MiR-155 affects antiviral effector and effector memory CD8 T cell

19 Pages·2012·0.41 MB·English

JVI Accepts, published online ahead of print on 5 December 2012
J. Virol. doi:10.1128/JVI.01742-12
Copyright © 2012, American Society for Microbiology. All Rights Reserved.

MiR-155 affects antiviral effector and effector
memory CD8 T cell differentiation*

Ching-Yi Tsai, S. Rameeza Allie, Weijun Zhang and Edward J. Usherwood
D
o
w

n
lo
Microbiology and Immunology Department, Geisel School of Medicine at a
d
e
d
Dartmouth College, 1 Medical Center Drive, Lebanon, NH 03756
f
r
o
m

h
t
t
* This work was supported by NIH grants R01CA069943 and R01AI076274. p
:
/
/
jv
i.
a
s
Running title: m
.
o
r
g
/

o
n
Mir-155 and effector and memory CD8 T cell differentiation
A
p
r
il
1
4
Corresponding author: ,
2
0
1
9

b
y
Dr. Edward J. Usherwood
g
u
e
Tel. [phone] s
t
Fax. [phone]
Email: [email protected]
1
1
2 Abstract
3 MicroRNAs are key regulators of the immune response, but their role in CD8 T
4 cell differentiation in vivo is not known. We show that miR-155 is important in
5 both effector and memory antiviral CD8 T cell responses. Without miR-155,
D
6 there was a weaker effector response, and a skewing toward memory precursor o
w
n
7 cells. At the memory stage, miR-155-deficient CD8 T cells preferentially lo
a
d
8 differentiated into central memory cells, and were capable of mounting a potent e
d

f
r
9 secondary response. o
m

h
t
10 tp
:
/
/
jv
i.
a
s
m
.
o
r
g
/

o
n

A
p
r
il
1
4
,

2
0
1
9

b
y

g
u
e
s
t
2
11 Introduction
12 For intracellular pathogens, a crucial part of long-term immunity is the generation
13 of memory CD8 T cells. Naïve T cells can be directed toward at least two
14 different fate-determining pathways after antigen exposure. One pathway, driven
15 by IL-12 and IL-2 signaling, upregulation of T-bet expression, resulting in terminal
D
16 differentiation toward short lived effector cells (SLECs). Alternatively, a pathway o
w
n
17 driven by lower levels IL-12, but stimulated by IL-21 or IL-10, leads to production lo
a
d
e
18 of the precursors of memory cells (MPECs), which display less effector activity d

f
r
o
19 (1). The ultimate repository for long-term memory is the central memory cell
m

h
20 (T ), and understanding how T cells can be directed toward this fate has t
cm tp
:
/
21 profound implications for vaccine design. Given the distinct transcriptional /jv
i.
a
22 profiles of effector and memory cells (2), it is an open question if there are master s
m
.
o
23 regulators of gene expression in these cells. MicroRNAs (miRNAs) are important r
g
/

o
24 post-transcriptional regulators in many developmental processes. Several n

A
25 miRNAs have been shown to be critical in many stages of hematopoetic p
r
il
1
26 development (3). While miRNAs have been shown to influence CD4 T cell and 4
,

2
27 regulatory T cell differentiation, it is currently unclear which miRNAs influence 0
1
9

28 CD8 T cell differentiation. b
y

g
29 Studies using CD8 T cells lacking dicer, an enzyme essential for the u
e
s
t
30 processing of all miRNAs, showed deficits in CD8 T cell accumulation and
31 survival (4). Other studies profiled miRNA expression following CD8 T cell
32 activation in vitro (5, 6). Upregulation of miR-21 and miR-155, was observed
33 which was interesting due to previous studies implicating roles in immune cell
3
34 function (7, 8). In another study, CD8 T cells cultured in vitro to resemble central
35 memory cells down-regulated miR-155 relative to effector CD8 T cells (9). While
36 these studies implied miR-155 involvement in CD8 T cell differentiation and
37 memory development, it is still unclear which processes are affected by miR-155
38 during T cell differentiation in vivo. Here we report that miR-155 plays a critical
D
39 role in the generation of memory CD8 T cells during virus infection in vivo. o
w
n
40 lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
jv
i.
a
s
m
.
o
r
g
/

o
n

A
p
r
il
1
4
,

2
0
1
9

b
y

g
u
e
s
t
4
41 Results and discussion
42
43 To determine the kinetics of miR-155 expression after CD8 T cell activation, we
44 simulated OT-I cells with antigen in vitro, and measured miR-155 expression by
45 PCR. Mir-155 expression peaked at 3 days post-stimulation, then declined at all D
o
w
46 subsequent timepoints (Figure 1). The decline in miR-155 was much more n
lo
a
47 marked in cells cultured with IL-15 when compared with cultures supplemented d
e
d
48 with IL-2, resulting in significantly lower levels of miR-155 at days 4, 5 and 6 after f
r
o
m
49 activation. Culture with IL-2 generates effector / effector memory phenotype CD8

h
t
t
p
50 T cells whereas IL-15 generates cells with central memory characteristics (10).
:
/
/
jv
51 These data therefore implied that sustained miR-155 expression was associated i.
a
s
m
52 with effector / effector memory cells, whereas central memory cells were
.
o
r
g
53 associated with lower miR-155 expression. /

o
n

A
54 Next we assessed the role of miR-155 in the antiviral CD8 T cell response in p
r
il
1
55 vivo. As miR-155 has been shown to affect the biology of multiple hematopoetic
4
,

2
56 cell types (11–13), we chose a system where CD8 T cell-intrinsic effects could be 0
1
9
57 measured. To measure the relevance of miR-155 for viral control, we b
y

g
58 constructed mixed bone marrow chimeric mice such that miR-155 was absent u
e
s
t
59 from the entire CD8 T cell compartment. Following infection with MHV-68, viral
60 titers in the lung were determined by standard plaque assay (13) at d14 post-
61 infection. Viral titers were significantly higher in chimeric mice lacking miR-155 in
62 CD8 T cells when compared with mice with intact miR-155 in CD8 T cells (Fig.
63 2A). In order to study the effect of miR-155 deficiency on the biology of the CD8
5
64 T cell compartment, without the potentially confounding factor of differences in
65 viral load, mixed bone marrow chimeric mice were generated, such that both WT
66 and miR-155-/- CD8 T cells were present in the same infected mouse (14). The
67 proportion and surface phenotype (CD127, KLRG-1, CD122 and CD62L) was
68 comparable between the miR-155-/- and WT CD8 T cells before infection in these
D
69 mice (data not shown). Upon infection with MHV-68, the epitope-specific primary o
w
n
70 CD8 T cell response from each population was measured. We observed an lo
a
d
71 attenuated effector response from miR-155-/- CD8 T cells at the acute phase (Fig. ed

f
r
o
72 2B, top). Phenotypic analysis showed markedly higher CD127 expression by
m

h
73 miR-155-/- cells, and lower KLRG1 expression (Fig. 2B, bottom), indicative of a t
t
p
:
/
74 skewing toward MPECs. Similar data was obtained from the lung and /jv
i.
a
75 mediastinal lymph nodes. These changes were also observed for the sub- s
m
.
o
76 dominant ORF6 epitope, showing this was not an epitope dependent r
g
/

o
77 phenomenon (data not shown). n

A
78 The miR-155 population also exhibited elevated expression of IL-21R and p
r
il
1
79 Bcl-2, markers indicative of memory cells (Fig 2C). Expression of transcription 4
,

2
80 factors associated with the activation of effector-associated genes, T-bet and 0
1
9

81 eomesodermin, was equivalent between WT and miR-155-/- cells (Fig. 2D). As b
y

g
82 expected, the frequency of CD8 T cells producing IFN-γ was reduced in the ue
s
t
83 absence of miR-155 (Fig. 2E). No difference was detected in the proportion
84 producing TNF-α or the levels of granzyme B produced (Fig. 2E). However we
85 detected a significantly elevated production of IL-2 (Fig. 2E), consistent with
86 phenotypic skewing toward the MPEC phenotype.
6
87 As our data indicated preferential generation of an MPEC phenotype, we
88 tested a later time post-infection (d28), after the resolution of the acute infection.
89 The overall magnitude of the memory response was decreased in the miR-155-/-
90 population, (Fig. 3A). Consistent with our previous research (15), the memory
91 CD8 T cell response was dominated by effector memory (CD62Llo) cells in the
D
92 WT population. However a significantly larger proportion of miR-155-/- cells were o
w
n
93 CD62Lhi, consistent between several different organs (Fig. 3A). Analysis of lo
a
d
e
94 effector functions revealed a decreased proportion of IFN-γ, but TNF-α and d

f
r
95 granzyme B production was unaffected (Fig. 3B). However miR-155-/- cells om

h
96 produced markedly more IL-2 than the equivalent WT cells (Fig. 3B). These tt
p
:
/
/
97 phenotypic and functional changes were also observed at day 60 post-infection, jv
i.
a
98 but with larger difference between the experimental groups with regard to CD62L s
m
.
o
99 expression (Fig. 4A and Fig. 4B). Collectively, these data indicated a significantly r
g
/

o
100 larger proportion of central memory cells within the miR-155-/- CD8 T population. n

A
p
101 This was consistent with our in vitro data, which associated lower miR-155 r
il
1
102 expression with central memory differentiation. 4
,

2
0
103 To test whether memory CD8 T cells remained functional in the absence 1
9

104 of miR-155-/-, chimeric mice were challenged with a recombinant vaccinia virus by

g
u
105 encoding the ORF61 epitope from MHV-68, 60 days post-MHV-68 infection. e
s
t
106 Both WT and miR-155-/- populations expanded equivalently (Fig. 5), indicating
107 the secondary proliferative capacity of central memory-skewed miR-155-/-
108 memory CD8 T cells was intact.
7
109 Little is known about the role played by specific miRNAs in CD8 T cell
110 immunity to viruses in vivo, and this study highlights a critical role for miR-155 in
111 this process. These data indicate that miR-155 plays an important role in the
112 generation and/or maintenance of both effector and effector memory CD8 T cells
113 during virus infection. Our data show the absence of miR-155 results in a
D
114 marked reduction in the effector response during the acute stage of infection, but o
w
n
115 the remaining cells have a higher proportion of MPECs. During the memory lo
a
d
e
116 phase of the response, in the absence of miR-155 more cells expressed CD62L d

f
r
o
117 and IL-2, indicative of a central memory phenotype. This was observed in
m

h
118 several organs, suggesting this represents a systemic change in the CD8 T cell t
t
p
:
/
119 phenotype rather than a redistribution of central or effector memory cells /jv
i.
a
120 between the WT and miR-155-/- populations. Interestingly, memory cells s
m
.
o
121 expanded similarly upon secondary viral challenge, indicating the defect r
g
/

o
122 observed during the primary response was not manifest during the recall n

A
123 response. As central memory CD8 T cells have been show to confer superior p
r
il
1
124 immunity both in vivo and in adoptive cell transfer studies, suppressing miR-155 4
,

2
125 may prove a useful way to preferentially generate these cells, and lead to better 0
1
9

126 immune therapies. b
y

g
u
e
s
t
8
Bibliography

127 1. Cui, W.,
128 Y. Liu, J. S. Weinstein, J. Craft, and S. M. Kaech. 2011. An Interleukin-21-
129 Interleukin-10-STAT3 Pathway Is Critical for Functional Maturation of Memory
130 CD8+ T Cells. Immunity 35: 792–805. D
131 2. Willinger, T., T. Freeman, H. Hasegawa, A. J. McMichael, and M. F. C. Callan. o
w
132 2005. Molecular Signatures Distinguish Human Central Memory from Effector n
133 Memory CD8 T Cell Subsets. J Immunol 175: 5895–5903. lo
a
134 3. Baltimore, D., M. P. Boldin, R. M. O’Connell, D. S. Rao, and K. D. Taganov. d
e
135 2008. MicroRNAs: new regulators of immune cell development and function. d
136 Nature Immunology 9: 839–845. f
r
o
137 4. Zhang, N., and M. J. Bevan. 2010. Dicer controls CD8+ T-cell activation,
m
138 migration, and survival. Proc Natl Acad Sci U S A 107: 21629–21634.
h
139 5. Wu, H., J. R. Neilson, P. Kumar, M. Manocha, P. Shankar, P. A. Sharp, and N. t
t
p
140 Manjunath. miRNA Profiling of Naïve, Effector and Memory CD8 T Cells. PLoS :
/
141 ONE 2. /jv
142 6. Salaun, B., T. Yamamoto, B. Badran, Y. Tsunetsugu-Yokota, A. Roux, L. i.
a
143 Baitsch, R. Rouas, H. Fayyad-Kazan, P. Baumgaertner, E. Devevre, A. s
m
144 Ramesh, M. Braun, D. Speiser, B. Autran, P. Martiat, V. Appay, and P. Romero.
.
o
145 Differentiation associated regulation of microRNA expression in vivo in human r
g
146 CD8+ T cell subsets. J Transl Med 9: 44–44. /

o
147 7. Rodriguez, A., E. Vigorito, S. Clare, M. V. Warren, P. Couttet, D. R. Soond, S. n
148 van Dongen, R. J. Grocock, P. P. Das, E. A. Miska, D. Vetrie, K. Okkenhaug, A. A
149 J. Enright, G. Dougan, M. Turner, and A. Bradley. 2007. Requirement of p
r
150 bic/microRNA-155 for normal immune function. Science 316: 608–611. il
1
151 8. Lu, T. X., J. Hartner, E.-J. Lim, V. Fabry, M. K. Mingler, E. T. Cole, S. H. Orkin, 4
,
152 B. J. Aronow, and M. E. Rothenberg. 2011. MicroRNA-21 Limits In Vivo Immune 2
153 Response-Mediated Activation of the IL-12/IFN-Γ Pathway, Th1 Polarization, 0
1
154 and the Severity of Delayed-Type Hypersensitivity. J Immunol 187: 3362–3373. 9

155 9. Almanza, G., A. Fernandez, S. Volinia, X. Cortez-Gonzalez, C. M. Croce, and M. b
y
156 Zanetti. Selected MicroRNAs Define Cell Fate Determination of Murine Central g
157 Memory CD8 T Cells. PLoS One 5. u
e
158 10. Manjunath, N., P. Shankar, J. Wan, W. Weninger, M. A. Crowley, K. Hieshima, s
t
159 T. A. Springer, X. Fan, H. Shen, J. Lieberman, and U. H. von Andrian. 2001.
160 Effector differentiation is not prerequisite for generation of memory cytotoxic T
161 lymphocytes. J Clin Invest 108: 871–878.
162 11. Lu, C., X. Huang, X. Zhang, K. Roensch, Q. Cao, K. I. Nakayama, B. R. Blazar,
163 Y. Zeng, and X. Zhou. 2011. miR-221 and miR-155 Regulate Human Dendritic
164 Cell Development, Apoptosis, and IL-12 Production Through Targeting of
165 P27kip1, KPC1, and SOCS-1. Blood 117: 4293–4303.
9
166 12. Forrest, A. R. R., M. Kanamori-Katayama, Y. Tomaru, T. Lassmann, N.
167 Ninomiya, Y. Takahashi, M. J. L. de Hoon, A. Kubosaki, A. Kaiho, M. Suzuki, J.
168 Yasuda, J. Kawai, Y. Hayashizaki, D. A. Hume, and H. Suzuki. 2009. Induction
169 of microRNAs, mir-155, mir-222, mir-424 and mir-503, promotes monocytic
170 differentiation through combinatorial regulation. Leukemia 24: 460–466.
171 13. He, M., Z. Xu, T. Ding, D.-M. Kuang, and L. Zheng. 2009. MicroRNA-155
172 Regulates Inflammatory Cytokine Production in Tumor-associated Macrophages
173 via Targeting C|[sol]|EBP|[beta]|. Cellular & Molecular Immunology 6: 343–352.
174 14. Allie, S. R., W. Zhang, S. Fuse, and E. J. Usherwood. 2011. Programmed
175 Death 1 Regulates Development of Central Memory CD8 T Cells after Acute D
176 Viral Infection. The Journal of Immunology 186: 6280 –6286. o
w
177 15. Obar, J. J., S. G. Crist, D. C. Gondek, and E. J. Usherwood. 2004. Different n
178 Functional Capacities of Latent and Lytic Antigen-Specific CD8 T Cells in Murine lo
a
179 Gammaherpesvirus Infection. J Immunol 172: 1213–1219. d
e
180 d
f
r
o
m

h
t
t
p
:
/
/
jv
i.
a
s
m
.
o
r
g
/

o
n

A
p
r
il
1
4
,

2
0
1
9

b
y

g
u
e
s
t
10