Transcriptional Repressor Blimp-1 Promotes CD8+ T Cell Terminal Differentiation and Represses the Acquisition of Central Memory T Cell Properties

Immunity

Article Transcriptional Repressor Blimp-1 Promotes CD8+ T Cell Terminal Differentiation and Represses the Acquisition of Central Memory T Cell Properties Rachel L. Rutishauser,1 Gislaˆine A. Martins,3,5 Sergey Kalachikov,2 Anmol Chandele,1 Ian A. Parish,1 Eric Meffre,1 Joshy Jacob,4 Kathryn Calame,3 and Susan M. Kaech1,* 1Department

of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA Genome Center 3Department of Biochemistry and Molecular Biophysics Columbia University College of Physicians and Surgeons, New York, NY 10032, USA 4Department of Microbiology and Immunology, Emory Vaccine Center, Yerkes National Primate Center, Emory University School of Medicine, Atlanta, GA 30322, USA 5Present address: Inflammatory Bowel Disease Center, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA *Correspondence: [email protected] DOI 10.1016/j.immuni.2009.05.014 2Columbia

SUMMARY

During acute infections, a small population of effector CD8+ T cells evades terminal differentiation and survives as long-lived memory T cells. We demonstrate that the transcriptional repressor Blimp-1 enhanced the formation of terminally differentiated CD8+ T cells during lymphocytic choriomeningitis virus (LCMV) infection, and Blimp-1 deficiency promoted the acquisition of memory cell properties by effector cells. Blimp-1 expression was preferentially increased in terminally differentiated effector and ‘‘effector memory’’ (Tem) CD8+ T cells, and gradually decayed after infection as central memory (Tcm) cells developed. Blimp-1-deficient effector CD8+ T cells showed some reduction in effector molecule expression, but primarily developed into memory precursor cells that survived better and more rapidly acquired several Tcm cell attributes, including CD62L and IL-2 expression and enhanced proliferative responses. These results reveal a critical role for Blimp-1 in controlling terminal differentiation and suppressing memory cell developmental potential in effector CD8+ T cells during viral infection. INTRODUCTION Memory T cells are a major component of long-term immunity because of their longevity and other unique properties that include the ability to maintain a high proliferative potential in order to expand robustly upon secondary infection, rapidly reexpress cytotoxic proteins and cytokines upon restimulation, and sustain memory T cell homeostasis in the absence of infection by undergoing cytokine-dependent cell division (Kaech and Wherry, 2007). A small population of CD8+ effector cytotoxic lymphocytes (CTLs) cells survive the effector-to-memory transition and acquire these hallmark memory properties, whereas the majority (80%–90%) of effector cells undergo a program of 296 Immunity 31, 296–308, August 21, 2009 ª2009 Elsevier Inc.

terminal differentiation in which they maintain effector functions but lose memory cell potential, longevity, and strong proliferative responses (Fearon et al., 2001; Klebanoff et al., 2006; Lanzavecchia and Sallusto, 2002). A major goal in the field of memory CD8+ T cell biology has been to understand the factors that determine the balance between memory cell potential and terminal differentiation. During some infections, surface protein expression can be used to distinguish the subpopulations of antigen-specific effector and memory cells that achieve a more terminally differentiated state from those that are more likely to acquire the memory cell properties outlined above. For example, during lymphocytic choreomeningitis virus (LCMV), Listeria, Toxoplasma gondii, and murine cytomegalovirus virus infections, terminally differentiated short-lived effector cells that express high amounts of the NK cell maker KLRG1 (killer cell lectin-like receptor G1) and low amounts of IL-7Ra (referred to as IL-7R) have a markedly shorter lifespan and reduced proliferative capacity in response to the homeostatic cytokines IL-7 and IL-15, or secondary antigenic challenge compared to effector cells with a KLRG1loIL-7Rhi phenotype (Huster et al., 2004; Joshi et al., 2007; Kaech et al., 2003; Sarkar et al., 2008; Schluns et al., 2000; Snyder et al., 2008; Voehringer et al., 2001; Wilson et al., 2008). In contrast, the KLRG1loIL-7Rhi effector cells survive the effector-to-memory transition better, have a longer lifespan, and acquire the ability to proliferate well in response to both homeostatic cytokines and antigenic rechallenge (Hikono et al., 2007; Joshi et al., 2007; Sarkar et al., 2008). In addition, memory cells that express high amounts of CD62L (referred to as central memory, Tcm), CD27, and CXCR3, which also produce IL-2 and express low amounts of CD43 and cytotoxic molecules, are more ‘‘functionally mature’’ memory cells (i.e., cells with enhanced proliferative capacity and longevity). These cells accumulate and dominate the memory population with time in lymphoid tissues, whereas end-stage cells with a CD62Llo (termed ‘‘effector memory,’’ Tem) KLRG1hiIL-7RloCD27loCXCR3loCD43hi phenotype typically decay but are often better maintained in peripheral tissues such as the liver and lung (Bachmann et al., 2005; Badovinac and Harty, 2007; Hikono et al., 2007; Kaech et al., 2002; Wherry et al., 2003). At the time of CD8+ T cell priming, decreasing the

Immunity Blimp-1 and Memory CD8+ T Cell Differentiation

duration or intensity of exposure to antigen and inflammation generates an activated cell population that acquires several phenotypic and functional characteristics associated with a more mature memory CD8+ T cell population (Badovinac et al., 2004, 2005; Badovinac and Harty, 2007; Cannarile et al., 2006b; Heffner and Fearon, 2007; Ichii et al., 2004; Intlekofer et al., 2008; Joshi et al., 2007; Sarkar et al., 2007). Some key signals involved in this process are the cytokines IFN-g and IL-12 and the transcriptional regulators T-bet, Id2, Bmi-1, Bcl6, and Bcl6b (Badovinac et al., 2004, 2005; Badovinac et al., 2004; Badovinac and Harty, 2007; Cannarile et al., 2006b; Heffner and Fearon, 2007; Ichii et al., 2004; Intlekofer et al., 2008; Joshi et al., 2007; Pearce and Shen, 2007; Sarkar et al., 2007; Takemoto et al., 2006). To better understand memory cell development and maturation, we focused on B lymphocyte-induced maturation protein1 (Blimp-1; encoded by Prdm1), a transcriptional repressor that is required for the terminal differentiation of B cells into plasma cells, as well as the terminal differentiation of other diverse cell types (Horsley et al., 2006; Magnusdottir et al., 2007; Martins and Calame, 2008; Ohinata et al., 2005; Shapiro-Shelef et al., 2003; Turner et al., 1994). Because it is a critical regulator of terminal differentiation, we hypothesized that Blimp-1 might positively regulate the formation of terminally differentiated CD8+ T cell subsets and potentially negatively regulate the process of memory cell maturation. Previously, Blimp-1 was found to play an important role in T cell homeostasis (Kallies et al., 2006; Martins et al., 2006). Blimp-1 deficiency in T cells during development results in a lethal inflammatory disease associated with excess accumulation of peripheral CD44hi CD62Llo-activated CD4+ and CD8+ T cells, and greater numbers of herpes simplex virus-specific memory CD8+ T cells form in these mice (Kallies et al., 2006; Martins et al., 2006). However, the precise role of Blimp-1 in the differentiation of antigenspecific effector and memory CD8+ T cells could not be properly assessed in these studies because of the added complications of ongoing inflammatory disease and poor regulatory T cell function in mice lacking Prdm1 expression in the entire T cell compartment. Another study demonstrated that Blimp-1 mRNA is preferentially expressed in the short-lived effector cell subset of LCMV antigen-specific CD8+ T cells, although no functional role for Blimp-1 in CD8+ T cell differentiation has been described (Intlekofer et al., 2007). In this study, we define a critical role for Blimp-1 in regulating the balance between terminal differentiation and memory cell development by showing that Blimp-1 not only controls the terminal differentiation of antigen-specific CD8+ T cells, but also that it affects the rate at which they acquire hallmark memory cell properties. RESULTS Blimp-1 Expression in Subsets of Effector and Memory CD8+ T Cells Using ‘‘P14 chimeric’’ mice, which contain a small number (5,000) of Thy1.1+ P14 CD8+ T cells that recognize the DbGP33-41 epitope of LCMV, we previously compared the gene expression profiles of naive and LCMV-specific effector and memory CD8+ T cells using DNA microarrays (Wherry et al., 2007). This analysis showed that Prdm1 mRNA was upregulated in day 8 effector CTLs compared to naive CD8+ T cells, but de-

cayed as memory T cells formed between days 40–100 postinfection (p.i.). Using quantitative real-time PCR (qRT-PCR), we observed a similar pattern of Prdm1 mRNA expression in LCMV-specific P14 CD8+ T cells (Figure 1A). To investigate Blimp-1 expression at the single-cell level, we infected BAC transgenic (tg) reporter mice that express YFP under the control of Prdm1 regulatory elements (Blimp-1-YFP) with LCMV. Although less than 1% of CD44loCD62Lhi naive CD8+ T cells expressed Blimp-1-YFP, Blimp-1 was upregulated in 80% of LCMV-specific effector CD8+ T cells and then progressively declined thereafter, both in terms of the percent of cells expressing Blimp-1-YFP as well as the amount expressed on a per-cell basis (i.e., the median fluorescence intensity (MFI) of YFP) (Figure 1B). Together, these data showed that Blimp-1 mRNA was induced in most effector CD8+ T cells during clonal expansion, but its expression gradually declined during the effector-tomemory transition because the memory T cell population functionally matured. Next, we explored whether Blimp-1 expression was differentially regulated between different subsets of effector and memory CD8+ T cells during LCMV infection. In agreement with previously published data, Blimp-1 mRNA, protein, and YFP reporter expression were increased 2- to 4-fold in terminally differentiated KLRG1hiIL-7Rlo short-lived effector cells (termed SLECs), compared to KLRG1loIL-7Rhi memory precursor effector cells (MPECs) (Figures 1C–1F) (Intlekofer et al., 2007). Heightened Blimp-1 expression continued to be maintained even in the memory population in KLRG1hiIL-7RloCD27loCD62Llo (Tem) cells (Figures 1C–1F). In contrast, the KLRG1loIL-7Rhi and CD27hiCD62Lhi (Tcm) subsets became preferentially enriched with Blimp-1-YFPlo cells between days 10–40 p.i. (Figure 1E, note the red highlighting in quadrant 3, left panels, and quadrant 2, right panels). These data show that Blimp-1 expression is extinguished in the population because functionally mature subsets of CD8+ Tcm cells that have increased longevity and proliferative responses to both antigen and homeostatic cytokines form (Hand et al., 2007; Hikono et al., 2007; Joshi et al., 2007; Wherry et al., 2003). Blimp-1 Deficiency Enhances the Survival of Effector Cells To investigate the function of Blimp-1 in effector and memory CD8+ T cell development, we generated mice that were deficient in Blimp-1 in CD8+ T cells by crossing mice containing a loxP-flanked Prdm1 gene (Prdm1flox/flox) (Martins et al., 2006) to transgenic mice expressing the Cre recombinase under the control of the human Granzyme B promoter (GzB-cre) (Jacob and Baltimore, 1999). For simplicity, we will refer to GzB-cre+; Prdm1flox/flox animals as ‘‘Prdm1 / ’’ and their littermate controls (GzB-cre+; Prdm1+/+ or GzB-cre ; Prdm1flox/flox) as ‘‘WT.’’ Unlike the mice in which Blimp-1 is deleted during thymic development (Kallies et al., 2006; Martins et al., 2006), in this system Blimp-1 is expressed normally until CD8+ T cells are activated and uninfected Prdm1 / mice did not demonstrate abnormal T cell activation (data not shown). Additionally, deletion efficiency reaches 85%–95% of virus-specific CD8+ T cells during LCMV infection (Figure S1 available online) (Chappell et al., 2006; Maris et al., 2003). Using these mice, we found that Blimp-1 deficiency markedly increased effector cell survival after infection. Eight days after Immunity 31, 296–308, August 21, 2009 ª2009 Elsevier Inc. 297

Immunity +

Blimp-1 and Memory CD8 T Cell Differentiation

A

B

Naive

4

4

10

Blimp-1-YFP

10 3

2

CD44

50 40

10 1

10 0

10 0 10

30

1

10

2

10

4

3

10

10

20

2

10 1

10 0

10 0 10

LCMV infected 4

0 8 45 150 Time after infection (days)

3

10 2

10 1

10 0 10 0 10

1

10

2

10

3

4

10

10

day 5

4

10

Blimp-1-YFP

10

1

10

CD8

10

0

0.4±0.06%

3

10

CD62L

D b GP33-41

2

10

3

4

10

10

3

10

4

3

1

3

1

1

10

2

10

3

4

10

10

100 0 10

3

10

4

10

100 0 10

1

10 0

1

10

2

10

3

10

4

10

10 0 10

1

10

2

10

3

10

4

10

CD8

D SLEC

C 80

Prdm1 mRNA (relative to naive)

2

1

2

10

30±7% 26±2

3

10

10

1

10

~day 200

10

2

10

10

100 0 10

104

63±4% 28±3

10

2

10

10

~day 60

10

79±4% 45±4

10

2

10

CD8

day 8

4

10

72±5% 65±3

IL-7R hi KLRG1lo IL-7R lo KLRG1hi

70 60

MPEC

Prdm1 mRNA (relative to naive)

10

Blimp-1

50 40

1.7±0.2x

30

Grp94

20 10 0

8 15 60 Time after infection (days)

E

F ~d40 29%

3

102

10 1

7%

10 0

10 0

10 1

18% 10 2

10 3

102

101

5%

100 100

10 4

49%

4%

10 1

102

103

10 1

46%

10 0

104

3%

23%

56%

18%

103

102

10 0

IL-7R

IL-7R

10 1

49% 10 2

10 3

10 4

102

101

10 0 10 0

CD27

10 1

10 2

10 3

10 4

CD27

8

80

10%

MFI 69

60

8%

6

MFI 75

# Cells

100

# Cells

Quadrant 1

1% 10 3

CD62L

4

17%

103

100

~d40 10 4

104

15%

KLRG1

2

KLRG1

1

d10

104

60% 10 3

very few cells

4

40 2

very few cells

0 100

102

13%

10 0

104

MFI 62

5

10 1

10 2

13%

10 3

104

MFI 61

4

6

20

35%

10

MFI 26

10

20

30

40

50

60

70

2

1

0

30

0

0

10 0

10 1

10 2

23%

10 3

10 4

10 0

10 1

10 2

10 3

54%

MFI 37

10 1

10 2

14%

10 3

10 4

MFI 54

60

100 6

10 1

102

44%

103

104

MFI 18

4

# Cells

# Cells

80

MFI 13

6

0 100

104

# Cells

20

4

40

10

2 2

0

20

0 10 1

10 2

12%

12

10 3

10 4

101

102

103

10 0

104 80

MFI 63

9

0 100

very few cells

6

3

0

10 1

10 2

14%

10 3

104

MFI 53

60

101

102

103

104

100

10 1

102

13%

103

10 4

MFI 57

10

40

5

20

0 100

0

15

# Cells

10 0

# Cells

0

Time after infection (days)

MFI 6

2

10

4

25

4

2

3

KLRG1hi KLRG1lo

50

6

4

3

80%

8

# Cells

# Cells

30

103

# Cells

2

101

75

0

20 0

Blimp-1-YFPhi (%)

d10 104

CD62L

Quadrants:

0 100

101

102

103

104

10 0

10 1

10 2

10 3

10 4

Blimp-1-YFP Figure 1. Blimp-1 Is Preferentially Expressed in Terminally Differentiated LCMV-Specific Effector and Memory CD8+ T Cells (A) Relative Prdm1 mRNA expression (±SEM) in naive or LCMV-specific P14 CD8+ T cells measured by qRT-PCR. n = 8 to 9 independent samples/time point. (B) Naive (CD44loCD62Lhi) or DbGP33-41-specific CD8+ T cells in the spleen of Blimp-1-YFP BAC tg mice were analyzed, and mean percentage (shown in black) and MFI (shown in green) of Blimp-1-YFPhi cells is shown (±SEM; n R 6 mice/time point). (C) Relative expression of Prdm1 mRNA (±SEM) in KLRG1loIL-7Rhi and KLRG1hiIL-7Rlo P14 CD8+ T cells in the spleen from days 8, 15, and 60 p.i. as measured by qRT-PCR. (D) Blimp-1 (or Grp94) protein expression in KLRG1hiIL-7Rlo (SLEC) or KLRG1loIL-7Rhi (MPEC) P14 effector CD8+ T cells purified by flow cytometry at 8–10 days p.i. Fold change represents average over 3 independent experiments (±SEM). (E and F) Blimp-1-YFP expression was analyzed in P14 BAC tg CD8+ T cells stained with antibodies to IL-7R and KLRG1 or CD27 and CD62L in the (E) blood or (F) spleen at days 10–40 p.i. n R 3 experiments each with 2 to 3 mice per time point. (E) Percentages of cells in each quadrant is shown. Histogram plots below each contour plot show the percent of Blimp-1-YFPlo cells and MFI of YFP in each quadrant. (F) Line graph shows the percent (±SEM) of Blimp-1-YFPhi cells in KLRG1hi or KLRG1lo DbGP33-41-specific CD8+ T cells in the spleen.

298 Immunity 31, 296–308, August 21, 2009 ª2009 Elsevier Inc.

Immunity Blimp-1 and Memory CD8+ T Cell Differentiation

WT

4

10 3

3

10

10

2

10

2

10

1

10

1

10

1

10

0

1

10

2

3

10

4

10

10

4

3

10

2

3

10

2

10

10

1

1

0

1

10

2

3

10

4

10

10

2

10

3

4

10

10

10 0 10

1

10

2

3

10

4

10

10

CD8

10 0 10

1

10

2

3

10

4

10

10

4

10

8.3±1.6%

10

0

0

1

10

4

2

10

1

10 0 10

4

10

3

10

10 0 10

1

10

10

3.7±0.7%

10

2

0

10 0 10

4

10

D b GP33-41

10

2

0

10.5±1%

3

10

10

10 0 10

B

Prdm1 -/-

4

10

9±1%

3

10

10

day 60

WT

4

10

12.5±1.4%

D b NP396-404

day 8

Prdm1-/-

4

10

11±2%

Number of LCMVspecific CD8 + T cells

A

10

6.7±1.5%

10

3

10

2

10

10.2±3.7%

3

2

10 1

1

10

10

0

1

2

10

10

3

4

10

10

10 0 10

1

10

2

3

10

*

10 5 5

10

D

60

110

150

E

day 8 p.i.

~day 35 p.i.

* p < 0.05

*

6

10 7

10

*

10 5

*

~day 150 p.i. WT Prdm1 -/-

*

* p < 0.05

* *

6

LN

BM

LV

LG

WT Prdm1 -/-

10 7

* p < 0.05

10 6

*

10 5

10 5

TOTAL SPL

Number of LCMVspecific CD8 + T cells

WT Prdm1 -/-

10 7

Number of LCMVspecific CD8 + T cells

Number of LCMVspecific CD8 + T cells

30

CD8

C

10

8

Time after infection (days)

4

10

* p < 0.05

10 6

0

10 0 10

WT Prdm1 -/-

* *

10 7

TOTAL SPL

LN

BM

LV

LG

F

TOTAL SPL

LN

* BM

LV

LG

G

103

WT 17±7%

103

102

MPEC 11±6%

7±3% 0 10 0 10

KLRG1

day 60

Prdm1 -/-

SLEC 3±2%

104

15±7%

102

101

104

104

1 10

2 10

13±5%

103

104

31±8%

101

104

1 10

2 10

1±1%

103

WT 0.6±1%

1.3±2%

103

102

102

101

79% 102

103

4 10

20±3%

100 100

10±3%

53%

103

103

103

102

102

102

102

101

101

101

101

2±1%

IL-7R

101

54±7% 102

103

104

0% 100 100

94±4% 101

102

103

104

101

63±6% 102

5%

103

100 100

15±5%

103

104

104

104

5±3%

1.6%

101

19%

100 100

104

104

103

101

MPEC 77±9%

5±3% 0 10 0 10

CD62L

day 8

SLEC 65±6%

25±4%

100 100

101

12±3% 102

103

4 10

74±6%

CD62L hi (%)

104

Prdm1-/-

90 80 70 60

Prdm1 -/WT

50 40 30 20 10 0

0

25

50

75

Time after infection (days) 8%

100 100

13±4% 101

102

103

104

CD27

Figure 2. Blimp-1 Deficiency Increases Effector CD8+ T Cell Survival and Prevents Terminal Effector Cell Differentiation (A) Representative plots of splenic DbGP33-41 and DbNP396-404 tetramer+ CD8+ T cells at 8 and 60 days after LCMV infection in WT and Prdm1 / mice (percent of tetramer+ cells CD8+ T cells ± SEM is indicated). (B) Prdm1 / (white bars) or WT littermate controls (black bars) were infected with LCMV, and numbers of LCMV-specific CD8+ T cells in the spleen (±SEM) were enumerated by IFN-g intracellular cytokine staining (ICCS) after 5 hr stimulation with NP396-404, GP33-41, and GP276-286 peptides. Note that numbers based on tetramer staining are similar to those based on ICCS. n = four experiments each with 2 to 3 animals per group per time point. (C, D, and E) Combined number (±SEM) of DbGP33-41-and DbNP396-404 tetramer+ CD8+ T cells in various tissues at days (C) 8, (D) 35, and (E) 150 p.i. in spleen (SPL), inguinal lymph nodes (LN), bone marrow (BM), liver (LV), and lung (LG). (F) Expression of IL-7R and KLRG1 (left plots) or CD62L and CD27 (right plots) in DbGP33-41 tetramer+ CD8+ T cells at day 8 or day 60 p.i. Mean percentages (±SEM) of 5–12 mice per time point. (G) Percent (±SEM) of CD62Lhi DbGP33-41-specific CD8+ T cells in the spleens of WT (black squares) or Prdm1 / (open squares) animals after LCMV infection.

LCMV infection, similar frequencies of LCMV-specific CD8+ T cells were observed in the spleens of Prdm1 / and WT mice (Figure 2A). The Prdm1 / mice also generated slightly, but not significantly, increased numbers of LCMV-specific effector CD8+ T cells in the spleen, similar numbers in several other tissues, and significantly more effector cells in the lymph nodes (LNs) and bone marrow (BM) as assessed by staining for both MHC class I tetramers and intracellular IFN-g after in vitro stimulation (Figures 2A–2C). Interestingly, the effector CD8+ T cell death that normally follows viral clearance was

substantially decreased in the Prdm1 / mice; initially 5-fold more LCMV-specific memory CD8+ T cells persisted in the spleen and other tissues (especially the LNs and BM) of the Prdm1 / animals (Figures 2B and 2D). BrdU-labeling studies during these time periods did not show more proliferation in Prdm1 / cells compared to WT cells, indicating that the Prdm1 / effector cells survived better than WT cells (data not shown). After 5 months, the number of LCMV-specific Prdm1 / CD8+ T cells declined to that found in WT mice (except in BM and LNs), indicating that the initial enhancement in survival was Immunity 31, 296–308, August 21, 2009 ª2009 Elsevier Inc. 299

Immunity +

Blimp-1 and Memory CD8 T Cell Differentiation

overridden by other, Blimp-1-independent, homeostatic control mechanisms (Figures 2B and 2E). Increased Memory Precursor Cell Formation in Prdm1 / Effector CD8+ T Cells Because Blimp-1 was preferentially expressed in more terminally differentiated subsets of effector and memory CD8+ T cells, we hypothesized that it might play a role in the formation of these cells. Indeed, Prdm1 / mice formed profoundly fewer effector cells with a KLRG1hiIL-7Rlo (SLEC) phenotype, and there was a corresponding increase in both the percentage and number of KLRG1loIL-7Rhi (MPEC phenotype), CD27hi, and CD62Lhi (Tcm) effector CD8+ T cells in the spleen, liver, and lungs (Figure 2F and data not shown). Normally, the WT memory population gradually accumulates CD62Lhi Tcm cells, but Prdm1 / mice accumulated a larger percentage of Tcm cells (and KLRG1loIL-7Rhi cells) at a faster rate than WT mice (Figures 2F and 2G). Effector and memory CD8+ T cells from the Prdm1 / animals had higher expression of CD95 (Fas) as previously described (Kallies et al., 2006), but similar expression of CD44, CD43, bcl-2, and CD122 compared to WT cells (data not shown). Thus, in the absence of Blimp-1, several phenotypic changes that normally occur during the effector-to-memory transition were accelerated, and a more mature-looking memory CD8+ T cell population with an increased number of Tcm cells developed at earlier time points after infection. In the GzB-cre system, Prdm1 is deleted in a proportion of granzyme B-expressing NK and virus-specific CD4+ T cells (40% and 10%, respectively) (Maris et al., 2003). Therefore, to specifically isolate the Blimp-1 deficiency to CD8+ T cells, we examined two additional experimental systems. First, Prdm1 was deleted in P14 CD8+ T cells using CD4-cre (CD4cre Prdm1 / = CD4-cre+; Prdm1flox/flox and WT littermate controls = CD4-cre+; Prdm1+/+), and small numbers of these cells were adoptively transferred into congenic B6 mice to generate chimeric mice that were subsequently infected with LCMV. The results from these experiments confirmed many of the phenotypes observed in the GzB-cre system, indicating that the aforementioned phenotypes were intrinsic to the Blimp-1-deficient CD8+ T cells (Figure S2A). However, genetic incompatibilities prevented long-term studies in these animals due to deletion of donor P14 cells. Therefore, we generated an additional experimental system involving mixed bone marrow chimeras (BMC) whereby bone marrow from Ly5.1+ Cd8a / animals was mixed in a 90:10 ratio with bone marrow from either Ly5.2+ GzB-cre ; Prdm1flox/flox (BMC WT) or Ly5.2+ GzB-cre+; Prdm1flox/flox (BMC Prdm1 / ) and used to reconstitute lethally irradiated Ly5.1+ recipient mice. Two months later, these mice were infected with LCMV. The phenotypes of antigen-specific effector and memory CD8+ T cells from these mice reconfirmed those found in the above systems (Figure S3A; data not shown). As in the GzB-cre system, the Prdm1 / effector cells from the mixed BMC also showed reduced cell death between days 7 and 35 p.i. compared to WT cells (Figure S3B). The Transcriptional Profile of Blimp-1-Deficient Effector Cells Resembles that of MPECs To better understand the mechanisms by which Blimp-1 regulates the balance between terminal differentiation and mainte300 Immunity 31, 296–308, August 21, 2009 ª2009 Elsevier Inc.

nance of memory cell potential, we compared the gene expression profiles of WT and Prdm1 / LCMV-specific effector CD8+ T cells using Illumina BeadChips. Of the 24,000 genes analyzed, 128 genes were significantly (p < 0.05) differentially expressed, and of these, 70% (87 genes) showed increased mRNA in the Prdm1 / effector CD8+ T cells compared to the WT cells (Table 1 and Table S1), in agreement with Blimp-1 being a transcriptional repressor (Huang, 1994). To further analyze the gene expression profile of the Prdm1 / effector cells, we categorized several of the differentially expressed genes into distinct functional groups (Table 1). Some potential Prdm1 gene targets, such as Bcl6, Tcf7, Tbx21 (encodes T-bet), and Kit, were then confirmed using qRT-PCR or flow cytometry (Figure 3). A number of the differentially expressed genes contain predicted Blimp-1 binding sites (BS): some are regulated by Blimp-1 in other cell types (Id2, Xcl1, and Cd83), and others are known direct targets of Blimp-1 (Bcl6, Id3, and Prdm1 itself) (Table 1, Column E) (G.M., K.C. and M. Cimmino unpublished data, Martins and Calame, 2008 and Gong and Malek, 2007). These data suggest that control of terminal differentiation by Blimp-1 may share common features across different cell types. Lastly, we compared the Prdm1 / effector cell gene signature to that of MPECs, as previously described by our lab using Affymetrix GeneChips (Joshi et al., 2007) (Table 1, compare columns A and D). Although the magnitude of the differential expression cannot be directly compared across the two different microarray platforms, similarity in terms of the relative increase or decrease in gene expression can be assessed. Of the 128 genes differentially expressed in Prdm1 / effector cells, 90% showed the same trend in expression in MPECs compared to SLECs (Table 1 and data not shown). Thus, it appears that Blimp-1 represses many of the genes that control memory CD8+ T cell potential and the development of memory CD8+ T cell properties. Blimp-1-Deficient CTLs Have Altered Effector Molecule Expression Because the mRNA transcripts of certain effector molecules were downregulated in Prdm1 / versus WT cells, we next sought to establish whether Blimp-1 deficiency might perturb CTL function during viral infection. First, we examined the ability of effector and memory WT and Prdm1 / CD8+ T cells to produce IFN-g, TNFa, and IL-2 after 5 hr in vitro stimulation with peptide (Figure 4A). The numbers of IFN-g-producing CD8+ T cells corresponded closely to those obtained with MHC class I tetramer staining between the two groups (compare Figures 2A and 4A), indicating no differences in the frequency of IFN-g+ virus-specific CD8+ T cells. In addition, similar frequencies of TNFa-producing CD8+ T cells were observed between the two groups (data not shown). However, a considerably larger percentage of Prdm1 / effector CD8+ T cells produced IL-2 compared to WT cells (Figure 4A), which translated into an overall increase in the percentage of polyfunctional, triple cytokineproducing (IFN-g, TNFa, and IL-2) cells in the Prdm1 / animals. Notably, though, the overall amount of IFN-g produced on a percell basis (MFI) was reduced in effector and memory Prdm1 / CD8+ T cells (Figure 4A). When the expression of granzyme B (GzB) protein was examined, we found that 50%–60% of the Prdm1 / effector CD8+

Immunity Blimp-1 and Memory CD8+ T Cell Differentiation

Table 1. Select Genes Significantly Differentially Expressed between WT and Prdm1

/

Effector CD8+ T Cells

A

B

C

D

E

Fold Change Prdm1 / /WTa

Gene Symbol

Gene Name

Fold Change IL-7Rhi/IL-7Rlo b

Predicted Blimp-1 BSc

Gzmm

granzyme M (lymphocyte met-ase 1)

1.6

Gzma

granzyme A

Effector Molecules 2.3 6.2

1.7

3 1

2.8

FasL

Fas ligand (tumor necrosis factor ligand superfamily, 6)

1.1

1

2.8

Prf1

perforin 1

2.1

0

Cytokine and Chemokine Receptors 13.4

Ccr7

chemokine (C-C motif) receptor 7

35.7

2

5.7

Ccr6

chemokine (C-C motif) receptor 6

8.3

3

2.8

Il7r

interleukin 7 receptor (CD127)

36.4

2

2.4

Sell

selectin, lymphocyte (CD62L)

11.3

Edg8

endothelial differentiation, sphingolipid G-protein-coupled receptor 8

7.8

20.5

4 0

6.5

Cx3cr1

chemokine (C-X3-C) receptor 1

5.6

0

3.5

Sema4a

semaphorin 4A

1.2

0

3.5

Cxcr6

chemokine (C-X-C motif) receptor 6

1.6

0

Cytokines, Chemokines, and Secreted Factors 8.5

Xcl1

chemokine (C motif) ligand 1

1.6

0

4.4

Cxcl10

chemokine (C-X-C motif) ligand 10

3.8

2

2.8

F2rl1

coagulation factor II (thrombin) receptor-like 1

5.7

2

2.4

Mif

macrophage migration inhibitory factor (Mif)

1.4

0

Costimulatory and Inhibitory Receptors 3.2

OX-40

tumor necrosis factor receptor subfamily, member 4

1.5

0

2.3

Ctla4

cytotoxic T-lymphocyte-associated protein 4

3.1

1

2.0

Tnfrsf25

tumor necrosis factor receptor superfamily 25

3.4

0

2.0

Cd83

CD83 antigen

3.7

Klrg1

killer cell lectin-like receptor subfamily G 1

9.2

Prdm1

PR domain containing 1 (Blimp-1), EXON 4 (not floxed)

N/A

1

6.9

Id3

inhibitor of DNA binding 3

11.3

4

6.4

Tcf7

transcription factor 7, T cell specific

11.1

0

2.4

Pou6f1

POU domain, class 6, transcription factor 1

5.8

0

2.1

Bcl6

B cell leukemia/lymphoma 6

2.6

Zfhx1b

zinc finger homeobox 1b

4.3

11.4

0 3

Transcription Factors

3.8

9.3

2.4

Prdm1

PR domain containing 1 (Blimp-1), EXON 8 (floxed)

2.2

Id2

inhibitor of DNA binding 2

2.2

Tbx21

T-box 21 (Tbet)

2.1 1.2 1.5

0 1 1 0 1

Cell Signaling (Including Cell-Cycle and Survival Factors) 3.1

Kit

kit oncogene

4.1

0

2.9

Cables1

Cdk5 and Abl enzyme substrate 1

3.5

1

2.9

Lrig1

leucine-rich repeats and Ig-like domains 1

3.9

2

2.8

Spi2a

serine proteinase inhibitor, clade A, member 3G (Serpina3g)

3.7

Ccnd3

cyclin D3

2.5

1.3

1 0

2.5

Anxa1

annexin A1

2.8

1

2.2

Socs2

suppressor of cytokine signaling 2

2.3

0

a

Data represent the average of four independent samples of LCMV-specific WT and Prdm1 / effector CD8+ T cells sorted at day 8 p.i. by flow cytometry and analyzed using Illumina Sentrix MouseRef-8 Expression BeadChips. Positive fold change values are more highly expressed in Prdm1 / cells (bold lettering); negative fold change values are more highly expressed in WT cells (full list in Table S1). All fold changes are statistically significant, p < 0.05. b Data from 3 independent samples of LCMV-specific P14 IL-7Rhi and IL-7Rlo effector CD8+ T cells sorted at day 7 p.i. by flow cytometry and analyzed using Affymetrix U430v2 genechips (Joshi et al., 2007). c Blimp-1 binding site (BS) predictions are based on algorithms from Genomatix software.

Immunity 31, 296–308, August 21, 2009 ª2009 Elsevier Inc. 301

Immunity +

Blimp-1 and Memory CD8 T Cell Differentiation

8.9x*

3 2

d150

30 20

Naive

d8

d8 WT

0

d8 SLEC

10

Prdm1 -/-

Tbx21 mRNA (relative to naive)

d8 MPEC

5.2x*

40

C

d150

B

d8 MPEC

d8 WT

0

d8 SLEC

1

Naive

Bcl6 mRNA (relative to naive)

4

d8 Prdm1 -/-

A

1.00 0.75

10x*

0.50

d150

d8 MPEC

Naive

d8 WT

0.00

d8 SLEC

0.25

d8 Prdm1 -/-

Tcf7 mRNA (relative to naive)

1.25

D 100

% of Max

80

WT 0.1% ± 0.1 Prdm1 -/- 5% ± 2

60

40

20

0 10

0

10

1

10

2

10

3

10

4

Kit

Figure 3. Blimp-1 Deficiency Alters the Expression of Genes Known to Regulate Effector and Memory CD8+ T Cell Differentiation (A–C) qRT-PCR analysis of Bcl6 (A), Tbx21 (B), and Tcf7 (C) mRNA expression in WT and Prdm1 / effector CD8+ T cells from day 8 p.i. or WT naive cells (CD44loCD62Lhi), SLEC (KLRG1hiIL-7Rlo), or MPEC (KLRG1loIL-7Rhi) from day 8 p.i., and memory CD8+ T cells from day 150 p.i. Fold change relative to naive cells (±SEM) are representative of two to three independent experiments each with 2 to 3 samples. *p < 0.05. (D) Percent (±SEM) Kit receptor positive DbGP33-41-tetramer+ CD8+ T cells from WT (gray histogram) and Prdm1 / (black line) animals 8 days after LCMV infection.

302 Immunity 31, 296–308, August 21, 2009 ª2009 Elsevier Inc.

T cells initially (day 5 p.i.) expressed GzB (compared to 90% in WT effector cells), but this rapidly decayed to near undetectable levels by day 7 p.i. (Figure 4B). Despite this profound reduction in GzB expression, cytotoxic activity of Prdm1 / CD8+ T cells was only marginally affected based on in vivo cytotoxicity assays, indicating that other cytotoxic pathways were unaffected by Blimp-1 deficiency (data not shown). The decreased GzB and increased IL-2 expression was also recapitulated in the P14 CD4-cre and BMC Prdm1 / systems (Figures S2B and S3A), although the depressed IFN-g staining was not found in the P14 CD4-cre system, perhaps due to differences in the polyclonal TCR repertoire versus that of the high affinity P14 TCR (data not shown). Despite the modest effects on CTL function in the GzB-cre Prdm1 / animals, the clearance of LCMV by plaque assay technique was slightly delayed by 1 to 2 days in the majority of Prdm1 / animals (Figure 4C and data not shown; note that virus was not detected in the spleen, liver, or kidney of Prdm1 / mice at day 15 p.i.). Nonetheless, the Prdm1 / memory CD8+ T cells were capable of providing a protective immune response because LCMV-immune Prdm1 / mice cleared a 2 infection with a more virulent strain of LCMV (cl. 13) that normally causes chronic infection and cannot be cleared in the absence of functional memory CD8+ T cells (as measured by serum viral titers at day 5 p.i., data not shown). Interestingly, restimulation of Prdm1 / memory CD8+ T cells did not ‘‘reverse’’ their phenotypes because the 2 Prdm1 / effector CD8+ T cells maintained a IL-7RhiCD27hiCD62LhiKLRG1loGzBlo phenotype compared to WT cells (Figure 4D). This indicated that, even with repeated stimulation, CD8+ T cells cannot bypass the need for Blimp-1 for terminal differentiation. Blimp-1 Regulates the Proliferative Responses of Virus-Specific CD8+ T Cells The ability to proliferate robustly in response to secondary infection and homeostatic cytokines is a hallmark property of memory cells (Bachmann et al., 2005; Badovinac and Harty, 2007; Hikono et al., 2007; Kaech et al., 2002; Wherry et al., 2003). Because Blimp-1 was preferentially expressed in memory T cell subsets that have relatively reduced proliferative responses, we first confirmed that Blimp-1 itself marked cells with reduced proliferative capacity by transferring equal numbers of sorted Blimp-1YFP+ and Blimp-1-YFP P14 memory T cells from the same chimeric mice into naive congenic recipient mice that were subsequently infected with recombinant Listeria-expressing GP33-41 (rLM33) and analyzing their numbers 7 days later (Figure 5A). As predicted, the Blimp-1-YFP+ cells expanded less than the Blimp-1-YFP population, indicating that Blimp-1 expression directly corresponds to memory T cells with decreased proliferative capacity. Next, we tested whether the absence of Blimp-1 also impacted the proliferative responsiveness of effector cells to antigen in vitro and in vivo. To do this, we first examined cell division by CFSE-dilution in purified effector cells from WT and Prdm1 / animals incubated in vitro with peptide for 48 hr. In these experiments, Prdm1 / CD8+ T cells underwent more extensive cell division than WT cells with less than 15% of WT effector cells dividing compared to >50% of Prdm1 / effector cells (Figure 5B). To test if similar results were found in vivo, equal numbers of day 8 LCMV P14 WT or CD4-cre Prdm1 / effector

Immunity Blimp-1 and Memory CD8+ T Cell Differentiation

A

Figure 4. Blimp-1 Deficiency Alters CTL Differentiation

GP33/34-41-specific WT

4

10 10

3

10

2

10

3

1

1

10

10

9.3%

0

10 0 10

1

2

10

3

10

4

10

10

4

1

2

10

3

10

10

4

10

10

2.1%

10

3

10

2

10

5.0%

MFI IFNWT 402±3 Prdm1-/- 210±3

3

2

10 1

1

10

10

4.2%

0

10 0 10

1

2

10

3

10

4

10

10

0

11%

0

10 0 10

1

2

10

3

10

10

*

25 20 15 10 5

4

10

IL-2

12%

0

10 0 10

45 40 35 30

MFI IFNWT 499±23 Prdm1-/- 446±21

2

10

day 60

4.9%

IL-2 + of IFN- +(%)

day 7

Prdm1-/-

4

10

0.8%

4

Effector day 8

10

IFN-

WT

4

10

0.5%

10

3

10

102

102

1

1

10

11%

0

10 0 10

1

2

10

3

10

10

8.9%

0

4

10

4

45 40 35 30

10 0 10

1

2

10

3

10

10

MFI IFNWT 541±21 Prdm1-/- 388±24

4

10

25 20 15 10 5

4

10

IL-2

1.8%

3

10

day 60

NP396-404-specific Prdm1 -/-

4

10

IL-2 + of IFN- +(%)

day 7

10

3.9%

10

3

10

102

102

1

10

3.0%

3

MFI IFNWT 351±23 Prdm1-/- 145±20

1

10

7.6%

0

10 0 10

1

2

10

3

10

10

7.7%

0

4

10

10 0 10

1

2

10

3

10

10

*

0

Effector day 8

4

10

IFN-

%GzB + WT 85% (±5) Prdm1-/- 48% (±2)

80

60

day 5

Memory day 60

C

100

Serum viral titer PFU/mL

B

Memory day 60

3000

40

20

(A) Prdm1 / or WT littermate controls were infected with LCMV and analyzed for IFN-g and IL-2 production at days 8 and 60 p.i. Representative plots showing frequency of GP33/34-41-specific and NP396-404-specific CD8+ T cells producing IFN-g and IL-2 based on ICCS after 5 hr stimulation with peptide (note that GP33-41 peptide activates both DbGP33-41- and KbGP34-41-specific CD8+ T cells; similar results observed for GP276-286specific CD8+ T cells). Mean (±SEM) IFN-g MFI is indicated next to plots. Bar graphs to right show the percent of IFN-g+ cells that coproduce IL-2. Note that no difference in TNFa was noted between the two groups of animals. *p < 0.05. (B) Granzyme B protein expression (percent GzB+ ± SEM) of WT (gray histogram) and Prdm1 / (black line) DbGP33-41-specific effector CD8+ T cells in the spleen 5 and 7 days after LCMV infection. (C) Serum LCMV viral titers in WT (black boxes) and Prdm1 / (white boxes) animals at various points after infection. n = 5–8 mice per group per time point. LOD, limit of detection. (D) Phenotype of 2 effector CD8+ T cells from LCMV immune WT (shaded) and Prdm1 / (black line) mice reinfected with LCMV-cl. 13 7 days prior. Virus was cleared in the serum in both groups by day 5 p.i.

Prdm1 -/WT

900 750 600 450 300 150 100

recall responses were not significantly different (Figure 5D). This result was not entirely unexpected since, by this time, day 7 WT and Prdm1 / memory CD8+ T cells had more similar phenotypes (Figures LOD = 50 0 2F, 2G, and 4A). These results suggest 5 8 15 that Blimp-1 acts to limit the proliferative Time after infection (days) potential of effector CD8+ T cells as they GzB terminally differentiate, but as Blimp-1 D expression naturally decays over time, the WT memory CD8+ T cells ‘‘catch up’’ to and attain similar proliferative responses to antigenic challenge as Prdm1 / cells. IL-7R CD27 CD62L KLRG1 To determine if Blimp-1 also regulated effector CD8+ T cell proliferative reWT sponses to homeostatic cytokines, WT Prdm1 -/and Prdm1 / LCMV-specific effector CD8+ T cells were CFSE-labeled and IL-2 GzB transferred into g-irradiated mice, and donor cells were examined 6 days later cells sorted from ‘‘chimeric’’ mice were transferred into new in the spleen. In a third group of mice, WT memory CD8+ naive congenic recipients that were then infected with recombi- T cells (from day 90 p.i.) were transferred for comparison. nant vaccinia virus expressing GP33-41 (rVV33). On a per-cell Similar to WT memory cells, Prdm1 / effector CD8+ T cells basis, the secondary expansion of Blimp-1-deficient effector proliferated more extensively than their WT effector counterparts cells was 4-fold greater than that of the WT effector CD8+ cells, (Figure 5E), even when the IL-7Rhi populations were compared indicating that Blimp-1 can suppress the ability of effector CD8+ directly (data not shown). In addition, larger numbers of donor T cells to proliferate when restimulated with antigen (Figure 5C). CD8+ T cells were recovered from animals that received However, when we performed similar experiments with the WT Prdm1 / versus WT effector cells (data not shown). Together, and Prdm1 / memory CD8+ T cells found 90 days p.i., their these data indicate that Blimp-1 antagonizes effector CD8+ 0

0

1

10

2

10

3

10

4

10

10

100

%GzB + WT 92% (±2) Prdm1-/- 14% (±3)

80

60

40

20

0

0

1

10

2

10

3

10

4

10

10

80

60

60

40

40

40

20

20

20

0

1 00

1 02

1 01

0

1 03

1 04

% of Max

80 60 40

0

1 04

1 03

40

20

1 00

10 1

1 02

1 03

1 04

0

1 00

10 1

1 02

1 03

1 04

80 60 40

20

0 0 10

1 02

1 01

60

100

% of Max

100

1 00

% of M ax

% of M ax

60

% of Max

1 00

80

% of Max

1 00

80

% of Max

1 00

80

% of Max

1 00

20

1

10

2

10

3

10

4

10

0 0 10

1

10

2

10

3

10

4

10

Immunity 31, 296–308, August 21, 2009 ª2009 Elsevier Inc. 303

Immunity +

Blimp-1 and Memory CD8 T Cell Differentiation

LCMV-specific P14 CD8 + T cells (x 10 5)

A 7.5

B

2.7x*

100

% div ided WT: 12% Prdm1-/-: 45%

Figure 5. Blimp-1 Expression Correlates with Cells of Lower Proliferative Capacity

(A) Blimp-1-YFP+ (black bar) or Blimp-1-YFP (white bar) Thy1.1+ P14 memory CD8+ T cells 60 5.0 from day 60 p.i. chimeric mice were sorted by 40 flow cytometry, and 25,000 cells were transferred 2.5 into naive Thy1.2+ B6 recipients that were subse20 quently infected with rLM33. The number (±SEM) 0 of donor cells recovered from the spleen 7 days 2 3 4 5 0 10 10 10 10 0 p.i. was calculated. *p < 0.05. Blimp-1-YFP+ Blimp-1-YFPCFSE (B) GzB-cre Prdm1 / (black line) or WT littermate (shaded) effector CD8+ T cells from days 8–10 p.i. C D were CFSE-labeled, stimulated with GP33-41 3.7x* 10 6 peptide, and analyzed for cell division 48 hr later. 7.5 NSD Data representative of four independent experiments each with 2 to 3 samples. 10 5 5.0 (C) Day 7 CD4-cre Prdm1 / (white bar) or WT littermate (black bar) P14 effector cells were puri10 4 2.5 fied from ‘‘chimeric’’ mice using FACS, and 25,000 cells were transferred into naive congenic recipients that were subsequently infected with 0 10 3 WT Prdm1-/rVV33. The number of secondary effector cells in WT Prdm1 -/the spleen (±SEM) was calculated 7 days postrVV33 infection. *p < 0.05. E 100 WT effector 100 Prdm1- /- effector 100 WT memory (D) 50,000 DbGP33-41-specific CD8+ T cells from PI = 1.26 PI = 1.91 PI = 1.75 GzB-cre Prdm1 / (white bar) or WT (black bar) 80 80 80 mice infected 90 days previously with LCMV 60 60 60 were transferred into naive Rag-1 / recipients 40 40 40 that were subsequently infected with rLM33. The number of secondary effector cells in the spleen 20 20 20 (±SEM) was calculated 7 days post-rLM33 infec5 4 3 2 1 0 5 4 3 2 1 0 5 4 3 2 1 0 0 0 0 0 10 2 10 3 10 4 10 5 0 102 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 tion. NSD, no significant difference. (E) Purified GzB-cre Prdm1 / or WT effector CFSE CD8+ T cells from day 10 p.i. or WT memory cells from day 90 p.i. were CFSE labeled, transferred into g-irradiated recipients, and analyzed for cell division 6 days later. Histograms were gated on DbGP33-41specific CD8+ T cells. Similar results were found for NP396-404-specific cells. Cell division numbers and PI, proliferation index (mean number of cell divisions), are indicated. Data are representative of three independent experiments each with 2 to 3 samples.

DbGP33-41-specific CD8 + T cells

% of Max

% of Max

% of Max

LCMV-specific P14 CD8 + T cells (x 10 5)

% of Max

80

T cell proliferative potential in response to antigenic and cytokine stimulation, and that the decline of Blimp-1 expression in the effector to memory transition directly contributes to the progressive increase of Tcm cells that acquire enhanced proliferative capacity and the ability to self-renew. DISCUSSION Formation and long-term survival of a functional memory CD8+ T cell population is critical for protective immunity, and this population is maintained for extended periods of time by long-lived, nonsenescent, self-renewing cells (Fearon et al., 2001; Kaech and Wherry, 2007; Lanzavecchia and Sallusto, 2002). How are T cells with these properties generated during an immune response? We propose that a small population of effector cells evades terminal differentiation during clonal expansion and acquires memory cell developmental potential. In this study, we asked whether Blimp-1, a transcriptional repressor known to regulate the terminal differentiation of diverse cell types, plays a functional role in regulating effector and memory CD8+ T cellfate decisions (Horsley et al., 2006; Magnusdottir et al., 2007; Ohinata et al., 2005; Shapiro-Shelef et al., 2003). Data from our laboratory and others indicated that Blimp-1 is preferentially expressed in more terminally differentiated effector populations and Tem cells (Intlekofer et al., 2007), and our studies here 304 Immunity 31, 296–308, August 21, 2009 ª2009 Elsevier Inc.

demonstrate that effector CD8+ T cells lacking Blimp-1 expression did not maintain normal expression of several cytolytic molecules and developed almost entirely into memory cell precursors that more rapidly acquired a Tcm cell phenotype and mature memory features such as enhanced survival, higher proliferative capacity, and increased IL-2 production. Thus, Blimp-1 suppresses the formation of both MPECs and Tcm cells, and hence, its activity balances whether an activated CD8+ T cell will terminally differentiate and acquire maximal CTL function or whether it will maintain memory cell potential. In general, after many acute infections such as LCMV, Listeria, or Sendai virus, the memory CD8+ T cell population evolves over time and slowly accumulates IL-7RhiKLRG1loCD62LhiCD27hi Tcm cells that display the functionally mature characteristics of enhanced survival and proliferation to antigen and homeostatic cytokines and increased IL-2 production compared to Tem cells (Bachmann et al., 2005; Hikono et al., 2007; Joshi et al., 2007; Pearce and Shen, 2007; Tripp et al., 1995; Wherry et al., 2003). In addition to demonstrating that this phenotypic and functional maturation of the memory population correlates with a loss of Blimp-1 expression, our studies suggest that Blimp-1 itself negatively regulates the acquisition of these mature memory features by effector cells. As suggested by our microarray data, which showed a high degree of similarity in global gene expression patterns between Blimp-1-deficient effector cells

Immunity Blimp-1 and Memory CD8+ T Cell Differentiation

and natural memory precursor effector cells, several target genes probably account for the regulation of memory cell properties by Blimp-1. In terms of effector CD8+ T cell survival, reduced cytotoxic molecule expression or increased expression of some protease inhibitors (such as Spi-6 and Spi2a) that block granzyme or cathepsin B activity have been proposed to enhance CTL survival (Ashton-Rickardt, 2005; Badovinac et al., 2000; Zhang et al., 2006, 2007). Indeed, Prdm1 / CTLs expressed lower amounts of granzyme B, granzyme A, fasL, and perforin, and increased amounts of Spi2a. In addition, Prdm1 / effector cell survival may also be enhanced by higher OX-40 expression because OX-40 signaling can augment memory T cell formation and its expression can be induced by IL-7 (Gaspal et al., 2005; Ruby et al., 2007). Finally, higher expression of lymph node homing receptors CD62L and CCR7 and retention in lymphoid organs may also contribute to the increased survival of the Prdm1 / cells by increasing their exposure to prosurvival cytokines at these sites (Link et al., 2007). Another critical memory feature that we found to be regulated by Blimp-1 is proliferative capacity. Populations of memory cells that naturally express high amounts of Blimp-1 have poorer proliferative responses to secondary recall challenge compared to populations with low Blimp-1 expression, and effector cells that genetically lack Blimp-1 expression proliferate more robustly to both antigen and homeostatic cytokines compared to their WT counterparts. In other cell types, Blimp-1 represses proliferative responses via direct transcriptional inhibition of the cell-cycle regulator myc (Horsley et al., 2006; Lin et al., 1997). Although our microarray studies did not show a marked difference in Myc mRNA expression between WT and Prdm1 / effector CD8+ T cells, past analyses demonstrate an inverse relationship between Myc and Prdm1 expression in effector and memory cells, suggesting the possibility that these factors may interact in CD8+ T cells (Wherry et al., 2007). A final feature of memory cells commonly observed is the ability to coproduce multiple cytokines (referred to as ‘‘polyfunctionality’’), and we found that a greater percentage of Prdm1 / effector and memory T cells were polyfunctional and coproduced IFN-g, TNFa, and IL-2 (albeit, average IFN-g expression was reduced in the GzB-cre Prdm1 / cells compared to WT cells, but no difference was observed in the CD4-cre P14 Prdm1 / cells). The increased IL-2 production is consistent with reports that Blimp-1 negatively regulates IL-2 expression in activated CD4+ and CD8+ T cells in vitro (Gong and Malek, 2007; Martins et al., 2008) and that the Il2 locus is a direct target of Blimp-1 repression in CD4+ T cells (Martins et al., 2008). One potential mechanism for the enhanced MPEC or Tcm phenotype seen in the Prdm1 / effector cells could be that Blimp-1 normally represses other transcriptional regulators that promote memory cell development (and suppress terminal differentiation), and therefore, in the absence of Blimp-1, these factors are expressed at higher amounts. One such transcription factor (TF), a candidate from our microarray data and studies in B cells, is the proto-oncogene Bcl6, which is a direct target of Blimp-1 in both B and CD4+ T cells (Shaffer et al., 2002; Cimmino et al., 2008). In germinal center B cells, Bcl6 and Blimp-1 mutually represses each other: Bcl6 inhibits Blimp-1 expression and promotes the formation of long-lived memory B cells (Martins and Calame, 2008; Shaffer et al., 2000; Toyama et al., 2002),

whereas Blimp-1 inhibits Bcl6 expression and promotes the formation of short-lived and long-lived antibody-secreting plasma cells (Shaffer et al., 2002; Shapiro-Shelef et al., 2003). In CD4+ T cells, Blimp-1 and Bcl-6 also mutually repress one another with Bcl-6-enhancing Th1 cell differentiation and Blimp-1-promoting Th2 cell differentiation (Cimmino et al., 2008). In CD8+ T cells, increased Bcl6 expression can augment both memory cell proliferative capacity as well as acquisition of a Tcm phenotype (Ichii et al., 2002, 2004). Mice deficient in Bcl6b, a close homolog of Bcl6, also demonstrate impaired secondary recall responses (Manders et al., 2005). Our gene expression studies found that Bcl6 is 2- to 3-fold increased IL-7Rhi MPECs versus IL-7Rlo SLECs. Moreover, Bach2, another repressor of Blimp-1 in B cells (Ochiai et al., 2006), is similarly upregulated 2- to 4-fold in IL-7Rhi MPECs (Joshi et al., 2007). Although it is not clear from these studies whether it is the amount of Bcl6 or Bcl6b or the amount of Blimp-1 expression that is directly responsible for the phenotypes observed, taken together, these data clearly indicate that the Blimp-1-Bcl6 axis plays an important role in memory CD8+ T cell maturation. The results from our microarray study also lend insight into how Blimp-1 may interact with other transcriptional regulators that are known to promote terminal differentiation. In particular, the mRNAs of the TFs T-bet (Tbx21) and inhibitor of DNA binding 2 (Id2) were reduced in Prdm1 / effector cells compared to WT cells. Similar to CD8+ T cells lacking Blimp-1, those lacking T-bet and Id2 form an increased frequency of IL-7Rhi and CD62Lhi cells (Cannarile et al., 2006a; Intlekofer et al., 2007; Joshi et al., 2007). Blimp-1 and T-bet appear to positively regulate each others’ expression in effector CD8+ T cells because CD8+ T cells lacking T-bet also produce less Prdm1 mRNA (data not shown; Intlekofer et al., 2005), although a different relationship may operate in activated CD4+ T cells because T-bet expression is actually elevated in the absence of Blimp-1 (Cimmino et al., 2008). A recent study has also found that XBP-1, a critical regulator of plasma cell formation that is downstream of Blimp-1 activity in B cells, enhances the formation of terminally differentiated KLRG1hi effector CD8+ T cells (Kamimura and Bevan, 2008). Our study provides a deeper understanding of the transcriptional network that governs effector cell fate decisions, and further exploration of the downstream targets and upstream regulators of Blimp-1 will help to better define this genetic pathway. Indeed, little is known about the upstream signals that regulate Blimp-1 expression in T cells. In B cells, IL-2, IL-5, IL-6, IL-10, and IL-21 can induce Blimp-1 expression, and STAT3 and STAT5, two transcription factors downstream of some of these cytokines, also regulate Blimp-1 and Bcl6 expression (Calame, 2008). In T cells, IL-2, IL-4, and IL-12 have been shown to induce Blimp-1 expression under certain in vitro conditions, but it is not yet known how this treatment relates to CD8+ T cell differentiation in vivo during immune responses (Gong and Malek, 2007). Thus, more work is needed to identify the signals that modulate Blimp-1 expression in vivo in both T and B cells. Improved understanding of Blimp-1 regulation could lead to new types of vaccines and treatments that modulate Blimp-1 activity to minimize certain aspects of Blimp-1-mediated effector CD8+ T cell terminal differentiation yet maximize other aspects of memory CD8+ T cell differentiation. Immunity 31, 296–308, August 21, 2009 ª2009 Elsevier Inc. 305

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EXPERIMENTAL PROCEDURES

REFERENCES

Mice, Infections, and Plaque Assays C57BL/6 (B6) mice were obtained from the NCI (Frederick, MD). Thy1.1+ P14 TCR transgenic (tg) mice and generation of ‘‘P14 chimeric mice’’ via transfer of 50,000 naive P14 CD8+ T cells (5,000 cells engraft) into B6 mice have been described previously (Joshi et al., 2007). Rag1 / animals were from the Jackson Laboratories (Bar Harbor, ME). Blimp-1-YFP BAC tg mice were either directly infected or crossed to the P14 strain to create Blimp-1-YFP P14 chimeric mice (see Supplemental Experimental Procedures for construct details). Prdm1flox mice were crossed to CD4-cre mice (Cimmino et al., 2008; Martins et al., 2006) or Granzyme B-cre mice (Jacob and Baltimore, 1999). Mice were infected with 2 3 105 pfu LCMV-Armstrong (i.p.), 2 3 106 pfu of LCMV clone 13 (i.v.), 2 3 104 cfu recombinant L. monocytogenes (i.v.) expressing the LCMV epitope GP33-41 (rLM33, a gift from Dr. H. Shen) (Kaech et al., 2003), or 2 3 104 pfu vaccinia virus expressing GP33-41 (i.p.; rVV33), kindly provided by Dr. R. Ahmed. Viral titers were measured by plaque assay (Wherry et al., 2003). All animal experiments were done with approved Institutional Animal Care and Use Committee protocols.

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Cell Isolations, Surface, and Intracellular Staining and Antibodies Lymphocytes were isolated as previously described (Joshi et al., 2007) or sorted using a FACSAria. For CFSE labeling and all other above methods, see Supplemental Experimental Procedures. Gene Expression Profiling, qRT-PCR, and Immunoblotting DNA microarray analysis was performed on four independent samples of day 8 WT or GzB-cre Prdm1 / effector cells FACS-purified based on CD8+ and MHC Class I tetramers DbGP33-41 and DbNP396-402 binding. RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA), followed by RNeasy mini elute Kit (QIAGEN, Valencia, CA). For hybridization and analysis information as well as qRT-PCR protocols, see Supplemental Experimental Procedures. Protein lysates from 1 3 106 sorted day 9 p.i. KLRG1hiIL-7Rlo or KLRG1loIL-7Rhi effector CD8+ T cells were processed and resolved by SDS-PAGE as described previously (Hand et al., 2007). Blimp-1 was detected by immunoblotting using rat anti-Blimp-1 antibody at 1:200 (clone 6D3, Santa Cruz Biotechnology Inc., Santa Cruz, CA), and expression was normalized to expression of Grp94 (Hand et al., 2007). Statistical Analysis Standard two-tailed t tests assuming normal variance were used for all statistical calculations. All error bars and variances represent standard error of the mean, and asterisks on all graphs represent p < 0.05. ACCESSION NUMBERS Microarray data have been deposited into the GEO, accession number GSE17211.

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SUPPLEMENTAL DATA

Heffner, M., and Fearon, D.T. (2007). Loss of T cell receptor-induced Bmi-1 in the KLRG1(+) senescent CD8(+) T lymphocyte. Proc. Natl. Acad. Sci. USA 104, 13414–13419.

Supplemental Data include three figures, one table, and Supplemental Experimental Procedures and can be found with this article online at http://www.cell. com/immunity/supplemental/S1074-7613(09)00323-9.

Hikono, H., Kohlmeier, J.E., Takamura, S., Wittmer, S.T., Roberts, A.D., and Woodland, D.L. (2007). Activation phenotype, rather than central- or effector-memory phenotype, predicts the recall efficacy of memory CD8+ T cells. J. Exp. Med. 204, 1625–1636.

ACKNOWLEDGMENTS We thank the members of the Kaech laboratory, Drs. J. Craft, R. Medzhitov, A. Poholek and T. Bondar for helpful comments and suggestions. We also thank Dr. Frank Constantini for the kind gift of ROSA26:EYFP mice. This work was supported by grants from the Burroughs Wellcome Fund and the NIH RO1AI066232 and R21-AI077075 (S.M.K.), RO1-AI50659 and RO1-AI43576 (K.C.), and MSTP TG 5T32GM07205 (R.L.R.). Received: December 23, 2008 Revised: April 23, 2009 Accepted: May 25, 2009 Published online: August 6, 2009

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