Low on-treatment levels of serum soluble CD8 (sCD8) predict better outcomes in advanced non-small cell lung cancer patients treated with atezolizumab.

BACKGROUND: Immunotherapy has changed the paradigm of treating non-small cell lung cancer (NSCLC). But, selecting patients who will achieve long-term benefits from treatment remains unsatisfactory. Here, we investigated the possible use of the soluble form of CD8 antigen (sCD8) in predicting durable disease control after PD-1/PD-L1 blockade. CD8 is a marker of the cytotoxic T lymphocytes. Its soluble form (sCD8) is secreted under activation of the immune system but also has immunosuppressive properties. The data about serum sCD8 in patients dosed with anti-PD-1/PD-L1 drugs are lacking. METHODS AND RESULTS: We included 42 NSCLC patients and collected samples at baseline and for the first 3 months of atezolizumab immunotherapy. The serum sCD8 concentrations were measured with the ELISA kit and correlated with treatment outcomes. Patients with durable (≥ 12 months) disease control presented lower serum sCD8 than those without long-term benefits. The sCD8 levels measured at the end of cycle 2 (sCD8.2) were the earliest time point that successfully differentiated patients (3.76 vs. 9.68 ng/mL, respectively, p < 0.001). Individuals with low sCD8.2 (≤ 4.09 ng/mL) presented longer progression-free survival (HR = 0.061, p < 0.001) and overall survival (HR = 0.104, p < 0.05) compared to individuals with high sCD8.2 (median values unreached vs. 4.4 months and 14.4 months for PFS and OS, respectively). CONCLUSIONS: Serum sCD8 could be an early biomarker of durable disease control after anti-PD-L1 treatment. Higher sCD8 in patients with worse outcomes could suggest the inhibitory effect of sCD8 on cytotoxic T-cells activation.

CD8 Binding of MHC-Peptide Complexes in cis or trans Regulates CD8+ T-cell Responses.

The coreceptor CD8αß can greatly promote activation of T cells by strengthening T-cell receptor (TCR) binding to cognate peptide-MHC complexes (pMHC) on antigen presenting cells and by bringing p56Lck to TCR/CD3. Here, we demonstrate that CD8 can also bind to pMHC on the T cell (in cis) and that this inhibits their activation. Using molecular modeling, fluorescence resonance energy transfer experiments on living cells, biochemical and mutational analysis, we show that CD8 binding to pMHC in cis involves a different docking mode and is regulated by posttranslational modifications including a membrane-distal interchain disulfide bond and negatively charged O-linked glycans near positively charged sequences on the CD8ß stalk. These modifications distort the stalk, thus favoring CD8 binding to pMHC in cis. Differential binding of CD8 to pMHC in cis or trans is a means to regulate CD8+ T-cell responses and provides new translational opportunities.

CD8αα homodimers function as a coreceptor for KIR3DL1.

Cluster of differentiation 8 (CD8) is a cell surface glycoprotein, which is expressed as 2 forms, αα homodimer or αß heterodimer. Peptide-loaded major histocompatibility complex class I (pMHC-I) molecules are major ligands for both forms of CD8. CD8αß is a coreceptor for the T cell receptor (TCR) and binds to the same cognate pMHC-I as the TCR, thus enabling or augmenting T cell responses. The function of CD8αα homodimers is largely unknown. While CD8αß heterodimer is expressed exclusively on CD8+ T cells, the CD8αα homodimer is present in subsets of T cells and human natural killer (NK) cells. Here, we report that the CD8αα homodimer functions as a coreceptor for KIR3DL1, an inhibitory receptor of NK cells that is specific for certain MHC-I allotypes. CD8αα enhances binding of pMHC-I to KIR3DL1, increases KIR3DL1 clustering at the immunological synapse, and augments KIR3DL1-mediated inhibition of NK cell activation. Additionally, interactions between pMHC-I and CD8αα homodimers regulate KIR3DL1+ NK cell education. Together, these findings reveal another dimension to the modulation of NK cell activity.

Identification of immunodominant CD8 epitope in the stalk domain of influenza B viral hemagglutinin.

Human infections by type B influenza virus constitute about 25% of all influenza cases. The viral hemagglutinin is comprised of two subunits, HA1 and HA2. While HA1 is constantly evolving in an unpredictable fashion, the HA2 subunit is highly conserved, making it a potential candidate for a universal vaccine. However, immunodominant epitopes in the HA2 subunit remain largely unknown. To delineate MHC Class I epitopes, we first identified 9-mer H-2Kd-restricted CD8 T cell epitopes in the HA2 domain by in silico analyses, followed by evaluating the immunodominance of these peptides in mice challenged with the virus. Of three peptides selected through in silico analysis, the universally conserved peptide, YYSTAASSL (B/HA2-190), possessed the highest predicted binding affinity to MHC Class I and was most effective in inducing IL-2 and TNF-α in mouse splenocytes. Importantly, the peptide demonstrated best capability of stimulating peptide-specific ex-vivo cytotoxicity against target cells. Taken together, this finding would be of value for assessment of cell-mediated immune responses elicited by vaccines based on the highly conserved HA2 stalk domain.

Structural insights into the evolution feature of a bony fish CD8αα homodimer.

The CD8αα homodimer structures of endotherms demonstrate that despite distinct diversity at the amino acid sequence level, a few conserved key amino acids ensure common structural features. The structure of CD8αα in ancient ectotherms, such as lower bony fish, remains unclear. In this study, the high-resolution structure of the grass carp (Ctenopharyngodon idella) CD8αα (Ctid-CD8αα) homodimer was determined using the single-wavelength anomalous diffraction (SAD) method. The structure of Ctid-CD8αα shows distinct differences from the known CD8αα structures of endotherms, including a distinct topological structure with shorter back ß sheets. The configuration and distribution of the hydrophobic core are different from those in endotherms. Interestingly, mutation of the key amino acid F32S, which is very common in fish and lies in the CDR loop region, leads to the absence of the typical cavity that binds to an epitope-MHC I (p/MHC I) in endotherms, yet Ctid-CD8αα can still specifically bind the grass carp peptide-Ctid-UAA-ß2m (p/UAA-ß2m). Our results indicate that during the evolutionary process, CD8αα has undergone dramatic changes that affect its dimeric structure and may use a new strategy to interact with p/MHC I.

The structural basis of chicken, swine and bovine CD8αα dimers provides insight into the co-evolution with MHC I in endotherm species.

It is unclear how the pivotal molecules of the adaptive immune system (AIS) maintain their inherent characteristics and relationships with their co-receptors over the course of co-evolution. CD8α, a fundamental but simple AIS component with only one immunoglobulin variable (IgV) domain, is a good example with which to explore this question because it can fold correctly to form homodimers (CD8αα) and interact with peptide-MHC I (p/MHC I) with low sequence identities between different species. Hereby, we resolved the crystal structures of chicken, swine and bovine CD8αα. They are typical homodimers consisting of two symmetric IgV domains with distinct species specificities. The CD8αα structures indicated that a few highly conserved residues are important in CD8 dimerization and in interacting with p/MHC I. The dimerization of CD8αα mainly depends on the pivotal residues on the dimer interface; in particular, four aromatic residues provide many intermolecular forces and contact areas. Three residues on the surface of CD8α connecting cavities that formed most of the hydrogen bonds with p/MHC I were also completely conserved. Our data propose that a few key conserved residues are able to ensure the CD8α own structural characteristics despite the great sequence variation that occurs during evolution in endotherms.

Spatiotemporal multistage consensus clustering in molecular dynamics studies of large proteins.

The aim of this work is to find semi-rigid domains within large proteins as reference structures for fitting molecular dynamics trajectories. We propose an algorithm, multistage consensus clustering, MCC, based on minimum variation of distances between pairs of Cα-atoms as target function. The whole dataset (trajectory) is split into sub-segments. For a given sub-segment, spatial clustering is repeatedly started from different random seeds, and we adopt the specific spatial clustering with minimum target function: the process described so far is stage 1 of MCC. Then, in stage 2, the results of spatial clustering are consolidated, to arrive at domains stable over the whole dataset. We found that MCC is robust regarding the choice of parameters and yields relevant information on functional domains of the major histocompatibility complex (MHC) studied in this paper: the α-helices and ß-floor of the protein (MHC) proved to be most flexible and did not contribute to clusters of significant size. Three alleles of the MHC, each in complex with ABCD3 peptide and LC13 T-cell receptor (TCR), yielded different patterns of motion. Those alleles causing immunological allo-reactions showed distinct correlations of motion between parts of the peptide, the binding cleft and the complementary determining regions (CDR)-loops of the TCR. Multistage consensus clustering reflected functional differences between MHC alleles and yields a methodological basis to increase sensitivity of functional analyses of bio-molecules. Due to the generality of approach, MCC is prone to lend itself as a potent tool also for the analysis of other kinds of big data.

Relative Movements of Domains in Large Molecules of the Immune System.

Molecular dynamics was used to simulate large molecules of the immune system (major histocompatibility complex class I, presented epitope, T-cell receptor, and a CD8 coreceptor.) To characterize the relative orientation and movements of domains local coordinate systems (based on principal component analysis) were generated and directional cosines and Euler angles were computed. As a most interesting result, we found that the presence of the coreceptor seems to influence the dynamics within the protein complex, in particular the relative movements of the two α-helices, Gα1 and Gα2.

Human adenovirus-specific γ/δ and CD8+ T cells generated by T-cell receptor transfection to treat adenovirus infection after allogeneic stem cell transplantation.

Human adenovirus infection is life threatening after allogeneic haematopoietic stem cell transplantation (HSCT). Immunotherapy with donor-derived adenovirus-specific T cells is promising; however, 20% of all donors lack adenovirus-specific T cells. To overcome this, we transfected α/ß T cells with mRNA encoding a T-cell receptor (TCR) specific for the HLA-A*0101-restricted peptide LTDLGQNLLY from the adenovirus hexon protein. Furthermore, since allo-reactive endogenous TCR of donor T lymphocytes would induce graft-versus-host disease (GvHD) in a mismatched patient, we transferred the TCR into γ/δ T cells, which are not allo-reactive. TCR-transfected γ/δ T cells secreted low quantities of cytokines after antigen-specific stimulation, which were increased dramatically after co-transfection of CD8α-encoding mRNA. In direct comparison with TCR-transfected α/ß T cells, TCR-CD8α-co-transfected γ/δ T cells produced more tumor necrosis factor (TNF), and lysed peptide-loaded target cells as efficiently. Most importantly, TCR-transfected α/ß T cells and TCR-CD8α-co-transfected γ/δ T cells efficiently lysed adenovirus-infected target cells. We show here, for the first time, that not only α/ß T cells but also γ/δ T cells can be equipped with an adenovirus specificity by TCR-RNA electroporation. Thus, our strategy offers a new means for the immunotherapy of adenovirus infection after allogeneic HSCT.

Complex assembly, crystallization and preliminary X-ray crystallographic analysis of the chicken CD8αα-BF2*0401 complex.

In the process of antigen presentation, the MHCI-CD8 complex is important for immune signal transduction by the activation of cytotoxic T cells. Here, the expression, purification, crystallization and X-ray analysis of the complex of the chicken MHC class I molecule BF2*0401 and CD8αα (CD8αα-BF2*0401) are reported. This complex was verified by SDS-PAGE analysis of a CD8αα-BF2*0401 crystal, which showed three bands corresponding to the molecular weights of BF2*0401, ß2-microglobulin and CD8α, respectively. The crystal of CD8αα-BF2*0401 diffracted to 2.8 Šresolution and belonged to space group P21, with unit-cell parameters a = 90.6, b = 90.8, c = 94.9 Å, ß = 98°. The Matthews coefficient and solvent content were calculated to be 2.88 Å(3) Da(-1) and ∼57.3%, respectively.

Complex assembly, crystallization and preliminary X-ray crystallographic analysis of the bovine CD8αα-BoLA-2*02201 complex.

In order to clarify the structural characteristics of the bovine MHC class I molecule (BoLA-I) complexed with CD8αα (CD8αα-BoLA-I), bovine CD8αα, BoLA-I (BoLA-2*02201) and ß2m were expressed and purified, and were then assembled with a peptide derived from Foot-and-mouth disease virus (FMDV-VP1YY9) and crystallized. The crystal diffracted to 1.7 Šresolution and belonged to space group P21, with unit-cell parameters a=53.9, b=103.8, c=61.8 Å, α=γ=90, ß=96°. The asymmetric unit contained one complex, with a Matthews coefficient of 2.41 Å3 Da(-1) and a solvent content of 48.9%. The rotation-function Z-score and translation-function Z-score for molecular replacement were 3.4 and 8.9, respectively. In addition, SDS-PAGE analysis of CD8αα-BoLA-I crystals showed three bands corresponding to the molecular weights of BoLA-I heavy chain, ß2m and CD8α. The structure of the CD8αα-BoLA-I complex should be helpful in obtaining insight into the interaction between bovine CD8αα and MHC class I molecules. Structure determination of BoLA-2*02201-FMDV-VP1YY9 will be useful in the design of vaccines for foot-and-mouth disease.

2D TCR-pMHC-CD8 kinetics determines T-cell responses in a self-antigen-specific TCR system.

Two-dimensional (2D) kinetic analysis directly measures molecular interactions at cell-cell junctions, thereby incorporating inherent cellular effects. By comparison, three-dimensional (3D) analysis probes the intrinsic physical chemistry of interacting molecules isolated from the cell. To understand how T-cell tumor reactivity relates to 2D and 3D binding parameters and to directly compare them, we performed kinetic analyses of a panel of human T-cell receptors (TCRs) interacting with a melanoma self-antigen peptide (gp100209 -217 ) bound to peptide-major histocompatibility complex in the absence and presence of co-receptor CD8. We found that while 3D parameters are inadequate to predict T-cell function, 2D parameters (that do not correlate with their 3D counterparts) show a far broader dynamic range and significantly improved correlation with T-cell function. Thus, our data support the general notion that 2D parameters of TCR-peptide-major histocompatibility complex-CD8 interactions determine T-cell responsiveness and suggest a potential 2D-based strategy to screen TCRs for tumor immunotherapy.

Chinese goose (Anser cygnoides) CD8a: cloning, tissue distribution and immunobiological in splenic mononuclear cells.

CD8 molecule is a cell membrane glycoprotein, which plays an important role in cell-mediated immunity. Here, we identified Chinese goose CD8α (goCD8α) gene for the first time. The full-length cDNA of goCD8α is 1459bp in length and contains a 711bp open reading frame. Phylogenetic analysis shows that the waterfowl CD8α formed a monophyletic group. Semi-quantitative RT-PCR analysis showed that transcripts of goCD8α mRNA were high in the immune-related organs and mucosal immune system in gosling, and high in thymus and spleen comparing to other immune-related tissues in goose. The obvious increase of CD8α expression was observed in spleen of acute new type gosling viral enteritis virus (NGVEV) infected bird, while the increase of CD8α were observed in the thymus, bursa of fabricius, and cecum of chronic infected bird. The CD8α mRNA transcription level in spleen mononuclear cells was significantly up-regulated when stimulated by phytohemagglutinin, but not by lipopolysaccharide in vitro.

Human Mycobacterium tuberculosis CD8 T Cell Antigens/Epitopes Identified by a Proteomic Peptide Library.

Identification of CD8(+) T cell antigens/epitopes expressed by human pathogens with large genomes is especially challenging, yet necessary for vaccine development. Immunity to tuberculosis, a leading cause of mortality worldwide, requires CD8(+) T cell immunity, yet the repertoire of CD8 antigens/epitopes remains undefined. We used integrated computational and proteomic approaches to screen 10% of the Mycobacterium tuberculosis (Mtb) proteome for CD8 Mtb antigens. We designed a weighting schema based upon a Multiple Attribute Decision Making:framework to select 10% of the Mtb proteome with a high probability of containing CD8(+) T cell epitopes. We created a synthetic peptide library consisting of 15-mers overlapping by 11 aa. Using the interferon-γ ELISPOT assay and Mtb-infected dendritic cells as antigen presenting cells, we screened Mtb-specific CD8(+) T cell clones restricted by classical MHC class I molecules (MHC class Ia molecules), that were isolated from Mtb-infected humans, against this library. Three novel CD8 antigens were unambiguously identified: the EsxJ family (Rv1038c, Rv1197, Rv3620c, Rv2347c, Rv1792), PE9 (Rv1088), and PE_PGRS42 (Rv2487c). The epitopes are B5701-restricted EsxJ24-34, B3905-restricted PE953-67, and B3514-restricted PE_PGRS4248-56, respectively. The utility of peptide libraries in identifying unknown epitopes recognized by classically restricted CD8(+) T cells was confirmed, which can be applied to other intracellular pathogens with large size genomes. In addition, we identified three novel Mtb epitopes/antigens that may be evaluated for inclusion in vaccines and/or diagnostics for tuberculosis.

Extracellular domains of CD8α and CD8ß subunits are sufficient for HLA class I restricted helper functions of TCR-engineered CD4(+) T cells.

By gene transfer of HLA-class I restricted T-cell receptors (TCRs) (HLA-I-TCR) into CD8(+) as well as CD4(+) T-cells, both effector T-cells as well as helper T-cells can be generated. Since most HLA-I-TCRs function best in the presence of the CD8 co-receptor, the CD8αß molecule has to be co-transferred into the CD4(+) T-cells to engineer optimal helper T-cells. In this study, we set out to determine the minimal part of CD8αß needed for optimal co-receptor function in HLA-I-TCR transduced CD4(+) T-cells. For this purpose, we transduced human peripheral blood derived CD4(+) T-cells with several HLA-class I restricted TCRs either with or without co-transfer of different CD8 subunits. We demonstrate that the co-transduced CD8αß co-receptor in HLA-I-TCR transduced CD4(+) T-cells behaves as an adhesion molecule, since for optimal antigen-specific HLA class I restricted CD4(+) T-cell reactivity the extracellular domains of the CD8α and ß subunits are sufficient.

The human CD8ß M-4 isoform dominant in effector memory T cells has distinct cytoplasmic motifs that confer unique properties.

The CD8 co-receptor influences T cell recognition and responses in both anti-tumor and anti-viral immunity. During evolution in the ancestor of humans and chimpanzees, the CD8B gene acquired two additional exons. As a result, in humans, there are four CD8ß splice variants (M1 to M4) that differ in their cytoplasmic tails. The M-1 isoform which is the equivalent of murine CD8ß, is predominantly expressed in naïve T cells, whereas, the M-4 isoform is predominantly expressed in effector memory T cells. The characteristics of the M-4 isoform conferred by its unique 36 amino acid cytoplasmic tail are not known. In this study, we identified a dihydrophobic leucine-based receptor internalization motif in the cytoplasmic tail of M-4 that regulated its cell surface expression and downregulation after activation. Further the M-4 cytoplasmic tail was able to associate with ubiquitinated targets in 293T cells and mutations in the amino acids NPW, a potential EH domain binding site, either enhanced or inhibited the interaction. In addition, the M-4 tail was itself mono-ubiquitinated on a lysine residue in both 293T cells and a human T cell line. When peripheral blood human T cells expressed CD8αß M-4, the frequency of MIP-1ß secreting cells responding to antigen presenting cells was two-fold higher as compared to CD8αß M-1 expressing T cells. Thus, the cytoplasmic tail of the CD8ß M-4 isoform has unique characteristics, which likely contributed to its selective expression and function in human effector memory T cells.

Trehalose preserves the integrity of lyophilized phycoerythrin-antihuman CD8 antibody conjugates and enhances their thermal stability in flow cytometric assays.

An increasing number of publications report on the efficacy of trehalose in preserving organisms, cells, and macromolecules from adverse environmental conditions such as extreme temperatures and dryness. Although the mechanism by which this disaccharide exerts its protection is still debated, the implementation of trehalose as stabilizer is becoming a praxis in several preparative protocols from the pharmaceutical industry. We tested the ability of trehalose in protecting R-Phycoerythrin (R-PE), a pigment-protein complex widely used as fluorescent marker, from thermal denaturation. Once embedded into a dried trehalose matrix, R-PE retains its optical absorption-emission characteristics even when exposed to 70°C for h or when subjected to freeze-drying. We subsequently examined the protection exerted by trehalose on freeze-dried antihuman CD8-RPE (CD8-RPE) conjugated antibodies. Flow cytometric analysis showed that colyophilized trehalose-CD8-RPE preparations can be exposed for 4 weeks at 45°C without significant loss of functionality. Remarkably, even following 4 weeks incubation at 70°C, the preparations are still able to specifically recognize CD8(+) lymphocyte populations. These results show that colyophilization with trehalose makes possible the preparation of antibody-based diagnostic kits which can withstand breaks in the "cold chain" distribution, particularly suited for use in less-developed countries of the tropical areas.

Long-term remission of diabetes in NOD mice is induced by nondepleting anti-CD4 and anti-CD8 antibodies.

Residual ß-cells found at the time of clinical onset of type 1 diabetes are sufficient to control hyperglycemia if rescued from ongoing autoimmune destruction. The challenge, however, is to develop an immunotherapy that not only selectively suppresses the diabetogenic response and efficiently reverses diabetes, but also establishes long-term ß-cell-specific tolerance to maintain remission. In the current study, we show that a short course of nondepleting antibodies (Abs) specific for the CD4 and CD8 coreceptors rapidly reversed clinical disease in recent-onset diabetic NOD mice. Once established, remission was maintained indefinitely and immunity to foreign antigens unimpaired. Induction of remission involved selective T-cell purging of the pancreas and draining pancreatic lymph nodes and upregulation of transforming growth factor (TGF)-ß1 by pancreas-resident antigen-presenting cells. Neutralization of TGF-ß blocked the induction of remission. In contrast, maintenance of remission was associated with tissue-specific immunoregulatory T cells. These findings demonstrate that the use of nondepleting Ab specific for CD4 and CD8 is a robust approach to establish long-term ß-cell-specific T-cell tolerance at the onset of clinical diabetes.

Single-color multitarget flow cytometry using monoclonal antibodies labeled with different intensities of the same fluorochrome.

BACKGROUND: We developed a single-color multitarget flow cytometry (SM-FC) assay, a single-tube assay with graded mean fluorescence intensities (MFIs). We evaluated the repeatability of SM-FC, and its correlation with multicolor flow cytometry (MFC), to assess its application as a routine FC assay. METHODS: We selected CD19, CD3, CD4, and CD8 as antigen targets to analyze a lymphocyte subset. MFIs were graded by adjusting monoclonal antibody (mAb) volumes to detect several cell populations. Dimly labeled mAb was prepared by decreasing mAb volume and the optimum diluted volume was determined by serial dilution. SM-FC repeatability was analyzed 10 times in 2 normal controls. The correlation between SM-FC and MFC was evaluated in 20 normal and 23 patient samples. RESULTS: CV values (0.8-5.0% and 1.3-4.1% in samples 1 and 2, respectively) acquired by SM-FC with CD3-fluorescein α-isothyocyanate (FITC)(dim)+CD4-FITC(bright) and with CD19-FITC(dim)+CD3-FITC(bright) showed good repeatability, comparable to that acquired by MFC (1.6-3.7% and 1.0-4.8% in samples 1 and 2, respectively). Excellent correlation was observed between the 2 methods in the 20 normal samples (B cells, T cells, non-T(helper) cells, and T(helper) cells; r(2)=0.87, 0.97, 0.97, and 0.98, respectively; P<0.05). There were also linear relationships between SM-FC with CD19-FITC(dim)+CD3-FITC(bright) and CD8-PE(dim)+CD4-PE(bright), and MFC, in the 23 patient samples (B cells, T cells, T(cytotoxic) cells, and T(helper) cells; r(2)≥0.98, 0.99, 0.99, and 0.99, respectively; P<0.05). CONCLUSIONS: The multicolor, single-tube SM-FC technique is a potential alternative tool for identifying a lymphocyte subset.

Mechanistic insight into pertussis toxin and lectin signaling using T cells engineered to express a CD8α/CD3ζ chimeric receptor.

Mammalian cell-surface receptors typically display N- or O-linked glycans added post-translationally. Plant lectins such as phytohemagluttinin (PHA) can activate the T cell receptor (TCR) and other cell-surface receptors by binding to glycans and initiating receptor cross-linking. Pathogenic microorganisms such as Bordetella pertussis also express proteins with lectin-like activities. Similar to plant lectins, pertussis toxin (PTx) can activate the TCR and bind to a variety of glycans. However, whether the lectin-like activity of PTx is responsible for its ability to activate TCR signaling has not been formally proven. Here we examined the ability of PTx and a panel of lectins to activate the TCR or a CD8α/CD3ζ chimeric receptor (termed CD8ζ). We demonstrate that CD8ζ rescues PTx-induced signaling events lacking in TCR null cells. This result indicates that CD8ζ can substitute for TCR and supports the hypothesis that PTxB (functioning as a lectin) stimulates signaling via receptor cross-linking rather than by binding to a specific epitope on the TCR. Moreover, PTx is able to activate signaling by binding either N-linked or O-linked glycan-modified receptors as the TCR displays N-linked glycans while CD8ζ displays O-linked glycans. Finally, studies with a diverse panel of lectins indicate that the signaling activity of the lectins does not always correlate with the biochemical reports of ligand preferences. Comparison of lectin signaling through TCR or CD8ζ allows us to better define the structural and functional properties of lectin-glycan interactions using a biologically based signaling readout.