Flow Sorting, Whole Genome Amplification and Next-Generation Sequencing as Combined Tools to Study Heterogeneous Acute Lymphoblastic Leukemia.

Next-generation sequencing (NGS) methods have been introduced for immunoglobulin (IG)/T-cell receptor (TR) gene rearrangement analysis in acute lymphoblastic leukemia (ALL) and lymphoma (LBL). These methods likely constitute faster and more sensitive approaches to analyze heterogenous cases of ALL/LBL, yet it is not known whether gene rearrangements constituting low percentages of the total sequence reads represent minor subpopulations of malignant cells or background IG/TR gene rearrangements in normal B-and T-cells. In a comparison of eight cases of B-cell precursor ALL (BCP-ALL) using both the EuroClonality NGS method and the IdentiClone multiplex-PCR/gene-scanning method, the NGS method identified between 29% and 139% more markers than the gene-scanning method, depending on whether the NGS data analysis used a threshold of 5% or 1%, respectively. As an alternative to using low thresholds, we show that IG/TR gene rearrangements in subpopulations of cancer cells can be discriminated from background IG/TR gene rearrangements in normal B-and T-cells through a combination of flow cytometry cell sorting and multiple displacement amplification (MDA)-based whole genome amplification (WGA) prior to the NGS. Using this approach to investigate the clonal evolution in a BCP-ALL patient with double relapse, clonal TR rearrangements were found in sorted leukemic cells at the time of second relapse that could be identified at the time of diagnosis, below 1% of the total sequence reads. These data emphasize that caution should be exerted when interpreting rare sequences in NGS experiments and show the advantage of employing the flow sorting of malignant cell populations in NGS clonality assessments.

HIV specific CD8+ TRM-like cells in tonsils express exhaustive signatures in the absence of natural HIV control.

Lymphoid tissues are an important HIV reservoir site that persists in the face of antiretroviral therapy and natural immunity. Targeting these reservoirs by harnessing the antiviral activity of local tissue-resident memory (TRM) CD8+ T-cells is of great interest, but limited data exist on TRM-like cells within lymph nodes of people living with HIV (PLWH). Here, we studied tonsil CD8+ T-cells obtained from PLWH and uninfected controls from South Africa. We show that these cells are preferentially located outside the germinal centers (GCs), the main reservoir site for HIV, and display a low cytolytic and a transcriptionally TRM-like profile distinct from blood CD8+ T-cells. In PLWH, CD8+ TRM-like cells are expanded and adopt a more cytolytic, activated, and exhausted phenotype not reversed by antiretroviral therapy (ART). This phenotype was enhanced in HIV-specific CD8+ T-cells from tonsils compared to matched blood suggesting a higher antigen burden in tonsils. Single-cell transcriptional and clonotype resolution showed that these HIV-specific CD8+ T-cells in the tonsils express heterogeneous signatures of T-cell activation, clonal expansion, and exhaustion ex-vivo. Interestingly, this signature was absent in a natural HIV controller, who expressed lower PD-1 and CXCR5 levels and reduced transcriptional evidence of T-cell activation, exhaustion, and cytolytic activity. These data provide important insights into lymphoid tissue-derived HIV-specific CD8+ TRM-like phenotypes in settings of HIV remission and highlight their potential for immunotherapy and targeting of the HIV reservoirs.

Single-cell profiling of environmental enteropathy reveals signatures of epithelial remodeling and immune activation.

Environmental enteropathy (EE) is a subclinical condition of the small intestine that is highly prevalent in low- and middle-income countries. It is thought to be a key contributing factor to childhood malnutrition, growth stunting, and diminished oral vaccine responses. Although EE has been shown to be the by-product of a recurrent enteric infection, its full pathophysiology remains unclear. Here, we mapped the cellular and molecular correlates of EE by performing high-throughput, single-cell RNA-sequencing on 33 small intestinal biopsies from 11 adults with EE in Lusaka, Zambia (eight HIV-negative and three HIV-positive), six adults without EE in Boston, United States, and two adults in Durban, South Africa, which we complemented with published data from three additional individuals from the same clinical site. We analyzed previously defined bulk-transcriptomic signatures of reduced villus height and decreased microbial translocation in EE and showed that these signatures may be driven by an increased abundance of surface mucosal cells-a gastric-like subset previously implicated in epithelial repair in the gastrointestinal tract. In addition, we determined cell subsets whose fractional abundances associate with EE severity, small intestinal region, and HIV infection. Furthermore, by comparing duodenal EE samples with those from three control cohorts, we identified dysregulated WNT and MAPK signaling in the EE epithelium and increased proinflammatory cytokine gene expression in a T cell subset highly expressing a transcriptional signature of tissue-resident memory cells in the EE cohort. Together, our work elucidates epithelial and immune correlates of EE and nominates cellular and molecular targets for intervention.

Irreversible depletion of intestinal CD4+ T cells is associated with T cell activation during chronic HIV infection.

HIV infection in the human gastrointestinal (GI) tract is thought to be central to HIV progression, but knowledge of this interaction is primarily limited to cohorts within Westernized countries. Here, we present a large cohort recruited from high HIV endemic areas in South Africa and found that people living with HIV (PLWH) presented at a younger age for investigation in the GI clinic. We identified severe CD4+ T cell depletion in the GI tract, which was greater in the small intestine than in the large intestine and not correlated with years on antiretroviral treatment (ART) or plasma viremia. HIV-p24 staining showed persistent viral expression, particularly in the colon, despite full suppression of plasma viremia. Quantification of mucosal antiretroviral (ARV) drugs revealed no differences in drug penetration between the duodenum and colon. Plasma markers of gut barrier breakdown and immune activation were elevated irrespective of HIV, but peripheral T cell activation was inversely correlated with loss of gut CD4+ T cells in PLWH alone. T cell activation is a strong predictor of HIV progression and independent of plasma viral load, implying that the irreversible loss of GI CD4+ T cells is a key event in the HIV pathogenesis of PLWH in South Africa, yet the underlying mechanisms remain unknown.

HIV infection drives interferon signaling within intestinal SARS-CoV-2 target cells.

SARS-CoV-2 infects epithelial cells of the human gastrointestinal (GI) tract and causes related symptoms. HIV infection impairs gut homeostasis and is associated with an increased risk of COVID-19 fatality. To investigate the potential link between these observations, we analyzed single-cell transcriptional profiles and SARS-CoV-2 entry receptor expression across lymphoid and mucosal human tissue from chronically HIV-infected individuals and uninfected controls. Absorptive gut enterocytes displayed the highest coexpression of SARS-CoV-2 receptors ACE2, TMPRSS2, and TMPRSS4, of which ACE2 expression was associated with canonical interferon response and antiviral genes. Chronic treated HIV infection was associated with a clear antiviral response in gut enterocytes and, unexpectedly, with a substantial reduction of ACE2 and TMPRSS2 target cells. Gut tissue from SARS-CoV-2-infected individuals, however, showed abundant SARS-CoV-2 nucleocapsid protein in both the large and small intestine, including an HIV-coinfected individual. Thus, upregulation of antiviral response genes and downregulation of ACE2 and TMPRSS2 in the GI tract of HIV-infected individuals does not prevent SARS-CoV-2 infection in this compartment. The impact of these HIV-associated intestinal mucosal changes on SARS-CoV-2 infection dynamics, disease severity, and vaccine responses remains unclear and requires further investigation.

Innate Lymphoid Cell Activation and Sustained Depletion in Blood and Tissue of Children Infected with HIV from Birth Despite Antiretroviral Therapy.

Innate lymphoid cells (ILCs) are important for response to infection and for immune development in early life. HIV infection in adults depletes circulating ILCs, but the impact on children infected from birth remains unknown. We study vertically HIV-infected children from birth to adulthood and find severe and persistent depletion of all circulating ILCs that, unlike CD4+ T cells, are not restored by long-term antiretroviral therapy unless initiated at birth. Remaining ILCs upregulate genes associated with cellular activation and metabolic perturbation. Unlike HIV-infected adults, ILCs are also profoundly depleted in tonsils of vertically infected children. Transcriptional profiling of remaining ILCs reveals ongoing cell-type-specific activity despite antiretroviral therapy. Collectively, these data suggest an important and ongoing role for ILCs in lymphoid tissue of HIV-infected children from birth, where persistent depletion and sustained transcriptional activity are likely to have long-term immune consequences that merit further investigation.

Recruited fibroblasts reconstitute the peri-islet membrane: a longitudinal imaging study of human islet grafting and revascularisation.

AIMS/HYPOTHESIS: Rapid and adequate islet revascularisation and restoration of the islet-extracellular matrix (ECM) interaction are significant factors influencing islet survival and function of the transplanted islets in individuals with type 1 diabetes. Because the ECM encapsulating the islets is degraded during islet isolation, understanding the process of revascularisation and engraftment after transplantation is essential and needs further investigation. METHODS: Here we apply a longitudinal and high-resolution imaging approach to investigate the dynamics of the pancreatic islet engraftment process up to 11 months after transplantation. Human and mouse islet grafts were inserted into the anterior chamber of the mouse eye, using a NOD.ROSA-tomato.Rag2-/- or B6.ROSA-tomato host allowing the investigation of the expansion of host vs donor cells and the contribution of host cells to aspects such as promoting the encapsulation and vascularisation of the graft. RESULTS: A fibroblast-like stromal cell population of host origin rapidly migrates to ensheath the transplanted islet and aid in the formation of a basement membrane-like structure. Moreover, we show that the vessel network, while reconstituted by host endothelial cells, still retains the overall architecture of the donor islets. CONCLUSIONS/INTERPRETATION: In this transplantation situation the fibroblast-like stromal cells appear to take over as main producers of ECM or act as a scaffold for other ECM-producing cells to reconstitute a peri-islet-like basement membrane. This may have implications for our understanding of long-term graft rejection and for the design of novel strategies to interfere with this process.

Immunodominant cytomegalovirus-specific CD8+ T-cell responses in sub-Saharan African populations.

More than 90% of children in Africa are infected with cytomegalovirus (CMV) by the age of 12 months. However, the high-frequency, immunodominant CD8+ T-cell responses that control CMV infection have not been well studied in African populations. We therefore sought to define the immunodominant CMV-specific CD8+ T-cell responses within sub-Saharan African study subjects. Among 257 subjects, we determined the CD8+ T-cell responses to overlapping peptides spanning three of the most immunogenic CMV proteins, pp65, IE-1 and IE-2, using IFN-γ ELISpot assays. A bioinformatics tool was used to predict optimal epitopes within overlapping peptides whose recognition was statistically associated with expression of particular HLA class I molecules. Using this approach, we identified 16 predicted novel CMV-specific epitopes within CMV-pp65, IE-1 and IE-2. The immunodominant pp65-specific, IE-1, IE-2 responses were all either previously well characterised or were confirmed using peptide-MHC tetramers. The novel epitopes identified included an IE-2-specific epitope restricted by HLA*B*44:03 that induced high-frequency CD8+ T-cell responses (mean 3.4% of CD8+ T-cells) in 95% of HLA-B*44:03-positive subjects tested, in one individual accounting for 18.8% of all CD8+ T-cells. These predicted novel CMV-specific CD8+ T-cell epitopes identified in an African cohort will facilitate future analyses of immune responses in African populations where CMV infection is almost universal during infancy.