Bi-allelic TCRα and β chain recombination affect thymic selection efficiency.

<p>T cell-depleted bone marrow was collected from WT C57BL/6 (CD45.1 and CD90.2) mice and combined with that of either TCRα^+/-, TCRβ^+/-, or TCRα^+/- TCRβ^+/- (all CD45.2 and CD90.2) mice in equal proportions and injected into lethally-irradiated C57BL/6 CD90.1 mice. Thymus, spleen, and lymph nodes were collected 10 weeks following transplantation. The ratios of WT:TCRα^+/-, WT:TCRβ^+/-, and WT:TCRα^+/- TCRβ^+/- (CD45.1:CD45.2) cells were determined by flow cytometric analysis of A) thymocytes at the indicated developmental stages and B) peripheral lymphocyte subsets. All ratios are adjusted to the peripheral B cell CD45.1:CD45.2 ratio in each mouse, as an internal control. The data plotted include the mean and SEM (n = 7–8 mice/group). Student’s <i>t</i>-test was used to determine <i>p</i> value, relative to the WT:WT control. *, **, and *** indicate <i>p</i><0.05, <i>p</i><0.01, and <i>p</i><0.001 respectively. Student’s <i>t</i>-test was used to determine <i>p</i> values of CD45.1:CD45.2 ratios pre/post α and β (DP_pre/DP_post and DN1/DP_pre respectively). ††† indicates <i>p</i><0.001 for pre/post selection comparisons.</p

The peripheral TCR repertoire remains diverse despite the absence of bi-allelic TCR rearrangements.

<p>TCRβ chain sequencing was performed on DNA purified from T cell-enriched splenocytes from WT and single TCR T cell mice. A) The number of unique productive TCRβ sequences in WT and single TCR T cell mice are similar (n = 3/genotype). Individual <i>Trbv</i> usage in B) productively and C) non-productively rearranged WT and single TCR T cell mice is displayed as a percentage of total unique sequences ranging from ~11,000 to ~35,000 sequences per mouse (n = 3/genotype). Results shown are mean +SEM. Two way ANOVA with Bonferroni posttest with a 95% confidence interval was used to determine <i>p</i> value. ** and *** indicate <i>p</i><0.01 and <i>p</i><0.001 respectively.</p

Bi-allelic TCR rearrangements do not alter self antigen-specific T cell repertoire.

<p>Splenocytes and lymph nodes were collected from naïve and MOG_35-55- or PLP_178-191-immunized WT and single TCR T cell mice. Cells were enriched for antigen-specific populations using tetramers and magnetic cell sorting. Antigen specific T cells were then stained and analyzed by flow cytometry. A) Representative flow cytometry plots and B) numbers of MOG_38-49:A^b- specific T cells in naïve (left) and immunized (right) WT and single TCR T cell mice. C) Representative flow cytometry plots and D) number of PLP_178-191:A^b-specific populations in naïve (left) and immunized (right) WT and single TCR T cell mice. C) Dual tetramer staining was used to identify the PLP_178-191:A^b-specific cells in naïve WT and single TCR T cell mice due to their rarity. In B and D, each symbol represents one mouse. No statistically significant differences between the two genotypes were present at either of the time points (Student’s t-test). Flow plots are in Log10 fluorescence scale.</p

Characterization of single TCR T cell mice.

<p>A) Splenocytes were collected from WT and single TCR T cell C57BL/6 mice and analyzed by flow cytometry for co-expression of Vα2 with Vα3.2 or Vα8.3 (top panels) or co-expression of Vβ6 with any of a panel of fourteen other Vβ proteins (bottom panels) in CD3⁺ CD4⁺ T cells. Representative flow plots from three independent experiments show the presence of dual TCRα and β populations in WT (boxed populations in left panels) that are absent in single TCR T cell mice (right panels). B) Flow cytometric analysis of splenocytes from WT and single TCR T cell mice reveals equivalent CD3 expression among CD4⁺ and CD8⁺ T cells. C) Developmental T cell stages (left) and peripheral T cell subsets (right) from WT and single TCR T cell mice were analyzed and enumerated by flow cytometry (n = 6). D) The number of γδ T cells in the lymph nodes and spleen of adult WT or single TCR T cell mice as determined by flow cytometry (B220^-, CD11b^-, CD11c^-, CD3⁺ and TCRγδ⁺). Results shown are mean +SEM (n = 3 for TCRα^+/- and TCRα^+/- TCRβ^+/-, n = 2 for TCRβ^+/-, and n = 6 for WT). Student’s <i>t</i>-test was used to determine <i>p</i> values; ***p<0.001. Flow cytometry plots are in Log10 fluorescence scale.</p

Bi-allelic TCR rearrangements do not impact the foreign antigen TCR repertoire.

<p>Splenocytes and lymph node cells were collected from naïve, 2W1S-, B8R-, or gp33-peptide immunized WT and single TCR T cell mice, followed by magnetic tetramer enrichment of antigen-specific populations. Antigen-specific T cells were then stained and analyzed by flow cytometry. A, C, E) Representative plots of naïve (left) and immunized (right) WT and single TCR T cell mice showing 2W1S:A^b-, B8R:K^b-, or gp33:D^b-specific populations respectively. B, D, F) Numbers antigen-specific T cells in naïve and antigen immunized WT and single TCR T cell mice. Each symbol represents one mouse. No statistically significant differences between the two genotypes were present at either of the time points (Student’s t-test). Flow plots are in Log10 fluorescence scale.</p

Dual TCR T cells are not required for initiation of EAE.

<p>EAE was induced in WT and single TCR T cell mice by immunization with 200 μg MOG_35-55 or 50 μg PLP_178-191 plus adjuvant. EAE severity scores were determined daily for up to 21 days post injection. A and B) Average EAE scores of MOG_35-55- and PLP_178-191- induced EAE in WT and single TCR T cell mice over time. Data points are depicted as the mean +/- SEM. One-way ANOVA was used to determine <i>p</i> values (n = 12-15/group). C and D) Percentage of WT or single TCR T cell mice with an EAE score equal to or greater than 1 in MOG_35-55- and PLP_178-191- induced EAE. The <i>p</i> values for Kaplan-Meier curves were calculated using log rank test with Prism software (GraphPad); no significant statistical difference was observed.</p

The Incubation Period of Primary Epstein-Barr Virus Infection: Viral Dynamics and Immunologic Events

<div><p>Epstein-Barr virus (EBV) is a human herpesvirus that causes acute infectious mononucleosis and is associated with cancer and autoimmune disease. While many studies have been performed examining acute disease in adults following primary infection, little is known about the virological and immunological events during EBV’s lengthy 6 week incubation period owing to the challenge of collecting samples from this stage of infection. We conducted a prospective study in college students with special emphasis on frequent screening to capture blood and oral wash samples during the incubation period. Here we describe the viral dissemination and immune response in the 6 weeks prior to onset of acute infectious mononucleosis symptoms. While virus is presumed to be present in the oral cavity from time of transmission, we did not detect viral genomes in the oral wash until one week before symptom onset, at which time viral genomes were present in high copy numbers, suggesting loss of initial viral replication control. In contrast, using a sensitive nested PCR method, we detected viral genomes at low levels in blood about 3 weeks before symptoms. However, high levels of EBV in the blood were only observed close to symptom onset–coincident with or just after increased viral detection in the oral cavity. These data imply that B cells are the major reservoir of virus in the oral cavity prior to infectious mononucleosis. The early presence of viral genomes in the blood, even at low levels, correlated with a striking decrease in the number of circulating plasmacytoid dendritic cells well before symptom onset, which remained depressed throughout convalescence. On the other hand, natural killer cells expanded only after symptom onset. Likewise, CD4+ Foxp3+ regulatory T cells decreased two fold, but only after symptom onset. We observed no substantial virus specific CD8 T cell expansion during the incubation period, although polyclonal CD8 activation was detected in concert with viral genomes increasing in the blood and oral cavity, possibly due to a systemic type I interferon response. This study provides the first description of events during the incubation period of natural EBV infection in humans and definitive data upon which to formulate theories of viral control and disease pathogenesis.</p></div

Viral genome detection during the incubation period.

<p>Quantitative viral load was determined by qPCR using DNA from oral wash cell pellets (A) or blood (B). Data are expressed as Log₁₀ viral genome copies/mL of sample. The dashed gray line represents the limit of detection. (C) and (D) show the time to the first positive measurement for each subject for viral genomes detected in the blood (D) by non-quantitative nested PCR (filled squares) or qPCR (filled inverted triangles), or in the oral cavity (C) by nested or qPCR (same results were obtained with both assays) (open circles). The theoretical presence of virus shown in (C) is the estimated time period in which study participants were initially exposed to oral virus. (E) In sequential samples from the incubation period, subjects were scored for which compartment viral genomes were first detected in: blood by nested PCR (blood (lo)), blood by qPCR (blood (hi)), oral, or a simultaneous positive in both compartments. (F) Shows an inset comparing blood and oral cavity for the time period close to symptom onset. The results for twenty-six subjects who had a sample collected within the first two weeks of symptom onset are shown.</p

Plasmacytoid DC declined in the circulation during the incubation period and remained depressed through convalescence.

<p>(A) Representative flow cytometry plots of pDC frequencies amongst non-lymphoid cells (CD3, CD56, CD14, CD20 negative) from samples collected at multiple timepoints for one subject (5524). (B) The percentage of pDC from 5524 over time. (C) Frequencies of pDC over time are shown for all subjects. (D) Frequencies of conventional DC (cDC) (CD11c⁺, HLA-DR⁺ cells) are shown over time for all subjects. (E) Numbers of pDC per mL of whole blood are shown for all subjects. (F) shows the percentage of pDC in samples where viral genomes were detected in the blood by nested PCR (Blood lo) or qPCR (Blood hi). Statistical analysis was performed using a one-way ANOVA with multiple test comparison. Light pink symbols indicate a significant difference (p<0.05) compared to pre-infection; darker pink symbols (p<0.001); red symbols (p<0.0001). Gray symbols indicate no statistical difference.</p

CD4⁺ Foxp3⁺ T cells transiently decline in the circulation at symptom onset during AIM.

<p>(A) Frequency of Foxp3⁺CD25⁺ cells amongst total CD4⁺ T cells data plotted over time for a representative individual (subject 7112). (B) Normalized frequency of Foxp3⁺ CD4 T cells over time in all subjects. Foxp3⁺ frequencies were normalized to each subject’s pre-infection baseline due to substantial variation in this population between individuals. (C) Number of CD4+ T cells per mL of whole blood over time. Statistics were performed using a one-way ANOVA with multiple test comparison. Gray values are not statistically different. Red value p<0.0001 compared to pre.</p