Out-of-Sequence Signal 3 Paralyzes Primary CD4(+) T-Cell-Dependent Immunity.

Primary T cell activation involves the integration of three distinct signals delivered in sequence: (1) antigen recognition, (2) costimulation, and (3) cytokine-mediated differentiation and expansion. Strong immunostimulatory events such as immunotherapy or infection induce profound cytokine release causing "bystander" T cell activation, thereby increasing the potential for autoreactivity and need for control. We show that during strong stimulation, a profound suppression of primary CD4(+) T-cell-mediated immune responses ensued and was observed across preclinical models and patients undergoing high-dose interleukin-2 (IL-2) therapy. This suppression targeted naive CD4(+) but not CD8(+) T cells and was mediated through transient suppressor of cytokine signaling-3 (SOCS3) inhibition of the STAT5b transcription factor signaling pathway. These events resulted in complete paralysis of primary CD4(+) T cell activation, affecting memory generation and induction of autoimmunity as well as impaired viral clearance. These data highlight the critical regulation of naive CD4(+) T cells during inflammatory conditions.

Increased antitumor effects using IL-2 with anti-TGF-ß reveals competition between mouse NK and CD8 T cells.

Because of increasing interest in the removal of immunosuppressive pathways in cancer, the combination of IL-2 with Abs to neutralize TGF-ß, a potent immunosuppressive cytokine, was assessed. Combination immunotherapy resulted in significantly greater antitumor effects. These were correlated with significant increases in the numbers and functionality of NK cells, NK cell progenitors, and activated CD8 T cells, resulting in the observed antitumor effects. Combination immunotherapy also was accompanied by lesser toxicities than was IL-2 therapy alone. Additionally, we observed a dual competition between NK cells and activated CD8 T cells such that, after immunotherapy, the depletion of either effector population resulted in the increased total expansion of the other population and compensatory antitumor effects. This study demonstrates the efficacy of this combination immunotherapeutic regimen as a promising cancer therapy and illustrates the existence of potent competitive regulatory pathways between NK cells and CD8 T cells in response to systemic activation.

Murine natural killer cell licensing and regulation by T regulatory cells in viral responses.

Natural killer (NK) cells show differential functionality based on their capability of binding to self-MHC consistent with licensing. Here we show in vivo confirmation of the physiologic effects of licensing with differential effects of NK subsets on anti-murine cytomegalovirus (anti-MCMV) responses after syngeneic hematopoietic stem cell transplantation (HSCT) or regulatory T-cell (Treg) depletion. After HSCT, depletion of licensed NK cells led to far greater viral loads in target organs early after infection compared with nondepleted and unlicensed depleted mice. There was a preferential expansion of licensed, C-type lectin-like activating receptor Ly49H+ NK cells with increased IFNγ production after infection in nondepleted mice post-HSCT and after Treg depletion. Adoptive transfer of licensed NK subsets into immunodeficient hosts provided significantly greater MCMV resistance compared with transfer of total NK populations or unlicensed subsets. In non-HSCT mice, only concurrent depletion of Tregs or TGF-ß neutralization resulted in detection of NK licensing effects. This suggests that licensed NK cells are the initial and rapidly responding population of NK cells to MCMV infection, but are highly regulated by Tregs and TGF-ß.

Murine NK-cell licensing is reflective of donor MHC-I following allogeneic hematopoietic stem cell transplantation in murine cytomegalovirus responses.

Natural killer (NK) cells express inhibitory receptors with varied binding affinities to specific major histocompatibility complex class I (MHC-I) haplotypes. NK cells can be classified as licensed or unlicensed based on their ability or inability to bind MHC-I, respectively. The role of donor vs host MHC on their development after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is not known. Following reciprocal MHC-disparate allogeneic transplants and during de novo NK-cell recovery, depletion of the licensed and not unlicensed population of NK cells as determined by the licensing patterns of donor MHC-I haplotypes, resulted in significantly increased susceptibility to murine cytomegalovirus (MCMV) infection. A corresponding expansion of the licensed Ly49H(+) NK cells occurred with greater interferon γ production by these cells than unlicensed NK cells in the context of donor MHC-I. Thus, NK licensing behavior to MCMV corresponds to the donor, and not recipient, MHC haplotype after allo-HSCT in mice.

Contrasting effects of anti-Ly49A due to MHC class I cis binding on NK cell-mediated allogeneic bone marrow cell resistance.

NK subsets have activating and inhibitory receptors that bind MHC-I. Ly49A is a mouse inhibitory receptor that binds with high affinity to H2(d) in both a cis- and trans-manner. Ly49A cis-associations limit trans-interactions with H2(d)-expressing targets as well as mAb binding. We demonstrate that cis-interactions affect mAb effector functions. In vivo administration of anti-Ly49A depleted NK cells in H2(b) but not H2(d) mice. Despite lack of depletion, in vivo treatment with anti-Ly49A reduced NK killing capabilities and inhibited activation, partially due to its agonistic effect. These data explain the previously described in vivo effects on bone marrow allograft rejection observed with anti-Ly49A treatment in H2(d)-haplotype mice. However, prior treatment of mice with poly(I:C) or mouse CMV infection resulted in increased Ly49A expression and Ly49A(+) NK cell depletion in H2(d) mice. These data indicate that, although Ly49 mAbs can exert similar in vivo effects in mice with different MHC haplotypes, these effects are mediated via different mechanisms of action correlating with Ly49A expression levels and can be altered within the same strain contingent on stimuli. This illustrates the marked diversity of mAb effector functions due to the regulation of the level of expression of target Ags and responses by stimulatory incidents such as infection.

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.

Human NK cells confer protection against HIV-1 infection in humanized mice.

The role of NK cells against HIV-1 infections remains to be elucidated in vivo. While humanized mouse models potentially could be used to directly evaluate human NK cell responses during HIV-1 infection, improved functional development of human NK cells in these hosts is needed. Here, we report the humanized MISTRG-6-15 mouse model, in which NK cells were quick to expand and exhibit degranulation, cytotoxicity, and proinflammatory cytokine production in nonlymphoid organs upon HIV-1 infection but had reduced functionality in lymphoid organs. Although HIV-1 infection induced functional impairment of NK cells, antiretroviral therapy reinvigorated NK cells in response to HIV-1 rebound after analytic treatment interruption. Moreover, a broadly neutralizing antibody, PGT121, enhanced NK cell function in vivo, consistent with antibody-dependent cellular cytotoxicity. Monoclonal antibody depletion of NK cells resulted in higher viral loads in multiple nonlymphoid organs. Overall, our results in humanized MISTRG-6-15 mice demonstrated that NK cells provided direct anti-HIV-1 responses in vivo but were limited in their responses in lymphoid organs.

Sustained Remission in Rheumatoid Arthritis: Time to Withdraw Treatment?

With increasing numbers of patients with rheumatoid arthritis achieving sustained remission, medication withdrawal is an important consideration to reduce polypharmacy and associated adverse events. An article from the journal Arthritis & Rheumatology (1) explores the treatment withdrawal options for patients on etanercept and methotrexate combination therapies and suggests methotrexate withdrawal has the least impact on disease worsening. There are limitations in the study, including the use of only one disease activity score and no assessment of radiographic progression, but, overall, the article provides a good framework for future studies on treatment withdrawal options and the possibility of medication reduction for patients.

Licensing delineates helper and effector NK cell subsets during viral infection.

Natural killer (NK) cells can be divided into phenotypic subsets based on expression of receptors that bind self-MHC-I molecules, a concept termed licensing or education. Here we show NK cell subsets with different migratory, effector, and immunoregulatory functions in dendritic cell and antigen (ag)-specific CD8+ T cell responses during influenza and murine cytomegalovirus infections. Shortly after infection, unlicensed NK cells localized in draining lymph nodes and produced GM-CSF, which correlated with the expansion and activation of dendritic cells, and resulted in greater and sustained ag-specific T cell responses. In contrast, licensed NK cells preferentially migrated to infected tissues and produced IFN-γ. Importantly, human NK cell subsets exhibited similar phenotypic characteristics. Collectively, our studies demonstrate a critical demarcation between the functions of licensed and unlicensed NK cell subsets, with the former functioning as the classical effector subset and the latter as the stimulator of adaptive immunity helping to prime immune responses.

Positive and negative regulation by NK cells in cancer.

Our understanding of NK biology has expanded immensely since the initial discovery of natural killer cells in 1975. New studies have uncovered various levels of immune regulation both on and by unique subsets of NK cells, which go well beyond simple receptor-ligand interactions between NK cells and target cancer cells. Distinct suppressor and effector populations of NK cells have been delineated in both viral and tumor models. Interactions between NK cells and dendritic cells, T cells, and B cells also dramatically alter the overall immune response to cancer. To exploit the diverse functional abilities of NK cell subsets for cancer immunotherapies, it is important to understand NK cell biology and NK regulator mechanisms.

Utilization of mouse models to decipher natural killer cell biology and potential clinical applications.

Natural killer (NK) cells represent a key component of innate immunity. The utility of mouse models to recapitulate the human immune response has been a matter of ongoing debate, especially with regard to NK cells. However, mouse models of NK cells have provided significant advancements in our understanding of the biology of the cells that bridge these species. Initial characterization of NK cell activity was in mouse hematopoietic stem cell transplantation models. Recent findings include uncovering functionally disparate subsets of NK cells based on unique inhibitory receptor expression patterns, the existence of memory-like NK cells, and immunoregulatory NK cells that affect hematopoiesis and T-cell function. In addition, the biology of these cells with regard to MHC-binding receptors that affect NK cell subset maturation and function in the context of licensing, the importance of cytokines such as IL-15 in their development and maintenance, and evidence of NK exhaustion have been initially studied in mice. Many of these findings have been validated in clinical studies and demonstrate the significant wealth of knowledge that can be obtained by mouse models. However, it is important to understand the limitations and conditions of the mouse models, particularly when studying NK cells in hematopoietic stem cell transplantation and cancer.