Transdifferentiation of Human Circulating Monocytes Into Neuronal-Like Cells in 20 Days and Without Reprograming

Despite progress, our understanding of psychiatric and neurological illnesses remains poor, at least in part due to the inability to access neurons directly from patients. Currently, there are in vitro models available but significant work remains, including the search for a less invasive, inexpensive and rapid method to obtain neuronal-like cells with the capacity to deliver reproducible results. Here, we present a new protocol to transdifferentiate human circulating monocytes into neuronal-like cells in 20 days and without the need for viral insertion or reprograming. We have thoroughly characterized these monocyte-derived-neuronal-like cells (MDNCs) through various approaches including immunofluorescence (IF), flow cytometry, qRT-PCR, single cell mRNA sequencing, electrophysiology and pharmacological techniques. These MDNCs resembled human neurons early in development, expressed a variety of neuroprogenitor and neuronal genes as well as several neuroprogenitor and neuronal proteins and also presented electrical activity. In addition, when these neuronal-like cells were exposed to either dopamine or colchicine, they responded similarly to neurons by retracting their neuronal arborizations. More importantly, MDNCs exhibited reproducible differentiation rates, arborizations and expression of dopamine 1 receptors (DR1) on separate sequential samples from the same individual. Differentiation efficiency measured by cell morphology was on average 11.9 ± 1.4% (mean, SEM, n = 38,819 cells from 15 donors). To provide context and help researchers decide which in vitro model of neuronal development is best suited to address their scientific question,we compared our results with those of other in vitro models currently available and exposed advantages and disadvantages of each paradigm

Classification of current anticancer immunotherapies

During the past decades, anticancer immunotherapy has evolved from a promising therapeutic option to a robust clinical reality. Many immunotherapeutic regimens are now approved by the US Food and Drug Administration and the European Medicines Agency for use in cancer patients, and many others are being investigated as standalone therapeutic interventions or combined with conventional treatments in clinical studies. Immunotherapies may be subdivided into “passive” and “active” based on their ability to engage the host immune system against cancer. Since the anticancer activity of most passive immunotherapeutics (including tumor-targeting monoclonal antibodies) also relies on the host immune system, this classification does not properly reflect the complexity of the drug-host-tumor interaction. Alternatively, anticancer immunotherapeutics can be classified according to their antigen specificity. While some immunotherapies specifically target one (or a few) defined tumor-associated antigen(s), others operate in a relatively non-specific manner and boost natural or therapy-elicited anticancer immune responses of unknown and often broad specificity. Here, we propose a critical, integrated classification of anticancer immunotherapies and discuss the clinical relevance of these approaches

HIV-Infected Spleens Present Altered Follicular Helper T Cell (Tfh) Subsets and Skewed B Cell Maturation.

Follicular helper T (Tfh) cells within secondary lymphoid organs control multiple steps of B cell maturation and antibody (Ab) production. HIV-1 infection is associated with an altered B cell differentiation and Tfh isolated from lymph nodes of HIV-infected (HIV+) individuals provide inadequate B cell help in vitro. However, the mechanisms underlying this impairment of Tfh function are not fully defined. Using a unique collection of splenocytes, we compared the frequency, phenotype and transcriptome of Tfh subsets in spleens from HIV negative (HIV-) and HIV+ subjects. We observed an increase of CXCR5+PD-1highCD57-Tfh and germinal center (GC) CD57+ Tfh in HIV+ spleens. Both subsets showed a reduced mRNA expression of the transcription factor STAT-3, co-stimulatory, regulatory and signal transduction molecules as compared to HIV- spleens. Similarly, Foxp3 expressing follicular regulatory T (Tfr) cells were increased, suggesting sustained GC reactions in chronically HIV+ spleens. As a consequence, GC B cell populations were expanded, however, complete maturation into memory B cells was reduced in HIV+ spleens where we evidenced a compromised production of B cell-activating cytokines such as IL-4 and IL-10. Collectively our data indicate that, although Tfh proliferation and GC reactions seem to be ongoing in HIV-infected spleens, Tfh "differentiation" and expression of costimulatory molecules is skewed with a profound effect on B cell maturation

Splenic Tfh subset harbor high amount of HIV-1 DNA integration.

<p>qRT-PCR quantification of HIV proviral DNA levels in GCTfh, Tfh, memory and naïve CD4⁺ T cell subsets isolated from splenocytes of five HIV⁺ ITP⁺ individuals as described in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140978#pone.0140978.s001" target="_blank">S1 Fig</a></b>. Symbols represent individual samples: horizontal bars represent mean; and error bars show SEM. Statistics were obtained using the non parametric Mann-Whitney test *p<0.05, **p<0.005, ***p<0.001.</p

Tfh cell subsets, including Tfr, are present in greater proportions in HIV⁺ spleens.

<p>(A) Splenocytes were stained for T helper cell markers and analyzed by flow cytometry: according to the expression of CD45RA and CCR7, CD4 T cells were subdivided into CD45RA⁺CCR7⁺ naïve, CD45RA^-CCR7^- effector memory (TEM), CD45RA⁺CCR7^- terminally differentiated effector memory (TEMRA) and CD45RA^-CCR7⁺ central memory (TCM) T cells. Regulatory T (Treg) cells were identified as CD4⁺ CD45RA^- Foxp3⁺ CD25⁺. HIV^-ITP^- n = 8, HIV⁺ITP^- n = 4, HIV^-ITP⁺ n = 3 and HIV⁺ITP⁺ n = 9. (B) Tfh cells were identified as CD3⁺ CD4⁺ CD45RA^- CCR7^- CXCR5⁺, GCTfh are the CD57⁺ subset of Tfh, and Tfr are Foxp3⁺ Tfh cells (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140978#pone.0140978.s001" target="_blank">S1 Fig</a></b>). The frequency of Tfh, GCTfh and Tfr cells is represented as the percentage of total memory CD4⁺ T cells. HIV^-ITP^- n = 8, HIV⁺ITP^- n = 4, HIV^-ITP⁺ n = 3 and HIV⁺ITP⁺ n = 9. Frequency of Treg and Foxp3⁺ Tfh (Tfr) were identified for HIV^-ITP^- n = 4, HIV^-ITP⁺ n = 3, HIV⁺ITP^- n = 3 and HIV⁺ITP⁺ n = 4. Statistics were obtained using the non parametric Mann-Whitney test *p<0.05, **p<0.005, ***p<0.001.</p

GC B cells accumulate whereas memory B cell compartment is reduced in HIV+ spleens.

<p>(A) Using flow cytometry, B cell maturation was assessed according to the expression of CD38 and IgD markers by CD19⁺ cells (34). The gating strategy is presented in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140978#pone.0140978.s001" target="_blank">S1 Fig</a></b>. In HIV chronically infected patients, B cell maturation is biased towards GC B cells and PC subsets as compared to HIV^- donors and ITP⁺ patients. (B) The transitional B cell population was identified as CD19⁺CD38⁺⁺IgD⁺CD27^-IgM⁺ and plasma cells as CD19⁺CD38⁺⁺IgD^-. In the box-and-whiskers plot, box size represents the limits of data for the second and third quartiles, with medians shown as bars. Whiskers define the minimum and maximum of the data presented. HIV^-ITP^- n = 8, HIV^-ITP⁺ n = 3, HIV⁺ITP^- n = 5 and HIV⁺ITP⁺ n = 8. Statistics were obtained using the non parametric Mann-Whitney test *p<0.05, **p<0.005, ***p<0.001.</p