We have examined the temporal sequence of events leading to the formation of hepatic granulomas after the intravenous injection of L. donovani amastigotes into BALB/c mice. Parasite ingestion by permissive Kupffer cells (KC) occurred promptly, and local KC aggregations were the foci about which granulomas were subsequently formed. Infected KC were recognized by the uptake of colloidal carbon and the expression of the macrophage-specific antigen recognized by F4/80 mAb. Peroxidase-positive granulocytes migrated rapidly and were followed by monocytes and L3T4+ T cells that enclosed the infected KC. Thereafter, Ly-2+ T cells were prominent members of the granulomatous lymphoid population. Parasites multiplied until 4 wk, and then a prompt reduction in infected cells occurred. This was associated with a sharp decline in the L3T4+ T cells of the granulomas and the maintenance of the Ly-2+ subset. In comparison, athymic nu/nu mice developed smaller, more slowly appearing granulomas that contained granulocytes and monocytes and exhibited progressive parasite replication. Upon rechallenge, the entire process was completed in 2 wk, and infected KC in the euthymic mice were never observed. We hypothesize that the effectiveness of the granulomatous response requires the destruction of parasitized host cells (KC), in a lymphokine rich environment. We further suggest that the Ly-2+ T cell serves as an important effector cell in this process, either by direct cytotoxicity or by supporting the cytotoxic potential of other cell types in the granuloma.

We examined human immunodeficiency virus (HIV) production in cultured mononuclear cells from 36 seropositive homosexual males. Production was detected by using an HIV p24 antigen ELISA assay in blood mononuclear cells in 54% of asymptomatic, 71% of acquired immunodeficiency syndrome (AIDS)-related complex, and 100% of AIDS patients. When the peripheral blood mononuclear cells were separated into monocytes and CD4' T cells, we found that the CD4' T-cell fraction was preferentially infected in the three clinical stages. The ability to isolate HIV from blood monocytederived macrophages was similar in the three stages (24-33%) and required coculture with phytohemagglutinin-stimulated lymphoblasts. Bone marrow and blood mononuclear cells cultured simultaneously yielded virus from both sources in 13 individuals. Again the prime source of virus was the nonadherent bone marrow mononuclear cells, which contained CD41

Transmission studies have suggested that an optimal human immunodeficiency virus type 1 (HIV-1) vaccine should induce both neutralzing antibodies and cytolytic T cells to eliminate free virus and infected cells. A phase I trial in healthy HIV-1-seronegative persons was conducted with a combination HIV-1 vaccine regimen (strain RIB) consiting of priming with a recombinant vaccinia (vac/env) virus expressing HIV-1 envelope and boosting with a gpl6O glycoprotein derived from a recombinant baculovirus (rgpl6O). T-cell and antibody responses detected after immunization with either vac/env alone or rgpl6O alone were generally oflow magnitude and transient, and no subject developed neutraling antibodies. In contrast, recipients of the combination regimen demonstrated in vitro T-cell proliferative responses to homologous HIV-1 antigens that were 3-to 10-fold higher than responses with either vaccine alone, and these responses were sustained for >18 months in 75% of recipients. Moreover, both CD8+ and CD4+ cytolytic T cells were detected. Antibody responses (titer, 1:800 to 1:102,400) to homologous HIV envelope developed in all recipients of the combination regimen, and neutralizing antibodies were detected in 7 of 13. Thus, immunization with a live virus vaccine followed by boosting with a soluble protein offers promise for inducing the broad immunity needed in an HIV vaccine.

The extent of latent HIV-1 infection in blood T cells and monocytes of 23 seropositive individuals was examined using DNA amplification (PCR) of HIV-1 sequences. Amplified DNA was found in at least one cell type in all seropositives tested, including 13 asymptomatic, 5 ARC, and 5 AIDS patients. Amplification with two or more primer sets from the gag, enm, LTR occurred in 21 (91%) patients' T cells and 17 (74%) patients' monocytes. However, amplification with the LTR primers in monocytes was uncommon.Among four patients tested, amplified DNA continued to be detected after a greater than one thousand-fold dilution (< 500 cells) of both T cell and monocyte lysates. Repeat analysis after 7-9 mo in five seropositives yielded similar findings in T cells and monocytes, but some variation in the efficacy of amplification with individual primers occurred. There was no difference in those 10 patients who were taking AZT, compared to those who were untreated.Our results indicate that a fraction (< 1%) of both T cells and monocytes in blood carry a latent infection in all stages of HIV-1 disease and can serve as reservoirs throughout AZT therapy. (J. Clin. Invest. 1991. 87:27-30.)

17Single-cell transcriptomic profiling enables the unprecedented interrogation of gene 18 expression heterogeneity in rare cell populations that would otherwise be obscured in 19bulk RNA sequencing experiments. The stochastic nature of transcription is revealed in 20 the bimodality of single-cell transcriptomic data, a feature shared across single-cell 21 expression platforms. There is, however, a paucity of computational tools that take 22 103 treatment groups are summarized with pairs of regression coefficients whose sampling 104 distributions are available through bootstrap or asymptotic expressions, enabling us to perform 105 complementary differential gene expression and gene set enrichment analyses (GSEA). We use 106 an empirical Bayesian framework to regularize model parameters, which helps improve 107 inference for genes with sparse expression, much like what has been done for bulk gene 108 expression 14 . Our GSEA approach accounts for gene-gene correlations, which is important for 109proper control of type I errors 15 . This GSEA framework is particularly useful for synthesizing 110 observed gene-level differences into statements about pathways or modules. Finally, our model 111yields single cell residuals that can be manipulated to interrogate cellular heterogeneity and 112 gene-gene correlations across cells and conditions. We have named our approach MAST for 113Model-based Analysis of Single-cell Transcriptomics. 114 115 We illustrate the method on two data sets. We first apply our approach to an experiment 116 comparing primary human non-stimulated and cytokine-activated Mucosal-Associated Invariant 117 T (MAIT) cells. MAST identifies novel expression signatures of activation, and the single-cell 118 residuals produced by the model highlights a population of MAIT cells showing partial activation 119 but no induction of effector function. We then illustrate the application of MAST to a previously-120 published complex experiment studying temporal changes in murine bone marrow-derived 121 dendritic cells subjected to LPS stimulation. We both recapitulate the findings of the original 122 publication and describe additional coordinated gene expression changes at the single-cell level 123 across time in LPS stimulated mDC cells. 124 125Results 126 127MAST can account for variation in the cellular detection rate. As discussed previously and 128 as shown on Figure 1 by principal component analysis (PCA), the cellular detection rate (CDR, 129 see Methods for exact definition), is an important source of variability. It is highly correlated with 130 the second principal component (PC, Pearson's rho=0.76 grouped, 0.91 stimulated, 0.97 non-131 stimulated) in the MAIT dataset and the first PC (rho=0.92 grouped, 0.97 non-stimulated, 0.92 132 LPS, 0.89 PAM, 0.92 PIC) in the mDC dataset. We observe larger CDR variability within 133 analysis. Nature methods 1-5 (2014).