The in vivo requirements for human natural killer (NK) cell development and differentiation into cytotoxic effectors expressing inhibitory receptors for self-major histocompatibility complex class I ...(MHC-I; killer Ig-like receptors KIRs) remain undefined. Here, we dissect the role of interleukin (IL)-15 in human NK cell development using Rag2(-/-)gamma c(-/-) mice transplanted with human hematopoietic stem cells. Human NK cell reconstitution was intrinsically low in this model because of the poor reactivity to mouse IL-15. Although exogenous human IL-15 (hIL-15) alone made little improvement, IL-15 coupled to IL-15 receptor alpha (IL-15R alpha) significantly augmented human NK cells. IL-15-IL-15R alpha complexes induced extensive NK cell proliferation and differentiation, resulting in accumulation of CD16(+)KIR(+) NK cells, which was not uniquely dependent on enhanced survival or preferential responsiveness of this subset to IL-15. Human NK cell differentiation in vivo required hIL-15 and progressed in a linear fashion from CD56(hi)CD16(-)KIR(-) to CD56(lo)CD16(+)KIR(-), and finally to CD56(lo)CD16(+)KIR(+). These data provide the first evidence that IL-15 trans-presentation regulates human NK cell homeostasis. Use of hIL-15 receptor agonists generates a robust humanized immune system model to study human NK cells in vivo. IL-15 receptor agonists may provide therapeutic tools to improve NK cell reconstitution after bone marrow transplants, enhance graft versus leukemia effects, and increase the pool of IL-15-responsive cells during immunotherapy strategies.
Hepatic infections by hepatitis B virus (HBV), hepatitis C virus (HCV) and Plasmodium parasites leading to acute or chronic diseases constitute a global health challenge. The species tropism of these ...hepatotropic pathogens is restricted to chimpanzees and humans, thus model systems to study their pathological mechanisms are severely limited. Although these pathogens infect hepatocytes, disease pathology is intimately related to the degree and quality of the immune response. As a first step to decipher the immune response to infected hepatocytes, we developed an animal model harboring both a human immune system (HIS) and human hepatocytes (HUHEP) in BALB/c Rag2-/- IL-2Rγc-/- NOD.sirpa uPAtg/tg mice. The extent and kinetics of human hepatocyte engraftment were similar between HUHEP and HIS-HUHEP mice. Transplanted human hepatocytes were polarized and mature in vivo, resulting in 20-50% liver chimerism in these models. Human myeloid and lymphoid cell lineages developed at similar frequencies in HIS and HIS-HUHEP mice, and splenic and hepatic compartments were humanized with mature B cells, NK cells and naïve T cells, as well as monocytes and dendritic cells. Taken together, these results demonstrate that HIS-HUHEP mice can be stably (> 5 months) and robustly engrafted with a humanized immune system and chimeric human liver. This novel HIS-HUHEP model provides a platform to investigate human immune responses against hepatotropic pathogens and to test novel drug strategies or vaccine candidates.
Natural killer (NK) cell development is thought to occur in the bone marrow. Here we identify the transcription factor GATA-3 and CD127 (IL-7R alpha) as molecular markers of a pathway of mouse NK ...cell development that originates in the thymus. Thymus-derived CD127+ NK cells repopulated peripheral lymphoid organs, and their homeostasis was strictly dependent on GATA-3 and interleukin 7. The CD127+ NK cells had a distinct phenotype (CD11b(lo) CD16- CD69(hi) Ly49(lo)) and unusual functional attributes, including reduced cytotoxicity but considerable cytokine production. Those characteristics are reminiscent of human CD56(hi) CD16- NK cells, which we found expressed CD127 and had more GATA-3 expression than human CD56+ CD16+ NK cells. We propose that bone marrow and thymic NK cell pathways generate distinct mouse NK cells with properties similar to those of the two human CD56 NK cell subsets.
Several distinct classes of surface receptors can, on ligand binding, transmit signals that modulate the survival, proliferation, and apoptosis of peripheral B, T, and natural killer (NK) cells. At ...the population level, dynamic changes in lymphocyte cell numbers are strictly regulated to maintain a steady state, a process referred to as homeostasis. Although several studies have investigated the signals that regulate B- and T-cell homeostasis, little is known about the mechanisms that control the survival and proliferation of peripheral NK cells. Using an adoptive transfer system, we have investigated the role of γc-dependent cytokines, in particular interleukin 7 (IL-7) and IL-15, and major histocompatibility complex (MHC) class I molecules in peripheral NK-cell homeostasis. We observed that IL-15 plays a dominant role in the survival of peripheral NK cells, via maintenance of the antiapoptotic factor Bcl-2. IL-15 availability, however, also plays an important role because endogenous NK cells in the recipient mice influence the behavior of adoptively transferred NK cells. Finally, although NK cells bear functional inhibitory Ly49 receptors for MHC class I molecules, the presence or absence of specific ligands on host cells did not influence the survival or homeostatic expansion of donor NK cells.
Steady-state numbers of peripheral lymphocyte are tightly controlled. For conventional T cells, signals delivered through the interaction of the T cell receptor (TCR) with antigen-loaded MHC ...molecules are required for the peripheral survival of naive T cells and for their homeostatic expansion in lymphopenic hosts. Cytokines, including IL-7, are also essential for survival of peripheral naive T cells. CD1d-restricted,$V\alpha14^+$natural killer (NK)-T cells are a specialized autoreactive T subset with immunoregulatory activity. The relative roles of TCR engagement and cytokine signaling in the peripheral homeostasis of$V\alpha14^+$NK-T cells were investigated. After adoptive transfer, the survival and expansion of peripheral$V\alpha14^+$NK-T cells was independent of CD1d expression in the host. In contrast, IL-15 (but not IL-7) was required for maintenance of peripheral CD1d-reactive$V\alpha14^+$T cells. Comparison of$V\alpha14^+$T cell transfers into NK-proficient vs. deficient hosts suggests that NK-T cells and NK cells compete for peripheral resources. Our results indicate that IL-15 maintains the homeostasis of peripheral$V\alpha14^+$NK-T cells. In contrast, TCR "tickling" of NK-T cells, if it occurs under steady-state conditions, does not by itself provide a sufficient signal for their peripheral survival.
Several cytokines (including IL-2, IL-7, IL-15, and IL-21) that signal through receptors sharing the common γ chain (γc) are critical for the generation and peripheral homeostasis of naive and memory ...T cells. Recently, we demonstrated that effector functions fail to develop in CD4⁺ T cells that differentiate in the absence of γc. To assess the role of γc cytokines in cell-fate decisions that condition effector versus memory CD8⁺ T cell generation, we compared the response of CD8⁺ T cells from γc⁺ or γc⁻ P14 TCR transgenic mice after challenge with lymphocytic choriomeningitis virus. The intrinsic IL-7-dependent survival defect of γc⁻ naive CD8⁺ T cells was corrected by transgenic expression of human Bcl-2. We demonstrated that although γc-dependent signals are dispensable for the initial expansion and the acquisition of cytotoxic functions following antigenic stimulation, they condition the terminal proliferation and differentiation of CD8⁺ effector T cells (i.e., KLRG1high CD127low short-lived effector T cells) via the transcription factor, T-bet. Moreover, the γc-dependent signals that are critical for memory T cell formation are not rescued by Bcl2 overexpression. Together, these data reveal an unexpected divergence in the requirement for γc cytokines in the differentiation of CD4⁺ versus CD8⁺ cytotoxic T lymphocytes.
After their initial antigen encounter in the secondary lymphoid organs, activated T cells must receive additional signals in the peripheral tissues to fully differentiate. Here, we provide evidence ...that γc cytokines are critical during this process. Using the Marilyn (Ml) T cell antigen receptor (TCR) transgenic model, we show that male skin grafts are tolerated in the absence of γc, but that Ml CD4⁺ T cells proliferate normally in response to antigen, traffic to the graft site and recruit an inflammatory response including natural killer (NK) cells, neutrophils, and macrophages that is independent of T cell γc expression. Whereas wild-type T cells demonstrate a progressive differentiation phenotype from the spleen to the tissues, skin-infiltrating effector T cells (CD44hiCD62Llo) from γFormula: see text mice were phenotypically abnormal with reduced ICOS, NKG2D, granzyme B, and IFN-γ expression. These defects could be mapped to deficiencies in IL-2 and, surprisingly, IL-15. These results define a late checkpoint in T cell differentiation in the tissues where γc cytokines, including IL-15, authenticate CD4⁺ T cell effector functions.
Humanized mice harboring human immune systems (HIS) represent a platform to study immune responses against pathogens and to screen vaccine candidates and novel immunotherapeutics. Innate and adaptive ...immune responses are suboptimal in HIS mice, possibly due to poor reconstitution of human antigen‐presenting cells, including dendritic cells (DCs). DC homeostasis is regulated by cytokine availability, and Flt3‐ligand (Flt3L) is one factor that conditions this process. Mouse myelopoiesis is essentially normal in most current HIS models. As such, developing mouse myeloid cells may limit human DC reconstitution by reducing available Flt3L and by cellular competition for specific “niches.” To address these issues, we created a novel HIS model that compromises host myeloid cell development via deficiency in the receptor tyrosine kinase Flk2/Flt3. In Balb/c Rag2−/−Il2rg−/−Flt3−/− (BRGF) recipients, human conventional DCs and plasmacytoid DCs develop from hCD34+ precursors and can be specifically boosted with exogenous Flt3L. Human DCs that develop in this context normally respond to TLR stimulation, and improved human DC homeostasis is associated with increased numbers of human NK and T cells. This new HIS‐DC model should provide a means to dissect human DC differentiation and represents a novel platform to screen immune adjuvants and DC targeting therapies.
Human Immune System (HIS) mice generate abundant human lymphocytes but suffer from poor human DC development. By eliminating the Flt3 receptor in recipient mice, a novel HIS mouse model has been validated that reduced endogenous mouse DC and allows for robust human DC development in response to exogenous Flt3 ligand.
Interferon-producing killer dendritic cells (IKDCs) are a recently described subset of CD11c(lo)B220(+) cells that share phenotypic and functional properties of DCs and natural killer (NK) cells ...(Chan, C.W., E. Crafton, H.N. Fan, J. Flook, K. Yoshimura, M. Skarica, D. Brockstedt, T.W. Dubensky, M.F. Stins, L.L. Lanier, et al. 2006. Nat. Med. 12:207-213; Taieb, J., N. Chaput, C. Menard, L. Apetoh, E. Ullrich, M. Bonmort, M. Pequignot, N. Casares, M. Terme, C. Flament, et al. 2006. Nat. Med. 12:214-219). IKDC development appears unusual in that cytokines using the interleukin (IL)-2 receptor beta (IL-2Rbeta) chain but not those using the common gamma chain (gamma(c)) are necessary for their generation. By directly comparing Rag2(-/-)gamma(c)(-/y), Rag2(-/-)IL-2Rbeta(-/-), Rag2(-/-)IL-15(-/-), and Rag2(-/-)IL-2(-/-) mice, we demonstrate that IKDC development parallels NK cell development in its strict IL-15 dependence. Moreover, IKDCs uniformly express NK-specific Ncr-1 transcripts (encoding NKp46), whereas NKp46(+) cells are absent in Ncr1(gfp/+)gamma(c)(-/y) mice. Distinguishing features of IKDCs (CD11c(lo)B220(+)MHC-II(+)) were carefully examined on developing NK cells in the bone marrow and on peripheral NK cells. As B220 expression was heterogeneous, defining B220(lo) versus B220(hi) NK1.1(+) NK cells could be considered as arbitrary, and few phenotypic differences were noted between NK1.1(+) NK cells bearing different levels of B220. CD11c expression did not correlate with B220 or major histocompatibility complex (MHC) class II (MHC-II) expression, and most MHC-II(+) NK1.1(+) cells did not express B220 and were thus not IKDCs. Finally, CD11c, MHC-II, and B220 levels were up-regulated on NK1.1(+) cells upon activation in vitro or in vivo in a proliferation-dependent fashion. Our data suggest that the majority of CD11c(lo)B220(+) "IKDC-like" cells represent activated NK cells.
Human immune system (HIS) mouse models provide a robust in vivo platform to study human immunity. Nevertheless, the signals that guide human lymphocyte differentiation in HIS mice remain poorly ...understood. Here, we have developed a novel Balb/c Rag2−/− Il2rg−/− SirpaNOD (BRGS) HIS mouse model expressing human HLA‐A2 and ‐DR2 transgenes (BRGSA2DR2). When comparing BRGS and BRGSA2DR2 HIS mice engrafted with human CD34+ stem cells, a more rapid emergence of T cells in the circulation of hosts bearing human HLA was shown, which may reflect a more efficient human T‐cell development in the mouse thymus. Development of CD4+ and CD8+ T cells was accelerated in BRGSA2DR2 HIS mice and generated more balanced B and T‐cell compartments in peripheral lymphoid organs. Both B‐ and T‐cell function appeared enhanced in the presence of human HLA transgenes with higher levels of class switched Ig, increased percentages of polyfunctional T cells and clear evidence for antigen‐specific T‐cell responses following immunization. Taken together, the presence of human HLA class I and II molecules can improve multiple aspects of human B‐ and T‐cell homeostasis and function in the BRGS‐based HIS mouse model.
MHC molecules are critical for T‐cell selection. To improve T‐cell function in human immune system (HIS) mice, human HLA‐A2 and HLA‐DR2 transgenes were expressed in BRGS hosts. Humanized BRGSA2DR2 HIS mice showed enhanced T‐cell development and improved antigen‐specific T‐ and B‐cell responses.
PDF
