Functional plasticity and robustness are essential characteristics of biological systems: Lessons learned from KLRG1‐deficient mice

Killer cell lectin‐like receptor G1 (KLRG1) receptor is considered to be a marker of terminally differentiated NK and T cells and is strongly induced by viral and other infections. KLRG1 is a C‐type lectin‐like inhibitory receptor, which interacts with members of the cadherin family of molecules leading to the inhibition of T‐ and NK‐cell function. A study in this issue of the European Journal of Immunology addresses the role of KLRG1 in the maturation and differentiation of NK and T cells in vivo. Using KLRG1‐deficient mice generated by homologous recombination, the study reveals that KLRG1 is dispensable for NK‐ and CD8⁺ T‐cell differentiation and function in vivo. This interesting finding is discussed in this Commentary in light of the plasticity and robustness of immune response mechanisms.     

Interaction of KLRG1 with E‐cadherin: New functional and structural insights

The killer cell lectin‐like receptor G1 (KLRG1) is an inhibitory receptor expressed by memory T cells and NK cells in man and mice. It is frequently used as a cell differentiation marker and members of the cadherin family are ligands for KLRG1. The present study provides new insights into the interaction of mouse KLRG1 with E‐cadherin. Firstly, we demonstrate that co‐engagement of KLRG1 and CD3/TCR in a spatially linked manner was required for inhibition arguing against the notion that KLRG1‐ligation per se transmits inhibitory signals. Secondly, experiments with T cells carrying Y₇F‐mutant KLRG1 molecules with a replacement of the tyrosine residue to phenylalanine in the single ITIM indicated that the inhibitory activity of KLRG1 is counteracted to some degree by increased interaction of KLRG1⁺ T cells with E‐cadherin expressing target cells. Thirdly, we demonstrate that deletion of the first or the second external domain of E‐cadherin abolished reactivity in KLRG1‐reporter cell assays. Finally, we made the intriguing observation that KLRG1 formed multimeric protein complexes in T cells in addition to the previously described mono‐ and dimeric molecules.     

TGF‐β downregulates KLRG1 expression in mouse and human CD8+ T cells

The inhibitory receptor killer cell lectin‐like receptor G1 (KLRG1) and the integrin α_E (CD103) are expressed by CD8⁺ T cells and both are specific for E‐cadherin. However, KLRG1 ligation by E‐cadherin inhibits effector T‐cell function, whereas binding of CD103 to E‐cadherin enhances cell–cell interaction and promotes target cell lysis. Here, we demonstrate that KLRG1 and CD103 expression in CD8⁺ T cells from untreated and virus‐infected mice are mutually exclusive. Inverse correlation of KLRG1 and CD103 expression was also found in human CD8⁺ T cells‐infiltrating hepatocellular carcinomas. As TGF‐β is known to induce CD103 expression in CD8⁺ T cells, we examined whether this cytokine also regulates KLRG1 expression. Indeed, our data further reveal that TGF‐β signaling in mouse as well as in human CD8⁺ T cells downregulates KLRG1 expression. This finding provides a rationale for the reciprocal expression of KLRG1 and CD103 in different CD8⁺ T‐cell subsets. In addition, it points to the limitation of KLRG1 as a marker for terminally differentiated CD8⁺ T cells if lymphocytes from tissues expressing high levels of TGF‐β are analyzed.     

KLRG1 impairs regulatory T‐cell competitive fitness in the gut

Immune homeostasis requires the tight, tissue‐specific control of the different CD4⁺ Foxp3⁺ regulatory T (Treg) cell populations. The cadherin‐binding inhibitory receptor killer cell lectin‐like receptor G1 (KLRG1) is expressed by a subpopulation of Treg cells with GATA3⁺ effector phenotype. Although such Treg cells are important for the immune balance, especially in the gut, the role of KLRG1 in Treg cells has not been assessed. Using KLRG1 knockout mice, we found that KLRG1 deficiency does not affect Treg cell frequencies in spleen, mesenteric lymph nodes or intestine, or frequencies of GATA3⁺ Treg cells in the gut. KLRG1‐deficient Treg cells were also protective in a T‐cell transfer model of colitis. Hence, KLRG1 is not essential for the development or activity of the general Treg cell population. We then checked the effects of KLRG1 on Treg cell activation. In line with KLRG1's reported inhibitory activity, in vitro KLRG1 cross‐linking dampened the Treg cell T‐cell receptor response. Consistently, lack of KLRG1 on Treg cells conferred on them a competitive advantage in the gut, but not in lymphoid organs. Hence, although absence of KLRG1 is not enough to increase intestinal Treg cells in KLRG1 knockout mice, KLRG1 ligation reduces T‐cell receptor signals and the competitive fitness of individual Treg cells in the intestine.     

Effector T‐cell differentiation during viral and bacterial infections: Role of direct IL‐12 signals for cell fate decision of CD8+ T cells

To study the role of IL‐12 as a third signal for T‐cell activation and differentiation in vivo, direct IL‐12 signaling to CD8⁺ T cells was analyzed in bacterial and viral infections using the P14 T‐cell adoptive transfer model with CD8⁺ T cells that lack the IL‐12 receptor. Results indicate that CD8⁺ T cells deficient in IL‐12 signaling were impaired in clonal expansion after Listeria monocytogenes infection but not after infection with lymphocytic choriomeningitis virus, vaccinia virus or vesicular stomatitis virus. Although limited in clonal expansion after Listeria infection, CD8⁺ T cells deficient in IL‐12 signaling exhibited normal degranulation activity, cytolytic functions, and secretion of IFN‐γ and TNF‐α. However, CD8⁺ T cells lacking IL‐12 signaling failed to up‐regulate KLRG1 and to down‐regulate CD127 in the context of Listeria but not viral infections. Thus, direct IL‐12 signaling to CD8⁺ T cells determines the cell fate decision between short‐lived effector cells and memory precursor effector cells, which is dependent on pathogen‐induced local cytokine milieu.     

Transferrin‘ activation: Bonding with transferrin receptors tunes KLRG1 function

The inhibitory C‐type lectin‐like immunoreceptor KLRG1 enables mature NK cells and differentiated T cells to sense cadherin‐expressing cells by ligating “classical” cadherins. Upon engagement of the KLRG1 ectodomain, an inhibitory signal emanates from the cytoplasmic immunoreceptor‐tyrosine‐based inhibition motif (ITIM), dampening functional responses of these lymphocytes. Malignancy‐associated loss of cadherins has been proposed to relieve KLRG1‐mediated inhibition of cytotoxic lymphocytes and thereby to contribute to tumor surveillance by an alternate mode of “missing self‐recognition”. In this issue of the European Journal of Immunology, Schweier et al. [Eur. J. Immunol. 2014. 44: 1851–1856] propose another intriguing mechanism that may relieve KLRG1‐mediated inhibition in the course of lymphocyte activation. Subsequent to identification of the transferrin receptor (TfR) as a component of a high molecular mass KLRG1 complex, they demonstrate that a fraction of mouse KLRG1 molecules undergoes disulfide‐bonding with TfRs and colocalises with the latter at the cell surface. In functional terms, high levels of TfRs such as those found on activated lymphocytes were found to be associated with decreased KLRG1 inhibitory function, indicating that TfRs may sequester KLRG1 from interacting with cadherins. Hence, this unexpected liaison between KLRG1 and TfR may represent a regulatory link between metabolic activation and responses of lymphocytes.     

KLRG1 activity is regulated by association with the transferrin receptor

The killer cell lectin‐like receptor G1 (KLRG1) is a cadherin‐binding inhibitory receptor expressed by NK cells and differentiated T cells. Here, we surprisingly found that a fraction of KLRG1 molecules expressed in the murine A5 T‐cell line and in IL‐2‐activated NK cells forms disulfide‐linked heteromers with the transferrin receptor (TfR). Fluorescence microscopy additionally revealed substantial colocalization of KLRG1 and TfR in intracellular compartments and on the cell surface. TfR expression in resting lymphocytes is known to be low but it is strongly upregulated in proliferating cells. Intriguingly, our data further demonstrate that the inhibitory activity of KLRG1 is decreased in T cells expressing high levels of TfR, indicating that association of KLRG1 with TfR hinders KLRG1‐mediated silencing. This implies that proliferating TfR^high KLRG1⁺ lymphocytes may respond strongly to activation signals even in the presence of KLRG1 ligands, whereas resting TfR^low cells may be efficiently silenced via KLRG1.     

Differential role of NK cells against Candida albicans infection in immunocompetent or immunocompromised mice

Little is known regarding the role of NK cells during primary and secondary disseminated Candida albicans infection. We assessed the role of NK cells for host defense against candidiasis in immunocompetent, as well as immunodeficient, hosts. Surprisingly, depletion of NK cells in immunocompetent WT mice did not increase susceptibility to systemic candidiasis, suggesting that NK cells are redundant for antifungal defense in otherwise immunocompetent hosts. NK‐cell‐depleted mice were found to be protected as a consequence of attenuation of systemic inflammation. In contrast, the absence of NK cells in T/B/NK‐cell‐deficient NSG (NOD SCID gamma) mice led to an increased susceptibility to both primary and secondary systemic C. albicans infections compared with T/B‐cell‐deficient SCID mice. In conclusion, this study demonstrates that NK cells are an essential and nonredundant component of anti‐C. albicans host defense in immunosuppressed hosts with defective T/B‐lymphocyte immunity, while contributing to hyperinflammation in immunocompetent hosts. The discovery of the importance of NK cells in hosts with severe defects of adaptive immunity might have important consequences for the design of adjunctive immunotherapeutic approaches in systemic C. albicans infections targeting NK‐cell function.     

Peripheral Thy1+ lymphocytes rearranging TCR‐γδ genes in LAT‐deficient mice

Linker for activation of T cells (LAT) is an adaptor molecule indispensable for development of αβ and γδ T lymphocytes. Surprisingly, using a new model of LAT‐deficient mice we found that despite arrested thymic development, a discrete population of cells with active Lat promoter, expressing Thy1 molecules, accumulated in peripheral lymphoid organs of homozygous (Lat^Inv/Inv) mutant mice. By measuring frequencies of TCR gene rearrangements in conjunction with a panel of cell surface Ag, we dissected two subsets of these Thy1⁺ cells. Thy1^dull cells expressed markers of NK lymphocytes and contained low frequency of TCR‐γ gene rearrangements without detectable TCR‐δ rearrangements. Thy1^high cells resembled immature CD44⁺CD25⁺ thymocytes and contained high frequency of non‐productive TCR‐γ and TCR‐δ rearrangements, indicating that cells displaying molecular signatures of commitment toward γδ T‐cell lineage can develop and populate lymphoid tissues of LAT‐deficient mice. Phenotypically similar Thy1^high cells were also found in lymph nodes of lymphocyte‐deficient (Rag2^?/?) mice but not in T lymphocyte proficient, heterozygous Lat^+/Inv mice suggesting that Thy1^high cells of LAT‐deficient mice identified in this study accumulate in peripheral lymphoid organs as a result of congenital lymphopenia.     

Skewed B cell differentiation affects lymphoid organogenesis but not T cell‐mediated autoimmunity

B cell receptor (BCR) signalling determines B cell differentiation and may potentially alter T cell‐mediated immune responses. In this study we used two transgenic strains of BCR‐deficient mice expressing Epstein–Barr virus latent membrane protein (LMP)2A in B cells, where either follicular and marginal zone differentiation (D_HLMP2A mice) or B‐1 cell development (V_HLMP2A mice) were supported, and evaluated the effects of skewed B lymphocyte differentiation on lymphoid organogenesis and T cell responses in vivo. Compared to wild‐type animals, both transgenic strains displayed alterations in the composition of lymphoid organs and in the dynamics of distinct immune cell subsets following immunization with the self‐antigen PLP_185–206. However, ex‐vivo T cell proliferation to PLP_185–206 peptide measured in immunized D_HLMP2A and V_HLMP2A mice was similar to that detected in immunized control mice. Further, clinical expression of experimental autoimmune encephalitis in both LMP2A strains was identical to that of wild‐type mice. In conclusion, mice with skewed B cell differentiation driven by LMP2A expression in BCR‐negative B cells do not show changes in the development of a T cell mediated disease model of autoimmunity, suggesting that compensatory mechanisms support the generation of T cell responses.     

KLRG1 binds cadherins and preferentially associates with SHIP-1

The killer cell lectin-like receptor G1 (KLRG1) is a unique inhibitory receptor expressed on a phenotypically mature subset of resting NK cells as well as subsets of T cells in naive mice. In vivo, pathogenic immune system activation induces dramatic changes in the expression patterns of KLRG1 among the different cell subsets. In order to enhance our understanding of KLRG1 signaling properties and to clarify the functions of KLRG1 on these cells, we identified the broadly expressed N-cadherin molecule as a ligand for KLRG1. We further demonstrate that a second member of this superfamily of adhesion molecules, E-cadherin, binds to KLRG1. Additionally, we show that upon phosphorylation of the immunoreceptor tyrosine-based inhibitory motif (ITIM) tyrosine, KLRG1 recruits both SHIP-1 and SHP-2 but not SHP-1. We also delineate the key KLRG1 ITIM amino acid residues required for optimal association with these phosphatases. Finally, we demonstrate that KLRG1 engagement can inhibit sub-optimal TCR signaling. Taken together, our results indicate that KLRG1 may differentially regulate NK cell and T cell functions through the association with different ligands as well as the recruitment of distinct phosphatases.     

Plasmacytoid dendritic cell‐induced migration and activation of NK cells in vivo

NK cells are cytotoxic cells of the innate immune system. They have been found to be critical in the defense against infections and also against some tumors. Recent studies have shown that NK cells require signals from accessory cells to induce their recruitment and activation at the site of infection or tumor growth. In this study, we examined whether plasmacytoid DC (pDC) could recruit and activate NK cells in vivo. When CpG‐stimulated pDC were injected i.p. to C57BL/6 mice, they efficiently recruited NK cells, a process that was dependent on NK cell CXCR3 and CD62L and in part on CCR5. NK cells isolated from the peritoneum of mice inoculated with TLR7/8 or TLR9‐stimulated pDC exhibited greater cytotoxicity against YAC‐1 tumor cells than NK cells recovered from mice inoculated with control pDC. The present results are discussed in relation to pDC‐induced NK cell migration and activation in vivo.     

The IFN regulatory factor 7‐dependent type I IFN response is not essential for early resistance against murine cytomegalovirus infection

IFN regulatory factor 7 (IRF7) has been described as the master regulator of type I IFN responses and has been shown to be critical for innate antiviral immunity in vivo. In addition to type I IFN, NK cell responses are involved in the control of viral replication during acute viral infection. To investigate the role of IRF7 in the context of a viral infection that induces a strong NK cell response, the murine cytomegalovirus (MCMV) infection model was used. WT, IRF7‐deficient and IRF3/IRF7‐double deficient mice were infected with MCMV. The systemic IFN‐α response to MCMV was entirely dependent on IRF7, but independent of IRF3. However, peak IFN‐β production during MCMV infection was not affected by the lack of IRF7 or both IRF7 and IRF3. Despite the complete lack of IFN‐α production IRF7‐ and IRF3/IRF7‐deficient mice were surprisingly efficient in controlling MCMV replication and were only modestly more susceptible to MCMV infection than WT mice. NK cell cytotoxicity was unimpaired and NK cell IFN‐γ production was enhanced in IRF7‐deficient mice correlating with increased levels of bioactive IL‐12. Owing to these compensatory mechanisms IRF7‐dependent antiviral immune responses were not essential for resistance against acute MCMV infection in vivo.     

DC therapy induces long‐term NK reactivity to tumors via host DC

We vaccinated mice with DC loaded with or without invariant NKT‐cell ligand α‐galactosylceramide and evaluated long‐term resistance against tumor challenge. When mice had been given either DC or DC/galactosylceramide and were challenged with tumor cells even 6–12 months later, both NK and NKT cells were quickly activated to express CD69 and produce IFN‐γ. The NK cells could resist a challenge with several different tumors in vivo. The activated NK and NKT cells could be depleted with anti‐NK1.1 treatment. In spite of this, the activated cells recovered, indicating that tumor‐responsive NK and NKT cells were being generated continuously as a result of vaccination with DC and were not true memory cells. The NK and NKT antitumor response in DC‐vaccinated mice depended on CD4⁺ T cells, but neither CD8⁺T cells nor CD4⁺CD25⁺ regulatory T cells. However, both vaccine DC and host DC were required for the development of long‐term, tumor reactive innate immunity. These results indicate that DC therapy in mice induces long‐lasting innate NK‐ and NKT‐cell activation through a pathway that requires host DC and CD4⁺ T cells and that the continued generation of active NK cells resists the establishment of metastases in vivo.     

IFN‐γ‐STAT1 signal regulates the differentiation of inducible Treg: Potential role for ROS‐mediated apoptosis

Regulatory CD4⁺ T cells are important for the homeostasis of immune cells, and their absence correlates with autoimmune disorders. However, how the immune system regulates Treg homeostasis remains unclear. We found that IFN‐γ‐deficient‐mice had more forkhead box P3 (FOXP3⁺) cells than WT mice in all secondary lymphoid organs except the thymus. However, T‐bet‐ or IL‐4Rα‐deficient mice did not show a similar increase. In vitro differentiation studies showed that conversion of naïve T cells into FOXP3⁺ cells (neo‐generated inducible Treg (iTreg)) by TGF‐β was significantly inhibited by IFN‐γ in a STAT‐1‐dependent manner. Moreover, an in vivo adoptive transfer study showed that inhibition of FOXP3⁺ iTreg generation by IFN‐γ was a T‐cell autocrine effect. This inhibitory effect of IFN‐γ on iTreg generation was significantly abrogated after N‐acetyl‐L ‐cysteine treatment both in vitro and in vivo, indicating that IFN‐γ regulation of iTreg generation is dependent on ROS‐mediated apoptosis. Therefore, our results suggest that autocrine IFN‐γ can negatively regulate the neo‐generation of FOXP3⁺ iTreg through ROS‐mediated apoptosis in the periphery.     

NCR1‐deficiency diminishes the generation of protective murine cytomegalovirus antibodies by limiting follicular helper T‐cell maturation

NKp46/NCR1 is an activating NK‐cell receptor implicated in the control of various viral and bacterial infections. Recent findings also suggest that it plays a role in shaping the adaptive immune response to pathogens. Using NCR1‐deficient (NCR1^gfp/gfp) mice, we provide evidence for the role of NCR1 in antibody response to mouse cytomegalovirus infection (MCMV). The absence of NCR1 resulted in impaired maturation, function and NK‐cell migration to regional lymph nodes. In addition, CD4⁺ T‐cell activation and follicular helper T‐cell (Tfh) generation were reduced, leading to inferior germinal center (GC) B‐cell maturation. As a consequence, NCR1^gfp/gfp mice produced lower amounts of MCMV‐specific antibodies upon infection, which correlated with lower number of virus‐specific antibody secreting cells in analyzed lymph nodes.