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Accurate quantification of rare genes from limited clinical samples is crucial for research purposes but is technically challenging, especially due to nucleic acid extraction. Using the commercially available genomic DNA (gDNA) extraction kits, which mostly include a DNA purification step through silica columns, magnetic beads or ethanol precipitation, are the preferred choice for many researchers. These kits, however, have a minimum cell number requirement for optimal DNA quality and yield. They are not ideal for use for clinical samples with limited cell numbers. Here, we report the development and validation of a novel crude lysate method for preparing DNA for the absolute quantification of rare genes, TRECs in our case, by droplet digital PCR (ddPCR), from infrequent cells, that removes the need for DNA extraction. Multiple optimization steps and analytical validation of this novel assay was performed on PBMCs extracted from the blood of healthy donors. The newly developed assay shows good agreement with standard ddPCR and has high accuracy, specificity, and reproducibility; additionally, it can also be applied to fixed and permeabilized cells. The assay has the potential to be used for quantification of other trace targets from limited cell samples.

T stem cell-like memory cells (T SCM cells) are considered to be essential for the maintenance of immune memory. The T SCM population has been shown to have the key properties of a stem cell population: multipotency, self-renewal and clonal longevity. Here we show that no single population has all these stem cell properties, instead the properties are distributed. We show that the human T SCM population consists of two distinct cell subpopulations which can be distinguished by the level of their CD95 expression (CD95int and CD95hi). Crucially, using long-term in vivo labelling of human volunteers, we establish that these are distinct populations rather than transient states of the same population. These two subpopulations have different functional profiles ex vivo , different transcriptional patterns, and different tissue distributions. They also have significantly different TREC content indicating different division histories and we find that the frequency of CD95hi T SCM increases with age. Most importantly, CD95hi and CD95int T SCM cells also have very different dynamics in vivo with CD95hi cells showing considerably higher proliferation but significantly reduced clonal longevity compared with CD95int T SCM . While both T SCM subpopulations exhibit considerable multipotency, no single population of T SCM cells has both the properties of self-renewal and clonal longevity. Instead, the “stemness” of the T SCM population is generated by the complementary dynamic properties of the two subpopulations: CD95int T SCM which have the property of clonal longevity and CD95hi T SCM which have the properties of expansion and self-renewal. We suggest that together, these two populations function as a stem cell population.

Inhibitory killer cell immunoglobulin-like receptors (iKIRs) are a family of inhibitory receptors that are expressed by natural killer (NK) cells and late-stage differentiated T cells. There is accumulating evidence that iKIRs regulate T cell-mediated immunity. Recently, we reported that T cell-mediated control was enhanced by iKIRs in chronic viral infections. We hypothesized that in the context of autoimmunity, where an enhanced T cell response might be considered detrimental, iKIRs would have an opposite effect. We studied Type 1 diabetes (T1D) as a paradigmatic example of autoimmunity. In T1D, variation in the Human Leucocyte Antigen (HLA) genes explains up to 50% of the genetic risk, indicating that T cells have a major role in T1D etiopathogenesis. To investigate if iKIRs affect this T cell response, we asked whether HLA associations were modified by iKIR genes. We conducted an immunogenetic analysis of a case-control T1D dataset (N = 11,961) and found that iKIR genes, in the presence of genes encoding their ligands, have a consistent and significant effect on protective HLA class II genetic associations. Our results were validated in an independent data set. We conclude that iKIRs significantly decrease HLA class II protective associations and suggest that iKIRs regulate CD4 + T cell responses in T1D.

Plasmodium infections are co-endemic with infections caused by other agents of acute febrile illnesses, such as dengue virus (DENV), chikungunya virus, Leptospira spp., and Orientia tsutsugamushi. However, co-infections may influence disease severity, treatment outcomes, and development of drug resistance. When we analyzed cases of acute febrile illness at the All India Institute of Medical Sciences, New Delhi, India, from July 2017 through September 2018, we found that most patients with malaria harbored co-infections (Plasmodium mixed species and other pathogens). DENV was the most common malaria co-infection (44% of total infections). DENV serotype 4 was associated with mild malaria, and Leptospira was associated with severe malaria. We also found the presence of P. knowlesi in our study population. Therefore, in areas with a large number of severe malaria cases, diagnostic screening for all 4 DENV serotypes, Leptospira, and all Plasmodium species should be performed.

Objective Acute kidney injury (AKI) is a frequent presentation in malaria infections. Several cases of AKI that are accompanied by clinical symptoms of malaria infection, such as fever, nausea, respiratory distress, and anemia remain undiagnosed due to challenges in accurate diagnosis using peripheral blood microscopy and rapid diagnostic tests that are currently used in clinical settings. This is particularly true for P. vivax and P. knowlesi infections. As a result, these patients are not able to receive anti-malarial therapy in a timely manner. The objective of the present study was to investigate if patients presenting with AKI harbored any of the five human Plasmodium species ( P. falciparum , P. vivax , P. knowlesi , P. malariae , and P. ovale ) within their renal tissues. Results We found that renal biopsies from malaria associated AKI patients harbor the human malaria parasites P. falciparum , P. vivax and P. knowlesi as mono- and mixed species infections. Presence of microvascular injury in a majority of the malaria associated AKI cases suggested vascular involvement of P. vivax and P. knowlesi . This research note also highlights P. knowlesi as an emerging pathogen in the Indian subcontinent.

Harnessing CD4+ T cell help in the lymph nodes through rational antigen design could enhance formation of broadly neutralizing antibodies (bNAbs) during experimental HIV immunization. This process has remained hidden due to difficulty with direct study, with clinical studies instead focusing on responses in the blood as a proxy for the secondary lymphoid tissue. To address this, lymph node cells (LNC) were collected using ultrasound guided fine needle aspiration of axillary lymph nodes from 11 HIV negative participants in an experimental HIV immunogen study (European AIDS Vaccine Initiative EAVI2020_01 study, NCT04046978). Cells from lymph node and blood (PBMC), were collected after intramuscular injection with HIV Env Mosaic immunogens based on HIV Envelope glycoprotein and combined with a liposomal toll-like receptor-4 adjuvant; monophosphoryl lipid A. Simultaneously sampled cells from both blood and lymph node in the same donors were compared for phenotype, function, and antigen-specificity. Unsupervised cluster analysis revealed tissue-specific differences in abundance, distribution, and functional response of LNC compared with PBMC. Monocytes were virtually absent from LNC, which were significantly enriched for CD4+ T cells compared with CD8+ T cells. T follicular helper cells with germinal center features were enriched in LNC, which contained specific CD4+ and CD8+ T cell subsets including CD4+ T cells that responded after a single injection with HIV Env Mosaic immunogens combined with adjuvant. Tissue-specific differences in response to an MHC-II dependent superantigen, staphylococcal enterotoxin B, indicated divergence in antigen presentation function between blood and lymph node. LNC are phenotypically and functionally distinct from PBMC, suggesting that whole blood is only a limited proxy of the T cell lymphatic response to immunization. HIV-specific CD4+ T cells in the lymph node are rapidly inducible upon experimental injection with HIV immunogens. Monitoring evolution of CD4+ T cell memory in LNC with repeated experimental HIV immunization could indicate the strategies most likely to be successful in inducing HIV-specific bNAbs.

Accurate quantification of rare genes from limited clinical samples is crucial for research purposes but is technically challenging. In particular, nucleic acid extraction for quantification of gene targets may lead to target loss. Here, we report the development and validation of a novel crude lysate ddPCR assay for the absolute quantification of rare genes, TRECs in our case, from infrequent cells, that removes the need for DNA extraction, hence minimizing the target loss. The analytical validation was performed on PBMCs extracted from the blood of healthy donors. Standard ddPCR was first optimized to detect TREC copies/cell and then applied to a crude lysate ddPCR assay. The assay was optimized by varying several steps. The optimised assay was directly compared to standard ddPCR and the performance of the assay quantified. The newly developed assay showed good agreement with the standard ddPCR assay in the range from 0.0003 to 0.01 TRECs/cell. The assay had a limit of quantification of <0.0003 TRECs/cell and a limit of detection of <0.0001 TRECs/cell; this performance is favourable compared to standard ddPCR. The intra-assay variation was low. This method can also be applied to fixed and permeabilized cells. The newly developed crude lysate ddPCR assay for quantifying rare targets from limited samples has high accuracy, specificity, and reproducibility; additionally, it eliminates the need for DNA extraction for absolute quantification. The assay has the potential to be used for quantification of other trace targets from small samples.

Inhibitory killer cell immunoglobulin-like receptors (iKIRs) are a family of inhibitory receptors that are expressed by natural killer cells and late-stage differentiated T cells. There is accumulating evidence that iKIRs regulate T cell-mediated immunity. Recently, we reported that T cell-mediated control was enhanced by iKIRs in chronic viral infections. We hypothesized that in the context of autoimmunity, where an enhanced T cell response might be considered detrimental, iKIRs would have an opposite effect. We studied Type 1 diabetes (T1D) as a paradigmatic example of autoimmunity. In T1D, variation in the Human Leucocyte Antigen (HLA) genes explains up to 50% of the genetic risk, indicating that T cells have a major role in T1D etiopathogenesis. To investigate if iKIRs affect this T cell response we asked whether HLA associations were modified by iKIR genes. We conducted an immunogenetic analysis of a case-control T1D dataset (N= 11,961) and found that iKIR genes, in the presence of genes encoding their ligands, have a consistent and significant effect on protective HLA class II genetic associations. Our results were validated in an independent data set. We conclude that iKIRs significantly decrease HLA class II protective associations and suggest that iKIRs regulate CD4+ T cell responses in T1D.