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 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.     

The NK receptor KLRG1 is dispensable for virus‐induced NK and CD8+ T‐cell differentiation and function in vivo

The killer cell lectin‐like receptor G1 (KLRG1) is expressed by NK and T‐cell subsets and recognizes members of the classical cadherin family. KLRG1 is widely used as a lymphocyte differentiation marker in both humans and mice but the physiological role of KLRG1 in vivo is still unclear. Here, we generated KLRG1‐deficient mice by homologous recombination and used several infection models for their characterization. The results revealed that KLRG1 deficiency did not affect development and function of NK cells examined under various conditions. KLRG1 was also dispensable for normal CD8⁺ T‐cell differentiation and function after viral infections. Thus, KLRG1 is a marker for distinct NK and T‐cell differentiation stages but it does not play a deterministic role in the generation and functional characteristics of these lymphocyte subsets. In addition, we demonstrate that E‐cadherin expressed by K562 target cells inhibited NK‐cell reactivity in transgenic mice over‐expressing KLRG1 but not in KLRG1‐deficient or WT mice. Hence, the inhibitory potential of KLRG1 in mice is rather weak and strong activation signals during viral infections may override the inhibitory signal in vivo.     

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.     

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.     

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 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.     

Diversification and senescence of Foxp3+ regulatory T cells during experimental autoimmune encephalomyelitis

The fate of Foxp3⁺ regulatory T (Treg) cells responding during autoimmunity is not well defined. We observed a marked elevation in KLRG1⁺ (where KLRG1 stands for killer cell lectin‐like receptor G1) CNS‐infiltrating Treg cells in experimental autoimmune encephalomyelitis (EAE), and assessed their origin and properties. KLRG1⁺ Treg cells showed increased activation marker expression, Foxp3 and CD25 levels, and more rapid cell cycling than KLRG1^? cells. KLRG1^? Treg cells converted into KLRG1⁺ cells and this was increased in autoimmune inflammation. Conversion was unidirectional; KLRG1⁺ Treg cells did not revert to a KLRG1^? state. KLRG1⁺ but notKLRG1^?Treg cells survived poorly, indicative of terminal differentiation. This was associated with diminished BCL2 and increased apoptosis of isolated cells. KLRG1 was also upregulated on iTreg cells after transfer and EAE induction or on iTreg cells developing spontaneously during EAE. KLRG1⁺ Treg cells produced more IL‐10 and had altered effector cytokine production compared with their KLRG1^? counterparts. Despite their differences, KLRG1⁺ and KLRG1^? Treg cells proved similarly potent in suppressing EAE. KLRG1⁺ and KLRG1^? populations were phenotypically heterogeneous, with the extent and pattern of activation marker expression dependent both on cellular location and inflammation. Our results support an extensive diversification of Treg cells during EAE, and associate KLRG1 with altered Treg‐cell function and senescence.     

Skin resident memory CD8+ T cells are phenotypically and functionally distinct from circulating populations and lack immediate cytotoxic function

The in‐depth understanding of skin resident memory CD8⁺ T lymphocytes (T_RM) may help to uncover strategies for their manipulation during disease. We investigated isolated T_RM from healthy human skin, which expressed the residence marker CD69, and compared them to circulating CD8⁺ T cell populations from the same donors. There were significantly increased proportions of CD8⁺CD45RA^–CD27^– T cells in the skin that expressed low levels of killer cell lectin‐like receptor G1 (KLRG1), CD57, perforin and granzyme B. The CD8⁺ T_RM in skin were therefore phenotypically distinct from circulating CD8⁺CD45RA^–CD27^– T cells that expressed high levels of all these molecules. Nevertheless, the activation of CD8⁺ T_RM with T cell receptor (TCR)/CD28 or interleukin (IL)‐2 or IL‐15 in vitro induced the expression of granzyme B. Blocking signalling through the inhibitory receptor programmed cell death 1 (PD)‐1 further boosted granzyme B expression. A unique feature of some CD8⁺ T_RM cells was their ability to secrete high levels of tumour necrosis factor (TNF)‐α and IL‐2, a cytokine combination that was not seen frequently in circulating CD8⁺ T cells. The cutaneous CD8⁺ T_RM are therefore diverse, and appear to be phenotypically and functionally distinct from circulating cells. Indeed, the surface receptors used to distinguish differentiation stages of blood T cells cannot be applied to T cells in the skin. Furthermore, the function of cutaneous T_RM appears to be stringently controlled by environmental signals in situ .     

Frequent expression of the natural killer cell receptor KLRG1 in human cord blood T cells: correlation with replicative history

The killer cell lectin-like receptor G1 (KLRG1) belongs to the family of inhibitory C-type lectins that are encoded in the NK gene complex. Similar to other inhibitory NK cell receptors, KLRG1 expression in adult peripheral blood lymphocytes is restricted to NK cells and to antigen-experienced T cells. Umbilical cord blood T cells are thought to represent an homogenous pool of naive T cells. Surprisingly, we identified substantial subsets of CD4 ( approximately 30%) and CD8 ( approximately 20%) alphabeta T cells in cord blood that expressed KLRG1. In contrast to T cells in adult, KLRG1(+) T cells in cord blood exhibited predominantly a naive CCR7(+)CD45RA(+) and CD11a(low) phenotype. After birth, KLRG1 expression in T cells from peripheral blood decreased rapidly to reappear in effector/memory T cells in adults. KLRG1(+) T cells in cord blood expressed a diverse T cell receptor beta (TCRbeta) repertoire and the cells proliferated normally, in contrast to KLRG1(+) T cells from adults. Finally, examination of T cell receptor excision circles (TREC) indicated that KLRG1 expression discriminated between cord blood T cells that differed in their post-thymic expansion rate. Thus, analysis of KLRG1 expression in cord blood revealed an unexpected heterogeneity of human T cells in newborns.     

P‐cadherin functional role is dependent on E‐cadherin cellular context: a proof of concept using the breast cancer model

P‐cadherin overexpression is associated with worse breast cancer survival, being a poor prognostic marker as well as a putative therapeutic target for the aggressive triple‐negative and basal‐like carcinomas (TNBCs). Previously, we have shown that P‐cadherin promotes breast cancer invasion of cells where membrane E‐cadherin was maintained; however, it suppresses invasion in models without endogenous cadherins, like melanomas. Here, we investigated if P‐cadherin expression would interfere with the normal adhesion complex and which were the cellular/molecular consequences, constituting, in this way, a new mechanism by which E‐cadherin invasive‐suppressor function was disrupted. Using breast TNBC models, we demonstrated, for the first time, that P‐cadherin co‐localizes with E‐cadherin, promoting cell invasion due to the disruption caused in the interaction between E‐cadherin and cytoplasmic catenins. P‐cadherin also induces cell migration and survival, modifying the expression profile of cells expressing wild‐type E‐cadherin and contributing to alter their cellular behaviour. Additionally, E‐ and P‐cadherin co‐expressing cells significantly enhanced in vivo tumour growth, compared with cells expressing only E‐ or only P‐cadherin. Finally, we still found that co‐expression of both molecules was significantly correlated with high‐grade breast carcinomas, biologically aggressive, and with poor patient survival, being a strong prognostic factor in this disease. Our results show a role for E‐ and P‐cadherin co‐expression in breast cancer progression and highlight the potential benefit of targeting P‐cadherin in the aggressive tumours expressing high levels of this protein. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Memory precursor phenotype of CD8+ T cells reflects early antigenic experience rather than memory numbers in a model of localized acute influenza infection

The mechanistic basis of memory T‐cell development is poorly defined. Phenotypic markers that define precursors at effector stages have been characterized for acute systemic infections with high antigen load. We asked whether such markers can identify memory precursors from early effectors (d6) to late memory (>d500) for two immunodominant CD8⁺ responses during the course of a localized low‐load influenza infection in mice. CD8⁺ T cells stained with the D^bNP₃₆₆ and D^bPA₂₂₄ tetramers were characterized as IL‐7Rα^hi, IL‐7Rα^hiCD62L^hi or IL‐7Rα^hiKLRG1^lo. While the D^bNP₃₆₆‐ and D^bPA₂₂₄‐specific responses were comparable in size, decay kinetics and memory precursor frequency, their expansion characteristics differed. This correlated with a divergence in the IL‐7Rα^hi, IL‐7Rα^hiCD62L^hi and IL‐7Rα^hiKLRG1^lo phenotypes on effector, but not naïve, CD8⁺ populations. That effect was abrogated by priming with viruses engineered to present equivalent levels of NP₃₆₆ and PA₂₂₄ peptides, indicating that memory phenotypes reflect early antigenic experience rather than memory potential. Thus, the IL‐7Rα^hiKLRG1^lo phenotype had a poor predictive value in identifying memory precursors in the spleen and at the site of infection. Greater consistency in influenza‐specific IL‐7Rα^hiKLRG1^loCD8⁺ T‐cell numbers was found in draining lymph nodes, suggesting that this may be the preferential site for memory establishment and maintenance following localized virus infections.     

PSGL‐1 and E/P‐selectins are essential for T‐cell rolling in inflamed CNS microvessels but dispensable for initiation of EAE

T‐cell migration across the blood‐brain barrier is a crucial step in the pathogenesis of EAE, an animal model for MS. Live cell imaging studies demonstrated that P‐selectin glycoprotein ligand‐1 (PSGL‐1) and its endothelial ligands E‐ and P‐selectin mediate the initial rolling of T cells in brain vessels during EAE. As functional absence of PSGL‐1 or E/P‐selectins does not result in ameliorated EAE, we speculated that T‐cell entry into the spinal cord is independent of PSGL‐1 and E/P‐selectin. Performing intravital microscopy, we observed the interaction of WT or PSGL‐1^?/? proteolipid protein‐specific T cells in inflamed spinal cord microvessels of WT or E/P‐selectin^?/? SJL/J mice during EAE. T‐cell rolling but not T‐cell capture was completely abrogated in the absence of either PSGL‐1 or E‐ and P‐selectin, resulting in a significantly reduced number of T cells able to firmly adhere in the inflamed spinal cord microvessels, but did not lead to reduced T‐cell invasion into the CNS parenchyma. Thus, PSGL‐1 interaction with E/P‐selectin is essential for T‐cell rolling in inflamed spinal cord microvessels during EAE. Taken together with previous observations, our findings show that T‐cell rolling is not required for successful T‐cell entry into the CNS and initiation of EAE.     

KLRG1 expression identifies short-lived Foxp3+ Treg effector cells with functional plasticity in islets of NOD mice

A progressive waning in Foxp3⁺ regulatory T (T_reg) cell function provokes autoimmunity in the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D), a cellular defect rescued by prophylactic IL-2 therapy. We showed that most islet-infiltrating T_reg cells express inducible T-cell co-stimulator (ICOS) in pre-diabetic NOD mice, and that ICOS⁺ T_reg cells display enhanced fitness and suppressive function in situ. Moreover, T1D progression is associated with decreased expansion and suppressive activity of ICOS⁺Foxp3⁺ T_reg cells, in islets, an observation consistent with the exacerbated T1D seen in NOD.BDC2.5 mice in which the ICOS pathway is abrogated. Here, we show that a large proportion of islet-resident T_reg cells express the KLRG1 marker of terminally differentiation, in contrast to islet-infiltrating ICOS^? T_reg or T_eff cells. We hypothesized that KLRG1 expression designates a subpopulation of ICOS⁺ T_reg cells in islets that progressively loses function, and contributes to the immune dysregulation observed at T1D onset. Indeed, KLRG1-expressing ICOS⁺ T_reg cells are prone to apoptosis, and have an impaired proliferative capacity and suppressive function in vitro and in vivo. T1D protective low-dose IL-2 treatment in vivo could not rescue the loss of KLRG1-expressing T_reg cells in situ. While the global pool of Foxp3⁺ T_reg cells displays some degree of functional plasticity in vivo, the KLRG1⁺ ICOS⁺ T_reg cell subset is particularly susceptible to lose Foxp3 expression and reprogram into Th1- or Th17-like effector T (T_eff) cells in the pancreas microenvironment. Overall, KLRG1 expression delineates a subpopulation of dysfunctional T_reg cells during T1D progression in autoantigen-specific TCR transgenic NOD mice.     

Cytomegalovirus vector expressing RAE‐1γ induces enhanced anti‐tumor capacity of murine CD8+ T cells

Designing CD8⁺ T‐cell vaccines, which would provide protection against tumors is still considered a great challenge in immunotherapy. Here we show the robust potential of cytomegalovirus (CMV) vector expressing the NKG2D ligand RAE‐1γ as CD8⁺ T cell‐based vaccine against malignant tumors. Immunization with the CMV vector expressing RAE‐1γ, delayed tumor growth or even provided complete protection against tumor challenge in both prophylactic and therapeutic settings. Moreover, a potent tumor control in mice vaccinated with this vector can be further enhanced by blocking the immune checkpoints TIGIT and PD‐1. CMV vector expressing RAE‐1γ potentiated expansion of KLRG1⁺ CD8⁺ T cells with enhanced effector properties. This vaccination was even more efficient in neonatal mice, resulting in the expansion and long‐term maintenance of epitope‐specific CD8⁺ T cells conferring robust resistance against tumor challenge. Our data show that immunomodulation of CD8⁺ T‐cell responses promoted by herpesvirus expressing a ligand for NKG2D receptor can provide a powerful platform for the prevention and treatment of CD8⁺ T‐cell sensitive tumors.     

The transduction pattern of IL‐12‐encoding lentiviral vectors shapes the immunological outcome

In situ modification of antigen‐presenting cells garnered interest in cancer immunotherapy. Therefore, we developed APC‐targeted lentiviral vectors (LVs). Unexpectedly, these LVs were inferior vaccines to broad tropism LVs. Since IL‐12 is a potent mediator of antitumor immunity, we evaluated whether this proinflammatory cytokine could enhance antitumor immunity of an APC‐targeted LV‐based vaccine. Therefore, we compared subcutaneous administration of broad tropism LVs (VSV‐G‐LV) with APC‐targeted LVs (DC2.1‐LV)‐encoding enhanced GFP and ovalbumin, or IL‐12 and ovalbumin in mice. We show that codelivery of IL‐12 by VSV‐G‐LVs or DC2.1‐LVs augments CD4⁺ or CD8⁺ T‐cell proliferation, respectively. Furthermore, we demonstrate that codelivery of IL‐12 enhances the CD4⁺ T_H1 profile irrespective of its delivery mode, while an increase in cytotoxic and therapeutic CD8⁺ T cells was only induced upon VSV‐G‐LV injection. While codelivery of IL‐12 by DC2.1‐LVs did not enhance CD8⁺ T‐cell performance, it increased expression of inhibitory checkpoint markers Lag3, Tim3, and PD‐1. Finally, the discrepancy between CD4⁺ T‐cell stimulation with and without functional CD8⁺ T‐cell stimulation by VSV‐G‐ and DC2.1‐LVs is partly explained by the observation that IL‐12 relieves CD8⁺ T cells from CD4⁺ T‐cell help, implying that a T_H1 profile is of minor importance for antitumor immunotherapy if IL‐12 is exogenously delivered.