Determinants of HIV-specific CD8 T-cell responses in HIV-infected pediatric patients and enhancement of HIV-gag-specific responses with exogenous IL-15

Cellular immune responses play a central role in controlling HIV-1 infection. HIV-specific IFN-gamma production by CD8 T cells was evaluated in 17 HLA-A2+ HIV-infected pediatric patients (age range 1 month to 16 years) in an ELISPOT assay. Most patients (15/17) exhibited responses to HIV-gag, followed by responses to envelope gp120, gp41, and V3 loop. Only 7 patients responded to all four antigenic peptides. Treatment-related immune reconstitution of CD4 T cells was associated with increase in gag-specific responses, but these declined with prolonged viral suppression. Exogenous IL-15 resulted in augmentation of HIV-gag-specific response in 71% of patients, while IL-2 and IL-7 had variable effects, augmenting responses in 25% patients. Thus, HIV-specific CD8 T-cell responses are dependent on both CD4 T-cell help and antigenic stimulation. The cytokine IL-15 may be a useful modality as adjunctive therapy to augment HIV-specific memory CD8 T cells

Simplified Fluorescent Multiplex PCR Method for Evaluation of the T-Cell Receptor Vβ-Chain Repertoire

Rationale: evaluation of the T-cell receptor (TCR) Vβ-chain repertoire by PCR-based CDR3 length analysis allows fine resolution of the usage of the TCR Vβ repertoire and is a sensitive tool to monitor changes in the T-cell compartment. A multiplex PCR method employing 24 labeled upstream Vβ primers instead of the conventionally labeled downstream Cβ primer is described. Method: RNA was isolated from purified CD4 and CD8 T-cell subsets from umbilical cord blood and clinical samples using TRI reagent followed by reverse transcription using a Cβ primer and an Omniscript RT kit. The 24 Vβ primers were multiplexed based on compatibility and product sizes into seven reactions. cDNA was amplified using 24 Vβ primers (labeled with tetrachloro-6-cardoxyfluorescein, 6-carboxyfluorescein, and hexachloro-6-carboxyfluorescein), an unlabeled Cβ primer, and Taqgold polymerase. The fluorescent PCR products were resolved on an automated DNA sequencer and analyzed using the Genotyper 2.1 software. Results: Vβ spectratypes of excellent resolution were obtained with RNA amounts of 250 ng using the labeled Vβ primers. The resolution was superior to that obtained with the labeled Cβ primer assay. Also the numbers of PCRs were reduced to 7 from the 12 required in the Cβ labeling method, and the sample processing time was reduced by half. Conclusion: The method described for T-cell receptor Vβ-chain repertoire analysis eliminates tedious dilutions and results in superior resolution with small amounts of RNA. The fast throughput makes this method suitable for automation and offers the feasibility to perform TCR Vβ repertoire analyses in clinical trials

Twelve-month observational study of children with cancer in 41 countries during the COVID-19 pandemic

Childhood cancer is a leading cause of death. It is unclear whether the COVID-19 pandemic has impacted childhood cancer mortality. In this study, we aimed to establish all-cause mortality rates for childhood cancers during the COVID-19 pandemic and determine the factors associated with mortality