Functional specific-T-cell expansion after first cytomegalovirus reactivation predicts viremia control in allogeneic hematopoietic stem cell transplant recipients.

The use of preemptive antiviral therapy to prevent cytomegalovirus (CMV) disease in allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients might result in over-treatment, inducing drug-related toxicity and viral resistance. A search for predictive markers is needed to determine requirement for antiviral therapy. Clinical follow-up, in combination with the use of streptamers (STs) and cytokine-intracellular staining, could help to identify patients at high risk for CMV reactivations. To study the immune response and reactivation control by CMV-specific CD8+ T-cell (CMV-CTL) populations, we monitored 25 patients who have undergone allo-HSCT by using ST multimer and intracellular cytokine staining. Our study has revealed that the presence of functional CMV-specific T cells, determined by early interferon γ production or by significant T-cell expansion after first CMV reactivation, correlated with short CMV viremia duration and low number of CMV reactivations. By contrast, the absence of functional CMV-CTLs does correlate with CMV recurrence. These results support that behavior of CMV-specific subpopulations after reactivation influences reactivations and can guide preemptive therapy.

Streptamer technology allows accurate and specific detection of CMV-specific HLA-A*02 CD8+ T cells by flow cytometry.

BACKGROUND: Multimer technology is widely used to screen antigen-specific immune recovery after allogeneic hematopoietic stem cell transplantation (allo-HSCT) as it enables identification, enumeration, phenotypic characterization and isolation of virus-specific T-cells. Novel approaches of multimerization might improve on classical tetramer staining; however, their use as standard monitoring technique to quantify antigen-specific cells has not been validated yet. We have compared two of these available multimeric complexes: pentamer and streptamer to select the best strategy for the incorporation into clinical monitoring practice. METHODS: CMVpp65495-503 -specific HLA-A*02:01 CD8+ T lymphocytes (CTLA *02:01 -CMVpp65495-503 ) were examined with pentamer and streptamer in peripheral blood cells of 77 healthy volunteers. Quantitative and qualitative analyses were performed to compare the precision and repeatability, sensitivity and accuracy and specificity of both technologies by flow cytometry. RESULTS: Standard deviation for both techniques was less than 0.05 showing that they are repetitive and precise. Both techniques significantly correlated at high frequencies (rSpearman = 0.9422; P < 0.0001) but it was lost at lower levels (<1%) of CTLA *02:01 -CMVpp65495-503 (rSpearman = 0.3351; P = 0.1376). Streptamer is more accurate for the detection of CTLA *02:01 -CMVpp65495-503 providing significantly closer values to the theoretical ones (P < 0.0001) as pentamer binds unspecifically to a notable proportion of non-CMV-specific CD8+ T-cells. CONCLUSION: Our results suggest that streptamer multimer provides precise, accurate and specific results to detect CTLA *02:01 -CMVpp65495-503 by flow cytometry. Streptamer multimer can be used not only for the monitoring of early CTLA *02:01 -CMVpp65495-503 reconstitution in immunosuppressed patients following allo-HSCT but also, in conjunction with its reversibility role, for the isolation of CTLA *02:01 -CMVpp65495-503 for its future use in adoptive immunotherapy. © 2016 International Clinical Cytometry Society.

The immune response to cytomegalovirus in allogeneic hematopoietic stem cell transplant recipients.

Approximately, up to 70 % of the human population is infected with cytomegalovirus (CMV) that persists for life in a latent state. In healthy people, CMV reactivation induces the expansion of CMV-specific T cells up to 10 % of the entire T cell repertoire. On the contrary, CMV infection is a major opportunistic viral pathogen that remains a leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Due to the delayed CMV-specific immune recovery, the incidence of CMV reactivation during post-transplant period is very high. Several methods are currently available for the monitoring of CMV-specific responses that help in clinical monitoring. In this review, essential aspects in the immune recovery against CMV are discussed to improve the better understanding of the immune system relying on CMV infection and, thereby, helping the avoidance of CMV disease or reactivation following hematopoietic stem cell transplantation with severe consequences for the transplanted patients.