Identification of Escape Mechanisms Involved in the Absence of Viral Control Despite Activation of Both Innate (natural killer and gammadelta T cells) and Virus-Specific T Cells in Children with CMV or EBV Disease Following Cord Blood Transplantation.

Cord blood transplantation (CBT) are being more and more used both for children and adults. However, one of the major limitation remains the low virus- CTL (cytotoxic T lymphocyte) response associated with the delayed immune recovery. Because the large majority of cord blood lymphocytes are naïve and so are not able to rapid clonal expansion, the monitoring of patients undergoing CBT provides a unique opportunity to 1) better understand the development of innate and adaptive human immune system and 2) identify the immune parameters associated to the viral response especially innate immunity (NK and gammadelta T cells) as well as T cells. Specifically, we have analysed their status of activation, function, and the expression of receptors (PD-1, 2B4, BTLA) recently demonstrated to be associated to immune escape.

We investigated children with severe CMV or EBV disease following CBT and compared them both with children who controlled viral-reactivation following CBT or allogenic bone marrow transplantation (BMT) and with healthy donors. All patients received myeloablative conditioning including ATG and achieved full donor chimerism. Moreover, none of the selected patients received corticosteroid at the time of disease. CMV and EBV-specific CD8 T cells were investigated using HLA class I pentamers or tetramers together with extensive phenotype analyses by multi parametric flow cytometry and functional assay were performed using IFN gamma ELISPOT assay.

Kinetic of immune recovery showed a dramatic increase of CD8 but no CD4 T cells in patients with CMV or EBV disease. All CD8 T cells were highly activated (CD69high, CD57 high, PD-1 high, BTLAlow, 2B4 high, CD28 high). Virus-specific CTL were present in variable proportion. The major population of EBV-CTL were differentiated in CD45RA-CD27-CD28- late effector memory (EM) with almost no effector memory CD45RA+ cells (TEMRA) nor EBV specific activated naïve T cell. This is in sharp contrast with EBV-CTL of either: 1) healthy volunteers with controlled viral infection, who were mainly in a CD45RA-CD27+CD28+ Central Memory (CM) and CD45RA-CD27+CD28- early EM stage of differentiation; 2) patients with EBV infection during post transplantation immunosuppression who have activated naïve and TEMRA EBV-CTL. CMV-CTL were also mainly in late EM but no TEMRA differentiation stages in contrast to healthy volunteers and patients who controlled their CMV infection after CBT. Virus- CTL were highly activated (High expression of 2B4, CD28, PD-1, DR) with an overexpression of inhibitory receptors as PD-1 and 2B4 but no BTLA.

The T cell response against viruses was highly variable in regard to different virus and patients as analysed by IFNg ELISPOT assay. CD4 T cells although low (<50 /mm3) were differentiated. The innate effectors gamma-delta T cells and NK cells were highly activated. Gamma-delta T cells belonged mainly to VD2- subset with a late EM and EMRA differentiation stage. VD2- subset were activated (CD69high, CD57high, CD8high) with low expression of BTLA. Their status was identical to CBT recipients with controlled viral infection. Similarly, NK cells were activated as determined by NKG2A, NKG2C, NKp30 and NKp46 expression. In addition, they were functional as demonstrated by the expression of CD57, which is associated to perforin and granzyme content.

Thus, these reports illustrate a full activation of innate effectors as well as virus-CTL during viral disease after CBT, despite lack of viral clearance. We have identified at least three mechanisms that could explain this apparent immune failure:

1. Profound CD4 T cell deficiency

2. Altered pattern of virus-specific CD8 T cells differentiation associated with overexpression of inhibitory receptors such as PD-1 and 2B4 but no BTLA

3. Deficiency in the generation of naive virus-specific CD8 T cells.

These data support for the first time the hypothesis that after CBT, the immune system although quantitatively insufficient, is highly activated and so that in-vitro expansion of patient self-lymphocytes or means to expand CD4 and CD8 T cells pools are a potential way to cure patients suffering from uncontrolled viral replication after CBT together with blockade of inhibitory pathways.

No relevant conflicts of interest to declare.