Live and let die at TEMRA

In this issue of Blood, Rensing-Ehl and colleagues¹  provide phenotypic and genomic data revealing a FAS-dependent, stage-specific developmental checkpoint in humans presenting with autoimmune lymphoproliferative syndrome (ALPS).

ALPS-FAS, a rare genetic disorder characterized by a benign lymphoproliferation and autoimmune cytopenia, is associated with dominant germ line,^2,3  somatic,⁴  or combined germ line and somatic mutations⁵  of the proapoptotic FAS gene. The hallmark of FAS deficiencies is the accumulation of a rare and unusual T-cell subset expressing neither CD4 nor CD8 coreceptors and thus called double-negative T cells (DNTs). These cells display markers of activated T cells, among which are human leukocyte antigen class II, CD69, and markers of naive T cells such as CD45RA, and they lack CD25 expression. DNTs also contain cytotoxic molecules such as granzymes and perforin.⁶  Accordingly, observations made in Fas-deficient mouse models and repertoire analysis performed on ALPS-FAS DNTs supported a peripheral CD8 T-cell origin.⁷  However, DNTs fail to produce interleukin-2 (IL-2) or interferon-γ or to proliferate upon T-cell receptor (TCR) stimulation but produce high amounts of IL-10 and soluble Fas-ligand, which are used as prediagnosis biomarkers.⁸  Moreover, they exhibit a paradoxical high rate of spontaneous apoptosis in vitro. They are thus regarded as anergic or senescent cells accumulating as a result of a missing FAS signal and dying through a FAS-independent mechanism after extensive in vivo proliferation at a so-far unknown developmental stage. This last feature has made them very difficult to study and contributed to the lack of definitive evidence with regard to their function and origin.

Rensing-Ehl et al used 3 different approaches and took elegant advantage of a recently described family in which patients carry a germ line mutation of the FAS initiation codon as well as a somatic loss of heterozygosity (sLOH) of the wild-type allele (ALPS-FAS-sLOH).¹  Indeed, mutations affecting the extracellular domain of FAS are most often associated with haploinsufficiency because the FAS mutant is not expressed at the cell surface. Such mutations display a partial clinical penetrance because healthy carriers of the germ line mutation are frequently observed among patients’ relatives. In addition to the germ line haploinsufficient mutations, somatic events affecting the second FAS allele have been identified in patients only.⁵  These somatic LOHs take place in hematopoietic progenitors (or at an earlier developmental stage during embryogenesis); they can be found in several lineages such as lymphocytes, monocytes, or even granulocytes, but usually remain undetectable in epithelial cells. In the present situation, the germ line initiation-codon mutation and the somatic LOH are leading to a complete FAS defect, both at the protein and RNA levels. This ALPS-FAS-sLOH case is, so far, the first example of human lymphocytes completely devoid of FAS molecules. This model offers the possibility to trace the disease-related T cells, notably the DNTs and their precursors, at both the cell-surface and genetic levels.

The first original observation of the present work comes from DNTs’ phenotypic analysis. Rensing-Ehl and collaborators found that these cells express a combination of surface markers usually observed on terminally differentiated effector memory CD45RA⁺ T cells (TEMRA cells). Nevertheless, DNTs can be distinguished from TEMRA cells by a high expression of CD27 and CD28 as well as by a lack of expression of KLRG1, an inhibitory receptor normally coexpressed with CD57 on TEMRA cells. Because the differentiation of TEMRA cells is governed by the presence of transcription factors such as Eomes and T-bet, the assessment of their expression was the next step of the study. Surprisingly, whereas Eomes is normally expressed, a complete lack of T-bet expression is observed in DNTs. These last observations correlate nicely with the absence of KLRG1 expression.

Moreover, this abnormal pattern of differentiation is also seen in ALPS-CD4⁺ or CD8⁺ single positive T cells, indicating that a proportion of single positive T cells has already acquired this “ALPS DNT-like” phenotype, pointing out DNT progenitors in both single positive T-cell subsets. This new concept is supported by the genetic approach in ALPS-FAS-sLOH patients because FAS-null cells accumulate both in CD4⁺ and CD8⁺ central memory cells. This is finally demonstrated by the identification of common TCR β CDR3 sequences (the variable sequence of the TCR that defines a clone identity) between CD4⁺ TEMRA cells and DNTs. The accumulation of DNT precursors at the TEMRA stage may reflect at least 2 nonexclusive differentiation pathways. On the one hand, the TEMRA stage can be a FAS-dependent checkpoint contributing to the elimination of highly proliferating or even autoreactive T cells. On the other hand, the DNT-like phenotype found in the TEMRA subset may reflect an aberrant proliferation of T cells that should have been eliminated through FAS at an earlier stage, possibly as early as the recent thymic emigrant stage or even in thymocytes. Previous studies on mice⁹  and our unpublished data showing a low content of recombination circles in patients’ DNTs do not support this last hypothesis, but it cannot be firmly ruled out. Further analysis of mouse models in light of the present findings might help answer these differentiation questions.

Previous studies supported the notion that DNTs mainly stemmed from CD8⁺ single positive T cells.^6,7  Thus the present observation indicates that the relative contribution of CD4⁺ and CD8⁺ T cells to the generation of the DNT subset may vary among ALPS patients or even in response to various stimuli. The nature of the stimuli leading to the generalized lymphoproliferation in ALPS-FAS patients is not clearly defined. Given that two-thirds of the patients developed autoimmune cytopenia, one may assume that self-antigens might be a main source of stimuli and that DNTs and their TEMRA precursors are highly enriched in self-reactive T cells. The anergic status of the DNTs precluded functional analysis, but now such studies can be contemplated, with the TEMRA cells or their immediate precursors most likely being easier to manipulate in vitro. Demonstrating the self-reactive nature of these cells is undoubtedly a future challenge. Therefore, if the TEMRA stage turns out to be a crucial checkpoint of self-reactive T cells, they can be of great help to clinicians who monitor treatment efficiency and potentially to define new therapeutic targets.