Primary cutaneous CD8⁺ and CD56⁺ T-cell lymphomas express HLA-G and killer-cell inhibitory ligand, ILT2

Primary cutaneous lymphomas comprise a spectrum of heterogeneous diseases characterized by clonal accumulation of lymphocytes initially restricted to the skin.¹  Clonal T-cell populations of primary cutaneous T-cell lymphomas transcribe and secrete T-helper 2 (T_h2) cytokines such as interleukin 10 (IL-10).²  Despite evidence of humoral and cellular antitumor immune response,^3-5  cutaneous T-cell lymphomas (CTCLs) eventually progress to systemic disease after a long history of quietness.

Human leukocyte antigen G (HLA-G) represents a nonclassical HLA class Ib molecule whose expression is restricted to immunoprivileged sites, such as the placenta.^6,7  HLA-G negatively affects almost every aspect of human immunity, inhibiting allogeneic T-cell and natural killer (NK)–cell cytotoxicity as well as T-cell proliferative response.^6,7  Ectopic expression of HLA-G in cancer is thought to enable tumor cells to escape host immunosurveillance.⁸  Being expressed in advanced stage of the disease, HLA-G and IL-10 are molecules implicated in the progression of CTCL.^2,9  One group of primary CTCLs is characterized by initially aggressive clinical behavior and poor outcome. Cytotoxic lymphomas of CD8⁺ or natural killer (NK)/T-cell phenotype represent rare entities that have only recently been recognized and characterized.^10,11  We have recently shown that cutaneous cytotoxic CD8⁺ and CD56⁺CD4⁺ lymphomas express HLA-G ligand, immunoglobulin-like transcript 2 (ILT2).¹²  We conducted a follow-up study investigating expression of HLA-G and IL-10 in these cytotoxic CD8⁺ and CD56⁺CD4⁺ lymphomas with respect to their killer cell inhibitory receptor (KIR) phenotype.

The following criteria were used to select patients from Lymphoma Registry of the Department of Dermatology in Zurich: diagnosis of lymphoproliferative disorder with cytotoxic phenotype with initial presentation in the skin; polymerase chain reaction evidence of T-cell receptor (TCR) rearrangement in biopsied lesion; no iatrogenic or HIV-induced immunosuppression. Patients' characteristics are presented in Table 1.

Both paraffin-embedded as well as frozen material was available from all 7 patients. Patients were extensively phenotyped with the use of a panel of antibodies (CD2, CD3, CD4, CD5, CD7, CD8, CD16, CD30, CD34, CD43, CD45RO, CD56, CD57, CD68, CD79a, and MAC383); only the positive staining is presented in Table 1. A monoclonal antibody recognizing ILT2 (F278) was kindly provided by Maria Christina Mingari (Advanced Biotechnology Center, Genoa, Italy); HLA-G (4H84) immunoglobulin G₁ (IgG₁) mouse monoclonal antibody was kindly provided by M. McMaster (University of California, San Francisco, CA). Antihuman IL-10 (E-10) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Immunohistochemistry was performed using the alkaline phosphatase–antialkaline phosphatase (APAAP) technique, as previously described.⁹  HLA-G staining was done according to the HLA-G workshop recommendations.¹³ 

Results of immunostaining for HLA-G and IL-10 are shown in Table 1 and Figure 1. All CD8⁺ lymphomas expressed HLA-G on a certain proportion of tumor cells. Two of 3 CD56⁺CD4⁺ lymphomas showed HLA-G immunoreactivity. It is of note that CD56⁺CD4⁺ lymphoma displayed stronger HLA-G immunoreactivity in comparison with CD8⁺ cases. IL-10 expression matched the expression pattern of HLA-G. IL-10 was either coexpressed or expressed in the vicinity of HLA-G⁺ cells. All 7 lymphoma cases, both CD56⁺CD4⁺ and CD8⁺, expressed the specific HLA-G ligand, ILT-2 (Table 1). Figure 1 shows that even though infiltrate cells strongly express ILT2 receptor, only a minority of cells actually express HLA-G. Evaluation of serial tissue sections revealed that intraepidermal atypical lymphocytes, a hallmark of CD8⁺ CTCL, expressed HLA-G as well as ILT2 in all cases (Figure 1D-E). Large blastoid lymphocytes, representing the major infiltrate component in CD56⁺ CTCL, preferentially expressed ILT2 with occasional HLA-G immunoreactivity (Figure 1A-B). Small-sized reactive lymphocytes displayed sporadic HLA-G positivity.

A recent study by Nikolova et al¹⁴  demonstrated increased cell surface expression of ILT2/CD85j in circulating Sézary cells. Sézary syndrome is an aggressive form of cutaneous T-cell lymphoma characterized by erythroderma, lymphadenopathy, and the presence of CD4⁺CD45RO⁺ Sézary cells in peripheral blood. ILT2 differs from other KIRs by virtue of its distribution on phagocytic and antigen-presenting cells, such as monocytes, macrophages, dendritic cells, and B lymphocytes.¹⁵  ILT2 is also expressed on some of the peripheral NK and T cells, in particular on CD8⁺ T cells with memory/effector phenotype.¹⁶  ILT2 is shown to down-regulate T-cell functions by inhibiting CD3/TCR–mediated activation of both CD4⁺ and CD8⁺ clones and by inhibiting antigen recognition by CD8⁺ cells.¹⁷  Nikolova et al¹⁴  found that ILT2-expressing Sézary cells, as compared with autologous reactive CD4⁺ lymphocytes, are resistant to CD3 monoclonal antibody–induced cell death. The expression of ILT2 inhibitory receptor may enable survival of the malignant clone by protecting them from activation-induced cell death, as has already been reported in normal circulating memory/effector CD8⁺ T cells expressing ILT2 and other KIRs.¹⁶  Saverino et al¹⁸  showed that the cross-linking of ILT2 receptor on T cells inhibits antigen-specific T-cell proliferation and induces production of immunosuppressive cytokines such as IL-10 and transforming growth factor (TGF)–β. IL-10 exerts a myriad of immunosuppressive effects through inhibition of IL-12 production and T_h1 switch, down-regulation of HLA and costimulatory molecules, together with compromised dendritic cell differentiation, maturation, and functionality.⁸  IL-10 is believed to affect the proliferation and/or cytotoxic phenotype in an autocrine manner in nasal NK-cell lymphomas.¹⁹  IL-10 is one of the cytokines implicated in the induction of HLA-G expression.^9,20  We have recently analyzed HLA-G and IL-10 expression in various subtypes of CTCL.²¹  Our study showed that in CD4⁺ CTCL, the expression of HLA-G associates with IL-10 expression in patients with high-grade histology and advanced disease stage II.¹  On the contrary, 2 cases of early stage CD8⁺ CTCL included in this study expressed HLA-G as well as IL-10.²¹  Our current results confirm these initial findings, demonstrating HLA-G and IL-10 expression in additional cases of early stage CD8⁺ CTCL. This early expression of HLA-G and IL-10 might be a parameter of aggressiveness of this lymphoma entity.

Apart from its inhibitory effects on immune response, HLA-G has the capability to modulate cytokine production, that is, to alter the T_h1/T_h2 balance toward T_h2 polarization. The recognition of membrane-bound HLA-G,²²  as well as incubation of peripheral blood mononuclear cells with purified HLA-G,²³  results in induction of the T_h2 shift. By modulating the cytokine microenvironment, HLA-G might be not only attracting immune cells bearing HLA-G–specific KIR ligand but up-regulating a specific KIR ligand on already present infiltrating cells. In contrast to the constitutive and stable KIR expression in mature NK cells,²⁴  the induction of a particular KIR repertoire in T cells is possible under specific conditions (eg, IL-10).²⁵  The expression of ILT2 on tumor cells may render tumor-specific T lymphocytes unable to recognize tumor cells, thus providing an additional immune-escape mechanism. Together with expression of KIRs, IL-10–induced HLA-G up-regulation could account for the growth/survival advantage of the dominant clone and contribute to the aggressive phenotype of these lymphoma entities.