Potential Involvement of Physiological TCR Gamma Delta Clones in Immune Surveillance of Preleukemic Cells
Authors: Zimmermannova Olga , Zaliova Marketa , Moorman Anthony V. , Fronkova Eva , Stary Jan , Trka Jan , Kalina Tomas , Zuna Jan 
Published: October 2019

Abstract
The mechanisms underlying the evolution and dynamics of preleukemic clone towards the overt disease are largely unknown. We examined this issue in slow onset leukemia (SOL) cases. SOLs represent a rare subgroup of acute lymphoblastic leukemias (ALLs) initially announced by clinical symptoms of fever, infections and anemia several weeks/months prior to ALL diagnosis. These cases can be indicated for bone marrow aspiration, thus providing us with a unique biological material to follow the disease before its own outbreak.
We collected 8 B-lineage ALL cases of SOL (incl. 1 hyperdiploid and 2 ETV6/RUNX1+ patients) with 1-3 prediagnostic samples (taken 6 weeks to 15 months prior to diagnosis). Using qPCR, NGS and SNP array we followed the dynamics of the preleukemic clone from the first symptoms to an overt disease. Strikingly, as we have shown previously, instead of an expected slow progression of blast growth, the preleukemic clones appeared to follow a specific pattern in dynamics, with initial high blast levels followed by a steep decrease, only to rise again towards the diagnosis of ALL.
Massive parallel sequencing of immunoglobulin/T-cell receptor repertoir provided deeper insight into the immune background of developing leukemias. We discovered significant proportion of several non-malignant T-cell clones bearing specific Vγ9Vδ2 rearrangements. The Vγ9Vδ2 cells showed opposite dynamics than the preleukemic blasts in the prediagnostic phase with initially low levels subsequently growing during the suppression of preleukemic clone followed by a final decrease before the ALL diagnosis. This finding raises a question whether the Vγ9Vδ2 cells can contribute to immune control of (pre-)ALL blasts.
The Vγ9Vδ2 cells, a major subset of circulating TCRγδ T-cells, can respond to phosphoantigens such as isopentenyl pyrophosphate (IPP), an intermediate product of the mevalonate pathway, often dysregulated in cancer cells. Vγ9Vδ2 cells are mentioned to play a pivotal role in tumor immune surveillance, which is attributed to their specific ability to exert tumor specific lysis, notably against epithelial carcinomas or some hematopoietic malignancies. However, their involvement in controlling ALL has not been reported yet.
To study its ability to kill the ALL blasts, we purified Vγ9Vδ2 cells and expanded them in the presence of IL2 (1000U/ml) or IL2 + IPP (15 μmol). After 10-14 days cultivation, the expression of intracellular granzyme B was measured within the Vγ9Vδ2 subset, reflecting its capacity to lyse the target tumor cells.
In a set of independent experiments, expanded Vγ9Vδ2 T-cells were co-cultivated with human ALL cell lines REH (ETV6/RUNX1+) and RS4;11 (MLL/AF4+) in effector: target ratios 10:1 and 1:3. In the subsequent cytotoxic assay, the viability of leukemic cells and the CD107a (LAMP-1, a marker of T-cell degranulation) expression on Vγ9Vδ2 T-cells were assessed.
We detected significantly higher levels of dying REH cells after the co-culture with the Vγ9Vδ2 lymphocytes which was more pronounced in IPP-expanded Vγ9Vδ2 (IL2+IPP: median increase 10.7x, range 5.5 to 29.4; IL-2 alone: 3.6x, range 2.2-5.0; both compared to control cells). Interestingly, the cytotoxicity was increased after IPP stimulation although the Vγ9Vδ2 cells displayed a seemingly lower potential to degranulate (median of granzyme B positivity: 23.6% and 30.6% in IL2+IPP and IL2 alone, respectively). The expression of CD107a was more pronounced in the Vγ9Vδ2 cells co-cultivated with REH. Importantly, no significant target lysis was achieved by co-cultivating of IPP-expanded Vg9Vd2 with RS4;11 cells (median increase 2.1x, range 0.9-4.0). This corresponds with our previous data showing that neither the increased level of Vγ9Vδ2 cells nor its divergent dynamics towards the leukemic clone have been observed in the preleukemic samples of patients with MLL+ secondary leukemia, suggesting a potential differential impact of various molecular aberrations on triggering anti-tumor immune response.
In conclusion, by following the preleukemic clone during the prediagnostic phase of the disease, we revealed a specific pattern in its dynamics which might result from the activation of protective anti-tumor mechanisms by certain leukemia subtypes. We showed that physiological TCRγδ T-cells might play an active role in immune surveillance in the preleukemic phase of ALL.
Support: IGA NT/12428-5, NT/14350-3, GAUK 618212

Disclosures
No relevant conflicts of interest to declare.