Shaping T Cell – B Cell Collaboration in the Response to Human Immunodeficiency Virus Type 1 Envelope Glycoprotein gp120 by Peptide Priming

<div><p>Prime-boost vaccination regimes have shown promise for obtaining protective immunity to HIV. Poorly understood mechanisms of cellular immunity could be responsible for improved humoral responses. Although CD4+ T-cell help promotes B-cell development, the relationship of CD4+ T-cell specificity to antibody specificity has not been systematically investigated. Here, protein and peptide-specific immune responses to HIV-1 gp120 were characterized in groups of ten mucosally immunized BALB/c mice. Protein and peptide reactivity of serum antibody was tested for correlation with cytokine secretion by splenocytes restimulated with individual gp120 peptides. Antibody titer for gp120 correlated poorly with the peptide-stimulated T-cell response. In contrast, titers for conformational epitopes, measured as crossreactivity or CD4-blocking, correlated with average interleukin-2 and interleukin-5 production in response to gp120 peptides. Antibodies specific for conformational epitopes and individual gp120 peptides typically correlated with T-cell responses to several peptides. In order to modify the specificity of immune responses, animals were primed with a gp120 peptide prior to immunization with protein. Priming induced distinct peptide-specific correlations of antibodies and T-cells. The majority of correlated antibodies were specific for the primed peptides or other peptides nearby in the gp120 sequence. These studies suggest that the dominant B-cell subsets recruit the dominant T-cell subsets and that T-B collaborations can be shaped by epitope-specific priming.</p></div

Response associated with T-B correlationsa.

a<p>Negative correlations indicated in parentheses.</p>b<p>Proliferation.</p>c<p>Bold underline indicates positive or negative correlation of same T and B cell specificity (not necessarily the same type of response) in multiple animal groups.</p>d<p>Antibodies that block CD4 binding to gp120.</p>e<p>Antibodies that crossreact with gp120_96ZM651.8.</p

Immunization schedule, treatment groups, and analyses of immune response.

<p>Immunization schedule, treatment groups, and analyses of immune response.</p

Correlation of antibody and bulk T-cell responses in the unprimed (A, B) and control-primed (C) mouse groups.

<p>Bar graphs indicate Pearson correlation coefficients for the bulk cytokine response with reactivity to the indicated antigens. Scatter plots illustrate the significant correlations (p<0.05). Bulk IL-6 and TNF-α correlated with gp120 and CD4-blocking antibodies, respectively, in the PBS-treated mouse group (A). Bulk IL-2 and IL-5 correlated with gp120_96ZM651.8-crossreactive and CD4-blocking antibodies in the IFA-treated mouse group (B). Bulk IL-2, IL-5, and IL-17 correlated with CD4-blocking antibodies in the control-primed group (C).</p

T-cell responses elicited by priming with peptide and immunization with gp120dss378.

<p>The groups of 10 mice are the same as in Figs. 3 and 4. Data are presented as mean +/− SEM for groups of ten mice. Bulk responses were compared using one-way ANOVA with Tukey’s post-test. Peptide-specific responses in gp120-peptide-primed groups were compared to those in the control-primed group using one-way ANOVA with Dunnett’s post-test. Asterisks indicate significant differences (*, p<0.05; and **, p<0.01). Bulk secretion of IL-2, IL-4, IL-5, and IL-6 (not shown) was enhanced by priming with a gp120 peptide. Bulk secretion of IL-17 was enhanced by priming with the control peptide. In contrast to the bulk response, peptide-2-specific IL-2 and IL-5 was reduced by peptide-2 priming. Likewise, peptide-28-specific IL-2 was reduced by peptide-29 priming.</p

Scatter plots illustrate selected T-B correlations.

<p>The groups of 10 mice are the same as in Figs. 3 and 4. A-E. In the IFA-treated mouse group, peptide-7 antibodies correlated with log-concentration of IL-2, IL-4, and IL-5 secreted in response to the indicated peptides. F, G. In two additional mouse groups, peptide-7 antibodies correlated with the IL-2 and IL-4 response to peptide 5. H. Priming with peptide 29 induced correlation of peptide-30 antibodies with IL-2 and IL-4 responses to peptide 5. I. In the peptide-29 group, correlations of peptide-7 antibodies with peptide-5 T-cell responses became negative. The peptide specificities of the correlated T-cell and B-cell responses are indicated in the graph titles, and the associated cytokines are indicated in the legends for individual graphs.</p

Epitope-specific T-B correlations in unprimed and control-primed mouse groups.

<p>The groups of mice are the same as in Fig. 2. Arrows connect <u>positively</u> correlated (<i>r</i>²>0.42 and <i>p</i><0.05) T-cell responses in the upper graph to B-cell responses in the lower graph. A unit in the <i>y</i>-axis indicates that at least one T-B correlation involved the indicated cell specificity. The T-cell specificities of only the correlations involving IL-2 and IL-5 are illustrated. The cytokine secreted in the T-cell response is indicated by the color of the bar, and the correlated B-cell specificity is indicated by the number next to the bar. [In a few cases, a single T-cell specificity and cytokine correlated with two B-cell specificities.] The B-cell specificities of all correlations (involving 9 cytokines or proliferation) are illustrated. Negative values indicate negative correlations. The line plots indicate epitope dominance as the number of responding mice. A positive T-cell response was identified by log[IL-2] greater than 2×SD for unstimulated wells, and a positive B-cell response was identified by A450 greater than 0.2. The analysis of correlation was limited to dominant T-cell and B-cell responses (see text). Positive correlations were more frequent than negative correlations. Many T-B pairings were observed in multiple mouse groups, e.g., T5–B7 in PBS-treated and IFA-treated groups and T39-B7 in IFA-treated and control-primed groups. Additional examples are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065748#pone-0065748-t001" target="_blank">Table 1</a>.</p

Aging and the negative geotaxis response (NGR) of adult Drosophila.

<p>The NGR of outcrossed wild-type control male and female flies (<i>w¹¹¹⁸/+</i>) was used to determining changes in average climbing index (CI, distance traveled within 5 seconds) between the ages of 1 and 4-weeks. *** P ≤ 0.001. See <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132768#pone.0132768.s001" target="_blank">S1 Fig</a></b> for the design of the NGR apparatus and additional details.</p

Aging and Autophagic Function Influences the Progressive Decline of Adult Drosophila Behaviors

<div><p>Multiple neurological disorders are characterized by the abnormal accumulation of protein aggregates and the progressive impairment of complex behaviors. Our Drosophila studies demonstrate that middle-aged wild-type flies (WT, ~4-weeks) exhibit a marked accumulation of neural aggregates that is commensurate with the decline of the autophagy pathway. However, enhancing autophagy via neuronal over-expression of <i>Atg8a</i> (Atg8a-OE) reduces the age-dependent accumulation of aggregates. Here we assess basal locomotor activity profiles for single- and group-housed male and female WT flies and observed that only modest behavioral changes occurred by 4-weeks of age, with the noted exception of group-housed male flies. Male flies in same-sex social groups exhibit a progressive increase in nighttime activity. Infrared videos show aged group-housed males (4-weeks) are engaged in extensive bouts of courtship during periods of darkness, which is partly repressed during lighted conditions. Together, these nighttime courtship behaviors were nearly absent in young WT flies and aged Atg8a-OE flies. Previous studies have indicated a regulatory role for olfaction in male courtship partner choice. Coincidently, the mRNA expression profiles of several olfactory genes decline with age in WT flies; however, they are maintained in age-matched Atg8a-OE flies. Together, these results suggest that middle-aged male flies develop impairments in olfaction, which could contribute to the dysregulation of courtship behaviors during dark time periods. Combined, our results demonstrate that as Drosophila age, they develop early behavior defects that are coordinate with protein aggregate accumulation in the nervous system. In addition, the nighttime activity behavior is preserved when neuronal autophagy is maintained (Atg8a-OE flies). Thus, environmental or genetic factors that modify autophagic capacity could have a positive impact on neuronal aging and complex behaviors.</p></div

Enhanced neural autophagy rescues male nighttime wakefulness.

<p>1 and 4-week old group-housed <i>Appl-Gal4/+</i>, <i>UAS-GFP-Atg8a/+</i> and <i>Appl-Gal4/ UAS-GFP-Atg8a</i> (Atg8a-OE) transgenic male flies were assayed using standard LD conditions. Yellow and black bars indicate day (8:00am to 8:00pm) and night (8:00pm to 8:00am) time periods, respectively. Activity is presented as an average per individual fly. (<b>A-B</b>) The average activity profiles of group-housed male flies at 1-week. (<b>C-D</b>) The average activity profiles of group-housed male flies at 4-weeks. **P ≤ 0.01 and *** P ≤ 0.001.</p