Germline Fas Mutations and ALPS
Germline mutations of the Fas gene are associated with the development of autoimmune lymphoproliferative syndrome (ALPS; Canale-Smith syndrome). The symptoms of the disease are similar to those in lpr mice [8]. Patients with ALPS usually have enlargement of the spleen and lymph nodes, various manifestations of autoimmunity, and elevation of double negative T cells. The autoimmune phenomena include thrombocytopenia and anemia. The anti-platelet antibodies associated with ALPS bind to the same platelet glycoprotein complexes as those of idiopathic thrombocytopenic purpura patients. Most ALPS patients have mutations in the death domain of the Fas gene. These mutations are dominant negative, which can be explained by the for a functional Fas receptor essential trimeric structure. The accumulation of double negative T cells is caused by loss of function of wild type Fas. This loss prevents the elimination of auto reactive T cells and the maintenance of normal lymphocyte homeostasis. The relationship between genotype, phenotype and disease penetrance is complex. Within a family, individuals with the mutation may have severe symptoms, moderate symptoms or even no symptoms at all. This suggests the contribution of one or more additional factors to the pathogenesis of ALPS. Treatment strategies remain mostly targeted at the disease manifestations. The diagnostic criteria for ALPS are summarised in table 2.

Table 2: Symptoms of Autoimmune Lymphoproliferative Syndrome

Required Hepatosplenomegaly & Lymphadenopathy Proliferation of double negative (DN) T cells Defective in vitro apoptosis (<50% of normal) Not required Autoimmune phenomena (anemia, thrombocytopenia) Family history

The Double Negative Lymphocytes
Elevation of CD4 and CD8 negative (double negative, DN) T cells is one of the most characteristic findings in patients suffering from autoimmune lymphoproliferative syndrome (ALPS). These DN T cells usually bear a b T cell receptors (TCR), are negative for CD45R0, but positive for CD45RA. Initially this phenotype was thought to be consistent with recent thymus emigrants. The expanded population of a b DN T cells from ALPS patients has a remarkable uniform phenotype. This is in contrast to the small a b DN T cell compartment in healthy individuals, that contains multiple immunophenotypically distinct subpopulations. Current data indicate that a b DN T cells from ALPS patients are derived from cytotoxic CD8 T cells, chronically activated in vivo, but anergic in vitro. Their anergic state may be related to persistent modifications of O-linked carbohydrates on cell surface molecules, such as CD43 and CD45, as well as to the increased levels of interleukin-10. The a b DN T cells are immunophenotypically and functionally similar to a b DN T cells that accumulate in lpr and gld mice, which bear genetic mutations in Fas (lpr) and FasL (gld). In these mice, TCRa b DN T cells express the B-cell-specific CD45R isoform B220. TCRa b DN T cells of ALPS patients, with either Fas or FasL mutations, also express B220. In addition, also similar to lpr/gld mice, they have an unusual population of B220-positive CD4(+) T cells [9].

In a study on the immunophenotypic findings in 166 members of 31 families with ALPS type Ia, the ALPS type Ia probands and relatives having both a Fas mutation and clinically proven ALPS showed significant expansion of CD8 T cells, TCR a b DN T cells, TCRgd DN T cells, HLA-DR positive CD3 T cells, CD8/CD57 T cells, and CD5 B cells. Relatives with Fas mutations, but without all the required criteria for ALPS, also had expansions of CD8 T cells, TCRab DN T cells, and TCRgd DN T cells. Interestingly, relatives without a Fas mutation and with no features of ALPS (n = 65) demonstrated a small but significant expansion of CD8(+) T cells, both DN T cell subsets, and CD5(+) B cells. As compared to unrelated healthy controls, lymphocyte subset alterations were greatest in the probands, followed by the relatives with mutations and ALPS. Probands and relatives with mutations and ALPS also showed a lower number of CD4/CD25 T cells that, in combination with an independent increase in HLA-DR positive T cells, provided a profile predictive of the presence of clinical ALPS. Because quantitative defects in apoptosis were similar in Fas mutation-positive relatives and in ALPS patients, factors other than modifiers of the Fas apoptosis pathway must contribute to disease penetrance in ALPS.

In a recent case report a 6-month-old girl was described with an enlarged liver and spleen, cervical lymphadenopathy, anemia, and thrombocytopenia since the age of 1 month [18]. Immunohistochemical analysis of an affected lymph node revealed a phenotype, i.e. presence CD3/CD57 DN T cell blasts, consistent with the usual ALPS phenotype. Remarkably, the DN T cells in the lymph node stained positive for TCR g d and not for TCRa b. By PCR and Southern blot analyses for the Immunoglobulin and TCR (a b and g d) genes on DNA isolated from the affected lymph node no evidence for a monoclonal population was found. Fisher et al reported one patient with an accumulation of DN TCR g d T cells in the circulation, and DN TCR a b T cells in the affected tissue [1]. These two cases demonstrate that ALPS can not only result in proliferation of TCRa b, but albeit infrequently, also of TCR g d DN T cells. It is important to distinguish this benign polyclonal proliferation from neoplastic g d + T cell proliferations such as hepatosplenic g d T cell lymphomas.

Although it is not known what triggers the accumulation of DN T cells in symptomatic ALPS patients, it may be that the nature of the initial trigger defines the phenotype of the T cells with respect to a b or g d TCR. Development of lymphadenopathy at a very young age of onset may also contribute to the accumulation of TCR g d DN T cells.

Usually, patients with ALPS also have an increase of CD5 positive polyclonal B-lymphocytes in the peripheral blood. The immunophenotype findings are summarized in table 3.

Table 3: Immunophenotype of double negative cells

Positive	Negative
CD2, CD3, TCR ab and/or gd CD45RA (B220) CD57 CD43 TIA-1, Perforin MIB-1	CD4 and CD8 CD45R0 CD56
Other Findings
Increase HLA class II T-cells Decrease CD4/CD25 subset Increase CD5 positive B cells Increased IL-10 levels

Cytokine Production by the Double Negative T Cells
Several studies have shown that DN T cells in ALPS patients respond poorly to mitogens or antigens and fail to produce cytokines on activation. This parallels the results of studies of DN T cells in lpr mice. An increase in circulating IL-10 levels was detected in ALPS patients as compared to normal individuals [10].

A possible cytotoxic T cell origin is supported by the expression of TIA-1 and perforin in DN T cells, which are normally associated with CD8+ cytotoxic T cells [11].

Histology of Lymph Nodes and Spleens Involved By ALPS
The enlarged lymph nodes show marked interfollicular and parafollicular hyperplasia. The expanded areas are populated by TCRab or gd DN T cells and blasts that are positive for CD3, CD57, CD45RA and TIA-1, but negative for CD45RO similar to the DN T cells in the peripheral blood. The infiltrate results from the combination of reduced apoptosis and increased proliferation, as measured by decreased in situ detection of DNA fragmentation and increased staining with MIB-1, respectively [12]. The proliferation may be extensive enough to suggest a diagnosis of malignant lymphoma. There is at least one case overdiagnosed as T cell lymphoma recorded in the literature. ALPS should be considered as a possible differential diagnosis in adult patients presenting with rare types of T-cell lymphomas [13]. Many of the lymphocytes and blasts express markers associated with cytotoxicity, such as perforin, TIA-1, and CD57, whereas CD25 is negative [12]. In addition, most lymph nodes exhibit florid follicular hyperplasia, often with focal progressive transformation of germinal centers, but in some cases follicular involution was seen. A polyclonal plasmacytosis is also frequently present. The spleen is markedly enlarged in virtually all patients, more than 10 time normal size. The marginal zone and the periarteriolar lymphocyte sheath are prominently involved with spilling over into the red pulp. DN T cells also can be observed in liver biopsies exhibiting portal triaditis. Taken together, the histopathological and immunophenotypic findings, particularly in lymph nodes and peripheral blood, are sufficiently distinctive to suggest a diagnosis of ALPS.

References

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