Human allogeneic melanoma-reactive T-helper lymphocyte clones: functional analysis of lymphocyte-melanoma
M Radrizzani1, B Benedetti, C Castelli
1Division of Experimental Oncology D, Istituto Nazionale Tumori, Milan, Italy.
Insights
Researchers isolated specific T-lymphocyte clones from melanoma patients that recognize melanoma-associated antigens (MAAs). These CD4+ T-cell clones, when activated by melanoma cells, produced cytokines and enhanced anti-tumor immunity, offering potential for new cancer diagnostics and therapies.
Area of Science:
- Immunology
- Oncology
- Cellular Biology
Background:
- Melanoma patients' immune responses are complex and involve T-lymphocytes.
- Identifying specific T-cell responses to melanoma-associated antigens (MAAs) is crucial for immunotherapy.
- Understanding T-cell receptor (TCR) interactions with tumor antigens and HLA molecules is key.
Purpose of the Study:
- To isolate and characterize CD4+ T-lymphocyte clones recognizing melanoma-associated antigens (MAAs).
- To investigate the specificity and functional properties of these T-cell clones.
- To explore the potential utility of these T-cell clones as reagents for MAA identification.
Main Methods:
- Isolation of CD4+ peripheral-blood lymphocytes (PBL) from a melanoma patient (9923).
- Co-culture of PBL with autologous accessory cells and allogeneic melanoma cells (Me 1811) and B-lymphoblastoid cell lines (LCLs).
- Phenotypic analysis (CD3, CD4, CD8, TCR alpha/beta, TCR gamma/delta), proliferation assays, and inhibition studies using monoclonal antibodies (MAbs).
Main Results:
- Fifty-five CD4+, TCR alpha/beta+ clones were generated.
- Eight clones proliferated specifically in response to allogeneic melanoma cells (Me 1811) but not to LCLs, suggesting MAA recognition.
- Clone 103 demonstrated specific reactivity to Me 1811, not other melanoma lines or cells, and its activation was inhibited by anti-TCR, anti-CD4, and anti-HLA-DR antibodies, indicating recognition of an MAA in conjunction with HLA-DR7.
Conclusions:
- A CD4+ T-cell clone (103) was identified that recognizes a melanoma-associated antigen (MAA) presented by HLA-DR7.
- This clone produced IL-2 and IFN-gamma upon stimulation and augmented anti-tumor cytotoxicity.
- Such T-cell clones represent valuable reagents for identifying and characterizing MAAs relevant to melanoma immunity.
Abstract:
Lymphocyte clones were isolated from CD4+ peripheral-blood lymphocytes (PBL) of melanoma (Me) patient 9923 (HLA-DR7, DQw2, w6), co-cultured for 30 days with autologous accessory cells, allogeneic Me (Me 1811) (HLA-DR7, DQw1, w2), IL-1 beta (2 U/ml) and IL-2 (15 IU/ml). The 55 clones tested displayed a CD3+, CD4+, CD8-, T-cell receptor (TCR) alpha/beta+, gamma/delta- phenotype. Twenty clones were assayed for proliferation in the presence of Me 1811 and B-lymphoblastoid cell line (LCL) 1811, both expressing HLA-class-I and -II (DR7 and DQw2 shared with patient 9923), intercellular adhesion molecule-1 (ICAM-1) and lymphocyte-function-associated antigen-3 (LFA-3) molecules. Eight clones were found to be reactive to Me 1811 but not to LCL 1811. Specificity analysis of these 8 clones revealed that each of them proliferated only to Me 1811, not to other 14 Me and 12 different LCL, suggesting recognition of melanoma-associated antigen (MAA) expressed on the stimulating Me. One clone (103) was analyzed in more detail. A wider specificity analysis showed that it reacted to Me 1811 but not to 10 other Me expressing or not HLA-DR7, 5 normal melanocyte cultures (2 of them typing HLA-DR7-positive when exposed to interferon-gamma--IFN-gamma), 4 tumors other than Me and 20 different LCL. Clones did not show proliferation in the presence of autologous Me cells. Clone proliferation in response to Me 1811 was significantly inhibited by monoclonal antibodies (MAbs) directed to CD3, TCR alpha/beta, TCR beta chain V12, CD4 and HLA-DR. Moreover, following stimulation with Me 1811, clone 103 showed increased surface expression of CD25 (IL-2 receptor) and CD71 (transferrin receptor) and produced significant amounts of IL-2 and IFN-gamma. The supernatant taken from co-culture of clone 103 with Me 1811 augmented the cytotoxicity of PBL 9923 and other allogeneic PBL against K562 and Me 1811. Thus, the lymphocyte clone 103 is a CD4+ Th clone which uses its CD3/TCR alpha/beta complex to recognize an MAA in conjunction with HLA-DR7. Availability of this type of reagent may prove useful to identify and characterize MAA recognized by T lymphocytes.
