Mcl-1 antagonizes bax/bak to promote effector cd4+ and cd8+ t-cell responses

ABSTRACT Members of the Bcl-2 family have critical roles in regulating tissue homeostasis by modulating apoptosis. Anti-apoptotic molecules physically interact and restrain pro-apoptotic

family members preventing the induction of cell death. However, the specificity of the functional interactions between pro- and anti-apoptotic Bcl-2 family members remains unclear. The

pro-apoptotic Bcl-2 family member Bcl-2 interacting mediator of death (Bim) has a critical role in promoting the death of activated, effector T cells following viral infections. Although

Bcl-2 is an important Bim antagonist in effector T cells, and Bcl-xL is not required for effector T-cell survival, the roles of other anti-apoptotic Bcl-2 family members remain unclear.

Here, we investigated the role of myeloid cell leukemia sequence 1 (Mcl-1) in regulating effector T-cell responses _in vivo_. We found, at the peak of the response to lymphocytic

choriomeningitis virus (LCMV) infection, that Mcl-1 expression was increased in activated CD4+ and CD8+ T cells. Retroviral overexpression of Mcl-1-protected activated T cells from death,

whereas deletion of Mcl-1 during the course of infection led to a massive loss of LCMV-specific CD4+ and CD8+ T cells. Interestingly, the co-deletion of Bim failed to prevent the loss of

Mcl-1-deficient T cells. Furthermore, lck-driven overexpression of a Bcl-xL transgene only partially rescued Mcl-1-deficient effector T cells suggesting a lack of redundancy between the

family members. In contrast, additional loss of Bax and Bak completely rescued Mcl-1-deficient effector T-cell number and function, without enhancing T-cell proliferation. These data suggest

that Mcl-1 is critical for promoting effector T-cell responses, but does so by combating pro-apoptotic molecules beyond Bim. SIMILAR CONTENT BEING VIEWED BY OTHERS C-MYC USES CUL4B TO

PRESERVE GENOME INTEGRITY AND PROMOTE ANTIVIRAL CD8+ T CELL IMMUNITY Article Open access 04 November 2023 BATF REGULATES PROGENITOR TO CYTOLYTIC EFFECTOR CD8+ T CELL TRANSITION DURING

CHRONIC VIRAL INFECTION Article 19 July 2021 HMGB2 REGULATES THE DIFFERENTIATION AND STEMNESS OF EXHAUSTED CD8+ T CELLS DURING CHRONIC VIRAL INFECTION AND CANCER Article Open access 13

September 2023 MAIN Bcl-2 family members have critical roles in immune system homeostasis. Indeed, genetic loss of the pro-apoptotic molecule Bcl-2-interacting mediator of death (Bim) or

loss of both Bax and Bak results in lymphadenopathy.1, 2 Conversely, genetic loss of the anti-apoptotic molecules Bcl-2 or myeloid cell leukemia sequence 1 (Mcl-1) results in profound

depletion of hematopoietic cells as well as developing thymocytes and peripheral T cells.3, 4, 5, 6 However, the specific interactions between pro- and anti-apoptotic Bcl-2 family members

and how these relationships control lymphocyte homeostasis remain unclear. During infection, T cells engage foreign antigen resulting in a proliferative expansion of antigen-specific

effector T cells. After antigen elimination, the majority of these effector T cells die by apoptosis, whereas some survive and become memory cells.7 This culling of activated T cells is

critical for restoring T-cell homeostasis, preventing autoimmunity, and promoting protective immune responses. Although initial work suggested a role for the death receptor pathway, more

recent work has shown a dominant role of the mitochondrial pathway of apoptosis, governed by Bcl-2 family members, in regulating the death of activated T cells.7, 8 Indeed, the BH3-only

molecule Bim, acting through Bax/Bak, is required for the apoptosis of most effector T cells.2, 9, 10, 11, 12, 13 Genetic ablation of Bim prevents the loss of effector CD4+ and CD8+ T cells

and enhances protective immunity.12, 14 However, the molecular mechanism(s) by which Bim is normally antagonized to promote effector T-cell survival remains unclear. The expression of Bcl-2

family members is dynamically regulated in activated T cells. During T-cell activation, the levels of Bcl-2 are decreased, whereas Bcl-xL expression is increased.13, 15, 16 However, despite

the normal induction of Bcl-xL, T cell-specific genetic deletion of Bcl-xL did not exacerbate the contraction of CD4+ or CD8+ T cell responses,17 suggesting that either Bcl-xL is not

required for effector T-cell survival or that it is redundant with other anti-apoptotic Bcl-2 family members. In the context of effector CD8+ T-cell subpopulations, KLRG-1hi cells have low

expression of Bcl-2, whereas KLRG-1low effector cells have high expression of Bcl-2.13 This high level of Bcl-2 expression in KLRG-1low cells is critical for their resistance to Bim-mediated

death and their survival into the memory compartment.13 Bcl-2 levels in activated T cells are controlled by IL-7 and IL-15 signaling through STAT5, a molecule essential for effector CD8+

T-cell survival.18 However, the role of other anti-apoptotic Bcl-2 family members in effector T-cell apoptosis remains unclear. Mcl-1 is another Bcl-2 family member that is highly expressed

in T cells.6, 19 Mcl-1 is critical for naive T-cell survival and Mcl-1 can efficiently bind to Bim _in vitro_ and _in vivo_.6 A recent report suggested that, at least _in vitro_, Mcl-1 may

be an important antagonist of Bim in naive T cells.20 Here, we investigated the role of Mcl-1 in maintaining activated T cells following lymphocytic choriomeningitis virus (LCMV) infection

in mice. The loss of Mcl-1 during viral infection led to a massive loss of antigen-specific CD4+ and CD8+ T cells. Notably, the loss of effector T cells in Mcl-1-deleted mice was restored by

the additional loss of Bax and Bak and was only partially restored by Bcl-xL overexpression, but was not restored by the additional loss of Bim. Together with our previous work on Bcl-2

being a major antagonist for Bim,13, 21 these data show a complex specificity between anti- and pro-apoptotic Bcl-2 family members in controlling T-cell fate. RESULTS DIVERGENT EXPRESSION OF

MCL-1 AND BCL-2 IN EFFECTOR CD4+ AND CD8+ T CELLS During T-cell activation, Bcl-2 levels are decreased in both CD4+ and CD8+ T cells, whereas Bcl-xL levels are increased.10, 19, 22, 23 A

recent report showed that Mcl-1 levels are increased following _in vitro_ T-cell activation,24 although whether or not Mcl-1 levels are changed in T cells activated _in vivo_ remains

unclear. We examined expression of Mcl-1 within antigen-specific CD4+ and CD8+ T cells after infection with LCMV. At 8 days after infection, Mcl-1 levels were increased within LCMV-sp. CD4+

and CD8+ T cells, as assessed by intracellular flow cytometric analysis of MHC-tetramer+ cells (Figure 1a). In contrast, Bcl-2 levels were decreased in both LCMV-sp. CD4+ and CD8+ T cells

(Figure 1b). Together, these data suggest that Mcl-1 could be a survival factor for activated T cells, particularly when Bcl-2 levels are low. MCL-1 IS CRITICAL FOR SURVIVAL OF ACTIVATED T

CELLS _IN VIVO_ As Mcl-1 levels were increased in activated T cells, we next determined whether sustained Mcl-1 overexpression can promote effector T-cell survival. To obtain a large

population of _in vivo_ activated T cells for retroviral transduction, we injected V_β_8.2 TCR Tg (V_β_DO) mice with staphylococcal enterotoxin B (SEB) and 24 h later, transduced purified

lymph node T cells with either empty retrovirus or retroviruses overexpressing Bcl-2 or Mcl-1 and tracked the survival of transduced (Thy1.1+), SEB-reactive, V_β_8-bearing T cells. Similar

to overexpression of Bcl-2, retroviral overexpression of Mcl-1 afforded significant protection of activated T cells after 24 h culture _in vitro_ (Figure 2a). We next determined whether

Mcl-1 was required for the generation of an effector T-cell response. Conventional Mcl-1-deficient embryos suffer from maturation defects at the blastomere stage along with defects in

blastocyst peri-implantation.25 Further, T-cell-specific deletion of Mcl-1 results in massive lymphopenia due to reduced survival of thymocytes and peripheral naive T cells.6 To avoid these

issues, we used a system in which Cre expression is controlled by the αIFN-inducible Mx1-promoter, to inducibly delete Mcl-1 during LCMV infection, as we have previously done to investigate

the role of STAT5 in effector T-cell survival.18 Mx1Cre-Mcl-1f/f and Mcl-1f/f mice were infected with LCMV and their T-cell response assessed using class I and class II MHC tetramers. At day

5 after infection, although the frequency of tetramer+ CD4+ and CD8+ T cells in Mx1Cre-Mcl-1f/f mice were slightly increased, the total numbers of tetramer+ T cells in Mx1Cre-Mcl-1f/f mice

were slightly decreased when compared with control mice (Figure 2b). This difference is likely due to a loss of total CD4+ and CD8+ T cells upon Mcl-1 deletion (Supplementary Figure 1). By

day 8, both the frequency and total numbers of LCMV-sp CD4+ and CD8+ T cells in Mx1Cre-Mcl-1f/f mice was significantly reduced when compared with control mice (Figure 2b). We next examined

the deletion of Mcl-1 in Mx1Cre-Mcl-1f/f mice by intracellular flow cytometry. Although Mcl-1 levels were decreased on day 5 after infection, by day 8 Mcl-1 levels in LCMV-sp. T cells were

similar between Mx1Cre-Mcl-1f/f mice and controls (Figure 2c). We considered that the Mcl-1-induced loss of T cells may prevent control of viral infection and drive T-cell exhaustion, so we

assessed viral load by plaque assay. On day 5 after infection, viral load was slightly higher in Mx1Cre-Mcl-1f/f mice, but by day 8, the virus was undetectable in the livers of both

Mx1cre-Mcl-1f/f and Mcl-1f/f mice (Supplementary Figure 2). Thus, Mx1Cre-induced deletion of Mcl-1 led to massive loss of LCMV-sp. T cells, and selection for Mcl-1-expressing cells (that

failed to delete Mcl-1) that were able to clear the infection. CONCOMITANT LOSS OF BIM DOES NOT RESCUE MCL-1-DEFICIENT LCMV-SP. T CELLS Mcl-1 can physically associate with Bim6 and has been

shown to antagonize Bim in T cells activated _in vitro_.20 To test whether additional loss of Bim could promote survival of Mcl-1-deficient cells, we bred Mcl-1-deficient mice to Bimf/f

mice.26 First, we administered polyI:C to groups of naive control, Mx1Cre-Mcl1f/f, and Mx1Cre-Mcl-1f/fBimf/f mice. PolyI:C induced a significant loss of CD4+ and CD8+ T cells in

Mx1Cre-Mcl1f/f mice and the additional loss of Bim failed to rescue this T-cell loss (Supplementary Figure 3). We next examined whether Mcl-1-antagonized Bim in effector T cells by infecting

the same groups of mice with LCMV. On day 8 after LCMV infection, the frequency and total numbers of CD4+ gp61-sp. and CD8+ gp33-sp.T cells were again decreased in Mx1Cre-Mcl-1f/f mice and

the additional loss of Bim was unable to restore LCMV-sp T-cell responses (Figures 3a and b). In Mx1Cre-Bimf/f mice, the numbers and frequency of LCMV-sp. CD4+ and CD8+ T cells were similar

to controls, consistent with our and others previous data showing that the total deletion of Bim does not increase LCMV-sp. T cells at the peak of the response.11, 12 We again failed to

detect a significant loss of Mcl-1 staining in LCMV-sp. CD4+ or CD8+ T cells in Mx1Cre-Mcl-1f/f mice (Figure 3c). To assess Bim deletion, we measured Bim expression within tetramer+ T cells

by intracellular flow cytometry using a Bim-specific antibody.27 Notably, we found substantial loss of Bim in CD4+gp61-sp and CD8+ gp33-sp T cells from Mx1Cre-Mcl-1f/f Bimf/f mice (Figure

3c). Thus, despite efficient deletion, this additional loss of Bim failed to restore either naive or LCMV-sp. CD4+ and CD8+ T-cell responses in mice that had inducibly deleted Mcl-1,

suggesting that Mcl-1 antagonizes additional pro-apoptotic molecules in T cells. BCL-XL OVEREXPRESSION PARTIALLY RESTORES CD4+, BUT NOT CD8+ T CELLS IN INDUCIBLE MCL-1-DELETED MICE To assess

whether overexpression of Bcl-xL could substitute for Mcl-1 deletion and rescue LCMV-sp. T-cell responses, we bred lck-Bcl-xL Tg mice to Mx1Cre-Mcl-1f/f mice, infected them with LCMV, and

assessed their T-cell responses on day 8 after infection. Bcl-xL levels, as assessed by intracellular flow cytometry were increased in both LCMV-sp. CD4+ and CD8+ T cells, although the

increase was greater in CD4+gp61-sp. T cells (Figure 4a). Surprisingly, the frequency and total numbers of CD8+ gp33-sp. T cells in Mx1Cre-Mcl-1f/f mice were not restored by expression of

ectopic Bcl-xL (Figures 4b and c). However, the frequency and total numbers of CD4+ gp61-sp. T cells were partially restored by Bcl-xL overexpression, but not back to control levels (Figures

4b and c). We observed a subtle (<20%) decrease in Mcl-1 levels in LCMV-sp. CD4+ and CD8+ T cells from lck-Bcl-xLMx1Cre-Mcl-1f/f mice (data not shown). Thus, even the overexpression of

Bcl-xL is unable to compensate for the Mcl-1 deletion in activated CD8+ T cells and only partially compensates in CD4+ T cells _in vivo._ LEVELS OF PUMA, BIM, AND NOXA ARE INCREASED IN

LCMV-SP. EFFECTOR T CELLS FROM MX1CRE-MCL1F/F MICE As concomitant loss of Bim was unable to restore LCMV-sp. T cells in Mx1Cre-Mcl-1f/f mice, we considered the possibility that Mcl-1 may

antagonize multiple BH3-only molecules. We found that the levels of Bim, Puma, and Noxa were all expressed in naive CD4+ and CD8+ T cells (Figure 5). Specificity of the Puma antibody was

confirmed on Puma-deficient mice (Supplementary Figure 4). Following LCMV infection, the levels of Bim and Puma were slightly decreased within effector CD4+ and CD8+ T cells (Figure 5),

whereas Noxa was increased in LCMV-sp. CD4+, but not CD8+, T cells (Figure 5). Nonetheless, activated T cells express several BH3-only molecules, and the loss of Bim alone is not sufficient

to restore T cells in Mcl-1-deficient mice. CONCOMITANT LOSS OF BAX AND BAK RESTORES MCL-1-DEFICIENT EFFECTOR T CELLS Neither the single additional loss of Bim nor the overexpression of

Bcl-xL, substantially restored effector T-cell responses in Mcl-1-deleted mice, suggesting that Mcl-1 may target multiple BH3-only molecules in a manner independent of Bcl-xL or that Mcl-1

targets the downstream apoptotic effectors Bax and Bak. Alternatively, we recently showed that an isoform of Mcl-1 contributes to mitochondrial respiration and ATP generation,28 raising the

possibility that Mcl-1 may function in a non-apoptotic manner to control T-cell responses. To distinguish between these possibilities, we generated mice lacking Bak that were also

conditionally deficient in Bax and Mcl-1. The additional loss of Bax and Bak would prevent the induction of apoptosis upon loss of Mcl-1, but would not rescue mitochondrial function.

Mx1Cre-Mcl-1f/f and Mx1Cre-Mcl-1f/fBaxf/fBak−/− mice (along with the respective controls) were infected with LCMV, killed at day 8 of infection and the numbers of LCMV-sp. T cells were

assessed with MHC tetramers. Inducible deletion of Mcl-1 resulted again in decreased CD4+ gp61-sp. and CD8+ gp33-sp. T-cell responses; however, in Mx1Cre-Mcl-1f/fBaxf/fBak−/− mice, the

frequency and total numbers of LCMV-sp. CD4+ and CD8+ T cells were restored (Figures 6a and b). Further, the loss of both Bax and Bak allowed the survival and persistence effector T cells

with decreased Mcl-1 expression indicating that the deletion of both pro-apoptotic effectors relieves the need of Mcl-1 (Figure 6c). To determine whether the restoration of Mcl-1-deleted

effector T cells by the additional loss of Bax and Bak was due to increased proliferation of these cells, we injected the mice with BrdU and assessed BrdU incorporation in tetramer+ T cells.

The frequency of LCMV-sp. CD4+ and CD8+ T cells that were BrdU+ was higher in Mx1Cre-Mcl-1f/fBaxf/fBak−/− mice compared with Mx1Cre-Baxf/fBak−/− mice (Figure 6d). However, the 2–4%

difference in the percentage of BrdU+ T cells is insufficient to account for the three- to sixfold increase in T cells rescued by the combined absence of Bax and Bak. Importantly, the

effector T cells retained functionality, as similar frequencies of IFN-_γ_/TNF-α-producing T cells were observed in mice that had or had not inducibly deleted all three genes (Supplementary

Figure 5). Further, virus was undetectable in the livers of mice with deletions of Mcl-1, Bax, and Bak on day 8 after infection (data not shown). Thus, the absence of both Bax and Bak

promoted the survival and functionality of effector CD4+ and CD8+ effector T cells following inducible Mcl-1 deletion. MIXED BONE MARROW (BM) CHIMERAS REVEAL A CELL INTRINSIC EFFECT OF MCL-1

ON T-CELL SURVIVAL One caveat of the Mx1Cre-system is that during the course of the response, Mcl-1 is being deleted in multiple type I interferon responsive tissues and not just in T

cells.5 To circumvent this issue, we generated mixed BM chimeras using BM from CD45.1+ B6.SJL-_Ptprc__a_ _Pepc__b_/BoyJ (referred to as CD45.1 congenics) and CD45.2+ Mx1Cre-Mcl-1f/f mice or

from CD45.1 congenics and Mcl-1f/f as controls at a 50 : 50 ratio into lethally irradiated CD45.1 congenic recipients (Figure 7a). Before infection, there was a reduced engraftment of

Mx1Cre-Mcl1f/f cells in the peripheral blood of mixed BM chimeras (Figure 7b). Nine weeks after reconstitution, mice were infected with LCMV and the numbers of control CD45.1 cells _versus_

Mx1Cre-Mcl-1f/f CD45.2, LCMV-sp. CD4+ and CD8+ T cells were enumerated on day 8 after infection. In control mice, the numbers of CD8+ gp33-sp. and CD4+ GP61-sp. T cells derived from CD45.2

Mcl-1f/f mice were slightly decreased compared with those derived from CD45.1 congenics (Figure 7c), likely because of the slightly lower CD45.2 chimerism observed in these animals (Figure

7b). In contrast, the numbers of CD8+ gp33-sp. and CD4+ gp61-sp. derived from CD45.2 Mx1Cre-Mcl-1f/f BM were decreased when compared the same cells derived from CD45.1 congenics (Figures 7c

and d). Although the chimerism was lower in this group (Figure 7b), there was a significant loss of both CD8+ gp33-sp. and CD4+ gp61-sp. derived from CD45.2 Mx1Cre-Mcl-1f/f BM compared with

their CD45.1 congenic controls (Figures 7c and d). The few tetramer+ cells emerging from the CD45.2 Mx1Cre-Mcl-1f/f BM exhibited a slight decrease in Mcl-1 levels compared with controls

(Figure 7e). Together, these data demonstrate that Mcl-1 is required in a cell intrinsic manner for generation of LCMV-sp. CD4+ and CD8+ T-cell responses. DISCUSSION T cells express multiple

pro- and anti-apoptotic Bcl-2 family members, however, the interactions between individual Bcl-2 family members and their specific roles in maintaining T-cell homeostasis has remained

unclear. Initial work, using BH3 peptides from BH3-only pro-apoptotic Bcl-2 family members indicated that Bim and Puma could bind to nearly all anti-apoptotic molecules, whereas Noxa and Bad

were more selective, Bad bound to Bcl-2, Bcl-xL, and Bcl-w but not A1 or Mcl-1 and Noxa had a higher affinity for Mcl-1 and A1 but not for Bcl-2, Bcl-xL, or Bcl-w.29 These data are

consistent with the function of ABT-737, a BH3-mimetic based on the BH3 domain of Bad, which targets Bcl-2, Bcl-xL, and Bcl-w, but not A1 or Mcl-1.30 We previously showed that that Mcl-1 is

a critical survival molecule for promoting naive T-cell survival _in vivo_6 and others have found that Mcl-1 is critical for activated and memory T-cell survival _in vitro_.24 Mcl-1 also

possesses another function, to ensure appropriate mitochondrial respiration,28 and it was possible that this function of Mcl-1 contributed to T-cell homeostasis. However, our ability to

rescue T-cell responses by the additional loss of Bax and Bak demonstrates that the anti-apoptotic function of Mcl-1 contributes significantly to its ability to maintain T-cell homeostasis.

Both biochemical and genetic experiments have suggested an interaction between Bim and Mcl-1 in naive T cells6, 20 and other cells,31, 32 although the _in vivo_ functionality of this

interaction has not been assessed. Our data show that the _in vivo_ deletion of Bim fails to rescue Mcl-1-deficient cells, whereas the loss of Bax and Bak is sufficient to rescue CD4+ and

CD8+ T-cell responses in Mcl-1-deleted mice. We envision three possible models by which Mcl-1 protects activated T cells from death. First, Mcl-1 may act downstream of Bim, targeting the

pro-apoptotic molecules Bax and/or Bak. In support of this model, it has been shown that Mcl-1 can antagonize Bak on the mitochondria.33, 34 Furthermore, the additional loss of Bak restored

most cells when Mcl-1 was deleted _in vitro_, but only when IL-7 is present.20 However, we have failed to observe rescue of Mcl-1-deleted hematopoietic cells by the loss of Bak alone (data

not shown). Nonetheless, it is possible that loss of Mcl-1 can directly lead to the spontaneous activation of both Bax and Bak in T cells. Second, it is possible that Mcl-1 inhibits a

pro-apoptotic BH3-only member that functions independently of Bim. Indeed, our data show that activated T cells express Puma and Noxa in addition to Bim; therefore, it is possible that with

the loss of Mcl-1, Puma, Noxa, and Bim can all facilitate the activation of Bax and Bak.29, 35 The third possibility is that Mcl-1 acts as to ‘tune’ Bim-mediated death. In this scenario, Bim

is largely inhibited by another anti-apoptotic Bcl-2 family member, such as Bcl-2, and Mcl-1 buffers excess Bim that is not antagonized by Bcl-2. Thus, in activated T cells, increased Mcl-1

expression may enhance resistance to death at a time when it is critical to balance pathogen clearance with immunopathology. Consistent with this concept, it has been shown that GSK-3_β_

inhibitors can maintain Mcl-1 levels and prolong activated T-cell survival.36 The loss of Bim failed to restore Mcl-1-deficient cells, so what normally restrains Bim in T cells? We recently

showed that the loss of naive, effector, and memory CD8+ T cells in Bcl-2-deficient or ABT-737-treated mice are largely rescued by additional Bim deficiency.13, 21 A critical component to

the sparing of effector CD8+ T cells is the action of the cytokines IL-7 and IL-15 that act to drive STAT5-dependent expression of Bcl-2.18 IL-4, IL-7, and IL-15 can induce an increase in

Mcl-1 protein levels in activated T cells20 and Mcl-1 levels are largely controlled post-translationally,37 one possibility is that cytokines regulate Mcl-1’s stability. However, when we

cultured T cells with cycloheximide, neither IL-7 nor IL-15 significantly stabilized Mcl-1 levels within effector CD8+ T cells (data not shown). Further, IL-7 can increase the survival of

Mcl-1-deleted T cells to a similar extent as Bcl-2 overexpression and IL-7 failed to further improve survival of Bcl-2 Tg/Mcl-1-deficient effector T cells.20 Together, these data suggest

that a major survival function of cytokines is to promote the transcriptional upregulation of Bcl-2 rather than to modulate the turnover of Mcl-1. In summary, these data suggest that

critical and specific interactions between Bcl-2 family members control T-cell homeostasis. This is important because of the potential development of Bcl-2 antagonists being developed as

therapeutics. As these drugs are developed for combating tumors, understanding the specificity of their interactions is crucial. One such drug, ABT-737 specifically antagonizes Bcl-2,

Bcl-xL, and Bcl-w, but not A1 or Mcl-1 and is effective at tumors that express high levels of Bcl-2 or Bcl-xL, but it is ineffective against tumors expressing high levels of Mcl-1.30, 38, 39

In tumors that do not express high levels of Bcl-2, Bcl-xL, or Bcl-w, other Bcl-2 family member antagonists may be more beneficial. However, a possible complication to these inhibitors is

that they may cause lymphopenia.21 Therefore, these inhibitors may also be exploited to target specific populations of T cells (activated effector cells) when these cells are wreaking havoc

(e.g., autoimmunity). Indeed, recent studies have exploited their use in mouse models of autoimmunity with substantial benefit.40, 41 For such therapeutics to have maximal efficiency with

minimal off-target effects, gaining knowledge of the specific interactions between Bcl-2 family members in specific populations of cells is essential. Despite a large amount of literature

showing biochemical interactions between Bim and Mcl-1, we clearly show that, _in vivo_, the additional loss of Bim fails to rescue the loss of cells imposed by Mcl-1 deficiency. These data

highlight the need to more carefully define the specific and antagonistic interactions between Bcl-2 family members that are operative _in vivo_. MATERIALS AND METHODS MICE Conditional

Mcl-1f/f mice6 were crossed to Mx1Cre mice (Jackson Laboratory, Bar Harbor, ME, USA); to Lck-human Bcl-xL Tg mice (Jackson Laboratory); to Baxf/fBak−/− mice;26 and to Bimf/f mice26 (a kind

gift from the late S. Korsmeyer). Puma-deficient mice were purchased from Jackson Laboratory. V_β_DO mice express the TCR_β_ chain from the DO11.10 TCR in a B10.D2 background.10, 42

B6.SJL-_Ptprc__a_ _Pepc__b_/BoyJ (BoyJ) were purchased from Jackson Laboratory. All animal protocols were reviewed and approved by our institutional animal care and use committees. VIRUS The

Armstrong-3 strain of LCMV, described previously,43 was grown in BHK-21 cells; the number of plaque-forming units (p.f.u.) was assayed on Vero cells as described.43 Mice were injected

intraperitoneally (i.p.) with 0.25 ml of LCMV (2 × 105 p.f.u.) diluted in balanced salt solution. Determination of liver viral load was determined by plaque assay on Vero cells as

described.43 MOLECULAR BIOLOGY Mouse Mcl-1 cDNA was purchased from Addgene (Cambridge, MA, USA) and subcloned into the retroviral plasmid MiT.44 MiT-Bcl-2 was generated as described.10

Retroviruses were generated by cotransfection of HEK293 with pCLEco and the MiT plasmid of interest by using calcium phosphate as described.10 After transduction, cells were stained with

various fluorescently labeled antibodies and live and dead cells were distinguished by their forward side scatter properties using a flow cytometer as described.10 MHC TETRAMERS Class II MHC

tetrameric staining reagents were created as described.45, 46 The methodology for preparation of MHC class I tetramers was modified from the protocol described by Altman and co-workers47

and were created as described previously.12, 48 For some experiments, I-Abgp66-77 tetramers were obtained from the NIH Tetramer Core Facility. We have observed no significant difference in

tracking the LCMV-sp. CD4+ T-cell response using homemade _versus_ NIH-generated LCMV-sp. class II tetramers. FLOW CYTOMETRY Spleens were harvested and 106 cells were stained with

fluorochrome-labeled antibodies (anti-CD4, anti-CD8, anti-CD44, anti-IL-7R and anti-KLRG-1) or intracellularly with antibodies against Bcl-xL (BD Transduction Labs, San Jose, CA, USA); Mcl-1

(Rockland Immunochemical, Gilbertsville, PA, USA); Bcl-2 (made in house from hybridoma 3F11); and Bim (Cell Signaling, Danvers, MA, USA), and data acquired using an LSRII flow cytometer (BD

Biosciences, San Jose, CA, USA). Data were analyzed with FacsDIVA software (BD Biosciences). Antigen-specific CD4+ T cells were identified by staining 2 × 106 spleen cells with I-Abgp61-80

tetrameric staining reagents for 2 h at 37 °C, gating away from CD16/32+ cells as described.12 LCMV-sp. CD8+ T cells were detected by staining 2 × 106 cells/well with either Dbgp33

tetrameric staining reagents for 90 min at 4 °C followed by cell surface marker staining as described.12 Intracellular cytokine staining was performed as described.12 Briefly, spleen cells

from mice were cultured at 37 °C for 4–5 h with or without various LCMV peptides (GP61-80 10 _μ_g/ml; GP33-41, at 1 _μ_g/ml) and BrefeldinA at 10 _μ_g/ml. After culture, cells were stained

for cell surface markers (CD4, CD8, CD44, antibodies from BD Pharmingen, San Jose, CA, USA) for 45 min at 4 °C. Cells were then washed, fixed, and permeabilized with 0.03% saponin and

stained intracellularly with PE-labeled anti-IFN-_γ_ or anti-IL-2 antibody (BD Pharmingen). A minimum of 5 × 105 events were acquired on a FacsCalibur flow cytometer and analyzed using

CellQuest software (BD Biosciences). MIXED BONE MARROW CHIMERAS BM from Mcl-1f/f Mx1Cre mice (CD45.2+) was mixed 1 : 1 with BM from B6.SJL-_Ptprc__a_ _Pepc__b_/BoyJ (CD45.1+) and 5 × 106

cells injected intravenously into lethally irradiated (1100 Rads) BoyJ recipients and allowed to engraft for 8 weeks at which time peripheral blood analyses indicated stable engraftment of

both CD45.1 and CD45.2 cell surface-positive cells at a 3 : 1 ratio. The chimeric mice were then infected as described. STATISTICAL ANALYSES Statistical analyses were performed using a

Student’s two-sample _t_-test with Minitab for Windows Software (Release 14), State College, PA, USA. ABBREVIATIONS * Bim: Bcl-2-interacting mediator of death * BM: bone marrow * LCMV:

lymphocytic choriomeningitis virus * Mcl-1: myeloid cell leukemia sequence 1 * p.f.u.: plaque forming units * SEB: staphylococcal enterotoxin B REFERENCES * Bouillet P, Metcalf D, Huang DC,

Tarlinton DM, Kay TW, Kontgen F _et al_. Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity. _Science_ 1999; 286:

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Immunol_ 2003; 170: 477–486. Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank the Hildeman and Opferman labs for helpful suggestions and comments. This

work was supported by Public Health Service Grants AI057753 and DK081175 (to DAH) and HL102175, the American Cancer Society 119130-RSG-10-255-01-LIB, a Cancer Center Support Grant

P30CA021765, and the American Lebanese Syrian Associated Charities of St. Judes Children’s Research Hospital (to JTO). AUTHOR CONTRIBUTIONS JTO and DAH designed the research; PT and BK

performed the research; DAH, JTO, BK, and PT analyzed and interpreted the data; PT performed the statistical analysis; and JTO and DAH wrote the manuscript. AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Division of Cellular and Molecular Immunology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine,

Cincinnati, Ohio, USA P Tripathi & D A Hildeman * Department of Biochemistry, St. Jude’s Children’s Research Hospital, Memphis, Tennessee, USA B Koss & J T Opferman Authors * P

Tripathi View author publications You can also search for this author inPubMed Google Scholar * B Koss View author publications You can also search for this author inPubMed Google Scholar *

J T Opferman View author publications You can also search for this author inPubMed Google Scholar * D A Hildeman View author publications You can also search for this author inPubMed Google

Scholar CORRESPONDING AUTHORS Correspondence to J T Opferman or D A Hildeman. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest. ADDITIONAL INFORMATION

Edited by C Borner Supplementary Information accompanies this paper on Cell Death and Differentiation website SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURES (PDF 306 KB) RIGHTS AND

PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Tripathi, P., Koss, B., Opferman, J. _et al._ Mcl-1 antagonizes Bax/Bak to promote effector CD4+ and CD8+ T-cell

responses. _Cell Death Differ_ 20, 998–1007 (2013). https://doi.org/10.1038/cdd.2013.25 Download citation * Received: 23 October 2012 * Revised: 26 February 2013 * Accepted: 28 February 2013

* Published: 05 April 2013 * Issue Date: August 2013 * DOI: https://doi.org/10.1038/cdd.2013.25 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this

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KEYWORDS * T cells * Mcl-1 * Bim * Bax * Bak * Bcl-2