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Intracellular Domain of CD3 zeta chain

Sequence and Features

Usage and Biology

T-cell surface glycoprotein CD3 zeta chain is part of the TCR-CD3 complex present on T-lymphocyte cell surface that plays an essential role in adaptive immune response. When antigen presenting cells (APCs) activate T-cell receptor (TCR), TCR-mediated signals are transmitted across the cell membrane by the CD3 chains CD3D, CD3E, CD3G and CD3Z. All CD3 chains contain immunoreceptor tyrosine-based activation motifs (ITAMs) in their cytoplasmic domain. Upon TCR engagement, these motifs become phosphorylated by Src family protein tyrosine kinases LCK and FYN, resulting in the activation of downstream signaling pathways [1,2].

CD3Z ITAMs phosphorylation creates multiple docking sites for the protein kinase ZAP70 leading to ZAP70 phosphorylation and its conversion into a catalytically active enzyme [2].

It plays an important role in intrathymic T-cell differentiation. Additionally, participates in the activity-dependent synapse formation of retinal ganglion cells (RGCs) in both the retina and dorsal lateral geniculate nucleus (dLGN) (By similarity).

Figure 1. Structure of CD3 zeta chain.

Background and detail description

Used in our project

The synthetic receptors were constructed to contain an scFv derived from an antibody recognizing the virus spike protein, CR3022, which has been reported to bind with the receptor-binding domain of the SARS-CoV-2 S glycoprotein with high affinity, and the CD8 transmembrane domain present in the aCD19 CAR for T cells. For the cytoplasmic domains, we used the common g subunit of Fc receptors (CARg), MEGF10 (CARMEGF10), MERTK (CARMERTK) and CD3z (CARz) in our study. These cytoplasmic domains are capable of promoting phagocytosis by macrophages[3]. More details and experimental results can be found in CAR-CD3 zeta(BBa_K4040017)

Used in the construction of CAR-Macrophages cells

Previous study has engineered phagocytes that recognize and ingest targets through specific antibody-mediated interactions. This strategy can be directed towards multiple extracellular ligands(CD19 and CD22) and can be used with several intracellular signaling domains that contain ITAM motifs (Megf10, FcRV, and CD3z). Previous work has suggested that spatial segregation between Src-family kinases and an inhibitory phosphatase, driven by receptor ligation, is sufficient to trigger signaling by the T cell receptor (Davis and van der Merwe, 2006; James and Vale, 2012) and FcR(Freeman et al., 2016). The CAR-Ps that we have developed may similarly convert receptor-ligand binding into receptor phosphorylation of ITAM domains through partitioning of kinases and phosphatases at the membrane-membrane interface.

Thus, although using CAR-Ps to enhance cross presentation of cancer antigen is an intriguing future avenue, such a strategy would likely require more optimization of the dendritic cell subset employed or the CAR-P receptor itself. Previous study has demonstrated that the CAR approach is transferrable to biological processes beyond T cell activation and that the expression of an engineered receptor in phagocytic cells is sufficient to promote specific engulfment and elimination of cancer cells.

Previous study has found that a chimeric adenoviral vector overcame the inherent resistance of primary human macrophages to genetic manipulation and imparted a sustained pro­inflammatory (M1) phenotype. CAR macrophages (CAR­Ms) demonstrated antigen­specific phagocytosis and tumor clearance in  vitro. In two solid tumor xenograft mouse models, a single infusion of human CAR­Ms decreased tumor burden and prolonged overall survival. Characterization of CAR­M activity showed that CAR­Ms expressed pro­inflammatory cytokines and chemokines, converted bystander M2 macrophages to M1, upregulated antigen presentation machinery, recruited and presented antigen to T cells and resisted the effects of immunosuppressive cytokines. In humanized mouse models, CAR­Ms were further shown to induce a pro­inflammatory tumor microenvironment and boost anti­tumor T cell activity[5].

CAR-P is a successful strategy for directing macrophages towards cancer targets, and can initiate whole cell eating and trogocytosis leading to cancer cell elimination(Figure 2)[4].

Figure 2.10,000 macrophages and 20,000 Raji B cells were incubated together for 44 hr. The number of Rajis was then quantified by FACS. 2–3 technical replicates were acquired each day on three separate days. The number of Rajis in each replicate was normalized to the average number present in the GFP-CAAX macrophage wells on that day. * indicates p<0.01, *** indicates p<0.0001 by two-tailed Fisher Exact Test(a and e) or by Ordinary one way ANOVA with Dunnet’s correction for multiple comparisons; error bars denote 95% confidence intervals.

CAR-M phagocytosis was an active process requiring Syk, non-muscle myosin IIA and actin polymerization, similarly to Fc receptor-mediated ADCP[5].

Co­localization and anti­tumor acti vity of CAR­M were evaluated by immunohistochemical analysis of explanted lungs, revealing the presence of multiple meta­ static tumor nests in this model (Fig. 3a), with a significant reduc­tion in metastatic tumor burden after CAR­M treatment (Fig. 3b)[5].

Figure 3. a, Lungs from mice with metastatic disease were assessed with dual immunohistochemical analysis (tumor: anti-GFP, brown; macrophage detection: anti-human CD68, red). Each panel shows low (left), medium (middle) and high (right) power view. The experiment was performed once. b, Quantification of GFP+ tumor cells in n = 5 random ×20 high-power fields per slice, with two slices per mouse and two mice per group. Analysis was done by an observer who was blinded to treatment group allocation. Data are presented as the mean, with statistical significance calculated using multiple two-sided t-tests.

Macrophage phenotype is plastic and can change in response to cytokines, pathogen-­associated molecular patterns, metabolic cues, cell–cell interactions and tissue­specific signals[6]. CAR­Ms maintained a pro­inflammatory phenotype within the human TME.

Using the humanized TME mouse model, the effect of CAR­Ms on the surrounding TME was interrogated at single­cell resolution. Adoptively transferred macrophages were removed from analysis by excluding all human male cells. The human TME grouped into two large clusters (cluster 0 and cluster 1) and one small cluster of cells that was excluded from analysis (cluster 2). Cluster 0 was enriched in the CAR­M­treated arm (86% CAR, 14% UTD), whereas cluster 1 was enriched in the UTD­treated arm (71% UTD, 29% CAR) (Fig. 4a). Gene expression analysis of cluster 0 revealed an enrichment of pro­inflammatory genes such as MHC­II and TNF , demonstrating that CAR­Ms remodeled the TME toward a pro­inflammatory state (Fig. 4b)[5].

Figure 4. a, Cluster plots demonstrating the phenotypic distinction of human TME after UTD versus CAR-M treatment. Cluster 0 was enriched in the CAR-M-treated arm (86% events from the CAR cohort), whereas cluster 1 was enriched in events from the UTD-treated arm (71% events from the UTD cohort). Cluster 2 was mixed and had few events. b, Gene expression heat map from each TME scRNAseq cluster from a. Pro-inflammatory genes were found in cluster 0 (annotated in red). Potential anti-inflammatory or M2-associated genes in cluster 1 are annotated in blue.n = 3 mice per group; the experiment was performed once.

CAR-Ms induced pro-inflammatory pathways such as interferon signaling, TH1 pathway and iNOS signaling in M2 macrophages (Fig. 5)[5].

Figure 5. Ingenuity pathway analysis of the transcriptome of in vitro-polarized M2A macrophages after exposure to conditioned media from UTD or CAR-M for 48 h; n = 3 technical replicates. Statistical significance was calculated using Fisher’s exact test.

Additionally, CAR­Ms induced activation and maturation markers in immature human dendritic cells (Fig. 6a) and directly induced the recruitment of both resting and activated human T cells in an in  vitro chemotaxis assay (Fig. 6b). Finally, having shown that CAR­Ms can exert a dominant effect on surrounding immune cells, we demonstrated that CAR­Ms maintained their anti­tumor activity in the presence of human M2 macrophages (Fig. 6c).

Figure 6. a, Human immature dendritic cells were treated with UTD or CAR-M conditioned media for 48 h, and activation markers CD86 and MHC-II (HLA-DR) were assessed by FACS. Data are represented as the mean ± s.e.m. from n = 3 technical replicates. Statistical significance was calculated using a two-tailed t-test. b, Chemotaxis of resting or activated CD3+ T cells by UTD or CAR-M conditioned media. Data represent T cells from n = 4 human donors, and statistical significance was calculated using a paired t-test. c, Incucyte-based killing assay of SKOV3 by CAR-M alone or in the presence of M0, M2a or M2c macrophages at an E:T:M2 ratio of 3:1:1. Data are represented as the mean ± s.e.m. of n = 3 technical replicates.

To test CAR­M cross­presentation of intracellular tumor antigens ingested during phagocytosis of whole tumor cells, we generated SKOV3 expressing NY­ESO­1 only (SKOV­NY), SKOV3 expressing HLA­A201 and NY­ESO­1 (SKOV­A201­NY) and CAR­Ms from an HLA­A201+ donor. TRAC knockout anti­NYESO1 TCR T cells were incubated with CAR­Ms (macrophage control), SKOV­NY (tumor control), SKOV­A2­NY (positive control) or CAR­Ms that were fed SKOV­NY for 48 h. CD8+ anti­NYESO1 T  cells were activated (as determined by CD69 induction and production of IFNγ) by CAR­Ms that ingested SKOV­NY (Fig. 6). Anti­NYESO1 T  cells were not activated by SKOV­NY or CAR­Ms alone, demonstrating that macro­phages were able to cross­present tumor­derived antigens after phago­cytosis, suggesting that CAR­Ms might lead to epitope spreading.

Figure 7. CAR-M cross-presentation assay showing CD69 induction (left) and IFNγ secretion (right) by anti-NYESO TCR+ T cells 24 h after co-culture with HLA-A201(+) CAR-M (CAR-M, blue), HLA-A201(-)NYESO(+) SKOV3 (SKOV NY , orange) or HLA-A201(+) CAR-M co-cultured with SKOV NY for 48 h (green). Anti-NYESO T cells exposed to HLA-A201(+)NYESO(+) SKOV3 alone were used as positive control (purple). Data are represented as the mean ± s.e.m. of n = 3 technical replicates. Statistical analysis was performed using ANOVA with multiple comparisons. For all panels, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. CM, conditioned media.

CAR-M eradicated SKOV3, a HER2+ ovarian cancer cell line, in a dose and time-dependent manner (Fig. 8a,b).

Figure 8. a, Incucyte-based killing assay of GFP+ SKOV3 by UTD or CAR-M after 48 h of co-culture at different E:T ratios. Statistical significance was calculated with one-way ANOVA with multiple comparisons, and data represent the mean ± s.e.m. of n = 3 technical replicates (representative of at least three individual experiments). b, Incucyte-based killing kinetics of UTD or CAR-M against SKOV3 at a 10:1 E:T ratio. Data are represented as the mean ± s.e.m. of n = 3 technical replicates (representative of at least three individual experiments). For all panels, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

In T cells, phosphorylated ITAMs in CD3z bind to tandem SH2 domains (tSH2) in the kinase ZAP70. Zap70 is not expressed in macrophages, but Syk, a phagocytic signaling effector and tSH2 domain containing protein, is expressed at high levels (Andreu et al., 2017). Previous work suggested that Syk kinase can also bind to the CD3z ITAMs (Bu et al., 1995), indicating that the CAR-T may promote engulfment through a similar mechanism as CAR-PFcRV. To quantitatively compare the interaction between SyktSH2and CD3z or FcRV in a membrane proximal system recapitulating physiological geometry, a study used a liposome-based assay (Figure 2 [Hui and Vale, 2014]). In this system, His10CD3z and His10-Lck (the kinase that phosphorylates CD3z) are bound to a liposome via NiNTA-lipids and the binding of labeled tandem SH2 domains to phosphorylated CD3z was measured using fluorescence quenching. Their results show that Syk-tSH2 binds the CD3z and FcRV with comparable affinity (~15 nM and ~30 nM respectively).

Collectively, these results demonstrate that the TCR CD3z chain can promote phagocytosis in a CAR-P, likely through the recruitment of Syk kinase[4].

Figure 9. Model of the liposome-based fluorescence quenching assay used to determine affinity between the Syk tSH2 domains and the receptor tails of CD3z and FcRV, two intracellular signaling domains that promote engulfment. Binding between the Syk tSH2 reporter (Syk tSH2), green, and a receptor tail, purple, was detected by rhodamine quenching of BG505 dye on the reporter (see Materials and methods). Kd was determined by assessing mean fluorescence quenching for the last 20 timepoints collected ~45 min after ATP addition over a receptor titration from 0 to 500 nM. Each point represents the mean ± SD from three independent experiments. Kd ± SE was calculated by nonlinear fit assuming one site specific binding.

Previous study has generated CAR-Ms targeting the solid tumor antigens mesothelin or HER2 and demonstrated phagocytosis of antigen-positive target cells (Fig.10a,b)[5]. Together, these data demonstrated that CD3ζ based CARs can direct anti-tumor phagocytic activity and provided support for subsequent efforts to translate this platform to primary human macrophages.

Figure 10. a,b, In vitro phagocytosis by UTD or CAR-meso-ζ THP-1 macrophages of mesothelin+ve K562 cells (a) or by CAR-HER2-ζ macrophages of HER2+ K562 cells (b).

References

[1]Barber EK, Dasgupta JD, Schlossman SF, Trevillyan JM, Rudd CE. The CD4 and CD8 antigens are coupled to a protein-tyrosine kinase (p56lck) that phosphorylates the CD3 complex. Proc Natl Acad Sci U S A. 1989 May;86(9):3277-81. doi: 10.1073/pnas.86.9.3277. PMID: 2470098; PMCID: PMC287114.

[2]Iwashima M, Irving BA, van Oers NS, Chan AC, Weiss A. Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science. 1994 Feb 25;263(5150):1136-9. doi: 10.1126/science.7509083. PMID: 7509083.

[3]Fu W, Lei C, Ma Z, Qian K, Li T, Zhao J, Hu S. CAR Macrophages for SARS-CoV-2 Immunotherapy. Front Immunol. 2021 Jul 23;12:669103. doi: 10.3389/fimmu.2021.669103. PMID: 34367135; PMCID: PMC8343226.

[4]Morrissey MA, Williamson AP, Steinbach AM, Roberts EW, Kern N, Headley MB, Vale RD. Chimeric antigen receptors that trigger phagocytosis. Elife. 2018 Jun 4;7:e36688. doi: 10.7554/eLife.36688. PMID: 29862966; PMCID: PMC6008046.

[5]Klichinsky M, Ruella M, Shestova O, Lu XM, Best A, Zeeman M, Schmierer M, Gabrusiewicz K, Anderson NR, Petty NE, Cummins KD, Shen F, Shan X, Veliz K, Blouch K, Yashiro-Ohtani Y, Kenderian SS, Kim MY, O'Connor RS, Wallace SR, Kozlowski MS, Marchione DM, Shestov M, Garcia BA, June CH, Gill S. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol. 2020 Aug;38(8):947-953. doi: 10.1038/s41587-020-0462-y. Epub 2020 Mar 23. PMID: 32361713; PMCID: PMC7883632.

[6]Mosser, D. M. & Edwards, J. P . Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 8, 958–969 (2008).