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An expanded peripheral T cell population to a cytotoxic T lymphocyte (CTL)-defined, melanocyte-specific antigen in metastatic melanoma patients impacts on generation of peptide-specific CTLs but does not overcome tumor escape from immune surveillance in metastatic lesions.

Anichini A, Molla A, Mortarini R, Tragni G, Bersani I, Di Nicola M, Gianni AM, Pilotti S, Dunbar R, Cerundolo V, Parmiani G - J. Exp. Med. (1999)

Bottom Line: In four out of nine patients, Melan-A/Mart-1(27-35)-specific CTL precursors (CTLp) were >/=1/2,000 peripheral blood lymphocytes and found mostly or only in the CD45RO(+) memory T cell subset.Furthermore, frequent lack of a "brisk" or "nonbrisk" CD3(+)CD8(+) T cell infiltrate or reduced/absent Melan-A/Mart-1 expression in several lesions and lack of HLA class I antigens were found in some instances.Thus, expansion of peripheral immune repertoire to Melan-A/Mart-1 takes place in some metastatic patients and leads to enhanced CTL induction after antigen-presenting cell-mediated selection, but, in most metastatic lesions, it does not overcome tumor escape from immune surveillance.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Oncology Human Tumor Immunobiology Unit, Istituto Nazionale per lo Studio e la Cura dei Tumori, 20133 Milan, Italy. Anichini@istitutotumori.mi.it

ABSTRACT
It is not known if immune response to T cell-defined human histocompatibility leukocyte antigen (HLA) class I-restricted melanoma antigens leads to an expanded peripheral pool of T cells in all patients, affects cytotoxic T lymphocyte (CTL) generation, and correlates with anti-tumor response in metastatic lesions. To this end, a limiting dilution analysis technique was developed that allowed us to evaluate the same frequency of peptide-specific T cells as by staining T cells with HLA-peptide tetrameric complexes. In four out of nine patients, Melan-A/Mart-1(27-35)-specific CTL precursors (CTLp) were >/=1/2,000 peripheral blood lymphocytes and found mostly or only in the CD45RO(+) memory T cell subset. In the remaining five patients, a low (<1/40,000) peptide-specific CTLp frequency was measured, and the precursors were only in the CD45RA(+) naive T cell subset. Evaluation of CTL effector frequency after bulk culture indicated that peptide-specific CTLs could be activated in all patients by using professional antigen-presenting cells as dendritic cells, but CTLp frequency determined the kinetics of generation of specificity and the final number of effectors as evaluated by both limiting dilution analysis and staining with HLA-A*0201-Melan-A/Mart-1 tetrameric complexes. Immunohistochemical analysis of 26 neoplastic lesions from the nine patients indicated absence of tumor regression in most instances, even in patients with an expanded peripheral T cell pool to Melan-A/Mart-1 and whose neoplastic lesions contained a high frequency of tetramer-positive Melan-A/Mart-1-specific T cells. Furthermore, frequent lack of a "brisk" or "nonbrisk" CD3(+)CD8(+) T cell infiltrate or reduced/absent Melan-A/Mart-1 expression in several lesions and lack of HLA class I antigens were found in some instances. Thus, expansion of peripheral immune repertoire to Melan-A/Mart-1 takes place in some metastatic patients and leads to enhanced CTL induction after antigen-presenting cell-mediated selection, but, in most metastatic lesions, it does not overcome tumor escape from immune surveillance.

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Immunohistochemical analysis of neoplastic lesions. Three examples of immunohistochemical analysis of neoplastic lesions from three patients are reported. Consecutive sections of paraffin-embedded tumor fragments were subjected to immunohistochemical staining with anti-CD3 (A, E, and I), anti-CD8 (B, F, and J) and anti–Melan-A/Mart-1 mAbs (C, G, and K) or conventionally stained with hematoxylin and eosin (D, H, and L). Patient 1 (Table , lesion 7), A–D: a subcutaneous lesion showed no evidence of tumor regression or necrosis but did show an intense intratumoral lymphocytic infiltrate (D) characterized by CD3+ (A) and CD8+ (B) cells in the presence of heterogeneous cytoplasmatic reactivity for Melan-A/Mart-1 in tumor cells (C). Patient 2 (lesion 9), E–H: a nodal metastasis with extended coagulative necrosis with strong cytoplasmic eosinophilia of cell shadows lacking nuclei and scattered granulocytes infiltrating the border of the necrotic area (H, right side). The left side of the lesion, containing vital tumor cells, showed a nonbrisk lymphocytic infiltrate (H) mostly characterized by CD3+ T cells (E) but with a few CD8+ lymphocytes (F). In this lesion, heterogeneous cytoplasmatic positivity for Melan-A/Mart-1 in neoplastic cells was confined to a small area (G). Patient 3 (lesion 16), I–L: a nodal metastasis without evidence of tumor regression and lacking intratumoral lymphocytes (L), either CD3+ (I) or CD8+ (J), showed intense cytoplasmatic reactivity for Melan-A/Mart-1 limited to a few neoplastic cells (K). Original magnification was 400 for all panels except for H, magnification 250).
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Figure 2: Immunohistochemical analysis of neoplastic lesions. Three examples of immunohistochemical analysis of neoplastic lesions from three patients are reported. Consecutive sections of paraffin-embedded tumor fragments were subjected to immunohistochemical staining with anti-CD3 (A, E, and I), anti-CD8 (B, F, and J) and anti–Melan-A/Mart-1 mAbs (C, G, and K) or conventionally stained with hematoxylin and eosin (D, H, and L). Patient 1 (Table , lesion 7), A–D: a subcutaneous lesion showed no evidence of tumor regression or necrosis but did show an intense intratumoral lymphocytic infiltrate (D) characterized by CD3+ (A) and CD8+ (B) cells in the presence of heterogeneous cytoplasmatic reactivity for Melan-A/Mart-1 in tumor cells (C). Patient 2 (lesion 9), E–H: a nodal metastasis with extended coagulative necrosis with strong cytoplasmic eosinophilia of cell shadows lacking nuclei and scattered granulocytes infiltrating the border of the necrotic area (H, right side). The left side of the lesion, containing vital tumor cells, showed a nonbrisk lymphocytic infiltrate (H) mostly characterized by CD3+ T cells (E) but with a few CD8+ lymphocytes (F). In this lesion, heterogeneous cytoplasmatic positivity for Melan-A/Mart-1 in neoplastic cells was confined to a small area (G). Patient 3 (lesion 16), I–L: a nodal metastasis without evidence of tumor regression and lacking intratumoral lymphocytes (L), either CD3+ (I) or CD8+ (J), showed intense cytoplasmatic reactivity for Melan-A/Mart-1 limited to a few neoplastic cells (K). Original magnification was 400 for all panels except for H, magnification 250).

Mentions: In patient 1, the primary lesion (lesion 1) and a satellitosis (lesion 2) were removed 2 mo before CTLp evaluation (Table ). The first lesion had an absent pattern of CD3+ T cells and Melan-A/Mart-1 antigen expressed on 20% of the neoplastic cells. The satellitosis was nonbrisk for CD3+ T cells, but CD8+ T cells represented only 30% of them and Melan-A/Mart-1 was not expressed. A subcutaneous lesion (lesion 3) isolated 1 wk before CTLp evaluation was Melan-A/Mart-1+ and expressed HLA-A2 on the tumor and 30% of CD8+ cells among the nonbrisk CD3+ infiltrate. However, no evidence of tumor destruction was observed, even though this same lesion contained a high frequency of Melan-A/Mart-1–specific CTLp (Table , Fig. 1). A lymph node metastasis (lesion 4) was almost completely negative for Melan-A/Mart-1 and absent for CD3+ T cells. In the same patient, in spite of an expanded T cell population to Melan-A/Mart-1 in peripheral blood, three additional subcutaneous metastases developed within 6 mo of CTLp analysis. Two of these lesions expressed Melan-A/Mart-1, but no evidence of tumor regression or destruction was found, although all lesions contained a brisk CD3+CD8+ infiltrate (Table , lesions 5–7; Fig. 2aFig. bFig. cFig. d).


An expanded peripheral T cell population to a cytotoxic T lymphocyte (CTL)-defined, melanocyte-specific antigen in metastatic melanoma patients impacts on generation of peptide-specific CTLs but does not overcome tumor escape from immune surveillance in metastatic lesions.

Anichini A, Molla A, Mortarini R, Tragni G, Bersani I, Di Nicola M, Gianni AM, Pilotti S, Dunbar R, Cerundolo V, Parmiani G - J. Exp. Med. (1999)

Immunohistochemical analysis of neoplastic lesions. Three examples of immunohistochemical analysis of neoplastic lesions from three patients are reported. Consecutive sections of paraffin-embedded tumor fragments were subjected to immunohistochemical staining with anti-CD3 (A, E, and I), anti-CD8 (B, F, and J) and anti–Melan-A/Mart-1 mAbs (C, G, and K) or conventionally stained with hematoxylin and eosin (D, H, and L). Patient 1 (Table , lesion 7), A–D: a subcutaneous lesion showed no evidence of tumor regression or necrosis but did show an intense intratumoral lymphocytic infiltrate (D) characterized by CD3+ (A) and CD8+ (B) cells in the presence of heterogeneous cytoplasmatic reactivity for Melan-A/Mart-1 in tumor cells (C). Patient 2 (lesion 9), E–H: a nodal metastasis with extended coagulative necrosis with strong cytoplasmic eosinophilia of cell shadows lacking nuclei and scattered granulocytes infiltrating the border of the necrotic area (H, right side). The left side of the lesion, containing vital tumor cells, showed a nonbrisk lymphocytic infiltrate (H) mostly characterized by CD3+ T cells (E) but with a few CD8+ lymphocytes (F). In this lesion, heterogeneous cytoplasmatic positivity for Melan-A/Mart-1 in neoplastic cells was confined to a small area (G). Patient 3 (lesion 16), I–L: a nodal metastasis without evidence of tumor regression and lacking intratumoral lymphocytes (L), either CD3+ (I) or CD8+ (J), showed intense cytoplasmatic reactivity for Melan-A/Mart-1 limited to a few neoplastic cells (K). Original magnification was 400 for all panels except for H, magnification 250).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195616&req=5

Figure 2: Immunohistochemical analysis of neoplastic lesions. Three examples of immunohistochemical analysis of neoplastic lesions from three patients are reported. Consecutive sections of paraffin-embedded tumor fragments were subjected to immunohistochemical staining with anti-CD3 (A, E, and I), anti-CD8 (B, F, and J) and anti–Melan-A/Mart-1 mAbs (C, G, and K) or conventionally stained with hematoxylin and eosin (D, H, and L). Patient 1 (Table , lesion 7), A–D: a subcutaneous lesion showed no evidence of tumor regression or necrosis but did show an intense intratumoral lymphocytic infiltrate (D) characterized by CD3+ (A) and CD8+ (B) cells in the presence of heterogeneous cytoplasmatic reactivity for Melan-A/Mart-1 in tumor cells (C). Patient 2 (lesion 9), E–H: a nodal metastasis with extended coagulative necrosis with strong cytoplasmic eosinophilia of cell shadows lacking nuclei and scattered granulocytes infiltrating the border of the necrotic area (H, right side). The left side of the lesion, containing vital tumor cells, showed a nonbrisk lymphocytic infiltrate (H) mostly characterized by CD3+ T cells (E) but with a few CD8+ lymphocytes (F). In this lesion, heterogeneous cytoplasmatic positivity for Melan-A/Mart-1 in neoplastic cells was confined to a small area (G). Patient 3 (lesion 16), I–L: a nodal metastasis without evidence of tumor regression and lacking intratumoral lymphocytes (L), either CD3+ (I) or CD8+ (J), showed intense cytoplasmatic reactivity for Melan-A/Mart-1 limited to a few neoplastic cells (K). Original magnification was 400 for all panels except for H, magnification 250).
Mentions: In patient 1, the primary lesion (lesion 1) and a satellitosis (lesion 2) were removed 2 mo before CTLp evaluation (Table ). The first lesion had an absent pattern of CD3+ T cells and Melan-A/Mart-1 antigen expressed on 20% of the neoplastic cells. The satellitosis was nonbrisk for CD3+ T cells, but CD8+ T cells represented only 30% of them and Melan-A/Mart-1 was not expressed. A subcutaneous lesion (lesion 3) isolated 1 wk before CTLp evaluation was Melan-A/Mart-1+ and expressed HLA-A2 on the tumor and 30% of CD8+ cells among the nonbrisk CD3+ infiltrate. However, no evidence of tumor destruction was observed, even though this same lesion contained a high frequency of Melan-A/Mart-1–specific CTLp (Table , Fig. 1). A lymph node metastasis (lesion 4) was almost completely negative for Melan-A/Mart-1 and absent for CD3+ T cells. In the same patient, in spite of an expanded T cell population to Melan-A/Mart-1 in peripheral blood, three additional subcutaneous metastases developed within 6 mo of CTLp analysis. Two of these lesions expressed Melan-A/Mart-1, but no evidence of tumor regression or destruction was found, although all lesions contained a brisk CD3+CD8+ infiltrate (Table , lesions 5–7; Fig. 2aFig. bFig. cFig. d).

Bottom Line: In four out of nine patients, Melan-A/Mart-1(27-35)-specific CTL precursors (CTLp) were >/=1/2,000 peripheral blood lymphocytes and found mostly or only in the CD45RO(+) memory T cell subset.Furthermore, frequent lack of a "brisk" or "nonbrisk" CD3(+)CD8(+) T cell infiltrate or reduced/absent Melan-A/Mart-1 expression in several lesions and lack of HLA class I antigens were found in some instances.Thus, expansion of peripheral immune repertoire to Melan-A/Mart-1 takes place in some metastatic patients and leads to enhanced CTL induction after antigen-presenting cell-mediated selection, but, in most metastatic lesions, it does not overcome tumor escape from immune surveillance.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Oncology Human Tumor Immunobiology Unit, Istituto Nazionale per lo Studio e la Cura dei Tumori, 20133 Milan, Italy. Anichini@istitutotumori.mi.it

ABSTRACT
It is not known if immune response to T cell-defined human histocompatibility leukocyte antigen (HLA) class I-restricted melanoma antigens leads to an expanded peripheral pool of T cells in all patients, affects cytotoxic T lymphocyte (CTL) generation, and correlates with anti-tumor response in metastatic lesions. To this end, a limiting dilution analysis technique was developed that allowed us to evaluate the same frequency of peptide-specific T cells as by staining T cells with HLA-peptide tetrameric complexes. In four out of nine patients, Melan-A/Mart-1(27-35)-specific CTL precursors (CTLp) were >/=1/2,000 peripheral blood lymphocytes and found mostly or only in the CD45RO(+) memory T cell subset. In the remaining five patients, a low (<1/40,000) peptide-specific CTLp frequency was measured, and the precursors were only in the CD45RA(+) naive T cell subset. Evaluation of CTL effector frequency after bulk culture indicated that peptide-specific CTLs could be activated in all patients by using professional antigen-presenting cells as dendritic cells, but CTLp frequency determined the kinetics of generation of specificity and the final number of effectors as evaluated by both limiting dilution analysis and staining with HLA-A*0201-Melan-A/Mart-1 tetrameric complexes. Immunohistochemical analysis of 26 neoplastic lesions from the nine patients indicated absence of tumor regression in most instances, even in patients with an expanded peripheral T cell pool to Melan-A/Mart-1 and whose neoplastic lesions contained a high frequency of tetramer-positive Melan-A/Mart-1-specific T cells. Furthermore, frequent lack of a "brisk" or "nonbrisk" CD3(+)CD8(+) T cell infiltrate or reduced/absent Melan-A/Mart-1 expression in several lesions and lack of HLA class I antigens were found in some instances. Thus, expansion of peripheral immune repertoire to Melan-A/Mart-1 takes place in some metastatic patients and leads to enhanced CTL induction after antigen-presenting cell-mediated selection, but, in most metastatic lesions, it does not overcome tumor escape from immune surveillance.

Show MeSH
Related in: MedlinePlus