Residual LCMV antigen in transiently CD4+ T cell-depleted mice induces high levels of virus-specific antibodies but only limited B-cell memory.

Infection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV) strain Armstrong (Arm) induces an acute infection with rapid virus clearance by CD8+ T cells independently of CD4+ T cell help. Residual viral antigen may, however, persist for a prolonged time. Here, we demonstrate that mice that had been transiently depleted of CD4+ T cells during acute LCMV Arm infection generated high levels of virus-specific IgG antibodies (Ab) after viral clearance. Robust induction of LCMV-specific IgG after transient CD4+ T cell depletion was dependent on Fcγ receptors but not on the complement receptors CD21/CD35. In contrast to the potent production of LCMV-specific IgG, the generation of LCMV-specific isotype-switched memory B cells after transient CD4+ T cell depletion was considerably reduced. Moreover, mice depleted of CD4+ T cells during acute infection were strongly impaired in generating a secondary LCMV-specific B cell response upon LCMV rechallenge. In conclusion, our data indicate that LCMV antigen depots after viral clearance were capable of inducing high levels of virus-specific IgG. They failed, however, to induce robust virus-specific B cell memory revealing a previously unappreciated dichotomy of specific Ab production and memory cell formation after priming with residual antigen.

Immunological tolerance to LCMV antigens differently affects control of acute and chronic virus infection in mice.

Cytotoxic T lymphocytes (CTLs) play a key role in the control of lymphocytic choriomeningitis virus (LCMV) infection. In C57BL/6 mice (H-2b ), the CTL response is mainly directed against epitopes from the LCMV glycoprotein (GP) and the nucleoprotein (NP) which represent the two major viral proteins. The role of GP- versus NP-derived epitopes for viral clearance was examined using transgenic (tg) mice ubiquitously expressing LCMV GP and NP, respectively. These mice lack GP- or NP-specific CTLs and show decreased levels of GP- or NP-specific antibodies as a result of tolerance induction. During acute LCMV infection, CTLs specific for GP- and NP-derived epitopes are generated with similar frequencies. Nonetheless, we found that lack of GP- but not of NP-specific CTLs abolished control of acute LCMV infection. In contrast, after high-dose or chronic LCMV infection, virus elimination was delayed to a similar extent in GP- and NP-tg mice. Thus, immunological tolerance to LCMV antigens differently affects virus clearance in acute and chronic infection settings. In addition, our data reveal that immunodominance of H-2b -restricted LCMV-specific CTL epitopes and their antiviral activity do not strictly correlate.

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.

Nucleoprotein-specific nonneutralizing antibodies speed up LCMV elimination independently of complement and FcγR.

CD8(+) T cells have an essential role in controlling lymphocytic choriomeningitis virus (LCMV) infection in mice. Here, we examined the contribution of humoral immunity, including nonneutralizing antibodies (Abs), in this infection induced by low virus inoculation doses. Mice with impaired humoral immunity readily terminated infection with the slowly replicating LCMV strain Armstrong but showed delayed virus elimination after inoculation with the faster replicating LCMV strain WE and failed to clear the rapidly replicating LCMV strain Docile, which is in contrast to the results obtained with wild-type mice. Thus, the requirement for adaptive humoral immunity to control the infection was dependent on the replication speed of the LCMV strains used. Ab transfers further showed that LCMV-specific IgG Abs isolated from LCMV immune serum accelerated virus elimination. These Abs were mainly directed against the viral nucleoprotein (NP) and completely lacked virus neutralizing activity. Moreover, mAbs specific for the LCMV NP were also able to decrease viral titers after transfer into infected hosts. Intriguingly, neither C3 nor Fcγ receptors were required for the antiviral activity of the transferred Abs. In conclusion, our study suggests that rapidly generated nonneutralizing Abs specific for the viral NP speed up virus elimination and thereby may counteract T-cell exhaustion.

Different inhibitory capacities of human and mouse KLRG1 are linked to distinct disulfide-mediated oligomerizations.

The killer cell lectin-like receptor G1 (KLRG1) is a cadherin-binding inhibitory receptor expressed by NK and T cells in humans and mice. Although structural and ligand-binding properties of human (h) and mouse (m) KLRG1 are very similar, KLRG1-mediated inhibition under physiological conditions is only observed with human lymphocytes. Using a well-defined in vitro system, we demonstrate here that mKLRG1 exhibits a significantly lower inhibitory capacity compared with the human homolog. Biochemical analyses further showed that mKLRG1 formed monomers and disulfide-linked dimers, trimers, and tetramers whereas hKLRG1 was exclusively present as disulfide-linked dimer. Mutational analysis revealed a crucial role of Cys(62) present in the stalk region of mKLRG1 but not of hKLRG1 for oligomer formation. Strikingly, mimicking hKLRG1 by replacement of Cys(62) in mKLRG1 by glutamine prevented tri- and tetramer formation and increased the inhibitory capacity. Furthermore, mutated mKLRG1 molecules that were unable to form disulfide-linked dimers at all or at a decreased level lacked inhibitory activity. These data indicate that only dimeric KLRG1 entities exhibit potent inhibitory capacities. The lower inhibitory capacity of mKLRG1 compared with hKLRG1 can thus be rationalized by a decreased proportion of dimeric entities, which can be pinpointed to a single amino acid.

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.

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 Y(7)F-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.

Efficacy of IL-2- versus IL-15-stimulated CD8 T cells in adoptive immunotherapy.

We determined the efficacy of in vitro expanded P14 TCR transgenic CD8 T cells to mediate tumor cell elimination and to protect against viral infection in mice. Contrary to previous studies, an adoptive transfer model without lymphodepletion, vaccination or cytokine treatment was used. Antigen-activated P14 T cells cultured in IL-2-containing medium for 7 days (P14IL-2) exhibited potent effector cell functions in vitro but did not confer protection against melanoma growth or viral infection. In contrast, P14 T cells cultured in IL-15 (P14IL-15) were highly effective in vivo although they displayed only moderate effector functions in vitro. Therapeutic efficacy correlated with the survival of the transferred T cells in the recipients: P14IL-2 cells disappeared rapidly whereas P14IL-15 cells persisted for prolonged time. Decreasing the IL-2 concentration in the culture media improved in vivo survival and efficacy but also lowered the cell yield of the cultures. Finally, we could extend the findings with monoclonal P14 T cells to polyclonal CD8 T cells. Thus, in vitro expansion of antigen-specific CD8 T cells in IL-15 allowed the generation of substantial numbers of T cells without inducing terminally differentiated effector cells that turned out to be unfavorable in the transfer model examined here.

Tumor-associated E-cadherin mutations affect binding to the killer cell lectin-like receptor G1 in humans.

The killer cell lectin-like receptor G1 (KLRG1) is expressed by NK cells and memory T cells in man and mice. Cadherins were recently identified as ligands for mouse KLRG1 but ligands for human KLRG1 have not yet been defined. In this study, we first demonstrate that human E-cadherin is a ligand for human KLRG1. This finding is remarkable because human and mouse KLRG1 show only an intermediate degree of homology (57% aa identity). In addition, we show that E-cadherin, expressed on K562 target cells, inhibited polyclonal human NK cells. Inhibition of NK cell function was observed consistently in three independent functional assays but the extent of inhibition was modest and required high expression of E-cadherin on target cells. E-cadherin function is often inactivated during development of human carcinomas and splice-site mutations resulting in in-frame loss of exon 8 or 9 occur frequently in diffuse type gastric carcinomas. Our experiments further revealed that interaction of human KLRG1 to E-cadherin was susceptible to these tumor-associated mutations and that KLRG1(+) NK cells were triggered more easily by K562 target cells carrying these mutations in comparison to target cells expressing wild-type E-cadherin. These results also indicate that the E-cadherin binding sites important for homophilic interaction are also involved in KLRG1 binding. Taken together, these data demonstrate that the main adhesion molecule of epithelial tissue, E-cadherin, is involved in regulation of NK cells in both humans and mice.

CD8 T cells specific for lymphocytic choriomeningitis virus require type I IFN receptor for clonal expansion.

The role of type I IFN signaling in CD8 T cells was analyzed in an adoptive transfer model using P14 TCR transgenic CD8 T cells specific for lymphocytic choriomeningitis virus (LCMV) but deficient in type I IFNR. In the present study, we demonstrate severe impairment in the capacity of P14 T cells lacking type I IFNR to expand in normal type I IFNR wild-type C57BL/6 hosts after LCMV infection. In contrast, following infection of recipient mice with recombinant vaccinia virus expressing LCMV glycoprotein, P14 T cell expansion was considerably less dependent on type I IFNR expression. Lack of type I IFNR expression by P14 T cells did not affect cell division after LCMV infection but interfered with clonal expansion. Thus, direct type I IFN signaling is essential for CD8 T cell survival in certain viral infections.

Cutting edge: identification of E-cadherin as a ligand for the murine killer cell lectin-like receptor G1.

The killer cell lectin-like receptor G1 (KLRG1) is expressed by NK cells and by T cells. In both humans and mice, KLRG1 identifies Ag-experienced T cells that are impaired in their proliferative capacity but are capable of performing effector functions. In this study, we identified E-cadherin as a ligand for murine KLRG1 by using fluorescently labeled, soluble tetrameric complexes of the extracellular domain of the murine KLRG1 molecule as staining reagents in expression cloning. Ectopic expression of E-cadherin in B16.BL6 target cells did not affect cell-mediated lysis by lymphokine-activated NK cells and by CD8 T cells but inhibited Ag-induced proliferation and induction of cytolytic activity of CD8 T cells. E-cadherin is expressed by normal epithelial cells, Langerhans cells, and keratinocytes and is usually down-regulated on metastatic cancer cells. KLRG1 ligation by E-cadherin in healthy tissue may thus exert an inhibitory effect on primed T cells.

Antibody to streptococcal cysteine proteinase as a seromarker of group A Streptococcal (Streptococcus pyogenes) infections.

Serological tests are commonly employed to aid the diagnosis of Streptococcus pyogenes infections, particularly when non-suppurative sequelae are suspected. Conventional laboratory practice is to measure antibody levels to various combinations of the extracellular group A Streptococcus (GAS) antigens streptolysin O (SLO), DNase B, streptokinase and hyaluronidase. Antibody to the extracellular cysteine proteinase streptococcal pyrogenic exotoxin B (SPE B) and its precursor zymogen is also produced in response to GAS infections. An indirect hemagglutination test for antibody to zymogen/SPE B was established and evaluated in serum samples from 168 patients with proven (n = 27) or suspected GAS (n = 141) infections, which were also screened for antibodies using the 4 conventional tests. For comparison, sera from 56 patients infected with a variety of other pathogens, as well as sera from 16 patients infected with either S. agalactiae or S. pneumoniae and 34 sera from healthy subjects, were tested. Statistical analysis confirmed that antibody to zymogen/SPE B is a serological marker that can discriminate GAS infections. It can be ranked with the anti-SLO titer, currently the most widely used test, as a marker of an antecedent GAS infection.