Regulation of human and mouse bystander T cell activation responses by PD-1.

Bystander activation of memory T cells occurs via cytokine signaling alone in the absence of T cell receptor (TCR) signaling and provides a means of amplifying T cell effector responses in an antigen-nonspecific manner. While the role of Programmed Cell Death Protein 1 (PD-1) on antigen-specific T cell responses is extensively characterized, its role in bystander T cell responses is less clear. We examined the role of the PD-1 pathway during human and mouse non-antigen-specific memory T cell bystander activation and observed that PD-1+ T cells demonstrated less activation and proliferation than activated PD-1- populations in vitro. Higher activation and proliferative responses were also observed in the PD-1- memory population in both mice and patients with cancer receiving high-dose IL-2, mirroring the in vitro phenotypes. This inhibitory effect of PD-1 could be reversed by PD-1 blockade in vivo or observed using memory T cells from PD-1-/- mice. Interestingly, increased activation through abrogation of PD-1 signaling in bystander-activated T cells also resulted in increased apoptosis due to activation-induced cell death (AICD) and eventual T cell loss in vivo. These results demonstrate that the PD-1/PD-Ligand 1 (PD-L1) pathway inhibited bystander-activated memory T cell responses but also protected cells from AICD.

Activation status dictates the function of unlicensed natural killer cells in mice and humans.

Natural killer (NK) cells are involved in innate defense against viral infection and cancer. NK cells can be divided into subsets based on the ability of different receptors to bind to major histocompatibility (MHC) class 1 molecules, resulting in differential responses upon activation in a process called "licensing" or "arming." NK cells expressing receptors that bind self-MHC are considered licensed due to an augmented effector lytic function capability compared with unlicensed subsets. However, we demonstrated that unlicensed NK subsets instead positively regulate the adaptive T-cell response during viral infections that are related to localization and cytokine production. In this study, the differential effects of the two types of NK subsets were contingent on the environment in viral infection and hematopoietic stem cell transplantation (HSCT) models. Infection of mice with high-dose (HD) murine cytomegalovirus (MCMC) led to a loss of licensing-associated differences, as compared with mice with low-dose (LD) infection: the unlicensed NK subset no longer localized in lymph nodes (LNs), but instead remained at the site of infection. Similarly, the patterns observed during HD infection paralleled the phenotypes of both human and mouse NK cells in an HSCT setting where NK cells exhibit an activated phenotype. However, in contrast to the effects of subset depletion in T-cell replete models, the licensed NK cell subsets still dominated antiviral responses after HSCT. Overall, our results highlight the intricate tuning of NK cells and how it affects overall immune responses with regard to licensing patterns and their dependency on the level of stimulation and activation status.

IL-2 and Anti-TGF-ß Promote NK Cell Reconstitution and Anti-tumor Effects after Syngeneic Hematopoietic Stem Cell Transplantation.

The failure of autologous hematopoietic stem cell transplantation (HSCT) has been associated with a profound immunodeficiency that follows shortly after treatment, which renders patients susceptible to opportunistic infections and/or cancer relapse. Thus, given the additional immunosuppressive pathways involved in immune evasion in cancer, strategies that induce a faster reconstitution of key immune effector cells are needed. Natural killer (NK) cells mediate potent anti-tumor effector functions and are the first immune cells to repopulate after HSCT. TGF-ß is a potent immunosuppressive cytokine that can impede both the development and function of immune cells. Here, we evaluated the use of an immunotherapeutic regimen that combines low dose of IL-2, an NK cell stimulatory signal, with TGF-ß neutralization, in order to accelerate NK cell reconstitution following congenic HSCT in mice by providing stimulatory signals yet also abrogating inhibitory ones. This therapy led to a marked expansion of NK cells and accelerated NK cell maturation. Following HSCT, mature NK cells from the treated recipients displayed an activated phenotype and enhanced anti-tumor responses both in vitro and in vivo. No overt toxicities or adverse effects were observed in the treated recipients. However, these stimulatory effects on NK cell recovery were predicated upon continuous treatment as cessation of treatment led to return to baseline levels and to no improvement of overall immune recovery when assessed at later time-points, indicating strict regulatory control of the NK cell compartment. Overall, this study still demonstrates that therapies that combine positive and negative signals can be plausible strategies to accelerate NK cell reconstitution following HSCT and augment anti-tumor efficacy.

Differential expression of the Ly49G(B6), but not the Ly49G(BALB), 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.

The TNF receptor-ligands 4-1BB-4-1BBL and GITR-GITRL in NK cell responses.

Interactions between several tumor necrosis factor (TNF)-TNF receptor (TNFR) superfamily members that are expressed by T cells and natural killer (NK) cells and various other cell types modulate immune responses. This review summarizes the current understanding of how the TNF ligand-TNFR interactions 4-1BBL with 4-1BB, and GITRL with glucocorticoid-induced TNFR-related (GITR) regulate NK cell mediated antitumor responses and discuss its therapeutic implications.

Mouse NK cell-mediated rejection of bone marrow allografts exhibits patterns consistent with Ly49 subset licensing.

Natural killer (NK) cells can mediate the rejection of bone marrow allografts and exist as subsets based on expression of inhibitory/activating receptors that can bind MHC. In vitro data have shown that NK subsets bearing Ly49 receptors for self-MHC class I have intrinsically higher effector function, supporting the hypothesis that NK cells undergo a host MHC-dependent functional education. These subsets also play a role in bone marrow cell (BMC) allograft rejection. Thus far, little in vivo evidence for this preferential licensing across mouse strains with different MHC haplotypes has been shown. We assessed the intrinsic response potential of the different Ly49(+) subsets in BMC rejection by using ß2-microglobulin deficient (ß2m(-/-)) mice as donors. Using congenic and allogeneic mice as recipients and depleting the different Ly49 subsets, we found that NK subsets bearing Ly49s, which bind "self-MHC" were found to be the dominant subset responsible for ß2m(-/-) BMC rejection. This provides in vivo evidence for host MHC class I-dependent functional education. Interestingly, all H2(d) strain mice regardless of background were able to resist significantly greater amounts of ß2m(-/-), but not wild-type BMC than H2(b) mice, providing evidence that the rheostat hypothesis regarding Ly49 affinities for MHC and NK-cell function impacts BMC rejection capability.

Hydrodynamic delivery of human IL-15 cDNA increases murine natural killer cell recovery after syngeneic bone marrow transplantation.

Immune deficiency immediately following bone marrow transplantation (BMT) increases susceptibility to opportunistic infections as well as tumor relapse. Natural Killer (NK) cells play important roles in the resistance to virally infected and transformed cells. Interleukin (IL)-15 has been shown to be essential for NK cell development and survival. We administered human (h) IL-15 cDNA (pIL-15) via hydrodynamic delivery to murine recipients undergoing congenic BMT to determine its effects on NK cell reconstitution. Hydrodynamic pIL-15 delivery resulted in high levels of hIL-15 protein in the serum that lasted for several days and then quickly declined. The appearance of hIL-15 was followed by a significant increase of mature donor-derived NK cells within the bone marrow, spleens, and livers of the treated recipients. No accumulation of immature NK cell progenitors was observed. The NK cells from IL-15-treated recipients displayed an activated phenotype and were lytically active toward tumor targets in vitro to a similar degree as did those cells from recipients treated with control plasmid. This suggests that the predominant effect of IL-15 was a quantitative increase in total NK cell numbers and not qualitative changes in NK cell functions. No toxicities or adverse effects were observed. Studies performed in transplanted mice bearing renal carcinoma tumors demonstrated that this mode of hIL-15 gene delivery resulted in increased antitumor responses. These results support the use of cytokine gene transfer-based regimens as a platform to augment NK cell recovery after BMT.

Mouse Ly49G2+ NK cells dominate early responses during both immune reconstitution and activation independently of MHC.

Natural killer (NK) cell subsets can be defined by the differential expression of inhibitory receptors for MHC class I molecules. Early after congenic HSCT, we found that Ly49G2(high) single-positive NK cells repopulated, displayed an activated phenotype, and were highly cytolytic. Over time, this subset was replaced with NK cells with a normal pattern of Ly49 expression. Treatment of mice with IL-2 also resulted in the rapid expansion of these Ly49G2(high) single-positive NK cells. Only the Ly49g (Klra7) Pro1 transcript was highly induced in both HSCT- and IL-2-treated recipients. MHC-independent expansion of the Ly49G2(+) subset was also observed after Listeria monocytogenes or mouse cytomegalovirus infection. Our data indicate that during reconstitution after HSCT and various activation stimuli, Ly49G2(+) NK cells represent the "first-responder" NK cells, which occur independently of NK-cell licensing via Ly49-MHC interactions. These data suggest that the inhibitory Ly49G2 receptor represents an activation marker on mouse NK cells under various conditions.

Hematopoietic progenitor cell regulation by CD4+CD25+ T cells.

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) possess the capacity to modulate both adaptive and innate immune responses. We hypothesized that Tregs could regulate hematopoiesis based on cytokine effector molecules they can produce. The studies here demonstrate that Tregs can affect the differentiation of myeloid progenitor cells. In vitro findings demonstrated the ability of Tregs to inhibit the differentiation of interleukin-3 (IL-3)/stem cell factor (colony-forming unit [CFU]-IL3)-driven progenitor cells. Inhibitory effects were mediated by a pathway requiring cell-cell contact, major histocompatibility complex class II expression on marrow cells, and transforming growth factor-beta. Importantly, depletion of Tregs in situ resulted in enhanced CFU-IL3 levels after bone marrow transplantation. Cotransplantation of CD4(+)FoxP3(+)(gfp) Tregs together with bone marrow was found to diminish CFU-IL3 responses after transplantation. To address the consequence of transplanted Tregs on differentiated progeny from these CFU 2 weeks after hematopoietic stem cell transplantation, peripheral blood complete blood counts were performed and examined for polymorphonuclear leukocyte content. Recipients of cotransplanted Tregs exhibited diminished neutrophil counts. Together, these findings illustrate that both recipient and donor Tregs can influence hematopoietic progenitor cell activity after transplantation and that these cells can alter responses outside the adaptive and innate immune systems.

Combination therapy using IL-2 and anti-CD25 results in augmented natural killer cell-mediated antitumor responses.

Interleukin (IL)-2 has been extensively examined to promote clinical T and natural killer (NK) cell responses. Regulatory T cells (Tregs) have been shown to regulate many aspects of the immune system, including NK cell-mediated responses. We have demonstrated that in vivo administration of IL-2 led to activation and expansion of both NK cells and immunosuppressive Tregs. Therefore, we attempted to augment NK cell antitumor effects by concurrently depleting Tregs using anti-CD25. Increased NK cell activation by IL-2 was found to be correlated with an increase in classical, short-term NK cell in vitro killing assays regardless of the depletion of Tregs. But when splenocytes of the treated mice were used in long-term tumor outgrowth experiments, we observed that prior depletion of Tregs from IL-2 administration led to improved antitumor effects compared with either treatment alone. Importantly, these in vitro data are correlated with subsequent in vivo survival of leukemia-bearing mice, in which co-treatment of IL-2 with anti-CD25 led to significantly improved survival compared with mice treated with either IL-2 alone or with Treg depletion. Prior depletion of NK1.1(+) cells, but not of CD8(+) cells, completely abrogated all antitumor effects mediated by IL-2 and anti-CD25 combination therapy. These findings demonstrate that superior NK cell-mediated antileukemic effects can be achieved with IL-2 administration and concurrent depletion of CD25(+) cells.

Sensitization of tumor cells to NK cell-mediated killing by proteasome inhibition.

Bortezomib is a proteasome inhibitor that has direct antitumor effects. We and others have previously demonstrated that bortezomib could also sensitize tumor cells to killing via the death ligand, TRAIL. NK cells represent a potent antitumor effector cell. Therefore, we investigated whether bortezomib could sensitize tumor cells to NK cell-mediated killing. Preincubation of tumor cells with bortezomib had no effect on short-term NK cell killing or purified granule killing assays. Using a 24-h lysis assay, increases in tumor killing was only observed using perforin-deficient NK cells, and this increased killing was found to be dependent on both TRAIL and FasL, correlating with an increase in tumor Fas and DR5 expression. Long-term tumor outgrowth assays allowed for the detection of this increased tumor killing by activated NK cells following bortezomib treatment of the tumor. In a tumor purging assay, in which tumor:bone marrow cell mixtures were placed into lethally irradiated mice, only treatment of these mixtures with a combination of NK cells with bortezomib resulted in significant tumor-free survival of the recipients. These results demonstrate that bortezomib treatment can sensitize tumor cells to cellular effector pathways. These results suggest that the combination of proteasome inhibition with immune therapy may result in increased antitumor efficacy.

Suppression of natural killer cell-mediated bone marrow cell rejection by CD4+CD25+ regulatory T cells.

Naturally occurring CD4(+)CD25(+) T regulatory (Treg) cells have been shown to inhibit adaptive responses by T cells. Natural killer (NK) cells represent an important component of innate immunity in both cancer and infectious disease states. We investigated whether CD4(+)CD25(+) Treg cells could affect NK cell function in vivo by using allogeneic (full H2-disparate) bone marrow (BM) transplantation and the model of hybrid resistance, in which parental marrow grafts are rejected solely by the NK cells of irradiated (BALB/c x C57BL/6) F(1) recipients. We demonstrate that the prior removal of host Treg cells, but not CD8(+) T cells, significantly enhanced NK cell-mediated BM rejection in both models. The inhibitory role of Treg cells on NK cells was confirmed in vivo with adoptive transfer studies in which transferred CD4(+)CD25(+) cells could abrogate NK cell-mediated hybrid resistance. Anti-TGF-beta mAb treatment also increased NK cell-mediated BM graft rejection, suggesting that the NK cell suppression is exerted through TGF-beta. Thus, CD4(+)CD25(+) Treg cells can potently inhibit NK cell function in vivo, and their depletion may have therapeutic ramifications for NK cell function in BM transplantation and cancer therapy.

Inhibition of acute graft-versus-host disease with retention of graft-versus-tumor effects by the proteasome inhibitor bortezomib.

Graft-versus-host disease (GVHD) represents a major hurdle impeding the efficacy of allogeneic bone marrow transplantation (BMT). Bortezomib is a proteasome inhibitor that was recently approved for treatment of myeloma. We found that bortezomib potently inhibited in vitro mixed lymphocyte responses and promoted the apoptosis of alloreactive T cells. Bortezomib given at the time of allogeneic BMT in mice resulted in significant protection from acute GVHD. Reductions in GVHD-associated parameters and biological evidence of proteasome inhibition were observed with this regimen but with no adverse effects on long-term donor reconstitution. Assessment of graft-versus-tumor responses in advanced leukemia-bearing mice demonstrated that only the combination of allogeneic BMT and T cells with bortezomib promoted significant increases in survival. Increased cytotoxic T cell killing of the tumor was also observed. Thus, the combination of proteasome inhibition with selective immune attack can markedly increase the efficacy of BMT in cancer.

The immunobiology of natural killer cells and bone marrow allograft rejection.

Natural killer (NK) cells mediate the acute rejection of bone marrow cell (BMC) allografts, but not solid tissue grafts, in lethally irradiated mice. However, the mechanisms underlying this capability for rejecting BMC remain unclear. NK cells express (1) inhibitory receptors specific for major histocompatibility complex (MHC) class I molecules and (2) activating receptors with diverse specificities. Inhibitory NK receptors confer to NK cells the ability to discriminate between MHC class I-positive and -negative target cells and are therefore involved in the control of NK cell tolerance to self, as well as in the elimination of cells that have downregulation of MHC class I molecules. Preclinical studies in mice have provided good evidence that subsets of NK cells that bear different combinations of both inhibitory and activating Ly49 receptors can interact with each other and target specific BMC rejection, as well as NK cell responses toward tumor cells. Recent clinical studies have also shown that the use of killer cell immunoglobulin-like receptor ligand incompatibility in patients with leukemia who received hematopoietic stem cell transplants correlated not only with the elimination of graft rejection, but also with eradication of tumor and prevention of graft-versus-host disease; this offers a significant advantage for survival. In this review, we attempt to bring together literature regarding the biology of NK cells and discuss the current issues in bone marrow transplantation and the potential clinical role of NK cell alloreactivity in the efficacy of this procedure for immunotherapy of cancer and infectious states.

IL-15-mediated induction of LFA-1 is a late step required for cytotoxic differentiation of human NK cells from CD34+Lin- bone marrow cells.

Optimal differentiation of cytotoxic NK cells is important to provide protective innate immunity to patients after bone marrow transplantation. In vitro differentiation of CD56(+)CD3(-) NK cells takes weeks and is supported by several cytokines, including IL-2, IL-7, and IL-15, and thus can be useful for immunotherapy. However, IL-2 therapy is problematic in vivo, and NK cells differentiated in vitro with only IL-7 lack cytotoxicity. We assessed whether human NK cells initially differentiated in vitro from CD34(+)Lin(-) bone marrow cells with IL-7 could acquire cytotoxicity after exposure to additional cytokines and what changes promoted cytotoxicity. The cells cultured with IL-7 already had granzyme B as well as perforin, as previously reported, the proteins of cytotoxic granules. The cells also lacked LFA-1. After 1 wk of secondary culture with either IL-2 or IL-15, but not with IL-12 or IL-18, the IL-7-cultured cells acquired cytotoxicity. IL-2 or IL-15 also induced LFA-1. Ab to the LFA-1 subunits CD11a and CD18 blocked lysis by the NK cells, indicating that the new LFA-1 correlated with, and was essential for, the cytotoxic function of the in vitro generated cells. The LFA-1 also participated in target cell binding by the in vitro differentiated cells. In this study, we demonstrated a new function for IL-15, the induction of LFA-1 in NK progenitor cells, and that IL-15 does more than merely support NK progenitor cell proliferation. The efficacy after only 1 wk of IL-15 administration is a positive practical feature that may apply to human therapy.

Human natural killer cell development in a xenogeneic culture system.

In vivo and in vitro xenogeneic models have shown the ability of a non-human environment in supporting human haemopoiesis. In the present study, we evaluated the effect of fetal sheep thymic stroma in the in vitro development of natural killer (NK) cells from human haemopoietic progenitors. CD34+HLA-DR+ (CD34+ DR+)Lin- and CD34+DR-Lin- bone marrow (BM) progenitors were cultured for 3 weeks with or without interleukin 2 (IL-2), in fetal sheep thymic stroma contact and transwell cultures. Both progenitors gave rise to NK cells, defined as CD45+CD56+ cells, in the presence or absence of IL-2; however, the percentage of NK cells originated in cultures with IL-2 was significantly higher. Direct contact with stroma seemed to be required for the most immature progenitors, CD34+DR-Lin-, to differentiate along the NK cell lineage. Functional assays revealed that only cells grown in the presence of IL-2 were cytolytic against K562 targets and, curiously, NK cells derived from CD34+DR-Lin- progenitors were more cytotoxic that NK cells derived from CD34+DR+Lin- progenitors. These studies suggest that the ability of fetal sheep thymic stroma in promoting the generation of human NK cells from haemopoietic progenitors may have relevance in terms of NK cell ontogeny and induction of tolerance in transplantation.