iNKT rearrangements in TCRα^−/− mice after intrathymic injection of DP^dim cells.

<p>The <i>X axis</i> shows days post injection, and the <i>Y axis</i> shows the percent of mice analyzed that had iNKT rearrangements. A. DN thymocytes were analyzed. The arrows and asterisks indicate statistically significant changes. Fisher’s exact test was used to compute the P value from contingency table (day 1& 3, P = 0.04; day 2&3, P = 0.03). B. Total splenocytes were analyzed. The correlation between the two linear regression lines was calculated based on the Zar’s method (P = 0.008). The actual data for each time point are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043509#pone-0043509-t001" target="_blank">Table 1</a>.</p

A model for the developmental dynamics of DN iNKT cells.

<p>The green box represents the thymus, and the red box the periphery. Filled ovals outlined in black identify cells transferred in the different experiments. Filled arrows and the timing next to them show the experimental findings. Open ovals represents specific compartments. Open arrows and the timing next to them are based on our interpretation. The gel insert shows the iNKT rearrangement in CD4⁺CD8^low and CD8⁺CD4^low cells.</p

Thymocytes with iNKT rearrangements are found in the DP^dim population.

<p>A. FACS profile of thymocytes based on CD4 and CD8 expression and defined gates for DP^dim and DP^med thymocytes. B. FACS profile of thymocytes based on CD3 expression and defined gates for CD3^neg, CD3^med and CD3^hi. C. FACS profile of thymocytes based on CD4 and CD8 expression through CD3^neg and CD3^med populations. Numbers inside DP^dim gates represent the number of collected cells. D. Rearrangement analysis of Vα14-Jα18 in different sorted DP populations. The asterisk shows the population (CD3^med DP^dim) in which iNKT cells are observed, and lane “M” represents the iNKT rearrangement in the spleen of wild type C57BL/6 mouse and serves as size marker.</p

DN iNKT cells can return to the thymus.

<p>DP^dim thymocytes were injected in the thymi of four TCR Cα^−/− mice and 6 days post transfer, the thymi were pooled and DN thymocytes were sorted. A.iNKT rearrangement from DN thymocytes 6 days post transfer of DP^dim. The newly generated DN day 6 thymocytes were transferred to the periphery of TCR Cα^−/− mice. B. iNKT rearrangements in the thymus at different days post peripheral transfer of day 6 DN thymocytes.</p

iNKT rearrangement in DN thymocytes and spleen samples obtained from TCRα^−/− mice at different days post DP^dim injection.

a<p>Data from 7 experiments are summarized.</p

Image_2_Role of cross-reactivity in cellular immune targeting of influenza A M158-66 variant peptide epitopes.jpg

The immunologic significance of cross-reactivity of TCR recognition of peptide:MHC complexes is still poorly understood. We have described TCR cross-reactivity in a system involving polyclonal CD8 T cell recognition of the well characterized influenza viral M158-66 epitope. While M158-66 is generally conserved between influenza A isolates, error-prone transcription generates stable variant RNA during infection which could act as novel epitopes. If packaged and viable, variant genomic RNA generates an influenza quasispecies. The stable RNA variants would generate a new transmissible epitope that can select a specific repertoire, which itself should have cross-reactive properties. We tested two candidate peptides in which Thr65 is changed to Ala (A65) or Ser (S65) using recall responses to identify responding T cell clonotypes. Both peptides generated large polyclonal T cell repertoires of their own with repertoire characteristics and cross-reactivity patterns like that observed for the M158-66 repertoire. Both substitutions could be present in viral genomes or mRNA at sufficient frequency during an infection to drive immunity. Peptides from the resulting protein would be a target for CD8 cells irrespective of virus viability or transmissibility. These data support the hypothesis that cross-reactivity is important for immunity against RNA virus infections.</p

DM mediated peptide exchange as function of reactant concentration.

<p>(a) Requirement of equimolar exchange peptide for initiating exchange. DM-mediated dissociation of the peptide HAS from DR1 measured in the presence of different concentration of unlabeled HA in excess as described in Results. The exchange peptide to complex ratio for each reaction is identified in the legend. Data points represent the mean and SD of three independent experiments, and lines represent the fit of the data to a five parameter double exponential decay function. (b) FP analysis of DM-catalyzed peptide binding to and release from DR. CLIP/DR complex at different concentrations (100, 300, 900 nM) was incubated with 3 fold DM and allowed to dissociate in absence of any free peptide. Simultaneously, loading of FAM-CLIP to an equimolar amount of DR at the same concentrations was measured. Reactions were set up in triplicates, and the average ±SDs are shown. Lines represent the fit of the data to either a five parameter double exponential decay or four parameters double exponential raise function. (c) Peptide release in the absence of exchange peptide is not a function of complex concentration. FP analysis of DM-catalyzed release of HAD from DR at four different concentrations in absence of any free peptide. At <i>t</i> = 1000 after steady state was reached, unlabeled peptide was added at an equimolar concentration to the complex at <i>t</i> = 0. Reactions were set up in triplicates, and the average values for each time point are shown. (d) DM-mediated binding is a function of reactant concentration. FP analysis of DM-catalyzed association of HAD to equimolar empty DR at the same four different concentrations as in panel B. Lines represent the fit of the data to a four parameter double exponential function. For (c) and (d), due to the small SD, error bars are hidden below data points.</p

Table_6_Role of cross-reactivity in cellular immune targeting of influenza A M158-66 variant peptide epitopes.docx

The immunologic significance of cross-reactivity of TCR recognition of peptide:MHC complexes is still poorly understood. We have described TCR cross-reactivity in a system involving polyclonal CD8 T cell recognition of the well characterized influenza viral M158-66 epitope. While M158-66 is generally conserved between influenza A isolates, error-prone transcription generates stable variant RNA during infection which could act as novel epitopes. If packaged and viable, variant genomic RNA generates an influenza quasispecies. The stable RNA variants would generate a new transmissible epitope that can select a specific repertoire, which itself should have cross-reactive properties. We tested two candidate peptides in which Thr65 is changed to Ala (A65) or Ser (S65) using recall responses to identify responding T cell clonotypes. Both peptides generated large polyclonal T cell repertoires of their own with repertoire characteristics and cross-reactivity patterns like that observed for the M158-66 repertoire. Both substitutions could be present in viral genomes or mRNA at sufficient frequency during an infection to drive immunity. Peptides from the resulting protein would be a target for CD8 cells irrespective of virus viability or transmissibility. These data support the hypothesis that cross-reactivity is important for immunity against RNA virus infections.</p

Table_1_Role of cross-reactivity in cellular immune targeting of influenza A M158-66 variant peptide epitopes.xlsx

The immunologic significance of cross-reactivity of TCR recognition of peptide:MHC complexes is still poorly understood. We have described TCR cross-reactivity in a system involving polyclonal CD8 T cell recognition of the well characterized influenza viral M158-66 epitope. While M158-66 is generally conserved between influenza A isolates, error-prone transcription generates stable variant RNA during infection which could act as novel epitopes. If packaged and viable, variant genomic RNA generates an influenza quasispecies. The stable RNA variants would generate a new transmissible epitope that can select a specific repertoire, which itself should have cross-reactive properties. We tested two candidate peptides in which Thr65 is changed to Ala (A65) or Ser (S65) using recall responses to identify responding T cell clonotypes. Both peptides generated large polyclonal T cell repertoires of their own with repertoire characteristics and cross-reactivity patterns like that observed for the M158-66 repertoire. Both substitutions could be present in viral genomes or mRNA at sufficient frequency during an infection to drive immunity. Peptides from the resulting protein would be a target for CD8 cells irrespective of virus viability or transmissibility. These data support the hypothesis that cross-reactivity is important for immunity against RNA virus infections.</p

Table_5_Role of cross-reactivity in cellular immune targeting of influenza A M158-66 variant peptide epitopes.docx

The immunologic significance of cross-reactivity of TCR recognition of peptide:MHC complexes is still poorly understood. We have described TCR cross-reactivity in a system involving polyclonal CD8 T cell recognition of the well characterized influenza viral M158-66 epitope. While M158-66 is generally conserved between influenza A isolates, error-prone transcription generates stable variant RNA during infection which could act as novel epitopes. If packaged and viable, variant genomic RNA generates an influenza quasispecies. The stable RNA variants would generate a new transmissible epitope that can select a specific repertoire, which itself should have cross-reactive properties. We tested two candidate peptides in which Thr65 is changed to Ala (A65) or Ser (S65) using recall responses to identify responding T cell clonotypes. Both peptides generated large polyclonal T cell repertoires of their own with repertoire characteristics and cross-reactivity patterns like that observed for the M158-66 repertoire. Both substitutions could be present in viral genomes or mRNA at sufficient frequency during an infection to drive immunity. Peptides from the resulting protein would be a target for CD8 cells irrespective of virus viability or transmissibility. These data support the hypothesis that cross-reactivity is important for immunity against RNA virus infections.</p