Phenotypic Mutation 'complementary' (pdf version)
Allelecomplementary
Mutation Type missense
Chromosome1
Coordinate60,951,650 bp (GRCm39)
Base Change T ⇒ C (forward strand)
Gene Ctla4
Gene Name cytotoxic T-lymphocyte-associated protein 4
Synonym(s) Ctla-4, Cd152, Ly-56
Chromosomal Location 60,948,184-60,954,991 bp (+) (GRCm39)
MGI Phenotype FUNCTION: This gene is a member of the immunoglobulin superfamily, and encodes a protein that functions as a negative regulator of T-cell responses. Alternatively spliced transcript variants encoding different isoforms have been described for this gene. [provided by RefSeq, Aug 2013]
PHENOTYPE: Mice homozygous for a knock-out allele exhibit lethality at 3 to 4 weeks of age, decreased T cell numbers, abnormal T cell physiology, inflammation in mutliple organs, abnormal thymus morphology, and lymph node hypoplasia. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_009843, NM_001281976; MGI:88556

MappedYes 
Amino Acid Change Tyrosine changed to Histidine
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000027164] [ENSMUSP00000095327]
AlphaFold no structure available at present
SMART Domains Protein: ENSMUSP00000027164
Gene: ENSMUSG00000026011
AA Change: Y60H

DomainStartEndE-ValueType
signal peptide 1 37 N/A INTRINSIC
IG 43 152 2.72e-5 SMART
transmembrane domain 162 184 N/A INTRINSIC
Predicted Effect probably damaging

PolyPhen 2 Score 0.998 (Sensitivity: 0.27; Specificity: 0.99)
(Using ENSMUST00000027164)
SMART Domains Protein: ENSMUSP00000095327
Gene: ENSMUSG00000026011
AA Change: Y60H

DomainStartEndE-ValueType
IG 43 152 2.72e-5 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000097720)
Meta Mutation Damage Score 0.5680 question?
Is this an essential gene? Possibly nonessential (E-score: 0.307) question?
Phenotypic Category Unknown
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All Mutations and Alleles(15) : Chemically induced (other)(1) Gene trapped(1) Radiation induced(1) Targeted(11) Transgenic(1)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL03169:Ctla4 APN 1 60953764 splice site probably benign
Congruent UTSW 1 60951695 missense probably damaging 1.00
zesty UTSW 1 60951872 missense probably benign 0.02
R0882:Ctla4 UTSW 1 60948397 missense probably benign
R2513:Ctla4 UTSW 1 60951723 missense probably damaging 1.00
R6130:Ctla4 UTSW 1 60951650 missense probably damaging 1.00
R6291:Ctla4 UTSW 1 60951837 missense probably benign
R6450:Ctla4 UTSW 1 60951872 missense probably benign 0.02
R7686:Ctla4 UTSW 1 60951752 missense probably benign
R8464:Ctla4 UTSW 1 60951686 missense probably damaging 0.98
R9167:Ctla4 UTSW 1 60951695 missense probably damaging 1.00
R9410:Ctla4 UTSW 1 60951911 missense probably damaging 1.00
X0023:Ctla4 UTSW 1 60951702 missense probably benign 0.02
Mode of Inheritance Unknown
Local Stock
Repository
Last Updated 2019-09-04 9:37 PM by Anne Murray
Record Created 2018-04-19 9:12 AM by Bruce Beutler
Record Posted 2018-09-07
Phenotypic Description

Figure 1. Complementary mice exhibit decreased frequencies of peripheral naive CD4 T cells in CD4 T cells. Flow cytometric analysis of peripheral blood was utilized to determine T cell frequency. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.

Figure 2. Complementary mice exhibit decreased frequencies of peripheral naive CD8 T cells in CD8 T cells. Flow cytometric analysis of peripheral blood was utilized to determine T cell frequency. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.
Figure 3. Complementary mice exhibit increased frequencies of peripheral effector memory CD4 T cells in CD4 T cells. Flow cytometric analysis of peripheral blood was utilized to determine T cell frequency. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.
Figure 4. Complementary mice exhibit increased frequencies of peripheral effector memory CD8 T cells in CD8 T cells. Flow cytometric analysis of peripheral blood was utilized to determine T cell frequency. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.
Figure 5. Complementary mice exhibit increased expression of CD44 on peripheral blood T cells. Flow cytometric analysis of peripheral blood was utilized to determine CD44 MFI. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.
Figure 6. Complementary mice exhibit increased expression of CD44 on peripheral blood CD4 T cells. Flow cytometric analysis of peripheral blood was utilized to determine CD44 MFI. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.

The complementary phenotype was identified among G3 mice of the pedigree R6130, some of which showed reduced frequencies of naive CD4 T cells in CD4 T cells (Figure 1) and naive CD8 T cells in CD8 T cells (Figure 2) with concomitant increased frequencies of effector memory CD4 T cells in CD4 T cells (Figure 3) and effector memory CD8 T cells in CD8 T cells (Figure 4). Expression of CD44 on peripheral blood T cells (Figure 5) and CD4 T cells (Figure 6) was increased.

Nature of Mutation

Figure 7. Linkage mapping of increased effector memory T cell frequency using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 50 mutations (X-axis) identified in the G1 male of pedigree R6130. Normalized phenotype data are shown for single locus linkage analysis without consideration of G2 dam identity. Horizontal pink and red lines represent thresholds of P = 0.05, and the threshold for P = 0.05 after applying Bonferroni correction, respectively.

Whole exome HiSeq sequencing of the G1 grandsire identified 50 mutations. All of the above anomalies were linked by continuous variable mapping to a mutation in Ctla4:  a T to C transition at base pair 60,912,491 (v38) on chromosome 1, or base pair 3,467 in the GenBank genomic region NC_000067. The strongest association was found with a recessive model of inheritance to the effector memory CD4 T cell phenotype, wherein 10 variant homozygotes departed phenotypically from 39 homozygous reference mice and 45 heterozygous mice with a P value of 1.635 x 10-17 (Figure 7).  

The mutation corresponds to residue 324 in the mRNA sequence NM_009843 within exon 2 of 4 total exons.


 

309 AGCTTTCCATGTGAATATTCACCATCACACAAC

55  -S--F--P--C--E--Y--S--P--S--H--N-

The mutated nucleotide is indicated in red. The mutation results in a tyrosine to histidine substitution at position 60 (Y60H) in the CTLA4 protein, and is strongly predicted by Polyphen-2 to be damaging (score = 0.998).

Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 8. Domain organization of CTLA4. The complementary mutation results in a tyrosine to histidine substitution at position 60 within the Ig-like V-type domain in the CTLA4 protein. The topology of CTLA4 is designated by above the brackets. Abbreviations: SP, signal peptide; TM, transmembrane doain

Ctla4 encodes cytotoxic T-lymphocyte antigen 4 (CTLA4; alternatively, CD152), a member of the immunoglobulin superfamily and the CD28 family of receptors. The CD28 family also includes CD28, ICOS, PD-1, and BTLA (1). All members of the CD28 family function in regulating T cell activation and tolerance.

CTLA4 is a single-pass transmembrane protein with an Ig-like V-type domain (Figure 8). Amino acids 134 to 139 in the V-type domain are required for interaction with the B7 family ligands CD80 (B7-1) and CD86 (B7-2) expressed on antigen-presenting cells (e.g., dendritic cells, macrophages, and B cells) (2). CTLA4 has a PI3K SH2-binding site as well as other putative SH2 domain-binding motifs and a SH3 domain-association sequence (3). The cytoplasmic domain of CTLA4, namely the unphosphorylated YVKM sequence at Tyr201, functions in the endocytosis of CTLA4 by associating with the medium chain subunit AP50 of the clathrin adaptor AP-2 (4;5). Phosphorylation of Tyr201 prevents binding to the AP-2 adapter complex, blocks endocytosis, and leads to retention of CTLA4 on the cell surface. CTLA4 is also phosphorylated at Tyr218. The Src family tyrosine kinases Fyn, Lyn (see the record for Lemon), and Lck (see the record for iconoclast) as well as Rlk (resting lymphocyte kinase) and JAK2 can phosphorylate both sites (6).

CTLA4 is expressed on the surface of T cells as a homodimer. Amino acids 46 to 50 and 150 to 155 mediate homodimerization. N-linked glycosylation at Asn108, Asn113, and Asn145 is essential for CTLA4 dimerization.

Alternative splicing of CTLA4 can produce a CTLA4 mRNA that skips the transmembrane domain-encoding exon 3 (7;8). The alternatively spliced CTLA4 encodes a soluble form of CTLA4 (sCTLA4) [(7); reviewed in (9)]]. The soluble CTLA4 transcript is expressed in lymph nodes, spleen, CD4 and CD8 T cells, B cells, and monocytes (7). sCTLA4 exhibits CD80/CD86 binding activity. Increased levels of sCTLA4 are found in patients with autoimmune diseases such as Graves' disease (8;10;11), Hashimoto’s thyroiditis (8;11), myasthenia gravis (12), systemic lupus erythematosus (13;14), systemic sclerosis (15), celiac disease (16), autoimmune pancreatitis (17), and rheumatoid arthritis (18). The mechanism by which sCTLA4 functions is unknown, but sCTLA4 may block the interaction of CD80/CD86 with CD28, inhibiting early T cell activation. Alternatively, sCTLA4 could complete for binding of CD80/CD86 with CTLA4, leading to reduced inhibitory signaling.

The complementary mutation results in a tyrosine to histidine substitution at position 60 (Y60H); Tyr is within the Ig-like V-type domain.

Expression/Localization

CTLA4 is expressed at high levels in spleen, thymus, and peripheral blood leukocytes (19). CTLA4 is expressed by both CD4+ and CD8+ T cells as well as on the surface of double-positive thymocytes (20;21). CD4+CD25+ regulatory T (Treg) cells constitutively express CTLA4, and TCR- or CD28-mediated conventional T cell activation induces CTLA4 expression. CTLA4 expression has also been detected in B cells, monocytes, granulocytes, CD34+ stem cells, and placental fibroblasts (22-25). The function of CTLA4 in non-T cells is unknown.

CTLA4 is predominantly localized in intracellular vesicles of Treg cells or activated conventional T cells. CTLA4 undergoes clathrin-mediated endocytosis in the absence of ligand binding (5;26). Interaction between CLTA4 and AP2 mediates rapid internalization. LRBA binding to CTLA4 putatively results in recycling of CTLA4 to the plasma membrane, and AP1 interaction with CTLA4 putatively mediates CTLA4 trafficking to lysosomal compartments.

Background
Figure 9. CTLA4 influence on TCR signaling. Stimulation of the TCR is triggered by MHC molecules on antigen presenting cells, which present antigen peptides to TCR complexes and induce a series of intracellular signaling cascades which regulate T-cell development, homeostasis, activation, acquisition of effector’s functions and apoptosis. CTLA-4 and CD28 receptors share two ligands, CD80 and CD86. CD28 associates with PI3K and Grb2 via SH2 domain binding to the Tyr–Val–Asn–Met (YMNM) motif. Activated CTLA4 binds to PI3K (Phosphatidylinositol 3-Kinase), the tyrosine phosphatases SHP1 and SHP2 as well as the serine/threonine phosphatase PP2A. Crosslinking of CTLA-4 reduces TCR-dependent activation of the mitogen-activated protein (MAP) kinases ERK (extracellular signal-regulated kinase) and JNK (Jun N-terminal kinase), as well as of the transcription factors nuclear factor-κB (NF-κB), AP-1 and NF-AT (nuclear factor of activated T cells). Ligation of CTLA-4 during TCR stimulation results in decreased cytokine production by T cells and cell-cycle arrest. See the text for more details.

Naïve T cells require two signals in order to proliferate and differentiate into effector T cells. The first signal is antigen-specific and occurs when the T cell receptor interacts with a peptide presented with MHC antigens. The second signal is costimulatory and is mediated by CD80/CD86 binding to CD28. The mechanism by which CD28 affects T cell activation is unknown. Activated CD28 putatively disassociates from the serine/threonine phosphatase PP2A (phosphatase 2A) and recruits PI3K (phosphatidylinositol 3-kinase) and GRB2 (growth factor receptor bound protein 2). PI3K activation would then induce the phosphorylation of phosphatidylinositol (PI) into phosphatidylinositol 3-phosphate (PIP3) and/or the activation of protein kinase B (PKB/Akt). Akt activation would subsequently result in NF-κB activation, BCL-XL upregulation, T-cell survival, and IL-2 production [reviewed in (27)].

CTLA4 is one of three inhibitory molecules (i.e., CTLA4, PD-1 [programmed death-1], and KIRs [killer inhibitory receptors)] expressed on the surface of T cells (Figure 9). CTLA4 inhibits T cell activation by reducing IL-2 production, reducing IL-2 receptor expression, and by arresting T cells at the G1 phase of the cell cycle (28;29). Little is known about CTLA4-associated signaling. It is unclear whether CTLA4 inhibits T cell responses by antagonizing CD28 (by scavenging CD80/CD86 and/or by sequestering intracellular molecules that can bind both receptors) and/or by directly/indirectly reducing TCR signals. CTLA4 can inhibit T cell responses in the absence of CD28 (30). Other studies showed that expression of a tailless CTLA4 did not prevent T cell activation or proliferation, indicating that the intracellular portion of CTLA4 does mediate inhibitory effects (31). CTLA4 putatively suppresses ERK or JNK activity downstream of the TCR (32). CTLA4 activation attenuates AP-1, NFAT and NF-κB nuclear transcription factor activity in activated CD4+ T cells and inhibits the DNA binding of AP-1 and NFAT complexes in the nucleus (33). CTLA4 also putatively recruits the tyrosine phosphatase SHP-2 through a YVKM motif in its cytoplasmic domain (34). SHP-2 would subsequently dephosphorylate factors involved in TCR signaling. However, studies showed that the tyrosine residue in the YVKM motif is not required for CTLA4 function (35;36).

Mutations in CTLA4 are associated with several human diseases related to immune dysregulation and autoimmunity (Table 1).

Human disease

OMIM

Patient symptoms

References

Autoimmune lymphoproliferative syndrome, type V  (alternatively, CHAI [CTLA-4 haploinsufficiency with autoimmune infiltration] disease)

#616100

Autoimmune thrombocytopenias and abnormal lymphocytic infiltration of nonlymphoid organs, including the lungs, brain, and gastrointestinal tract, resulting in enteropathy; loss of circulating B cells and/or immunoglobulin levels

(37;38)

{Susceptibility to celiac disease-3}

#609755

Malabsorption resulting from inflammatory injury to the mucosa of the small intestine

(39-41)

{Insulin-dependent diabetes mellitus-12}

#601388

Elevated blood glucose levels

(41-43)

{Susceptibility to Graves disease-1}

%27500

Constitutive activation of the thyrotropin receptor and increased levels of thyroid hormone

(44;45)

{Hashimoto thyroiditis}

#140300

Hypothyroidism

(8;46)

{Susceptibility to systemic lupus erythematosus}

#152700

Autoimmune disease that induces inflammation and subsequent injury of multiple organs

(47;48)

Anti-CTLA4 antibodies have been developed for the use against metastatic melanoma, prostate cancer, ovarian cancer, and renal cancer. Blocking CLTA4 with anti-CTLA4 increases antitumor responses by permitting the immune system to respond to an antigen.

Putative Mechanism

Ctla4-deficient (Ctla4-/-) mice exhibited enlarged lymph nodes and spleens due to accumulation of T-cells as well as higher serum immunoglobin concentrations compared to wild-type mice (49;50). Ctla4-/- mice showed an increased frequency of double-negative T cells with a concomitant reduced number of single-positive T cells (51). Ctla4-/- mice showed increased frequencies of IFN-gamma, IL-17, IL-2, and IL-4 Foxp3-CD4+ T cells in the spleen and lymph nodes (52). Ctla4-/- mice died at 3 to 4 weeks of age due to myocardial failure due to lymphocytic infiltration (49-54).

The phenotype of the complementary mice indicate loss of CTLA4-associated function; however, lethality was not observed in the complementary mice, indicating that some residual CTLA4 function may remain.

Primers PCR Primer
complementary_pcr_F: TGAGCTTGCAGGAGTTCATC
complementary_pcr_R: ATGAGTTCCACCTTGCAGAG

Sequencing Primer
complementary_seq_F: TCCAAGATGAACCTCCCCTGG
complementary_seq_R: AACAGCTCTCAGTCCTTGGATGG
Genotyping

PCR program

1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40x
6) 72°C 10:00
7) 4°C hold


The following sequence of 407 nucleotides is amplified (chromosome 1, + strand):


1   tgagcttgca ggagttcatc caagatgaac ctcccctggc ctcaggtgtg gcctaatagt
61  tcaaaccgtg gatgatcatg agcccactaa gtgccctttg gactttccat gtcagccata
121 caggtgaccc aaccttcagt ggtgttggct agcagccatg gtgtcgccag ctttccatgt
181 gaatattcac catcacacaa cactgatgag gtccgggtga ctgtgctgcg gcagacaaat
241 gaccaaatga ctgaggtctg tgccacgaca ttcacagaga agaatacagt gggcttccta
301 gattacccct tctgcagtgg tacctttaat gaaagcagag tgaacctcac catccaagga
361 ctgagagctg ttgacacggg actgtacctc tgcaaggtgg aactcat


Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red.

References
Science Writers Anne Murray
Illustrators Diantha La Vine
AuthorsJin Huk Choi, Xue Zhong, and Bruce Beutler