wei, G., Yurkowski, D. & Bretscher, P. elimintion of Leishmni mjor in mice: implictions for immune memory, vccintion, nd rectivtion disese. J. Immunol. 167, Belkid, Y., Piccirillo, C.A., Mendez, S., Shevch, E.M. & Scks, D.L. CD4 + CD25 + regultory T cells control Leishmni mjor persistence nd immunity. Nture 420, (2002). 11. Sllusto, F., Lenig, D., Forster, R., Lipp, M. & Lnzvecchi, A. Two susets of memory T lymphocytes with distinct homing potentils nd effector functions. Nture 401, (1999). 12. Msopust, D., Vezys, V., Mrzo, A.L. & Lefrncois, L. Preferentil locliztion of effector memory cells in nonlymphoid tissue. Science 291, Reinhrdt, R.L., Khoruts, A., Meric, R., Zell, T. & Jenkins, M.K. Visulizing the genertion of memory CD4 T cells in the whole ody. Nture 410, Romn, E. et l. CD4 effector T cell susets in the response to influenz: heterogeneity, migrtion, nd function. J. Exp. Med. 196, (2002). 15. Seder, R.A. & Ahmed, R. Similrities nd differences in CD4 + nd CD8 + effector nd memory T cell genertion. Nt. Immunol. 4, (2003). 16. Kech, S.M., Hemy, S., Kersh, E. & Ahmed, R. Moleculr nd functionl profiling of memory CD8 T cell differentition. Cell 111, (2002). 17. Wherry, E.J. et l. Linege reltionship nd protective immunity of memory CD8 T cell susets. Nt. Immunol. 4, (2003). 18. Brdley, L.M., Wtson, S.R. & Swin, S.L. Entry of nive CD4 T cells into peripherl lymph nodes requires L-selectin. J. Exp. Med. 180, (1994). 19. Jung, T.M., Glltin, W.M., Weissmn, I.L. & Diley, M.O. Down-regultion of homing receptors fter T cell ctivtion. J. Immunol. 141, (1988). 20. Picker, L.J. et l. Differentil expression of homing-ssocited dhesion molecules y T cell susets in mn. J. Immunol. 145, (1990). 21. Zph, C. & Scott, P. Th1 cell-medited resistnce to cutneous infection with Leishmni mjor is independent of P- nd E-selectins. J. Immunol. 171, (2003). 22. Titus, R.G., Gueiros-Filho, F.J., de Freits, L.A. & Beverley, S.M. Development of sfe live Leishmni vccine line y gene replcement. Proc. Ntl. Acd. Sci. USA 92, (1995). 23. Brodskyn, C., Beverley, S.M. & Titus, R.G. Virulent or virulent (dhfr-ts ) Leishmni mjor elicit predominntly type-1 cytokine response y humn cells in vitro. Clin. Exp. Immunol. 119, (2000). 24. Mocci, S. & Coffmn, R.L. Induction of Th2 popultion from polrized Leishmni-specific Th1 popultion y in vitro culture with IL-4. J. Immunol. 154, (1995). 25. Mocci, S. & Coffmn, R.L. The mechnism of in vitro T helper cell type 1 to T helper cell type 2 switching in highly polrized Leishmni mjor-specific T cell popultions. J. Immunol. 158, (1997). 26. Iezzi, G., Scheidegger, D. & Lnzvecchi, A. Migrtion nd function of ntigenprimed nonpolrized T lymphocytes in vivo. J. Exp. Med. 193, Wng, X. & Mosmnn, T. In vivo priming of CD4 T cells tht produce interleukin NATURE MEDICINE VOLUME 10 NUMBER 10 OCTOBER

7 (IL)-2 ut not IL-4 or interferon (IFN)-gmm, nd cn susequently differentite into IL-4- or IFN-gmm-secreting cells. J. Exp. Med. 194, Lnzvecchi, A. & Sllusto, F. Progressive differentition nd selection of the fittest in the immune response. Nt. Rev. Immunol. 2, (2002). 29. Doyle, A.M. et l. Induction of cytotoxic T lymphocyte ntigen 4 (CTLA-4) restricts clonl expnsion of helper T cells. J. Exp. Med. 194, Gry, D. A role for ntigen in the mintennce of immunologicl memory. Nt. Rev. Immunol. 2, (2002). 31. Zinkerngel, R.M. On nturl nd rtificil vccintions. Annu. Rev. Immunol. 21, (2003). 32. Swin, S.L., Hu, H. & Huston, G. Clss II-independent genertion of CD4 memory T cells from effectors. Science 286, (1999). 33. Murli-Krishn, K. et l. Persistence of memory CD8 T cells in MHC clss I-deficient mice. Science 286, (1999). 34. London, C.A., Perez, V.L. & As, A.K. Functionl chrcteristics nd survivl requirements of memory CD4 + T lymphocytes in vivo. J. Immunol. 162, (1999). 35. Bunce, C. & Bell, E.B. CD45RC isoforms define two types of CD4 memory T cells, one of which depends on persisting ntigen. J. Exp. Med. 185, (1997). 36. Grci, S., DiSnto, J. & Stockinger, B. Following the development of CD4 T cell response in vivo: from ctivtion to memory formtion. Immunity 11, (1999). 37. Hrertson, J., Biedermn, E., Bennett, K.E., Kondrck, R.M. & Brdley, L.M. Withdrwl of stimultion my initite the trnsition of effector to memory CD4 cells. J. Immunol. 168, (2002). 38. Flynn, J.L. Immunology of tuerculosis nd implictions in vccine development. Tuerculosis (Edin.) 84, (2004). 39. Desrosiers, R.C. Prospects for n AIDS vccine. Nt. Med. 10, (2004). 40. Scott, P., Perce, E., Ntovitz, P. & Sher, A. Vccintion ginst cutneous leishmnisis in murine model. I. Induction of protective immunity with solule extrct of promstigotes. J. Immunol. 139, (1987). 41. Lyons, A.B. & Prish, C.R. Determintion of lymphocyte division y flow cytometry. J. Immunol. Methods 171, (1994) VOLUME 10 NUMBER 10 OCTOBER 2004 NATURE MEDICINE