Effect of the Immune Modulators BM 12.531 (Azimexone) and BM 41.332 on the Subsets of T-Lymphocytes in Mice

The immunomodulating 2-cyanaziridine derivatives BM 12.531 (azimexone) and BM 41.332 have no effects on the total amount of T-lymphocytes in the spleen of mice but ingrease dose-dependently the percentage of Ly1^--, 2⁺, 3⁺ T-lymphocytes (killer/suppressor) and decrease the percentage of Ly1⁺, 2^--, (helper) cells. These investigations were carried out by means of specific monoclonal antibodies and fluoreqcence-activated cell sorting. The increase in Ly2⁺-cells is mainly due to increased suppressor activity.     

Gr‐1 antibody induces STAT signaling,macrophage marker expression and abrogation of myeloid‐derived suppressor cell activity in BM cells

The Gr‐1 (RB6‐8C5) Ab binds with high affinity to mouse Ly‐6G molecules and to a lower extent to Ly‐6C and has been widely used for cell depletion in infected or tumor‐bearing mice. Here we found that Gr‐1 treatment of BM cells in vitro and in vivo showed no depleting effects. The epitope recognized by the Gr‐1 Ab overlapped with Ly‐6G (1A8 Ab) but not Ly‐6C (ER‐MP20 Ab). In vitro the Gr‐1 Ab transmitted signals via STAT‐1, STAT‐3 and STAT‐5 into BM cells, similar to GM‐CSF. In healthy mice injected with the Gr‐1 Ab, the Ab remained attached to the surface of myeloid cells for at least four days. Gr‐1 Ab induced myeloid cell expansion, upregulation of macrophage markers, but not the DC marker CD11c. Suppressor activity of two distinct Gr‐1^high and Gr‐1^low expressing BM‐myeloid‐derived suppressor cell subsets was transiently ablated by Gr‐1 Ab injection. Depleting effects of Gr‐1 Ab could only be observed on inflammatory Ly‐6C^intLy‐6G^high neutrophils from the peritoneal cavity, which occurred via apoptosis and was associated with the absence of Mcl‐1 expression. Together, Gr‐1 Ab induces signals leading to myelopoiesis and affects myeloid‐derived suppressor cell activity, suggesting functional roles for Ly‐6C/G molecules in macrophage differentiation and neutrophil apoptosis.     

Fate Mapping Reveals Origins and Dynamics of Monocytes and Tissue Macrophages under Homeostasis

1. Download : Download high-res image (219KB)
2. Download : Download full-size image

Highlights? Most tissue macrophages are established before birth and are self-maintaining ? Ly6C⁺ monocytes are obligatory precursors of Ly6C^? monocytes in steady state ? Ly6C⁺ monocytes negatively control lifespan of Ly6C^? monocytes as Csf-1 sink     

S1PR5 is pivotal for the homeostasis of patrolling monocytes

Patrolling Ly6C^? monocytes are blood‐circulating cells that play a role in inflammation and in the defense against pathogens. Here, we show that similar to natural killer (NK) cells, patrolling monocytes express high levels of S1PR5, a G‐coupled receptor for sphingosine‐1 phosphate. We found that S1pr5^?/? mice lack peripheral Ly6C^? monocytes but have a normal number of these cells in the bone marrow (BM). Various lines of evidence exclude a direct contribution of S1PR5 in the survival of Ly6C^? monocytes at the periphery. Rather, our data support a role for S1PR5 in the egress of Ly6C^? monocytes from the BM. In particular, we observed a reduced frequency of patrolling monocytes in BM sinusoids of S1PR5 KO mice. Unexpectedly, S1P was not a chemoattractant for patrolling monocytes and had no significant effect on their viability in vitro. Moreover, the disruption of S1P gradients in vivo did not alter Ly6C^? monocyte trafficking and viability. These data suggest that S1PR5 regulates the trafficking of monocytes via a mechanism independent of S1P gradients.     

The kinetics of cell surface receptor expression in children perinatally exposed to polychlorinated biphenyls

Exposure to polychlorinated biphenyls (PCBs) during pre-natal and early life can alter normal immune system development. Blood specimens from newborns, 6-, and 16-month-old infants were collected in the Michalovce and Svidnik/Stropkov districts, areas with, respectively, high and low environmental PCB contamination, and lymphocyte receptor expression was evaluated by multi-color flow cytometry. The results indicate that the percentage of lymphoid dendritic cells (DC) and naïve/resting T-lymphocytes were significantly increased at 6-months in Michalovce as compared to the same cell types in cord blood samples (p?<?0.001), whereas natural regulatory T-lymphocytes and suppressor inducer T-lymphocytes were reduced (p?<?0.001). Overall, a positive linear correlation of terminally differentiated effector memory (TEM) T-lymphocyte population with age, but a negative linear correlation for myeloid DC from birth to 6-months in both regions were found. Michalovce samples indicated significantly higher expression of memory T-lymphocytes (birth, 6^th, and 16^th month), TEM T-lymphocytes (birth and 6^th month), and lymphoid DC (6^th month) compared to the Svidnik/Stropkov regions. After adjustment for relevant covariates, such as maternal age, parity, season of birth, breastfeeding, birth weight, and gender, the myeloid DC, suppressor inducer T-lymphocytes, truly naïve helper/inducer T-lymphocytes, and TEM T-lymphocytes remained significantly different between districts in cord blood samples. The multivariate analysis models for 6- and 16-month samples showed district differences in all cellular determinants, except for lymphoid DC and macrophage-like cells. This study provides the first evidence that pre-natal and early post-natal exposure to PCBs affects the dynamics of cell surface receptor expression on lymphoid DC and DC-like cells, suggesting impaired immunologic development following pre-natal and early post-natal PCB exposure.     

Tumor‐induced myeloid‐derived suppressor cell subsets exert either inhibitory or stimulatory effects on distinct CD8+ T‐cell activation events

Tumor growth coincides with an accumulation of myeloid‐derived suppressor cells (MDSCs), which exert immune suppression and which consist of two main subpopulations, known as monocytic (MO) CD11b⁺CD115⁺Ly6G^?Ly6C^high MDSCs and granulocytic CD11b⁺CD115^?Ly6G⁺Ly6C^int polymorphonuclear (PMN)‐MDSCs. However, whether these distinct MDSC subsets hamper all aspects of early CD8⁺ T‐cell activation — including cytokine production, surface marker expression, survival, and cytotoxicity — is currently unclear. Here, employing an in vitro coculture system, we demonstrate that splenic MDSC subsets suppress antigen‐driven CD8⁺ T‐cell proliferation, but differ in their dependency on IFN‐γ, STAT‐1, IRF‐1, and NO to do so. Moreover, MO‐MDSC and PMN‐MDSCs diminish IL‐2 levels, but only MO‐MDSCs affect IL‐2Rα (CD25) expression and STAT‐5 signaling. Unexpectedly, however, both MDSC populations stimulate IFN‐γ production by CD8⁺ T cells on a per cell basis, illustrating that some T‐cell activation characteristics are actually stimulated by MDSCs. Conversely, MO‐MDSCs counteract the activation‐induced change in CD44, CD62L, CD162, and granzyme B expression, while promoting CD69 and Fas upregulation. Together, these effects result in an altered CD8⁺ T‐cell adhesiveness to the extracellular matrix and selectins, sensitivity to FasL‐mediated apoptosis, and cytotoxicity. Hence, MDSCs intricately influence different CD8⁺ T‐cell activation events in vitro, whereby some parameters are suppressed while others are stimulated.     

Chronic Trichuris muris infection alters hematopoiesis and causes IFN‐γ‐expressing T‐cell accumulation in the mouse bone marrow

Proinflammatory cytokines produced during immune responses to infectious stimuli are well‐characterized to have secondary effects on the function of hematopoietic progenitor cells in the BM. However, these effects on the BM are poorly characterized during chronic infection with intestinal helminth parasites. In this study, we use the Trichuris muris model of infection and show that Th1 cell‐associated, but not acute Th2 cell‐associated, responses to chronic T. muris infection cause a major, transient expansion of CD48^?CD150^? multipotent progenitor cells in the BM that is dependent on the presence of adaptive immune cells and IFN‐γ signaling. Chronic T. muris infection also broadly stimulated proliferation of BM progenitor cells including CD48^?CD150⁺ hematopoietic stem cells. This shift in progenitor activity during chronic T. muris infection correlated with a functional increase in myeloid colony formation in vitro as well as neutrophilia in the BM and peripheral blood. In parallel, we observed an accumulation of CD4⁺, CD8⁺, and CD4^?CD8^? (double negative) T cells that expressed IFN‐γ, displaying activated and central memory‐type phenotypes in the bone marrow during chronic infection. Thus, these results demonstrate that Th1 cell‐driven responses in the intestine during chronic helminth infection potently influence upstream hematopoietic processes in the BM via IFN‐γ.     

Elicitation of Anti-Vesicular Stomatitis Virus Cytotoxic T Lymphocytes by Using Purified Viral and Cellular Antigens Incorporated into Phospholipid Vesicles

We evaluated the minimal molecular and cellular requirements for elicitation of anti-vesicular stomatitis virus (VSV) cytotoxic T lymphocytes (CTL). The results indicated that lipid vesicles containing the purified major surface glyco-protein of VSV (G protein) and purified H-2K^k glycoproteins elicited specific H-2K^k-restricted anti-VSV CTL. These antiviral CTL were shown to be Ly 1⁻,2⁺. However, both Ly 1⁺,2⁻ and Ly1⁻,2⁺ T-cell subpopulations were shown to be required for elicitation of these CTL.     

CD11b+ Ly6Chi Ly6G? Immature Myeloid Cells Recruited in Response to Salmonella enterica Serovar Typhimurium Infection Exhibit Protective and Immunosuppressive Properties

Immature myeloid cells in bone marrow are a heterogeneous population of cells that, under normal conditions, provide tissues with protective cell types such as granulocytes and macrophages. Under certain pathological conditions, myeloid cell homeostasis is altered and immature forms of these cells appear in tissues. Murine immature myeloid cells that express CD11b and Ly6C or Ly6G (two isoforms of Gr-1) have been associated with immunosuppression in cancer (in the form of myeloid-derived suppressor cells) and, more recently, infection. Here, we found that CD11b⁺ Ly6C^hi Ly6G⁻ and CD11b⁺ Ly6C^int Ly6G⁺ cells accumulated and persisted in tissues of mice infected with Salmonella enterica serovar Typhimurium (S. Typhimurium). Recruitment of CD11b⁺ Ly6C^hi Ly6G⁻ but not CD11b⁺ Ly6C^int Ly6G⁺ cells from bone marrow into infected tissues depended on chemokine receptor CCR2. The CD11b⁺ Ly6C^hi Ly6G⁻ cells exhibited a mononuclear morphology, whereas the CD11b⁺ Ly6C^int Ly6G⁺ cells exhibited a polymorphonuclear or band-shaped nuclear morphology. The CD11b⁺ Ly6C^hi Ly6G⁻ cells differentiated into macrophage-like cells following ex vivo culture and could present antigen to T cells in vitro. However, significant proliferation of T cells was observed only when the ability of the CD11b⁺ Ly6C^hi Ly6G⁻ cells to produce nitric oxide was blocked. CD11b⁺ Ly6C^hi Ly6G⁻ cells recruited in response to S. Typhimurium infection could also present antigen to T cells in vivo, but increasing their numbers by adoptive transfer did not cause a corresponding increase in T cell response. Thus, CD11b⁺ Ly6C^hi Ly6G⁻ immature myeloid cells recruited in response to S. Typhimurium infection exhibit protective and immunosuppressive properties that may influence the outcome of infection.     

Human tonsil,blood and bone marrow in vivo-induced B cells capable of spontaneous and high-rate immunoglobulin secretion in vitro: Differences in the requirements for factors and for adherent and bone marrow stromal cells,as well as distinctive adhesion molecule expression

Human B cells capable of spontaneous IgG secretion are commonly found in circulation and in lymphoid tissues such as tonsil and bone marrow (BM). The present study compares the mechanisms that regulate tonsil, blood and BM B cells capable of spontaneous IgG secretion. The BM cell subset produced IgG during a markedly longer period of time (14 days) than did tonsil and blood cell subsets (2–3 days). Blood and BM, but not tonsil, B cell IgG secretion depended on the presence of adherent cells, as demonstrated by adherent cell depletion and re-addition experiments. Stromal BM cells supported linear IgG secretion by non-adherent BM cells for 2 weeks, but were unable to prolong the short-term IgG secretion by tonsil and blood cells. Different factors induced IgG secretion in each of the three B cell populations as optimal IgG secretion by tonsil, blood or BM cell subsets required either tumor necrosis factor-α, interleukin-6 or fibronectin + interleukin-6, respectively. Finally, these populations also showed differences in the expression of adhesion molecules; the tonsilar cell subset was PNA^+/? CD44⁺ CD49d⁺ CD49e^? Leu-8^+/?, the blood cell subset was PNA^? CD44^+/? CD49d⁺ CD49e^? Leu-8⁺ and the BM cell subset was PNA^? CD44^+/? CD49d⁺ CD49e^? Leu-8^?. These results suggest that the mechanisms controlling the final differentiation and the expression of adhesion molecules in these B lymphocytes exhibit territorial specificity.     

Fully functional monocytic MDSC generation from the murine HoxB8 cell line

Myeloid-derived suppressor cells (MDSC) play a crucial role in controlling T-cell responses, but their development and suppressor mechanisms are not fully understood. To study the molecular functions of MDSC, a large number of standardized cells are required. Traditionally, bone marrow (BM) has been used to generate myeloid cell types, including MDSC. In this study, we demonstrate that a previously described protocol for generating monocytic MDSC (M-MDSC) from murine BM with GM-CSF can be fully transferred to BM cells that are conditionally transformed with HoxB8 gene (HoxB8 cells). HoxB8 cells have an extended lifespan and efficiently differentiate into MDSC that are quantitatively and qualitatively comparable to M-MDSC from BM cells. Flow cytometric analyses of LPS/IFN-γ activated cultures revealed the same iNOS⁺ and/or Arg1⁺ PD-L1^high M-MDSC subsets in similar frequencies from BM or HoxB8 cells. In vitro suppression of CD4⁺ and CD8⁺ T-cell proliferations was also largely comparable in their efficacy and its iNOS- or Arg1-dependent suppressor mechanisms, which was confirmed by the similar amounts of nitric oxide (NO) secretion measured from the suppressor assay. Therefore, our data suggest that murine M-MDSC generation from HoxB8 cells with GM-CSF can be used to substitute BM cultures.     

FTY720 suppresses the development of colitis in lymphoid‐null mice by modulating the trafficking of colitogenic CD4+ T cells in bone marrow

2‐Amino‐2‐(2‐[4‐octylphenyl]ethyl)‐1,3‐propanediol hydrochloride (FTY720) suppresses T‐cell egress from LN, thereby preventing pathogenic T cells from migrating toward disease sites. However, little is known about whether FTY720 could control the trafficking of T cells without the presence of lymphoid tissues. Here we demonstrate that FTY720 treatment suppresses the recirculation of CD4⁺ T cells in splenectomized (SPX) lymphotoxin‐α^?/? (LT‐α^?/?) mice that lack LN and spleen, as shown by peripheral blood (PB) lymphopenia in FTY720‐treated SPX LT‐α^?/? mice. In a short‐term transfer experiment, the cell number of transferred Ly5.1⁺CD4⁺ T cells recovered from host FTY720‐treated SPX LT‐α^?/? mice (Ly5.2⁺) was markedly decreased in PB, but conversely increased in BM. Notably, FTY720 treatment prevented the development of colitis that is otherwise induced in untreated SPX LT‐α^?/?×RAG‐2^?/? mice upon transfer of colitic lamina propria CD4⁺ T cells. In such mice, the number of CD4⁺ T cells in PB or lamina propria of FTY720‐treated SPX LT‐α^?/?×RAG‐2^?/? recipients was significantly reduced, but that in the BM was significantly increased as compared with untreated control mice. Altogether, the present results indicate that FTY720 treatment may offer an additional role to direct trafficking of CD4⁺ T cells in BM, resulting in the prevention of colitis.     

Adult BM generates CD5+ B1 cells containing abundant N‐region additions

The self‐renewing capacity of B1 cells infers homeostatic regulation; however, previous work suggests the low level of N‐region addition characterizing B1 cells early in life increases with age, which implies that the B1‐cell population is not a closed system. To explore this, we evaluated N‐region addition in CD5⁺ B1 cells generated from adult BM. Adult BM cells were marked with GFP introduced by mouse stem cell virus transduction, and were then adoptively transferred into lethally irradiated recipients. Within 2–3 months, we found GFP‐marked CD5⁺ B cells in the peritoneal cavities of recipients, which we demonstrate here meet a variety of criteria for B1‐cell traits including Mac‐1 surface expression; annexin, elfin, and Pax‐5 gene expression; mitogenic responsiveness to phorbol ester; and spontaneous immunoglobulin secretion. Notably, we found by single‐cell PCR that this population of BM‐derived CD5⁺ B1 cells expressed immunoglobulin with abundant N‐region addition (and little V_H11/V_H12 skewing), unlike CD5⁺ B1 cells obtained from unmanipulated animals but reminiscent of B2 cells. Further, we confirmed that native CD5⁺ B1 cells from older mice contain more N‐region additions than native CD5⁺ B1 cells from younger mice. These results suggest that adult BM progenitors contribute to the peritoneal CD5⁺ B1‐cell pool over time.     

Differential Expression of the Ly49GB6, but Not the Ly49GBALB,Receptor Isoform during Natural Killer Cell Reconstitution after Hematopoietic Stem Cell Transplantation

Inhibitory natural killer (NK) cell receptors specific for major histocompatibility complex class I (MHC-I) molecules include Ly49 receptors in mice and killer immunoglobulin-like receptors (KIR) in humans. The “licensing” or “arming” models imply that engagement of these receptors to self MHC-I molecules during NK cell development educates NK cells to be more responsive to cancer and viral infection. We recently reported that hematopoietic stem cell transplantation (HSCT) induced rapid and preferential expansion of functionally competent Ly49G⁺, but not other Ly49 family, NK cells independent of NK cell licensing via Ly49–MHC-I interactions. We now extend these studies to evaluate expression of the two Ly49G receptor isoforms Ly49G^B6 and Ly49G^BALB, using mice with different MHC-I haplotypes that express one or both of the isoforms. NK cells from CB6F₁ (H-2^bxd) hybrid mice express two different alleles for Ly49G receptor, Ly49G^B6 and Ly49G^BALB. We found that CB6F₁ mice had more Ly49G^B6+ NK cells than Ly49^BALB+ NK cells, and that only Ly49G^B6+ NK cells increased in relative numbers and in Ly49G mean fluorescence intensity values after HSCT similar to the B6 parental strain. We further observed that Ly49G⁺ NK cells in BALB/c (H-2^d) and BALB.B (H-2^b) mice, which have the same background genes, recover slowly after HSCT, in contrast to Ly49G⁺ NK cells in B6 (H-2^b) recipients. The difference in expression of Ly49G^B6 relative to Ly49G^BALB was linked to differences in the activity of the Pro1 promoter between the two alleles. Thus, we conclude that the Ly49G^B6 receptor dominates Ly49G expression on NK cells after HSCT in strains in which that allele is expressed. The data suggest that Ly49 allelic polymorphism within a particular Ly49 family member can differentially affect NK cell recovery after HSCT depending on the background genes of the recipient, not on the MHC-I haplotype.     

Expansion of CD11b+Ly-6C+ myeloid-derived suppressor cells (MDSCs) driven by galectin-9 attenuates CVB3-induced myocarditis

Galectin-9 is known to play a role in the modulation of innate and adaptive immunity to ameliorate CVB3-induced myocarditis. In the present study, we found that galectin-9 induced the expansion of CD11b⁺Ly-6C⁺ myeloid-derived suppressor cells (MDSCs) in the heart from CVB3-infected mice. Adoptive transfer of CD11b⁺Ly-6C⁺ MDSCs significantly alleviated myocarditis accompanied by increased Th2 and Treg frequency and anti-inflammatory cytokines expression in the heart tissue. Moreover, Ly6C⁺ MDSCs, but not Ly6G⁺ cells, expressed Arg-1 and NOS2, and suppressed CD4⁺ T cell proliferation in vitro in an Arg-1-dependent mechanism; an event that was reversed with treatment of either an Arg-1 inhibitor or addition of excess l-arginine. Furthermore, Ly6C⁺ MDSCs co-expressed higher levels of F4/80, Tim-3, and IL-4Rα, and had the plasticity to up-regulate NOS2 or Arg-1 in response to IFN-γ or IL-4 treatment. The present results indicate that galectin-9 expands CD11b⁺Ly-6C⁺ MDSCs to ameliorate CVB3-induced myocarditis.     

Expression of the Ly49A gene in murine natural killer cell clones is predominantly but not exclusively mono-allelic

The Ly49 family of natural killer (NK) cell receptors are major histocompatibility complex (MHC) class I-specific inhibitory receptors that are distributed to overlapping NK cell subsets. Extending earlier studies of polyclonal NK cell populations, we have employed an analysis of short-term NK cell clones from Ly49A heterozygous mice, to demonstrate that the Ly49A gene is usually expressed from one or the other allele in each Ly49A⁺ cell. However, we also detected a small percentage of clones that expressed both Ly49A alleles. The possibility that the colonies exhibiting bi-allelic Ly49A gene expression had been inoculated with more than one cell was ruled out by parallel analysis of clones isolated from mixtures of NK cells from Ly49A homozygous mice. The frequency of bi-allelic Ly49A⁺ clones suggested that the two Ly49A alleles in an NK cell are chosen for expression independently. These data are consistent with the proposal that mono-allelic Ly49A gene expression may arise as a consequence of a stochastic Ly49 gene activation mechanism. Analysis of Ly49A⁺ clones from MHC-different mice demonstrated that class I-deficient mice harbored a greater number of bi-allelic Ly49A⁺ cells than did H-2^d mice, which express a Ly49A ligand. Although the numbers were insufficiently large for a clear assignment, H-2^b mice may harbor an intermediate number of bi-allelic Ly49A⁺ NK cells. The effects of MHC expression on the prevalence of bi-allelic Ly49A⁺ cells suggest that an MHC-dependent education process modifies the Ly49 repertoire.     

The migration of bone marrow-derived non-hematopoietic tissue-committed stem cells is regulated in an SDF-1-, HGF-, and LIF-dependent manner

Introduction: Recently we identified in bone marrow (BM) by employing chemotactic isolation to SDF-1 gradient combined with real time RT-PCR analysis a mobile population of CXCR4⁺ BM mononuclear cells that express mRNA for various markers of early tissue-committed stem cells (TCSCs). In this study we evaluated whether TCSCs respond to other motomorphogens, such as hepatocyte growth factor (HGF) and leukemia inhibitory factor (LIF). Materials and Methods: We again employed chemotactic isolation combined with real-time RT-PCR analysis to assess whether murine and human BM contain TCSCs that respond to HGF and LIF gradients. We also evaluated expressions of HGF and LIF in damaged organs. Results: We noted that the number of TCSCs is highest in BM from young (1- to 2-month-old) mice and decreases in 1-year–old animals. Murine and human TCSCs 1) respond to HGF and LIF gradients in addition to an SDF-1 gradient, 2) reside in populations of BM-derived non-hematopoietic CD45⁻cells, and 3) are released (mobilized) from BM into the peripheral blood (PB) during tissue injury (e.g. after partial body irradiation). Conclusions: These findings further support our theory of the BM as a “hideout” for TCSCs and we suggest that their presence in BM tissue should be considered before experimental evidence is interpreted simply as transdifferentiation/plasticity of hematopoietic stem cells. Since we demonstrated that not only SDF-1, but also HGF and LIF are upregulated in damaged tissues, we postulate that CXCR4⁺ c-Met⁺ LIF-R⁺ TCSC could be mobilized from the BM into the PB, from which they are subsequently chemoattracted to damaged organs, where they play a role in tissue repair/regeneration.     

Impaired NK‐cell education diminishes resistance to murine CMV infection

Ly49G2 (G2⁺) NK cells mediate murine (M)CMV resistance in MHC D^k‐expressing mice. Bone marrow transplantation (BMT) studies revealed that G2⁺ NK cell‐mediated MCMV resistance requires D^k in both hematopoietic and nonhematopoietic cells. As a Ly49G2 ligand, D^k in both cell lineages may contribute to lysis of virus‐infected cells. Alternatively, cellular differences in self‐MHC D^k may have affected NK‐cell education, and consequently NK cell‐mediated viral clearance. We investigated the D^k‐licensing effect on BM‐derived NK cells in BMT recipients by analyzing cytokines, cytotoxicity and MCMV resistance. In BMT recipients with lineage‐restricted D^k, G2⁺ NK‐cell reactivity and cytotoxicity was diminished in comparison to BMT recipients with self‐MHC in all cells. Reduced G2⁺ NK‐mediated MCMV resistance in BMT recipients with lineage‐restricted self‐MHC indicates that licensing of G2⁺ NK cells is related to NK‐cell reactivity and viral control. Titrating donor BM with self‐MHC‐bearing hematopoietic cells, as well as adoptive transfer of mature G2⁺ NK cells into BMT recipients with self‐MHC in non‐hematopoietic cells only, enhanced NK‐cell licensing and rescued MCMV resistance. This disparate self‐MHC NK‐cell education model would suggest that inadequately licensed NK cells corresponded to inefficient viral sensing and clearance.