Intracerebral injection of human mesenchymal stem cells impacts cerebral microvasculature after experimental stroke: MRI study

Stroke, the leading cause of disability, lacks treatment beyond thrombolysis. The acute injection of human mesenchymal stem cells (hMSCs) provides a benefit which could be mediated by an enhancement of angiogenesis. A clinical autologous graft requires an hMSC culture delay incompatible with an acute administration. This study evaluates the cerebral microvascular changes after a delayed injection of hMSCs. At day 8 after middle cerebral artery occlusion (MCAo), two groups of rats received an intracerebral injection in the damaged brain of either 10 μL of cell suspension medium (MCAo‐PBS, n = 4) or 4 × 10⁵ hMSCs (MCAo‐hMSC, n = 5). Two control groups of healthy rats underwent the same injection procedures in the right hemisphere (control‐PBS, n = 6; control‐hMSC, n = 5). The effect of hMSCs on the microvasculature was assessed by MRI using three parameters: apparent diffusion coefficient (ADC), cerebral blood volume (CBV) and vessel size index (VSI). At day 9, eight additional rats were euthanised for a histological study of the microvascular parameters (CBV, VSI and vascular fraction). No ADC difference was observed between MCAo groups. One day after intracerebral injection, hMSCs abolished the CBV increase observed in the lesion (MCAo‐hMSC: 1.7 ± 0.1% versus MCAo‐PBS: 2.2 ± 0.2%) and delayed the VSI increase (vasodilation) secondary to cerebral ischaemia. Histological analysis at day 9 confirmed that hMSCs modified the microvascular parameters (CBV, VSI and vascular fraction) in the lesion. No ADC, CBV or VSI differences were observed between control groups. At the stroke post‐acute phase, hMSC intracerebral injection rapidly and transiently modifies the cerebral microvasculature. This microvascular effect can be monitored in vivo by MRI. Copyright © 2012 John Wiley & Sons, Ltd.     

<Emphasis Type="Italic">In vivo</Emphasis> MR Imaging of Tissue-engineered Human Mesenchymal Stem Cells Transplanted to Mouse: a Preliminary Study

Current progress integrating stem cell biology and tissue engineering techniques has been invaluable to clinical applications. Prior to the application of celluar transplantation technique to patients, we need to establish techniques that can monitor their tissue biodistribution non-invasively. In this study, we proposed an imaging modality using MRI to not only monitor implanted scaffold in vivo, but also to track transplanted cells and behavior around the implant. For this purpose, human bone marrow-derived mesenchymal stem cells (hMSCs) were labeled with superparamagnetic iron oxide (Feridex) and then labeled hMSCs were cultured in a gelatin sponge used as a scaffold to support cell growth and proliferation. Histological assessment and MTT assay showed that cell labeling with MR contrast agent did not harm cell viability. Also, Feridex-labeled hMSCs showed a significant decrease in T2 signal intensity, even within the gelatin sponge in vitro. After implanting the sponge/cell complex in vivo, we could visualize cellular behavior around the implant over time using a noninvasive MRI modality and this finding was correlated with histological study, which illustrates the potential of a new approach proposed here for in vivo monitoring of implanted cell-based tissue-engineered product.     

In vivo real-time visualization of mesenchymal stem cells tropism for cutaneous regeneration using NIR-II fluorescence imaging

Mesenchymal stem cells (MSCs) have shown great potential for cutaneous wound regeneration in clinical practice. However, the in vivo homing behavior of intravenously transplanted MSCs to the wounds is still poorly understood. In this work, fluorescence imaging with Ag₂S quantum dots (QDs) in the second near-infrared (NIR-II) window was performed to visualize the dynamic homing behavior of transplanted human mesenchymal stem cells (hMSCs) to a cutaneous wound in mice. Benefiting from the desirable spatial and temporal resolution of Ag₂S QDs-based NIR-II imaging, for the first time, the migration of hMSCs to the wound was dynamically visualized in vivo. By transplanting a blank collagen scaffold in the wound to help the healing, it was found that hMSCs were slowly recruited at the wound after intravenous injection and were predominantly accumulated around the edge of wound. This resulted in poor healing effects in terms of slow wound closure and thin thickness of the regenerated skin. In contrast, for the wound treated by the collagen scaffold loaded with stromal cell derived factor-1α (SDF-1α), more hMSCs were recruited at the wound within a much shorter time and were homogenously distributed across the whole wound area, which enhances the re-epithelialization, the neovascularization, and accelerates the wound healing.     

Systemically administered human bone marrow-derived mesenchymal stem home into peripheral organs but do not induce neuroprotective effects in the MCAo-mouse model for cerebral ischemia

Mesenchymal stem cells (MSC) from bone marrow induce neuroprotective effects and improve clinical symptoms in animal models for acute cerebral ischemia. So far only few data are available from the murine system. Moreover, no data exist regarding neuroprotective effects depending on the application route. Because most preclinical trials regarding restorative therapy in stroke are performed in mice, we aimed to investigate the neuroprotective capacities of human MSC (hMSC) in the middle cerebral artery occlusion (MCAo)-mouse model of cerebral ischemia. As systemic transplantation of MSC could provide a gentle therapeutic procedure for the (mostly elderly) stroke patients, we analyzed effects of this application at a clinically relevant time point. Bone marrow-derived hMSCs were administered intravenously 24 h after MCAo. Mortality and clinical outcome of the transplanted mice did not differ from PBS-treated controls. After 3 and 7 days hMSC were robustly detected in lung, spleen, kidney and intestine, but not in the brain. MRI measurements revealed no differences in infarct size in hMSC injected animals compared to controls. In the neurogenic subventricular zone and the dentate gyrus no significant increase of endogenous cell proliferation was detected following systemic hMSC transplantation. This data further prove the week neurogenic and neuroprotective effect and the limitations of systemically administered hMSCs in cerebral ischemia.     

Transfer stamping of human mesenchymal stem cell patches using thermally expandable hydrogels with tunable cell-adhesive properties

Development of stem cell delivery system with ability of control over mutilineage differentiation and improved engraft efficiency is imperative in regenerative medicine. We herein report transfer stamping of human mesenchymal stem cells (hMSCs) patches using thermally expandable hydrogels with tunable cell-adhesive properties. The hydrogels were prepared from functionalized four arm copolymer of Tetronic^®, and the cell adhesion on the hydrogel was modulated by incorporation of fibronectin (FN) or cell-adhesive peptide (RGD). The resulting hydrogels showed spontaneous expansion in size within 10 min in response to the temperature reduction from 37 to 4°C. The adhesion and proliferation of hMSCs on FN-hydrogels were positively tunable in proportion to the amount of FN within hydrogels with complete monolayer of hMSCs (hMSC patch) being successfully achieved. The hMSC patch on the hydrogel was faced to the target substrate, which was then easily detached and re-attached to the target when the temperature was reduced from 37°C up to 4°C. We found that the transfer stamping of cell patch was facilitated at lower temperature of 4°C relative to 25°C, with the use of thinner hydrogels (0.5 mm in thickness relatively to 1.0 or 1.5 mm) and longer transfer time (>15 min). Notably, the hMSC patch was simply transferred from the hydrogel to the subcutaneous mouse skin tissue within 15 min with cold saline solution being dropped to the hydrogel. The hMSC patch following osteogenic or adipogenic commitment was also achieved with long-term culture of hMSCs on the hydrogel, which was successfully detached to the target surface. These results suggest that the hydrogels with thermally expandable and tunable cell-adhesive properties may serve as a universal substrate to harvest hMSC patch in a reliable and effective manner, which could potentially be utilized in many cell-sheet based therapeutic applications.     

Mesenchymal Stem Cells Overexpressing Interleukin‐35 Propagate Immunosuppressive Effects in Mice

To explore generation of interleukin (IL)‐35‐expressing mouse adipocyte‐derived mesenchymal stem cells (Ad‐MSCs) using lentiviral vector and their potential immunosuppressive effects in mice. Ad‐MSCs were isolated and cultured in vitro and transfected with a lentivirus vector for overexpression of the therapeutic murine IL‐35 gene. IL‐35 expression in transfected MSCs (IL‐35‐MSCs) was quantified by enzyme‐linked immunosorbent assay (ELISA). The lymphocytes subsets after one‐way mixed lymphocyte culture and in vivo intravenous transplantation were analysed by flow cytometry to evaluate the immunosuppressive effects of IL‐35‐MSCs. ELISA was performed to examine IL‐10, IL‐17A and IL‐35 expression in lymphocyte culture. Mouse Ad‐MSCs were isolated and cultured. IL‐35 was expressed in the MSC supernatant and serum after IL‐35 transduction into Ad‐MSCs by lentiviral vector transfection in vitro and in vivo. The percentage of CD4⁺ CD25⁺ T regulatory (Treg) cells in mice treated with IL‐35‐MSCs significantly increased. IL‐35‐MSCs upregulated the CD4⁺ CD25⁺ Treg cells in the allogeneic mixed lymphocyte reaction system, and lowered the percentage of CD4⁺ T cells compared with the other two control groups (P < 0.01). IL‐17A expression significantly decreased and IL‐10 expression significantly increased in IL‐35‐MSCs and MSCs when compared by ELISA to the control groups (P < 0.01). IL‐35‐transduced Ad‐MSCs in vivo can enhance proliferation of CD4⁺ CD25⁺ Treg cells and suppress the function of effector T cells such as T helper (Th) 1, Th2 and Th17 cells and may reduce the development of allograft rejection. Our data suggest that transduced Ad‐MSCs overexpressing IL‐35 may provide a useful approach for basic research on cell‐based immunotolerance therapy for inducing transplantation tolerance.     

Mesenchymal stem cells support migration, extracellular matrix invasion, proliferation, and survival of endothelial cells in vitro

We investigated effects of the paracrine factors secreted by human mesenchymal stem cells (hMSCs) on endothelial cell migration, extracellular matrix invasion, proliferation, and survival in vitro. Human mesenchymal stem cells were cultured as a monolayer or as three-dimensional aggregates in hanging drops (hMSC spheroids). We performed analysis of paracrine factors in medium conditioned by a monolayer of hMSCs and hMSC spheroids. Concentrations of vascular endothelial growth factor (VEGF), basic fibroblast growth factor, angiogenin, procathepsin B, interleukin (IL)-11, and bone morphogenic protein 2 were increased 5-20 times in medium conditioned by hMSC spheroids, whereas concentrations of IL-6, IL-8, and monocyte hemoattractant protein-1 were not increased. Concentrations of VEGF and angiogenin in medium conditioned by hMSC spheroids showed a weak dependence on the presence of serum, which allows serum-free conditioned medium with elevated concentrations of angiogenic cytokines to be obtained. Medium conditioned by hMSC spheroids was more effective in stimulation of umbilical vein endothelial cell proliferation, migration, and basement membrane invasion than medium conditioned by a monolayer of hMSCs. This medium also promotes endothelial cell survival in vitro. We suggest that culturing of hMSCs as three-dimensional cellular aggregates provides a method to concentrate proangiogenic factors secreted by hMSCs and allows for reduction of serum concentration in conditioned medium. Our data support the hypothesis that hMSCs serve as trophic mediators for endothelial cells. Disclosure of potential conflicts of interest is found at the end of this article.     

Gadolinium-chelate nanoparticle entrapped human mesenchymal stem cell via photochemical internalization for cancer diagnosis

To improve the gadolinium (Gd) internalization efficiency in stem cells, gadolinium-chelate nanoparticles were prepared from a pullulan derivative (pullulan-deoxycholic acid (DOCA)-diethylene triamine pentaacetic (DTPA)-Gd conjugate; PDDG) and then the PDDG was entrapped into human mesenchymal stem cells (hMSCs) by the photochemical-internalization (PCI) method for cancer diagnosis via the cancer homing property of hMSCs. The internalization efficiency of Gd in hMSCs was significantly increased to 98 ± 4 pg Gd/cell from 32 ± 2 pg Gd/cell via the PCI method. Moreover, the Gd-entrapped hMSCs revealed a low exocytosis ratio of gadolinium-chelate nanoparticles during cell division in vitro and a high cellular labeling efficiency for at least 21 days in vivo. The cancer-targeting and diagnosis effect of the Gd-entrapped hMSCs were confirmed in a small CT26 tumor-bearing mice model. The stem cells detected an early tumor (∼3 mm³) within 2 h using 4.7-T MR and optical imaging. The results demonstrated that the PCI-mediated internalization of Gd-incorporated nanoparticles into hMSCs is a promising protocol for efficient cell labeling and tracking.     

The effects of clinically used MRI contrast agents on the biological properties of human mesenchymal stem cells

This study was undertaken to compare the labeling efficiencies of three iron‐oxide based MRI contrast agents [Feridex, Resovist and monocrystalline iron oxide (MION)] and to evaluate their effects on the biological properties of human mesenchymal stem cells (hMSCs). The hMSCs were cultivated for 1 and 7 days after 24‐h labeling with iron oxide nanoparticles (12.5 µg Fe/mL) in the presence of poly‐L‐lysine (0.75 µg/mL). The hMSCs were labeled more efficiently with use of Feridex, Resovist as compared to MION. No significant differences were observed in terms of viability and proliferation of labeled hMSCs. The level of Oct‐4 mRNA increased in labeled hMSCs at day 1 and the cellular phenotype changed from CD45‐/CD44+/CD29+ to CD45low/CD44+/CD29+ at day 7, which closely resembles the phenotype of fresh bone marrow‐derived hMSCs. Our study has demonstrated that the Feridex or Resovist is the preferred labeling agent for hMSCs. There was a change in Oct‐4 and CD45 expression after labeling. Copyright © 2010 John Wiley & Sons, Ltd.     

体外定向诱导人骨髓间质干细胞分化为成骨细胞的研究

目的:建立体外定向诱导成人骨髓间质干细胞(MSC)分化为成骨细胞的模型。方法:采用Ficoll-Paque淋巴细胞分离液分离成人MSC,体外扩增,流式细胞仪检测细胞表面抗原的表达,应用地塞米松、β-甘油磷酸钠、vitaminC定向诱导MSC分化为成骨细胞,检测碱性磷酸酶(AP)活性、钙沉积、骨桥蛋白的表达鉴定成骨细胞,比较P3和P10代MSC成骨分化的能力。结果:MSC在体外扩增原代可获得(5-6)×10⁵个细胞,10代可获得2×10¹⁰个细胞。流式细胞仪检测结果显示CD29、CD44、CD59、CD105、CD166表达阳性,CD11a、CD14、CD33、CD34、CD45、CD38、CD80、CD86、CD117表达为阴性。加入成骨诱导剂,细胞形态发生变化,AP活性增强,骨桥蛋白表达阳性,钙沉积逐渐出现。P3和P10代的MSC均有良好的分化为成骨细胞的能力。结论:成人骨髓间质干细胞在体外可分化为成骨细胞。     

Chondrogenesis of hMSC in affinity-bound TGF-beta scaffolds

Herein we describe a bio-inspired, affinity binding alginate-sulfate scaffold, designed for the presentation and sustained release of transforming growth factor beta 1 (TGF-β1), and examine its effects on the chondrogenesis of human mesenchymal stem cells (hMSCs). When attached to matrix via affinity interactions with alginate sulfate, TGF-β1 loading was significantly greater and its initial release from the scaffold was attenuated compared to its burst release (>90%) from scaffolds lacking alginate-sulfate. The sustained TGF-β1 release was further supported by the prolonged activation (14 d) of Smad-dependent (Smad2) and Smad-independent (ERK1/2) signaling pathways in the seeded hMSCs. Such presentation of TGF-β1 led to hMSC chondrogenic differentiation; differentiated chondrocytes with deposited collagen type II were seen within three weeks of in vitro hMSC seeding. By contrast, in scaffolds lacking alginate-sulfate, the effect of TGF-β1 was short-term and hMSCs could not reach a similar differentiation degree. When hMSC constructs were subcutaneously implanted in nude mice, chondrocytes with deposited type II collagen and aggrecan typical of the articular cartilage were found in the TGF-β1 affinity-bound constructs. Our results highlight the fundamental importance of appropriate factor presentation to its biological activity, namely - inducing efficient stem cell differentiation.     

骨髓间充质干细胞在肿瘤血管生成过程中的作用

目的 研究人骨髓间充质干细胞(hMSC)在肿瘤血管生成过程中的作用.方法 通过梯度密度离心法及贴壁筛选法分离、培养hMSC;利用pLEGFP-N1逆转录病毒载体获得增强型绿色荧光蛋白(EGFP)标记的hMSC(hMSC-EGFP);流式细胞仪检测hMSC-EGFP的免疫表型及分化能力;通过建立BALB/C裸鼠实体瘤模型(分别皮下接种乳腺癌细胞系MCF-7及hMSC-EGFP细胞与MCF-7混悬液)来观察hMSC在肿瘤血管生成过程中的作用;检测肿瘤细胞和内皮细胞条件培养基对hMSC细胞生长和迁移的影响;体外诱导hMSC向内皮细胞分化,观察其对人脐静脉内皮细胞(HUVEC)迁移的影响.结果 hMSC-EGFP与hMSC形态相似,均呈成纤维细胞样;二者具有相似的免疫表型,在条件基质作用下,均能被诱导分化为成骨细胞及脂肪细胞;hMSC能促进肿瘤血管生成,单纯接种MCF-7组肿瘤平均血管密度为5.33±1.42,混悬液组为13.67±1.53,差异有统计学意义(P<0.05);大部分血管是由hMSC植入体内引起的宿主源性血管新生,只有少数血管的内皮细胞是由hMSC植入体内分化而来的;肿瘤细胞和内皮细胞能够通过其旁分泌作用促进hMSC的生长和迁移;经内皮诱导2周后,hMSC呈CD31阳性;hMSC能促进HUVEC的迁移.结论 MSC具有促进肿瘤血管生成的作用.     

Single cell‐derived clones from human adipose stem cells present different immunomodulatory properties

Human adipose mesenchymal stem cells are a heterogeneous population, where cell cultures derived from single‐cell‐expanded clones present varying degrees of differential plasticity. This work focuses on the immunomodulatory/anti‐inflammatory properties of these cells. To this end, five single‐cell clones were isolated (generally called 1.X and 3.X) from two volunteers. Regarding the expression level of the lineage‐characteristic surface antigens, clones 1·10 and 1·22 expressed the lowest amounts, while clones 3·10 and 3·5 expressed more CD105 than the rest and clone 1·7 expressed higher amounts of CD73 and CD44. Regarding cytokine secretion, all clones were capable of spontaneously releasing high levels of interleukin (IL)‐6 and low to moderate levels of IL‐8. These differences can be explained in part by the distinct methylation profile exhibited by the clones. Furthermore, and after lipopolysaccharide stimulation, clone 3.X produced the highest amounts of proinflammatory cytokines such as IL‐1β, while clones 1·10 and 1·22 highly expressed IL‐4 and IL‐5. In co‐culture experiments, clones 1.X are, together, more potent inhibitors than clones 3.X for proliferation of total, CD3⁺T, CD4⁺T and CD8⁺T lymphocytes and natural killer (NK) cells. The results of this work indicate that the adipose stem cell population is heterogeneous in cytokine production profile, and that isolation, characterization and selection of the appropriate cell clone is a more exact method for the possible treatment of different patients or pathologies.     

Human Mesenchymal Stem/Stromal Cells Cultured as Spheroids are Self-activated to Produce Prostaglandin E2 that Directs Stimulated Macrophages into an Anti-inflammatory Phenotype

Culturing cells in three dimension (3D) provides an insight into their characteristics in vivo. We previously reported that human mesenchymal stem/stromal cells (hMSCs) cultured as 3D spheroids acquire enhanced anti-inflammatory properties. Here, we explored the effects of hMSC spheroids on macrophages that are critical cells in the regulation of inflammation. Conditioned medium (CM) from hMSC spheroids inhibited lipopolysaccharide-stimulated macrophages from secreting proinflammatory cytokines TNFα, CXCL2, IL6, IL12p40, and IL23. CM also increased the secretion of anti-inflammatory cytokines IL10 and IL1ra by the stimulated macrophages, and augmented expression of CD206, a marker of alternatively activated M2 macrophages. The principal anti-inflammatory activity in CM had a small molecular weight, and microarray data suggested that it was prostaglandin E2 (PGE2). This was confirmed by the observations that PGE2 levels were markedly elevated in hMSC spheroid-CM, and that the anti-inflammatory activity was abolished by an inhibitor of cyclooxygenase-2 (COX-2), a silencing RNA for COX-2, and an antibody to PGE2. The anti-inflammatory effects of the PGE2 on stimulated macrophages were mediated by the EP4 receptor. Spheroids formed by human adult dermal fibroblasts produced low levels of PGE2 and displayed negligible anti-inflammatory effects on stimulated macrophages, suggesting the features as unique to hMSCs. Moreover, production of PGE2 by hMSC spheroids was dependent on the activity of caspases and NFκB activation in the hMSCs. The results indicated that hMSCs in 3D-spheroid cultures are self-activated, in part by intracellular stress responses, to produce PGE2 that can change stimulated macrophages from a primarily proinflammatory M1 phenotype to a more anti-inflammatory M2 phenotype. STEM Cells2012;30:2283-2296.     

miR-486-5p通过抑制端粒酶活性促进人骨髓间充质干细胞衰老

目的:研究年龄相关microRNA-486-5p(miR-486-5p)对人骨髓间充质干细胞(h MSCs)衰老的调控作用。方法:通过microRNA芯片和real-time PCR检测供体年龄对h MSCs中miR-486-5p表达的影响;通过转染miR-486-5p模拟物或抑制物,过表达miR-486-5p或抑制其表达;用β-半乳糖苷酶染色检测miR-486-5p对h MSCs衰老的影响;通过siRNA研究沉默信息调节因子1(SIRT1)对h MSCs端粒酶逆转录酶(TERT)、端粒酶活性及衰老的影响。结果:随供体年龄增加,h MSCs中miR-486-5p的表达增加。过表达miR-486-5p可促进h MSCs衰老。相反,抑制miR-486-5p的表达可减少h MSCs的衰老。SIRT1及TERT随供体年龄增加而表达下降,直接抑制SIRT1的表达可减少TERT表达,抑制端粒酶活性,促进细胞衰老。同时抑制miR-486-5p和SIRT1的表达,使miR-486-5p失去对h MSCs端粒酶活性及衰老的调控作用。结论:miR-486-5p通过抑制SIRT1,减少h MSCs端粒酶活性,促进h MSCs衰老。     

Telomerase promoter reprogramming and interaction with general transcription factors in the human mesenchymal stem cell

The human adult mesenchymal stem cell (hMSC) does not express telomerase and has been shown to be the target for neoplastic transformation after transduction with hTERT. These findings lend support to the stem cell hypothesis of cancer development but by supplying hTERT, the molecular events required to upregulate hTERT expression in cancer development are missed. Therefore, the hMSC is ideal for the identification of molecular mechanisms regulating telomerase gene expression in stem cells. This study shows that the repression of hTERT expression in hMSC is chromatin based and that modifications of the chromatin environment lead to reactivation of telomerase gene expression. It is shown that repression of hTERT expression in hMSCs is due to promoter-specific histone hypoacetylation coupled with low Pol II and TFIIB trafficking. This repression is overcome by treatment with Trichostatin A (TSA), an HDAC inhibitor, concomitant with increases in promoter-specific histone acetylation and increases in Pol II and TFIIB tracking. hTR expression is also increased in TSA-treated hMSCs, concomitant with changes in Pol II and TFIIB dynamics.     

Interleukin (IL)-21-independent pathogen-specific CD8+ T-cell expansion, and IL-21-dependent suppression of CD4+ T-cell IL-17 production

Although interleukin‐21 (IL‐21) potently activates and controls the differentiation of immune cells after stimulation in vitro, the role for this pleiotropic cytokine during in vivo infection remains poorly defined. Herein, the requirement for IL‐21 in innate and adaptive host defence after Listeria monocytogenes infection was examined. In the innate phase, IL‐21 deficiency did not cause significant defects in infection susceptibility, or in the early activation of natural killer and T cells. In the adaptive phase, L. monocytogenes‐specific CD8⁺ T cells expand to a similar magnitude in IL‐21‐deficient mice compared with control mice. Interestingly, the IL‐21‐independent expansion of L. monocytogenes‐specific CD8⁺ T cells was maintained even in the combined absence of IL‐12 and type I interferon (IFN) receptor. Similarly, L. monocytogenes‐specific CD4⁺ T cells expanded and produced similar levels of IFN‐γ regardless of IL‐21 deficiency. Unexpectedly however, IL‐21 deficiency caused significantly increased CD4⁺ T‐cell IL‐17 production, and this effect became even more pronounced after L. monocytogenes infection in mice with combined defects in both IL‐12 and type I IFN receptor that develop a T helper type 17‐dominated CD4⁺ T‐cell response. Despite increased CD4⁺ T‐cell IL‐17 production, L. monocytogenes‐specific T cells re‐expanded and conferred protection against secondary challenge with virulent L. monocytogenes regardless of IL‐21 deficiency, or combined defects in IL‐21, IL‐12, and type I IFN receptor. Together, these results demonstrate non‐essential individual and combined roles for IL‐21, IL‐12 and type I IFNs in priming pathogen‐specific CD8⁺ T cells, and reveal IL‐21‐dependent suppression of IL‐17 production by CD4⁺ T cells during in vivo infection.