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Bcl-3 is an atypical member of the IκB family that modulates NF-κB activity in nuclei. lpr mice carry the lpr mutation in Fas, resulting in functional loss of this death receptor; they serve as models for lupus erythematosus and autoimmune lymphoproliferation syndrome (ALPS). To explore the biologic roles of Bcl-3 in this disease model, we generated BL6/lpr mice lacking Bcl-3. Unlike lpr mice on an MRL background, BL6/lpr mice present with very mild lupus- or ALPS-like phenotypes. Bcl-3 KO BL6/lpr mice, however, developed severe splenomegaly, dramatically increased numbers of double negative T cells - a hallmark of human lupus, ALPS, and MRL/lpr mice - and exhibited inflammation in multiple organs, despite low levels of autoantibodies, similar to those in BL6/lpr mice. Loss of Bcl-3 specifically in T cells exacerbated select lupus-like phenotypes, specifically organ infiltration. Mechanistically, elevated levels of Tnfα in Bcl-3 KO BL6/lpr mice may promote lupus-like phenotypes, since loss of Tnfα in these mice reversed the pathology due to loss of Bcl-3. Contrary to the inhibitory functions of Bcl-3 revealed here, this regulator has also been shown to promote inflammation in different settings. Our findings highlight the profound, yet highly context-dependent roles of Bcl-3 in the development of inflammation-associated pathology., (Published 2020. This article is a U.S. Government work and is in the public domain in the USA.)

Natural killer (NK) cells are affected by infection with human cytomegalovirus (HCMV) manifested by increased expression of the HLA-E binding activating receptor NKG2C. We here show that HCMV seropositivity was associated with a profound expansion of NKG2C(+) CD56(dim) NK cells in patients with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. Multi-color flow cytometry revealed that the expanded NKG2C(+) CD56(dim) NK cells displayed a highly differentiated phenotype, expressed high amounts of granzyme B and exhibited polyfunctional responses (CD107a, IFN-γ, and TNF-α) to stimulation with antibody-coated as well as HLA-E expressing target cells but not when stimulated with IL-12/IL-18. More importantly, NKG2C(+) CD56(dim) NK cells had a clonal expression pattern of inhibitory killer cell immunoglobulin-like receptors (KIRs) specific for self-HLA class I molecules, with predominant usage of KIR2DL2/3. KIR engagement dampened NKG2C-mediated activation suggesting that such biased expression of self-specific KIRs may preserve self-tolerance and limit immune-pathology during viral infection. Together, these findings shed new light on how the human NK-cell compartment adjusts to HCMV infection resulting in clonal expansion and differentiation of educated and polyfunctional NK cells., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

CD1d-restricted NKT cells include CD4(+) and DN subsets, with an additional CD8(+) subset that is present in humans but not in mice. The molecular regulation of CD4/CD8 expression by NKT cells, and differentiation of these NKT-cell subsets, is poorly understood. The transcription factors GATA3 and ThPOK regulate lineage commitment of conventional MHC class II-restricted CD4(+) T cells; however, their role in CD4/CD8 expression by CD1d-restricted NKT cells is less clear. A new study in this issue of the European Journal of Immunology demonstrates a key role for ThPOK in differentiation of NKT-cell subsets. This study reveals that GATA3 and ThPOK are necessary for the development of CD4(+) NKT cells. Furthermore, ThPOK-deficient mice generate an unusual population of CD8(+) NKT cells, which are absent in control mice. This study sheds new light on the underlying molecular events leading to the emergence of distinct NKT-cell subsets.

While most CD4(+) T cells are MHC class II-restricted, a small subset, including the CD1d-restricted 'invariant' NKT (iNKT) cells, are selected on non-classical MHC-I or MHC-I-like molecules. We previously showed that the sequential activity of two zinc finger transcription factors, Gata3 and Thpok, promotes the differentiation of conventional, MHC II-restricted thymocytes into CD4(+) T cells. In the current study, we show that a Gata3-Thpok cascade is required for the differentiation of CD4(+) iNKT cells. Gata3 is required for iNKT cells to express Thpok, whereas Thpok is needed for proper NKT cell differentiation, and notably for NKT cells to maintain CD4 and terminate CD8 expression. These findings identify the sequential activity of Gata3 and Thpok as a hallmark of CD4(+) T-cell differentiation, regardless of MHC restriction.

Human peripheral blood NK cells may be divided into two main subsets: CD56(bright)CD16(-) and CD56(dim)CD16(+). Since TGF-β is known to influence the development of many leukocyte lineages, its effects on NK cell differentiation either from human CD34(+)Lin(-) hematopoietic progenitor/stem cells in vitro or from peripheral blood NK cells were investigated. TGF-β represses development of NK cells from CD34(+) progenitors and inhibits differentiation of CD16(+) NK cells. Moreover, TGF-β also results in conversion of a minor fraction of CD56(bright)CD16(+) cells found in peripheral blood into CD56(bright)CD16(-) cells, highlighting a possible role of the former as a developmental intermediate and of TGF-β in influencing the genesis of NK subsets found in blood.

Of the three nonclassical class I antigens expressed in humans, HLA-F has been least characterized with regard to expression or function. In this study, we examined HLA-F expression focusing on lymphoid cells, where our previous work with homologous cell lines had demonstrated surface HLA-F expression. HLA-F protein expression was observed by Western blot analysis in all resting lymphocytes, including B cells, T cells, NK cells, and monocytes, all of which lacked surface expression in the resting state. Upon activation, using a variety of methods to activate different lymphocyte subpopulations, all cell types that expressed HLA-F intracellularly showed an induction of surface HLA-F protein. An examination of peripheral blood from individuals genetically deficient for TAP and tapasin expression demonstrated the same activation expression profiles for HLA-F,but with altered kinetics post-activation. Further analysis of CD41+CD25+1 Treg showed that HLA-F was not upregulated on the major fraction of these cells when they were activated,whereas CD41+CD25- T cells showed strong expression of surface HLA-F when activated under identical conditions. These findings are discussed with regard to possible functions for HLA-F and its potential clinical use as a marker of an activated immune response.

Immunosuppression following severe sepsis remains a significant human health concern, as long-term morbidity and mortality rates of patients who have recovered from life-threatening septic shock remain poor. Mouse models of severe sepsis indicate this immunosuppression may be partly due to alterations in myeloid cell function; however, the effect of severe sepsis on subsequent CD4(+) T-cell responses remains unclear. In the present study, CD4(+) T cells from mice subjected to an experimental model of severe sepsis (cecal ligation and puncture (CLP)) were analyzed in vitro. CD4(+)CD62L(+) T cells from CLP mice exhibited reduced proliferative capacity and altered gene expression. Additionally, CD4(+)CD62L(+) T cells from CLP mice exhibit dysregulated cytokine production after in vitro skewing with exogenous cytokines, indicating a decreased capability of these cells to commit to either the T(H)1 or T(H)2 lineage. Repressive histone methylation marks were also evident at promoter regions for the T(H)1 cytokine IFN-gamma and the T(H)2 transcription factor GATA-3 in naïve CD4(+) T cells from CLP mice. These results provide evidence that CD4(+) T-cell subsets from post-septic mice exhibit defects in activation and effector function, possibly due to chromatin remodeling proximal to genes involved in cytokine production or gene transcription.

Natural killer (NK) cells (CD56(+)/CD3(-)) in the circulation of cancer patients were reported to have low NK activity and undergo spontaneous apoptosis. A possible relationship between apoptosis and impaired NK activity was studied by Annexin V-binding and NK-cell assays performed with peripheral blood mononuclear cells of patients with head and neck cancer (HNC), breast cancer (BC) and normal controls (NC). Cells stained with Annexin V (Anx) and antibodies to CD56, CD3, CD95, CD25, CD122 or CD132 were examined by flow cytometry. NK activity was tested against K562 targets in 4-h (51)Cr-release assays. The ratio of CD56(dim)/CD56(bright) NK cells was significantly different in patients vs. controls (10 vs. 16; p<0.01). A significantly greater percentage of CD56(dim) NK cells bound Anx in HNC patients (27+/-17%, median +/- SD) or BC (46+/-18%) than in NC (15+/-18%, p<0.04 and p<0.0002, respectively). CD56(dim) NK cells were preferentially targeted for apoptosis. NK activity was significantly lower in patients with HNC and BC than in NC (p<0.009). An inverse correlation between NK activity and the percent of Anx(+)CD56(dim) NK cells was observed in cancer patients (p =0.002) but not in NC. In patients, circulating CD56(dim) NK cells were targeted for apoptosis, leading to low levels of NK activity.

Pathogenesis of autoimmune diseases like systemic lupus erythematosus (SLE) is unresolved. Dysregulation of programmed cell death is discussed as a pathogenetic factor. We have previously shown that increased in vitro apoptosis of cultured peripheral blood mononuclear cells (PBMC) is nonspecific for SLE. Importantly, however, in recent experiments with SLE PBMC from patients with infections and fever in vitro apoptosis was strongly accelerated. We therefore hypothesized that regulation of apoptosis might be disturbed in activated SLE lymphocytes. Thus, we generated phytohemagglutinine (PHA)/IL-2 stimulated lymphoblasts in vitro. These lymphoblasts readily undergo apoptosis after culture in cytokine-free medium, and can be rescued by addition of gammac-chain cytokines IL-2, -4, -7, or -15. In lymphoblasts from 60 SLE patients tested in comparison to lymphoblasts from normal donors cultured in parallel, we found significant hyporesponsiveness to gammac-chain cytokines in SLE cells. Minor differences were also seen in lymphoblasts from patients with other systemic autoimmunopathies (mixed connective tissue disease, vasculitis, n=49)and in lymphoblasts from patients with other autoimmune diseases (mainly rheumatoid or reactive arthritis, myositis, n=44). In patients with high erythrocyte sedimentation rate (> 25 mm/h), TNF-alpha (> 6.5 pg/ml) or IL-12 (> 4.7 pg/ml) serum levels or detectable IFN-gamma concentrations hyporesponsiveness to gammac-chain cytokines was even more pronounced in SLE lymphoblasts, but not in lymphoblasts from the other groups. Moreover, increased apoptosis was seen in lymphoblasts from SLE patients with decreased complement (C)4 or elevated dsDNA antibody levels. In conclusion, these data suggest that in SLE patients with increased inflammatory activity and/or Th1 dominance signaling through gammac-chain cytokine receptors is deteriorated, leading to facilitated apoptosis of activated lymphocytes and enlarged onflow of apoptotic material.

E2A, HEB and E2-2 genes encode a group of basic helix-loop-helix (bHLH) transcription factors that are structurally and functionally similar. Deletion of the genes encoding either of these proteins leads to early lethality and a block in B lymphocyte development. Evidence for a function in T lymphocyte development has, however, only been reported for E2A and HEB. To further elucidate the role of E2-2 at developmental stages that have proven difficult to study due to the early lethality phenotype of mice defective in E2-2, we generated and analyzed mice conditionally mutated in the E2-2 gene. These mice are mosaic with respect to E2-2 expression, consisting of cells with either one functional and one null mutated E2-2 allele or two null mutated alleles. Using this experimental model, we find that cells with a homozygous null mutated E2-2 gene are under-represented in B lymphocyte as well as T lymphocyte cell lineages as compared to other hematopoietic or non-hematopoietic cell lineages. Our data suggests that E2-2 deficiency leads to a partial block in both B and T lymphocyte development. The block in T cell development appears to occur at an early stage in differentiation, since skewing in the mosaicism is observed already in CD4+8+ double-positive thymocytes.

Omenn's syndrome (OS) is characterized by erythrodermia, hepatosplenomegaly, lymphadenopathy, hypereosinophilia and elevated IgE levels associated with increased susceptibility to severe infections. Peripheral blood T cells, though usually present in normal number, show an activated phenotype (including an increased expression of CD95/Fas), a Th2 pattern of cytokine secretion and defective proliferative response to mitogens. In this report, we demonstrate that T cells from patients with OS undergo an excessive cell death in vitro resulting from two mechanisms. First, a substantial number of peripheral blood lymphocytes from OS patients die in unstimulated cultures (p = 0.009 vs. healthy controls). This spontaneous apoptosis is associated with reduced expression of bcl-2 gene product (p < 0.05) and can be prevented by addition of interleukin (IL)-2 (which also prevents down-modulation of bcl-2), while is independent from CD95 signaling. Second, lymphocytes from OS patients are highly susceptible to activation-induced cell death (AICD) induced with mitogens. This mechanism is largely independent from IL-2, while it can be significantly inhibited blocking CD95 with an IgG2b monoclonal antibody (mAb). The dependence of AICD from signals transduced via CD95 was confirmed showing that cross-linking CD95 with an IgM mAb induces a higher cell death in purified CD4+ CD45R0+ cells from OS patients than in controls (comparable for CD95 expression). Both mechanisms of cell death observed in this study result from lymphocyte hyperactivation occurring in vivo in these patients and may contribute to functional T cell defects of OS.

LEC rats are known to show a maturational arrest in the development of CD4+8+ to CD4+8- cells in the thymus. Despite the blockade of maturation of CD4+8-thymocytes, CD4+ T cells were observed in peripheral lymphoid organs, and these cells exhibit a defect in interleukin-2 (IL-2) production upon concanavalin A (Con A) stimulation. Although peripheral CD4+ cells in normal rat highly expressed CD45RC (CD45RChigh), the level of CD45RC expression was low (CD45RClow) in LEC rat peripheral CD4+ cells. However, CD4+ cells from both strains highly expressed CD45 when those cells were stained by pan-CD45 mAb, suggesting that LEC rat CD4+ cells are deficient in expression of the CD45RC isoform, but not of CD45 molecules. When backcross rats from (F344 x LEC)F1 x LEC were examined, the phenotype for CD45 expression pattern in CD4+ cells was clearly correlated with IL-2 production level in response to Con A stimulation. Thus, CD45RClow cells exhibit a defect in IL-2 production, while CD45RChigh cells show normal IL-2 production. Protein tyrosine phosphatase (PTPase) activity in the membrane fraction of LEC rat CD4+ cells was threefold higher than that of normal rat CD4+ cells. Con A stimulation led to an increase in tyrosine phosphorylation levels, especially 100- and 40-kDa proteins, in normal rat CD4+ cells. In LEC rat CD4+ cells, however, the level of tyrosine phosphorylation in those proteins were very low. These results suggest that an elevated CD45 PTPase activity is responsive for a defect in IL-2 production in LEC rat peripheral CD4+ T cells.