Combination therapy of HIFα inhibitors and Treg depletion strengthen the anti‐tumor immunity in mice

Hypoxia‐inducible factor 1 alpha (HIF1α), under hypoxic conditions, is known to play an oxygen sensor stabilizing role by exerting context‐ and cell‐dependent stimulatory and inhibitory functions in immune cells. Nevertheless, how HIF1α regulates T cell differentiation and functions in tumor settings has not been elucidated. Herein, we demonstrated that T‐cell‐specific deletion of HIF1α improves the inflammatory potential and memory phenotype of CD8+ T cells. We validated that T cell‐specific HIF1α ablation reduced the B16 melanomas development with the indication of ameliorated antitumor immune response with enhanced IFN‐γ+ CD8+ T cells despite the increase in the Foxp3+ regulatory T‐cell population. This was further verified by treating tumor‐bearing mice with a HIF1α inhibitor. Results indicated that HIF1α inhibitor also recapitulates HIF1α ablation effects by declining tumor growth and enhancing the memory and inflammatory potential of CD8+ T cells. Furthermore, a combination of Treg inhibitor with HIF1α inhibitor can substantially reduce tumor size. Collectively, these findings highlight the notable roles of HIF1α in distinct CD8+ T‐cell subsets. This study suggests the significant implications for enhancing the potential of T cell‐based antitumor immunity by combining HIF1α and Tregs inhibitors.


Introduction
CD8 + T cells play critical roles in various biological processes and diseases, including adaptive immune response and cancer [1,2].Cognate stimuli clonally expand and differentiate CD8 + naïve T cells into distinct effector T cells and memory T-cell subsets [3], which possess short-lived cytotoxic effects and the capacity to persist and respond to repeated threats, respectively.However, uncovering and understanding the factors controlling the differentiation and the functions of these cells in cancer have been and remain the subject of intense study.Cellular metabolism is critical in regulating the expansion, differentiation and functions of T cells in various contexts, including the defense against malignancies [4][5][6][7].Naïve T cells are generally thought to be metabolically quiescent.However, activation of naïve T cells initiates the glycolytic switch by signaling cascades triggering at antigen receptors and co-stimulatory molecules.But it has been reported that IL-2, which plays a vital role in regulatory T-cell biology, can further sustain such metabolic activity [8,9].In many cells, growth factors and cytokines control glucose metabolism, and the hypoxia-inducible factor 1 alpha (HIF1α) is known to be necessary for switching to glycolysis to ensure the survival of cells and tissues under hypoxic conditions [10].During normoxia, prolyl-hydroxylases help the HIF1α subunits to hydroxylate at a specific site where two conserved prolines exist.After this stabilization, Von Hippel-Lindau (VHL) protein leads to the ultimate HIF1α proteasomal degradation through ubiquitination [11,12].Even though the hypoxic microenvironment in inflamed tissues and tumors is associated with immunosuppression and T cell differentiation [13][14][15], a few investigations validated that hypoxic conditions and HIF1 facilitate the differentiation of CD8 + T cells into the cytotoxic T cells more efficiently [16,17].It has been demonstrated that independent of HIF1α, the initial glycolytic switch in CD8 + T cells is mediated by c-Myc [18], and HIF1α is involved in CD8 + T cell metabolism and migration [19].However, the role of HIF1α in the differentiation and function of immune cells in cancerous condition is still not completely understood.
Here we report that mice lacking HIF1α, specifically in their T cells, more effectively controlled the growth of implanted melanomas and developed fewer metastatic tumors in their lung tissues than their WT counterparts.Additionally, we determined that the tumors of mice with HIF1α-deficient T cells improved the production of IFN-γ, which is known to promote inflammation and tumor cell death.These protective anti-tumor effects of genetic HIF1α blockade were effectively recapitulated by chemical inhibition of HIF1α, and further amplified upon simultaneous depletion of regulatory T cells in a novel combinational treatment strategy.In all, our results suggest that the development of HIF1α targeting approaches in cancer therapy provides effective strategies to improve antitumor immunity and achieve optimal control and eradication of tumors.

T-cell-specific HIF1α ablation significantly delays B16F10 melanoma growth
To explore the role of HIF1α in the T cell in response to the tumor growth, we generated T-cell-specific HIF-1 knockout (T-HIF1α −/− ) mice, which was challenged with subcutaneous (s.c.) injection of an aggressive melanoma line (B16F10).Our results show that mice lacking HIF1α in their T cells developed tumors that grew significantly slower than those implanted in WT mice (Fig. 1A).Despite the improved tumor growth control in the T-HIF1 −/− group, flow cytometric analysis revealed that these mice harbored elevated accumulation of Foxp3 + Tregs among the tumor-infiltrated lymphocytes (TILs) compared with their WT controls (76.4% vs. 47.3%).Similarly, increased Treg proportions were seen in the spleens and tumor-draining lymph nodes (dLN) of T-HIF1α −/− mice (Fig. 1B, C).These observations were in line with those of prior studies of HIF1α deficiency [20] that seem incongruous at first glance with more effective antitumor immune responses.In contrast, flow cytometric analysis also demonstrated a significantly enhanced frequency of IFN-γ-secreting CD8 + cells in TILs (89.0% vs. 47.9%) and lymphoid tissues of T-HIF1α −/− mice (Fig. 1D).To further verify if the decrease in tumor size and antitumor immune response in T-HIF1 −/− group is due to the increase in the activity of CD8 + T cells, we conducted the in-vivo cytotoxic CD8 T cells (CTLs) assay.The results indicated that antigen-specific cytotoxic responses of transferred T cells were significantly improved as T-HIF1α −/− CD8 + Tcell killing of p15Epeptide-pulsed splenocytes robustly enhanced (Fig. 1E).
This unexpected observation potentially accounted for the slower tumor growth in the T-HIF1α −/− mice.As reported previously, frequencies of endogenous IL-17-producing CD4 + and CD8 + T cells were low in this tumor model [21,22].Having observed pronounced stunting of in vivo tumor growth in mice specifically lacking HIF1α in their T cell compartment (T-HIF1α −/− mice), we then set out to confirm this result in a complementary model of metastatic tumor growth.Here B16F10 melanoma cells were implanted into age-and sex-matched WT and T-HIF1α −/− mice by tail vein injection (1.25×10 5 cells per mouse).Twenty-one days later, the mice were sacrificed to harvest the lungs, spleens, and lymph nodes.In line with the results of the s.c.tumor model, T-cell-specific HIF1α deficiency suppressed the profound formation of lung metastasis resulting from i.v.B16F10 challenge.Compared with WT mice, T-HIF1α −/− mice showed significantly fewer lung tumor nodules and lower lung weights (Fig. 2A, B).Again, a more robust accumulation of Foxp3 + Tregs was seen in the spleen and tumor-bearing lungs of the T-HIF1 −/− group (68.3% vs. 43.8%,Fig. 2C, D) alongside higher frequencies of IFN-γ-producing CD8 + T cells (Fig. 2E, F) in these tissues compared with WT controls.Taken together, these in vivo results suggest that HIF1α in T-cells negatively affects antitumor immune responses.They also implicate a robust enhancement in the antitumor immune response upon HIF1α ablation in these cells that may limit tumor progression in terms of systemically elevated suppressor cell populations.

IFN-γ and T-bet mRNA levels are elevated in HIF1α-deficient T cells
We next set out to determine the underlying mechanism behind the antitumor effect associated with T cell-specific HIF1α ablation and to test whether the chemical inhibition of HIF1α can also accomplish this mechanism.Given the role of HIF1α in metabolic pathways and T-cell lineage fate decisions, we suspected that the absence of HIF1α may impact molecular events occurring during T-cell activation with yet undefined consequences for T-cell function.To test this notion, CD8 + T cells were harvested from the spleens and lymph nodes of WT and T-HIF1α −/− mice and activated by using the anti-CD3 and anti-CD28 antibodies for four days.Total RNA was extracted, and cDNA was generated to quantify key transcripts by qRT-PCR.As shown in Fig. 3A, T-HIF1α −/− derived CD8 + T cells considerably increased the levels of IFN-γ mRNA upon stimulation.Correspondingly, the transcript encoding the transcriptional factor T-bet, which is known to control the expression of IFN-γ, was also found to be higher in T-HIF1α −/− CD8 + T cells (Fig. 3B).Similar results were seen in CD8 + T cells treated with HIF1α inhibitor Acriflavin in vitro (Fig. 3C, D).These results indicate that the enhanced T cell responses to tumors seen upon HIF1α loss are likely intrinsically rooted in effects on T cell activation and differentiation.

Cytotoxic CD8 + T cells differentiation is altered by HIF-1 deficiency
Cytotoxic CD8 + T cells (CTLs) exhibit a potent tumor cell eradicating mechanism that can play a central role in exerting robust antitumor effects we observed in the absence of T cell-specific HIF-1.Upon TCR activation, CD8 + T cells differentiate into effector or memory T cell subsets which are discernable by their regulated expression of the phenotypic markers CD127 (the IL-7 receptor) and KLRG1 (lectin-like receptor G1).Enhanced CD127 level exhibited the effector CD8 + T cells destined to differentiate into memory T cells, while effector cytotoxic CD8 + cells display a KLRG-1 high /CD127 low phenotype [23,24].Since HIF-1 and relevant metabolic pathways have been previously implicated in the regulation of effector and memory T-cell differentiation [25], the levels of CD127 + KLRG1 + cells were assessed in the WT and T-HIF-1 −/− mice-derived splenic cells followed by PMA and ionomycin stimulation.As shown in Fig. 4A, WT and T-HIF-1 −/− derived CD8 + T cells display similar patterns of CD127 + KLRG1 + expression (Fig. 4A, B) in unstimulated cells.Yet, 6 h after exposure to PMA and ionomycin, a significant increased population of cells exhibiting KLRG-1 high has been observed in CD8 + CD127 + cells from T-HIF-1 −/− mice, but not in CD8 + CD127 + cells from WT mice upon this short-term activation (Fig. 4A, B).While activation of CD8 + T cells with anti-CD3 and anti-CD28 antibodies for 4 days elevated the KLRG1 + effector cell fraction of both groups.As expected, the expansion of these cells was significantly pronounced in CD8 + T cells lacking HIF-1 (Fig. 4C).Similarly, we also identified the population of CD127 + KLRG1 + cells from the TILs of WT and T-HIF-1 −/− mice bearing B16F10 tumors.The gating strategy used to identify CD127 + and KLRG1 + cells in TILs is highlighted in Supporting information Fig. S1.Data indicated an increase in the frequency of CD127 lo cells in TILs of T-HIF-1 −/− mice, which represents an increase in the effector population of CD8 + T cells in   T-HIF-1 −/− mice (Supporting information Fig. S2A-C).These data suggested that HIF-1 deficiency may drive CD8 + T cells differentiation toward an effector T-cell fate.Supporting this, intracellular staining of molecules known to be essential for CTL effector function, including granzyme B, TNFα, and IFN-γ, were all found to be elevated in T-HIF-1 −/− CD8 + T cells after 4 days of in vitro stimulation (Fig. 4D-F).This suggests that T cells-specific HIF-1 −/− cells reduced the exhaustion function of CD8 + T cells, whereas an increase in IFN-γ + and TNFα + CD8 + T cells shows that there was an increase in the effector function of CD8 + T cells.Thus, HIF-1 may discourage certain effector functions of CD8 + T cells, which can destroy and target tumor cells.Moreover, inhibiting this HIF1 molecule may result in a multifaceted enhancement of antitumor immunity.

Targeting Treg amplifies the antitumor effects of HIF1α inhibition
In order to explore whether the aforementioned effects of genetic HIF1α deletion in T cells can be achieved pharmacologically, we treated mice, carrying established s.c.B16F10 tumors, with a previously characterized small molecule inhibitor of HIF1α activity, Acriflavine (ACF) [26].Similat to genetic knockout, ACF treatment substantially delayed tumor progression relative to control mice receiving vehicle injections (Supporting information Fig. S3).Furthermore, treatment of WT-derived CD8 + T cells with ACF during in vitro activation resulted in an up-regulation of IFN-γ and T-bet transcripts reminiscent of that seen in the T cellspecific HIF1α knockouts (Fig. 3C, D).These results indicate that therapeutic agents inhibiting the HIF1α may synergies the potent antitumor effects and enhance antitumor T-cell immunity.
In light of the substantial anti-tumor effect of HIF1α inhibition, we next explored avenues to achieve even more potent efficacy through rationally designed combinational regimens.In our experiments described above, significantly elevated proportions of CD4 + Foxp3 + Tregs were seen to accumulate in the tumors and lymphoid tissues of T-HIF1α −/− or ACF-treated mice.This suggests a potentially counterproductive side effect of targeting HIF1α in cancer since these tumor-abetting cells are notorious suppressors of anti-tumor immunity.We hypothesized that simultaneous depletion of Tregs alongside HIF1α targeting should provide an optimal anti-tumor effect.To test this, we administered ACF to C57BL/6 mice carrying s.c.B16F10 melanomas, either alone, as described above, or in combination with a periodic, lowdose injection of cyclophosphamide (Cytoxan, a Treg-depleting agent being explored in the clinic) [27,28].Consistent with our earlier observation, ACF-treated mice displayed markedly sloweddown rates of tumor growth compared with vehicle-treated control mice.A similar reduction in tumor growth was seen upon Cytoxan monotherapy.Importantly, in support of our hypothesis, combining the HIF1α inhibitor with periodic low-dose Cytoxan further suppressed tumor development to a level barely detectable for much of the experiment's duration (Fig. 5A, B).TIL analysis revealed that significantly more CD8 + T cells infiltrated the tumors of mice treated with ACF and Cytoxan, but this was more pronounced in the mice treated with the combination of two therapies (Fig. 5C-E).
Further, flow cytometric analysis revealed that mice treated with Cytoxan showed significantly reduced numbers of Foxp3+ Tregs among TILs and SPL.However, ACF significantly improved the Foxp3 + Tregs among TILs, SPL, and dLN (Fig. 5F; Supporting information Fig. S4).Despite the reduced tumor size after treatment of mice with ACF + Cytoxan, there was no difference in the population of Foxp3 + Tregs among TILs, SPL, and dLN (Fig. 5F; Supporting information Fig. S4).Moreover, flow cytometric analysis demonstrated a significantly enhanced frequency of IFN-γsecreting CD8 + cells in TILs, SPL, and dLN in mice co-treated with ACF and CYTOXAN (Fig. 5G; Supporting information Fig. S5).Thus, we conclude that combinational targeting of HIF1α and Treg is a viable strategy to enhance CD8 + T-cell-mediated antitumor immune responses.

Deletion of HIF1α in CD8 + T cells promotes infiltration of CD8 + T cells into solid tumors
As indicated earlier (Figs. 1 and 2), T cell-specific HIF1α knockout provokes anti-tumor immunity and delayed tumor development, indicating a HIF1α role in T-cell responses against tumors.Growing evidence suggests the use of CD8 + T cells as anticancer agents (i.e. in adoptive T-cell therapies).It offers a strong correlation between CD8 + T-cell tumor infiltration and both patient survival and favorable responses to immunotherapy [29].Therefore, we investigated the impact of HIF1α ablation on the infiltration of OVA-expressing tumors by adoptively transferred T cells.To determine the ability of HIF1α −/− CD8 + T cells to infiltrate tumors, we challenged WT mice with T-HIF1α −/− OT1 CD8 + T cells or OT1 CD8 + T cells through tail vein injection.Our results show that mice injected with OT1 CD8 + T cells significantly reduced tumor development (Fig. 6A, B), whereas mice injected with T-HIF1α −/− OT1 CD8 + T cells further strengthened the anti-tumor effects by effectively reducing tumor size as compared with WT mice and WT mice treated with OT1 CD8 + T cells (Fig. 6A, B).Meanwhile, flow cytometric analysis exhibited that mice treated with T-HIF1α −/− OT1 CD8 + T cells failed to show a significant change in the number of Foxp3 + Tregs among TILs, SPL and dLN (Fig. 6C; Supporting information Fig. S6).Nevertheless, mice treated with T-HIF1 −/− OT1 CD8 + T cells significantly boosted the population of IFN-γ + CD8 + cells in TILs, SPL, and dLN (Fig. 6D; Supporting information Fig. S7).
Additionally, we collected polyclonal CD8 + T cells from WT tdTomato + mice on a C57BL/6 background and congenic T-HIF1α −/− mice crossed to an EYFP reporter line, as illustrated in Fig. 6E.CD8 + T cells from these donor mice were mixed at a 1-to-1 ratio before i.v.injection into WT recipient mice which were injected with s.c.B16F10 cells on the same day.The relative frequencies and absolute numbers of tdTomato + (WT) and EYFP + T-HIF1α −/− CD8 + T cells were then measured in recipient mouse tumors 21 days later by flow cytometry (Fig. 6F).Our analysis revealed that T-HIF-1 −/− CD8 + T cells were found in both elevated proportions (with ∼35% of all CD8 + TILs being EYFP + compared with almost undetectable numbers of tdTomato+ cells) in the recovered TIL and greater absolute numbers compared with T cells from WT mice suggestive of an enhanced capacity to infiltrate and thrive in the tumor microenvironment (Fig. 6G).These data are the first to our knowledge to conclusively show that an intrinsic enhancement of tumor CD8+ T cell presence can result from HIF1α deletion in the condition of adoptive cell transfer.

Discussion
Hypoxia exerts a critical role in various pathological and inflammatory conditions through HIF1α.Both hypoxia and HIF1α have previously testified to modify several aspects of CD8 + T cells, including their fate, functions, and biology.However, the role of HIF1α in the function of CD8 + T cells is still controversial.Therefore, we designed this study to elaborate on the role of HIF1α in CD8 + T cells.We found that T-cell-specific knockout of HIF1α improves the inflammatory potential and memory phenotype of CD8 + T cells.
Several studies have implicated HIF1α as an important facilitator of CD8 + activation.Some investigations stipulate that HIF-1α may augment the functional capability of CD8 + T cells.For instance, mice developed with VHL-deficient CD8 + T cells exhibited upregulated HIF1α and HIF2α and controlled B16 tumor development [17].In our present study, we specifically deleted HIF-1α in T cells, and dissected its role in CD8 + T cell in vitro and in vivo.In other studies, however, stabilization of HIF1α by VHLdeletion or hypoxia induced an exhaustion-resistant and highly inflammatory CD8+ T cell phenotype [17].Likewise, in another contrasting study identifying the function of HIF1α in T cells using HIF1α fl/fl dlck CRE mice exhibited elevated tumor growth and depleted effector differentiation of CD8 + T cells [30].In the same way, deletion of VHL, HIF negative regulator, in CD8 + T cells stimulated the differentiation of memory CD8 + T cells in HIF-1α/HIF-2α-dependent manner.Meanwhile, the activity of higher antitumor potency of CD8 + T cells with constitutive HIF1α activity [31].Conversely, our study used T cell-specific HIF1 depletion to understand the functions of HIF1 in CD8 + T cells.It established a negative role for HIF1α on specific elements of differentiating CD8 + T cell and antitumor function by improving the effector functions of CD8 + T cells.It is noteworthy that earlier studies showed the role of lymphocyte protein tyrosine kinase gene (lck) and VHL in early and innate immune responses [32][33][34].This may influence the activity of HIF1α during the progression of tumors in hypoxic conditions and the activity of various T cells subsets, particularly CD8 + T cells.
Besides, it has been well established that CD8 + T cells undergo terminally exhausted differentiation under hypoxic conditions to ultimately induce the immune suppressive microenvironment [35,36].This notion was further verified by a recent study [37] indicating that the accumulation of terminally exhausted CD8 + T cells in the tumors is highly associated with suppressive functions, mainly in the hypoxic microenvironment in HIF-1α dependent manner.Meanwhile, the expression of CD39 is necessary for terminally exhausted CD8 + T cells to perform necessary suppressive functions under hypoxic conditions [37].Similar to these findings, our study identified that the ablation of HIF1α in T cells exerted an anti-tumor effect by increasing the numbers of Tregs and enhancing the population of IFN-γ + CD8 + effector T cells.
During hypoxia, exhaustion-resistant CD8 + T cells were deficit in KLRG1, identification marker for terminally differentiating T cells into effector CTLs with short-life, revealed improved granzyme B, exhibiting surface markers, that is, LAG3 and CTLA-4 upon stimulation, and alleviated involvement of transcription factors, that is, T-bet and TCF-1 [17].KLRG1 has been regarded as a marker of short-lived effector CD8 + T cells.However, it was indicated that both KLRG1 hi and KLRG1 lo effector CD8 + T cells exert similar functions, whereas T-bet regulates the activation of KLRG1 hi IL-7R lo effector CD8 + T cells [38].Meanwhile, in our study, CD8 + T cells from T-HIF1α −/− mice displayed enhanced IFN-γ and Tbx21 (T-bet) mRNA levels and the potential to effectively participate in the antitumor response.While the reasons for these discordant conclusions are currently unclear, and the existence of this conflict demands a further in-depth study of HIF1α expression and activity across the likely dynamic metabolic life cycle of T cells in various tissues.
Overall, this study concludes that HIF1α play a substantial role in CD8 + T cell function.We found that T cell-specific deletion of HIF1α significantly reduces tumor development.Meanwhile, we highlighted that, regardless of the elevated Foxp3 + Treg, T-HIF1α −/− mice developed B16 melanomas with slightly lower aggressiveness and increased memory and inflammatory potential of CD8 + T cells.Furthermore, we also suggest that combinational therapy using Treg and HIF1α inhibitors can delay tumor development (Fig. 7).Taken together, our findings endorse a negative role for HIF1α in shaping effective T responses to tumors while demonstrating the potential utility of HIF1α blocking agents and Tregs inhibitors in treating aggressive cancers.

Ethics statement
The current investigation was conducted in strict compliance with the recommendations highlighted in the Guide for the Care and Use of Laboratory Animals.The institutional Animal Care and Use Committee of SIAT and JHU scrutinized and authorized all invivo experimental protocols.All mice were housed in a specific pathogen-free facility per institutional guidelines.

Cell culture and mouse tumor challenge models
B16F10-murine melanoma cells (ATCC ® CRL-6475 TM ) were grown in DMEM formulated with sodium pyruvate, L-glutamine, glucose, 10% FBS, and penicillin and streptomycin.At 70% confluence, cells were divided.All the in-vitro experiments were conducted on less than a fifth passage.T cells were grown in RPMI

T-cell isolation and in vitro activation
Using an insulin syringe plunger, murine spleens collected from WT or T-HIF-1 −/− mice were segregated by a 70 μm mesh (Falcon) and washed with PBS.Single-cell suspension containing splenocytes were processed with ACK red blood cell lysis solution and stained for flow cytometric sorting.For intracellular staining, cells were stimulated using the phorbol 12-myristate 13acetate (10 ng/mL; Sigma), ionomycin (1 mg/mL; Sigma), and brefeldin A (5 mg/mL; Sigma) for 6 h.For the enrichment of CD4 + or CD8 + T-cells, magnetic beads (Miltenyi Biotec or STEM-CELL Technologies) were utilized.A naive CD4 + T-cell isolation kit (Miltenyi Biotec) was utilized to sequester naive CD4 + T cells.
CD8+ or CD4+ cells were activated with anti-CD3 and anti-CD28 (1 mg/mL each) to induce the expression of T-bet and IFN-γ.

Tumor digestion
Isolated B16 tumors were dissociated mechanically, digested for 30 min in 2 mg/mL collagenase type I (Worthington) and DNase I (Sigma) prepared in serum-free media at 37˚C in a shaker and passed through a 70 μm mesh (Falcon) with the help of insulin syringe plunger.Cells were washed with PBS and processed with ACK red blood cell lysis buffer (Gibco) to obtain tumor single-cell suspensions for flow cytometry analysis.

Quantitative real-time RT-PCR
For total RNA extraction, including a small RNA fraction, cultured cells were digested with TRIzol reagent (Invitrogen).Then, quantitative real-time PCR analysis (SYBR green; TaKaRa) was performed after the reverse transcriptase kit (TaKaRa) for cDNA synthesis.

Immunofluorescence staining
For immunofluorescence, 10 mm thick frozen sections were fixed with acetone and blocked with 1% BSA for 30 min at room temperature.Anti-CD8-FITC, and anti-CD45 primary antibodies were incubated for 1 h at 4°C.Alexa Fluor 568 conjugate antibody was used as the secondary antibody.4', 6-diamidino-2-phenylindole was used as the nuclear counterstain.Positive cells in 10 randomly selected fields at X40 magnification were quantitated in a blinded manner.The microscope was Nikon, Eclipse E800.The camera was Nikon, DS-Qi1Mc.The software was NIS-Element AR 3.0

Statistical analyses
In our study, data were ventured as mean ± SD or SEM as designated.The Shapiro-Wilk test analyzed data obtained during experimental processes for normal distribution.To determine statistically significant differences, a student t-test was employed by using GraphPad Prism.

Figure 3 .
Figure 3. HIF-1-deficient T cells improve the IFN-γ and T-bet mRNA levels.To measure the level of IFN-γ and T-bet mRNA, qRT-PCR was used.Primary cells were isolated from the indicated mice and activated with CD3 and CD28 antibodies or treated with Acriflavine for the respective experiments.(A, B) Expression levels of IFN-γ and T-bet mRNA in CD8 + T cells from WT and T-HIF1 −/− mice following in vitro activation with antibodies against CD3 and CD28 were assessed by qRT-PCR.(C, D) Relative IFN-γ and T-bet mRNA levels of WT CD8 cells, and the cells treated with HIF-1 inhibitor acriflavine were evaluated by qRT-PCR.For mRNA expression normalization, the housekeeping gene GAPDH was used.Data shown as ± SEM (3 replicates).Values show *p < 0.05; **p < 0.02 using unpaired student t-test.

Figure 7 .
Figure 7.An integrated strategy targeting HIF-1 to strengthen anti-tumor immune response.Reducing HIF-1 level (1) impedes the levels of genes correlated to Th17, including tumor-endorsing gene IL-17.Curbing HIF-1 also elevates the amount of highly proinflammatory subset of CD8 + T cells which raises proficient immune responses against tumors.Interestingly, HIF-1 suppression also increases the frequency of potentially suppressive Foxp3 + T cells.Meanwhile, instantaneous Treg cell inhibition or depletion (2) hinders this counterproductive potentiality or further escalate immunity against tumors.
Flow cytometryIsolated cell suspensions were stained with the LIVE/DEAD TM Fixable Near-IR Stain (Invitrogen).Cells were then washed and resuspended with staining buffer (BD) and buffer containing Fc block (BD), respectively, and incubated at 4˚C for 5 min.Predetermined concentrations of surface marker antibodies, including those recognizing CD45, CD3, CD4, and CD8 (Biolegend), were used for cell staining and incubated for 15 min or 30 min at room temperature or 4˚C, respectively.Then, BD stain buffer was used to wash the cells, and the cells were resuspended in PBS for flow cytometric analysis.Meanwhile, BD Cytofix/Cytoperm fixation/permeabilization kit (BD) was utilized for cell fixation and permeabilization after staining dead cells and cell surface for intracellular cytokine staining.We utilized eBioscience Foxp3/transcription factor staining buffer to stain transcription factor.BD LSRII Flow cytometer, available at Boston University School of Medicine Flow Cytometry Facility or the University of Maryland School of Medicine Center for Innovative Biomedical Resources, Flow Cytometry Shared Service, was utilized for flow cytometry, and FlowJo (TreeStar) was used for analysis.Finally, AccuCount fluorescent particles (Spherotech) were utilized in the tumor suspension to evaluate the tumor-infiltrating T cells by flow cytometry.