Regulatory T Cells Suppress T Cell Activation at the Pathologic Site of Human Visceral Leishmaniasis

Suppression of T cell response is thought to be involved in the pathogenesis of visceral leishmaniasis (VL). Regulatory T cell (Treg) mediated immune-suppression is reported in animal models of Leishmania infection. However, their precise role among human patients still requires pathologic validation. The present study is aimed at understanding the frequency dynamics and function of Treg cells in the blood and bone marrow (BM) of VL patients. The study included 42 parasitologically confirmed patients, 17 healthy contact and 9 normal bone marrow specimens (NBM). We show i) the selective accumulation of Treg cells at one of the disease inflicted site(s), the BM, ii) their in vitro expansion in response to LD antigen and iii) persistence after successful chemotherapy. Results indicate that the Treg cells isolated from BM produces IL-10 and may inhibit T cell activation in IL-10 dependent manner. Moreover, we observed significantly higher levels of IL-10 among drug unresponsive patients, suggesting their critical role in suppression of immunity among VL patients. Our results suggest that IL-10 plays an important role in suppression of host immunity in human VL and possibly determines the efficacy of chemotherapy

Genome sequence of the tsetse fly (Glossina morsitans):Vector of African trypanosomiasis

Tsetse flies are the sole vectors of human African trypanosomiasis throughout sub-Saharan Africa. Both sexes of adult tsetse feed exclusively on blood and contribute to disease transmission. Notable differences between tsetse and other disease vectors include obligate microbial symbioses, viviparous reproduction, and lactation. Here, we describe the sequence and annotation of the 366-megabase Glossina morsitans morsitans genome. Analysis of the genome and the 12,308 predicted protein-encoding genes led to multiple discoveries, including chromosomal integrations of bacterial (Wolbachia) genome sequences, a family of lactation-specific proteins, reduced complement of host pathogen recognition proteins, and reduced olfaction/chemosensory associated genes. These genome data provide a foundation for research into trypanosomiasis prevention and yield important insights with broad implications for multiple aspects of tsetse biology.IS

Details of visceral leishmaniasis patients monitored in the study.

<p>Details of visceral leishmaniasis patients monitored in the study.</p

<i>L. donovani</i> antigen driven induction of FoxP3⁺ cells in BM-MNCs of visceral leishmaniasis (VL) patients: A i–ii

<p>) Representative plot shows increased frequency of FoxP3⁺ cells (%) in MNCs upon <i>in vitro</i> stimulation with <i>LD</i> antigen (whole cell lysate, WCL). <b>A iii–iv</b>) Representative plot shows increase in the positivity of Ki67 (an intra nuclear cells proliferating antigen) among gated CD4⁺FoxP3⁺ cells upon antigen stimulation (30.8%) as compared to unstimulated cells (16.9%). <b>B i</b>) Scatter plot representing the frequency of Foxp3⁺ Treg cells in gated CD4⁺ cells BM-MNCs (<i>n = 13</i>) of VL patients upon <i>in vitro</i> stimulation with <i>LD</i> antigen. Significant increase in the frequency of FoxP3⁺ Treg cells from BM-MNCs of VL patients occur upon antigen stimulation (<i>p = 0.003, paired t test</i>). Horizontal line in dot plot depicts median value. <b>B ii</b>) Plot shows increase in the frequency of FoxP3⁺ cells in BMMNCs of VL patients upon stimulation (% increase = (frequency of Treg cell in stimulated culture- unstimulated culture)/frequency of Treg in unstimulated×100).</p

Comparative densitometric analysis of the band intensity of FoxP3 mRNA.

<p>Ratio of relative intensity shows 2.7 times increase in band intensity of FoxP3 mRNA in patient PBMCs as compared to healthy PBMCs. FoxP3 band intensity is 2.28 times higher among patient's BMMNCs compared to their PBMCs. In cured cases, band intensity is 2.75 times higher than its pre-treatment level. Set I experiments were executed with higher amount of DNA compared to set II, due to extremely lower expression of FoxP3 in healthy subjects. (*Between Patient's PBMCs and BMMNCs at pretreatment level, # Between Patient's BMMNCs at post treatment level, ** r = average intensity of FoxP3 bands/GAPDH bands).</p

Enrichment of Treg cells at the disease site of visceral leishmaniasis (VL) patients:

<p><b>A</b>) <b>Enumeration of FoxP3⁺ Treg cells:</b> On gated lymphocytes, expression of CD4 and CD25 was defined and subsequently expression of FoxP3 was observed among gated CD4⁺CD25⁺ cells. Overlay histogram shows FoxP3⁺ cells are exclusively present among CD4⁺CD25⁺ T cells (thin line) and absent among CD4⁺CD25⁻ population (solid line). <b>B</b>) <b>Increased frequency of CD4⁺CD25⁺ cells at disease sites:</b> Dual positive cells were enumerated among the MNCs isolated from the peripheral blood, bone marrow (BM) of VL patients (<i>n = 14</i>), peripheral blood of HCs (<i>n = 13</i>) and normal bone marrow (NBM; <i>n = 9</i>). CD4⁺CD25⁺ cells were significantly enriched in BM-MNCs as compared to PBMCs of VL patients (<i>p = 0.016, unpaired t test</i>). A statistically non-significant increase is also observed in PBMCs of VL patients in comparison with that of HCs (<i>p = 0.060, Unpaired t test</i>). Horizontal lines in dot plot depict median value. <b>C</b>) <b>Increased frequency of CD4⁺CD25⁺FoxP3⁺ cells in BM of VL patients:</b> Significant increase in frequency of CD4⁺CD25⁺FoxP3⁺ cells in PBMCs of patients (<i>n = 14</i>) as compared to that of HCs (<i>n = 13</i>; <i>p = 0.009</i>, <i>unpaired t test</i>). CD4⁺CD25⁺FoxP3⁺ cells are further enriched in BM-MNCs at disease site (<i>n = 15</i>) as compared to their autologus PBMCs (n = 14; <i>p = 0.001, paired t test</i>) and NBM (<i>n = 9</i>; <i>p>0.000</i>, <i>unpaired t test</i>). Horizontal lines in dot plot depict median values. Asterix values are enrichment ratios {ER = (Mean of Treg in BM- blood)/Mean of Treg in blood ×100) of FoxP3⁺ Treg cells in the NBM (<i>ER = 18.26.%</i>) and VL patient's BM (<i>ER = 186.93%</i>) relative to peripheral blood. <b>D</b>) <b>Increased expression of FoxP3 mRNA in PBMCs of patients:</b> (i) Photograph depicts increased expression of FoxP3 mRNA in PBMCs of VL patients than that of HCs. Three individual experiments are shown herewith. (ii) Relative density analysis shows increase in FoxP3 mRNA in patient's PBMCs (<i>p = 0.018, unpaired t test</i>). However, GAPDH expression remains unchanged in both the group. <b>E</b>) <b>CD4⁺FoxP3⁺CD39⁺ cells are increased in the VL-BM:</b> Data shows higher frequency of CD4⁺FoxP3⁺CD39⁺ cells at the disease sites of VL patients (iv; 6.5%) as compared to NBM (iii; 0.96%). Contour plots i and ii show fluorescence minus one (FMO) staining for iii and iv respectively. <b>F</b>) <b>FoxP3⁺ (Treg) and FoxP3⁻ (Teff) cells within gated CD4⁺CD25⁺ cells:</b> i) Bar diagram showing significant increase in the frequency of Treg cells in the BM-MNCs (disease site) of VL patients (n = 14) than that of PBMCs (<i>p = 0.001</i>, <i>paired t test</i>). However, the frequency of Teff cells at disease site (BMMNCs) is decreased as compared to peripheral compartment (PBMCs) (Mean±SEM, <i>p = 0.023, paired t test</i>). ii) FACS plot shows <i>in vivo</i> proliferating (Ki67⁺; an intra nuclear cells proliferating antigen) CD4⁺FoxP3⁻ (Teff) cells in blood and BM of freshly diagnosed VL patients. iii) Significant reduction in the proliferating Teff cells obtained from the disease site of VL patients was observed (<i>n = 3</i>; Mean±SD, <i>p = 0.049, unpaired t test</i>).</p