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Product details

Synonyms = T cell immunoreceptor with Ig and ITIM domains , VSIG9 , VSTM3 , WUCAM

Antibody type = Recombinant Rabbit monoclonal / IgG

Clone = HMV322

Positive control = Tonsil: A moderate to strong TIGIT staining should be seen in a subset of germinal centre cells (CD4+ follicular T helper cells located in the germinal centre periphery orientated towards the tonsil surface epithelium). Subsets of other lymphocytes should also be positive but less intense.

Negative control = Tonsil: TIGIT staining should be absent in all epithelial cells and in most lymphocytes.

Cellular localization = Nucleus

Reactivity = Human

 

 

Application = Immunohistochemistry
Dilution = 1:100 – 1:200
Intended Use = Research Use Only

Relevance of Antibody

TIGIT is a pivotal target in immune-oncology.

Biology Behind

TIGIT (T cell immunoreceptor with Ig and ITIM domains) is a transmembrane glycoprotein of the poliovirus receptor (PVR) family which is coded by the TIGIT gene at 3q13.31. TIGIT acts as an inhibitory immune receptor (immune checkpoint) which can bind to CD155 with high affinity, and to CD112 with lower affinity. TIGIT expression is restricted to some CD8+ cytotoxic T cells, CD4+ T helper cells, FOXP3+ regulatory T cells, and NK cells. The highest level of expression occurs in CD4+ follicular T helper cells located in the germinal centre periphery orientated towards the tonsil surface epithelium. TIGIT expression has a limiting effect on antitumoral immune reactions. TIGIT inhibition, by either genetic ablation or blocking antibodies, increases T-cell activation and proliferation in response to stimulation and consequently results in reduced tumor growth in experimental models. Various compounds targeting TIGIT have been developed (i.e. tiragolumab, domvanalimab, vibostolimab, etigilimab, m6223, ociperlimab) and are currently evaluated in clinical trials, mostly in combination with other immune checkpoint inhibitors.

Staining Pattern in Normal Tissues

Images describing the TIGIT staining pattern in normal tissues obtained by the antibody HMV322 are shown in our “Normal Tissue Gallery”.

Brain Cerebrum Weak to moderate, nuclear staining of a subset of (probaly glial) cells (crossreactivity).
Cerebellum Negative.
Endocrine Tissues Thyroid Negative.
Parathyroid Negative.
Adrenal gland Negative.
Pituitary gland Negative.
Respiratory system Respiratory epithelium Negative.
Lung Negative.
Gastrointestinal Tract Salivary glands Negative.
Esophagus Negative.
Stomach Negative.
Duodenum Negative.
Small intestine Negative.
Appendix Negative.
Colon Negative.
Rectum Negative.
Liver Negative.
Gallbladder Negative.
Pancreas Negative.
Genitourinary Kidney Negative.
Urothelium Negative.
Male genital Prostate Negative.
Seminal vesicles Negative.
Testis Negative.
Epididymis Negative.
Female genital Breast Negative.
Uterus, myometrium Negative.
Uterus, ectocervix Negative.
Uterus endocervix Negative.
Uterus, endometrium Negative.
Fallopian Tube Negative.
Ovary Negative.
Placenta early Negative.
Placenta mature Negative.
Amnion Negative.
Chorion Negative.
Skin Epidermis Negative.
Sebaceous glands Negative.
Muscle/connective tissue Heart muscle Negative.
Skeletal muscle Negative.
Smooth muscle Negative.
Vessel walls Negative.
Fat Negative.
Stroma Negative.
Endothelium Negative.
Bone marrow/ lymphoid tissue Bone marrow Negative.
Lymph node Most TIGIT positive lymphocytes are in the interfollicular area while the strongest TIGIT staining occurs in the few labeled lymphocytes in germinal centres.
Spleen Weak to moderate TIGIT staining of a subset of lymphocytes.
Thymus Weak to moderate TIGIT staining of a subset of lymphocytes.
Tonsil Variable levels of TIGIT staining in subsets of follicular and interfollicular lymphocytes. The highest level of expression occurs in CD4+ follicular T helper cells located in the germinal centre periphery orientated towards the tonsil surface epithelium.
Remarks Some TIGIT positive lymphocytes can occur in all tissues.

These findings are largely consistent with the TIGIT RNA data described in the Human Protein Atlas (Tissue expression TIGIT). TIGIT staining by HMV

322 is most prominent in lymphatic tissues.

 

Positive control = Tonsil: A moderate to strong TIGIT staining should be seen in a subset of germinal centre cells (CD4+ follicular T helper cells located in the germinal centre periphery orientated towards the tonsil surface epithelium). Subsets of other lymphocytes should also be positive but less intense.

Negative control = Tonsil: TIGIT staining should be absent in all epithelial cells and in most lymphocytes.

 

Normal tissue gallery

Staining Pattern in Relevant Tumor Types

TIGIT is expressed in a variable subset of tumor associated T-cells. TIGIT expression may also occur in some T-cell lymphomas.

The TCGA findings on TIGIT RNA expression in different tumor categories have been summarized in the Human Protein Atlas.

 

 

Cancer tissue gallery

Compatibility of Antibodies

No data available at the moment

Protocol Recommendations

IHC users have different preferences on how the stains should look like. Some prefer high staining intensity of the target stain and even accept some background. Others favor absolute specificity and lighter target stains. Factors that invariably lead to more intense staining include higher concentration of the antibody and visualization tools, longer incubation time, higher temperature during incubation, higher temperature and longer duration of the heat induced epitope retrieval (slide pretreatment). The impact of the pH during slide pretreatment has variable effects and depends on the antibody and the target protein.

 

All images and data shown here and in our image galleries are obtained by the manual protocol described below. Other protocols resulting in equivalent staining are described as well.

 

Manual protocol

Freshly cut sections should be used (less than 10 days between cutting and staining). Heat-induced antigen retrieval for 5 minutes in an autoclave at 121°C in pH 7,8 Target Retrieval Solution buffer. Apply HMV322 at a dilution of 1:150 at 37°C for 60 minutes. Visualization of bound antibody by the EnVision Kit (Dako, Agilent) according to the manufacturer’s directions.

Potential Research Applications

  • The role of TIGIT as a drug target is under scrutiny.
  • The predictive role of TIGIT analysis in tissues is unknown.
  • The downstream signaling of TIGIT is not fully understood.

Evidence for Antibody Specificity in IHC

There are two ways how the specificity of antibodies can be documented for immunohistochemistry on formalin fixed tissues. These are: 1. Comparison with a second independent method for target expression measurement across a large number of different tissue types (orthogonal strategy), and 2. Comparison with one or several independent antibodies for the same target and showing that all positive staining results are also seen with other antibodies for the same target (independent antibody strategy). 

 

Orthogonal validation: Comparison with RNA expression data is not well suited to validate immunohistochemical stainings of cell types such as hematolymphoid cells which occur in virtually all organs. Nevertheless, in agreement with data from three independent RNA screening studies, including the Human Protein Atlas (HPA) RNA-seq tissue dataset, the FANTOM5 project, and the Genotype-Tissue Expression (GTEx) project, which are all summarized in the Human Protein Atlas (Tissue expression TIGIT), TIGIT immunostaining with HMV322 revealed by far the highest fractions of positive cells in lymphoid tissues.

 

Comparison of antibodies: The validity of TIGIT immunostaining of a subset of lymphocytic cells is strongly supported by a similar distribution of positive lymphocytic cells found by another commercially available independent antibody (termed “validation antibody”). That the nuclear staining of a subset cells of the cerebrum which was seen by HMV322, but not by the validation antibody, clarifies that this nuclear staining is an antibody specific cross-reactivity of our HMV322. Because this is only seen in the brain, we consider this a tolerable cross-reactivity, especially if other tissues than brain tissues should be analyzed.

 

Normal tissue gallery