Inhibitory LILRBs (LILRB1–4): Leukocyte immunoglobulin-like receptors and targets for tumor microenvironment reprogramming 

• Gene Information: LILRB1–4 (encoding CD85j, CD85d, CD85a, and CD85k, also known as ILT2/4/5/3) are protein-coding genes clustered on chromosome 19q13.42. They encode leukocyte immunoglobulin-like receptor subfamily B members, a class of ITIM-bearing cell-surface transmembrane receptors functioning as next-generation myeloid immune checkpoints. 
• Protein Expression: LILRB1–4 are predominantly expressed on myeloid lineages, specifically tumor-associated macrophages, dendritic cells, and monocytes. While LILRB2–3 remain myeloid-restricted and LILRB4 marks monocytic AML blasts, LILRB1 uniquely extends to subsets of T and NK cells. 
• Signaling Pathway: LILRB1–4 transduce inhibitory signals by binding cognate ligands (e.g., MHC-I, ANGPTLs, APOE, fibronectin) and engaging intrinsic cytoplasmic ITIM motifs to recruit SHP-1/2 phosphatases, dampening downstream activating kinase cascades. 
• Therapeutic Inhibition: LILRB-targeted antagonist antibodies, bispecifics, ADCs, and CAR-T cells reprogram immunosuppressive myeloid niches and block leukemic infiltration, restoring macrophage phagocytosis and antitumor immunity. Combination with anti-PD-(L)1 therapies or chemotherapy is investigated in clinical trials to overcome resistance.

• Human LILRB1 was specifically detected on B cells and NK cells in LILRB2/LILRB3/LILRB1/LILRB4 humanized transgenic mice but not wild-type mice.

Strain specific human LILRB1 expression analysis in LILRB2/LILRB3/LILRB1/LILRB4 humanized transgenic mice by flow cytometry. Blood cells were collected from wild-type C57BL/6 mice (+/+) and transgenic B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice (Tg). Human LILRB1 expression was analyzed by flow cytometry using anti-human LILRB1 antibody (eBioscience™, 17-5129-41).

• Human LILRB1 was specifically detected on macrophages and DCs in LILRB2/LILRB3/LILRB1/LILRB4 humanized transgenic mice but not wild-type mice.

Strain specific hLILRB1 expression analysis in LILRB2/LILRB3/LILRB1/LILRB4 humanized transgenic mice by flow cytometry. Blood cells were collected from wild-type C57BL/6 mice (+/+) and transgenic B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice (Tg). Human LILRB1 expression was analyzed by flow cytometry using anti-human LILRB1 antibody (eBioscience™, 17-5129-41).

• Human LILRB2 was specifically detected on granulocytes in LILRB2/LILRB3/LILRB1/LILRB4 humanized transgenic mice but not wild-type mice.

Strain specific hLILRB2 expression analysis in  LILRB2/LILRB3/LILRB1/LILRB4 humanized transgenic mice by flow cytometry. Blood cells were collected from wild-type C57BL/6 mice (+/+) and transgenic B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice (Tg). Human LILRB2 expression was analyzed by flow cytometry using anti-human LILRB2 antibody (R&D, FAB2078P).

• Human LILRB2 was specifically detected on macrophages and DCs in LILRB2/LILRB3/LILRB1/LILRB4 humanized transgenic mice but not wild-type mice.

Strain specific hLILRB2 expression analysis in LILRB2/LILRB3/LILRB1/LILRB4 humanized transgenic mice by flow cytometry. Blood cells were collected from wild-type C57BL/6 mice (+/+) and transgenic B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice (Tg). Human LILRB2 expression was analyzed by flow cytometry using anti-human LILRB2 antibody (R&D, FAB2078P).

• Human LILRB3 was specifically detected on T cells, B cells, NK cells and granulocytes in LILRB2/LILRB3/LILRB1/LILRB4 humanized transgenic mice but not wild-type mice.

Strain specific hLILRB3 expression analysis in LILRB2/LILRB3/LILRB1/LILRB4 humanized transgenic mice by flow cytometry. Blood cells were collected from wild-type C57BL/6 mice (+/+) and transgenic B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice (Tg). Human LILRB3 expression was analyzed by flow cytometry using anti-human LILRB3 antibody (R&D, FAB1806P-100).

• Human LILRB3 was specifically detected on monocytes, macrophages and DCs in LILRB2/LILRB3/LILRB1/LILRB4 humanized transgenic mice but not wild-type mice.

Strain specific hLILRB3 expression analysis in LILRB2/LILRB3/LILRB1/LILRB4 humanized transgenic mice by flow cytometry. Blood cells were collected from wild-type C57BL/6 mice (+/+) and transgenic B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice (Tg). Human LILRB3 expression was analyzed by flow cytometry using anti-human LILRB3 antibody (R&D, FAB1806P-100).

• Human LILRB4 was specifically detected on B cells in LILRB2/LILRB3/LILRB1/LILRB4 humanized transgenic mice but not wild-type mice.

Strain specific hLILRB4 expression analysis in LILRB2/LILRB3/LILRB1/LILRB4 humanized transgenic mice by flow cytometry. Blood cells were collected from wild-type C57BL/6 mice (+/+) and transgenic B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice (Tg). Human LILRB4 expression was analyzed by flow cytometry using anti-human LILRB4 antibody (R&D, FAB24251P).

• Human LILRB4 was specifically detected on macrophages and DCs in LILRB2/LILRB3/LILRB1/LILRB4 humanized transgenic mice but not wild-type mice.

Strain specific hLILRB4 expression analysis in LILRB2/LILRB3/LILRB1/LILRB4 humanized transgenic mice by flow cytometry. Blood cells were collected from wild-type C57BL/6 mice (+/+) and transgenic B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice (Tg). Human LILRB4 expression was analyzed by flow cytometry using anti-human LILRB4 antibody (R&D, FAB24251P).

Analysis of leukocytes cell subpopulation in spleen

Analysis of the spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice (n=3, 8-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. (A) Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated. (B) Results of FACS analysis. The percentages of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes, and macrophages in B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice were similar to those in the C57BL/6 mice, demonstrating that LILRB1, LILRB2, LILRB3, and LILRB4 humanized does not change the overall development, differentiation or distribution of these cell types in the spleen. Values are expressed as mean ± SEM.

Analysis of T cell subpopulation in spleen

Analysis of the spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice (n=3, 8-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. (A) Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated. (B) Results of FACS analysis. The percentages of CD4+ T cells, CD8+ T cells, and Tregs in B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice were similar to those in the C57BL/6 mice, demonstrating that introduction of LILRB1, LILRB2, LILRB3, and LILRB4 does not change the overall development, differentiation or distribution of these T cell subtypes in the spleen. Values are expressed as mean ± SEM.

Analysis of leukocytes cell subpopulation in LNs

Analysis of the thymus leukocyte subpopulations by FACS. LNs (Lymph nodes) were isolated from female C57BL/6 and B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice (n=3, 8-week-old). Flow cytometry analysis of the LNs was performed to assess leukocyte subpopulations. (A) Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated. (B) Results of FACS analysis. The percentages of T cells, B cells, and NK cells in B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4)mice were similar to those in the C57BL/6 mice, demonstrating that LILRB1, LILRB2, LILRB3, and LILRB4 humanized does not change the overall development, differentiation or distribution of these cell types in the LNs. Values are expressed as mean ± SEM.

Analysis of T cell subpopulation in LNs

Analysis of the thymus T cell subpopulations by FACS. LNs (Lymph nodes) were isolated from female C57BL/6 and B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice (n=3, 8-week-old). Flow cytometry analysis of the LNs was performed to assess leukocyte subpopulations. (A) Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated. (B) Results of FACS analysis. The percent of CD4+ T cells, CD8+ T cells, and Tregs in B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice were similar to those in the C57BL/6 mice, demonstrating that introduction of LILRB1, LILRB2, LILRB3, and LILRB4 does not change the overall development, differentiation or distribution of these T cell subtypes in the LNs. Values are expressed as mean ± SEM.

Analysis of leukocytes cell subpopulation in blood

Analysis of the blood leukocyte subpopulations by FACS. Blood leukocytes were isolated from female C57BL/6 and B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice (n=3, 8-week-old). Flow cytometry analysis of the blood leukocytes were performed to assess leukocyte subpopulations. (A) Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated here. (B) Results of FACS analysis. The percentages of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes, and macrophages in B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice were similar to those in the C57BL/6 mice, demonstrating that LILRB1, LILRB2, LILRB3, and LILRB4 humanized does not change the overall development, differentiation or distribution of these cell types in the blood. Values are expressed as mean ± SEM.

Analysis of T cell subpopulation in blood

Analysis of the blood T cell subpopulations by FACS. Blood leukocytes were isolated from female C57BL/6 and B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice (n=3, 8-week-old). Flow cytometry analysis of the blood leukocytes were performed to assess leukocyte subpopulations. (A) Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated here. (B) Results of FACS analysis. The percentages of CD4+ T cells, CD8+ T cells, and Tregs in B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice were similar to those in the C57BL/6 mice, demonstrating that introduction of LILRB1, LILRB2, LILRB3, and LILRB4 does not change the overall development, differentiation or distribution of these T cell subtypes in the blood. Values are expressed as mean ± SEM.

Analysis of leukocytes cell subpopulation in BM

Analysis of the bone marrow leukocyte subpopulations by FACS. Bone marrow leukocytes were isolated from female C57BL/6 and B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice (n=3, 8-week-old). Flow cytometry analysis of the bone marrow leukocyte was performed to assess leukocyte subpopulations. (A) Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated. (B) Results of FACS analysis. The percentages of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes, and macrophages in B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice were similar to those in the C57BL/6 mice, demonstrating that LILRB1, LILRB2, LILRB3, and LILRB4 humanized does not change the overall development, differentiation or distribution of these cell types in the bone marrow. Values are expressed as mean ± SEM.

Analysis of T cell subpopulation in BM

Analysis of the bone marrow T cell subpopulations by FACS. Bone marrow leukocytes were isolated from female C57BL/6 and B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice (n=3, 8-week-old). Flow cytometry analysis of the bone marrow leukocyte was performed to assess leukocyte subpopulations. (A) Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated. (B) Results of FACS analysis. The percentages of CD4+ T cells, CD8+ T cells, and Tregs in B-Tg(hLILRB2/hLILRB3/hLILRB1/hLILRB4) mice were similar to those in the C57BL/6 mice, demonstrating that introduction of LILRB1, LILRB2, LILRB3, and LILRB4 does not change the overall development, differentiation or distribution of these T cell subtypes in the bone marrow. Values are expressed as mean ± SEM.

hLILRB1 expression in CD45+ cells

Strain specific hLILRB1 expression analysis in B-Tg(hLILRB1/hLILRB2/hLILRB3/hLILRB4) mice by flow cytometry. Heart, liver, spleen, lung, kidney, large intestine, small intestine, stomach, brain, ovary, and skin were collected from B-Tg(hLILRB1/hLILRB2/hLILRB3/hLILRB4) mice, analyzed by flow cytometry with anti-hLILRB1 antibody.

hLILRB2 expression in CD45+ cells

Strain specific hLILRB2 expression analysis in B-Tg(hLILRB1/hLILRB2/hLILRB3/hLILRB4) mice by flow cytometry. Heart, liver, spleen, lung, kidney, large intestine, small intestine, stomach, brain, ovary, and skin were collected from B-Tg(hLILRB1/hLILRB2/hLILRB3/hLILRB4) mice, analyzed by flow cytometry with anti-hLILRB2 antibody.

hLILRB3 expression in CD45+ cells

Strain specific hLILRB3 expression analysis in B-Tg(hLILRB1/hLILRB2/hLILRB3/hLILRB4) mice by flow cytometry. Heart, liver, spleen, lung, kidney, large intestine, small intestine, stomach, brain, ovary, and skin were collected from B-Tg(hLILRB1/hLILRB2/hLILRB3/hLILRB4) mice, analyzed by flow cytometry with anti-hLILRB3 antibody.

hLILRB4 expression in CD45+ cells

Strain specific hLILRB4 expression analysis in B-Tg(hLILRB1/hLILRB2/hLILRB3/hLILRB4) mice by flow cytometry. Heart, liver, spleen, lung, kidney, large intestine, small intestine, stomach, brain, ovary, and skin were collected from B-Tg(hLILRB1/hLILRB2/hLILRB3/hLILRB4) mice, analyzed by flow cytometry with anti-hLILRB4 antibody.

Protein expression in T cells of bone marrow

Strain specific hLILRB1/4 expression analysis in B-Tg(hLILRB1/hLILRB2/hLILRB3/hLILRB4) mice by flow cytometry. Bone marrow cells were collected from wild-type mice and B-Tg(hLILRB1/hLILRB2/hLILRB3/hLILRB4) mice, analyzed by flow cytometry with anti-LILRB1 antibody or anti-LILRB4 antibody.