Stochastic expansions maintain the clonal stability of CD8+ T cell populations undergoing memory inflation driven by murine cytomegalovirus

CMV is an obligate and persistent intracellular pathogen that continually drives the production of highly differentiated virus-specific CD8+ T cells in an Ag-dependent manner, a phenomenon known as memory inflation. Extensive proliferation is required to generate and maintain inflationary CD8+ T cell populations, which are counterintuitively short-lived and typically exposed to limited amounts of Ag during the chronic phase of infection. An apparent discrepancy therefore exists between the magnitude of expansion and the requirement for ongoing immunogenic stimulation. To address this issue, we explored the clonal dynamics of memory inflation. First, we tracked congenically marked OT-I cell populations in recipient mice infected with murine CMV (MCMV) expressing the cognate Ag OVA. Irrespective of numerical dominance, stochastic expansions were observed in each population, such that dominant and subdominant OT-I cells were maintained at stable frequencies over time. Second, we characterized endogenous CD8+ T cell populations specific for two classic inflationary epitopes, M38 and IE3. Multiple clonotypes simultaneously underwent Ag-driven proliferation during latent infection with MCMV. In addition, the corresponding CD8+ T cell repertoires were stable over time and dominated by persistent clonotypes, many of which also occurred in more than one mouse. Collectively, these data suggest that stochastic encounters with Ag occur frequently enough to maintain oligoclonal populations of inflationary CD8+ T cells, despite intrinsic constraints on epitope display at individual sites of infection with MCMV

Transient increase in IL-10-producing cells after natalizumab initiation and high frequency in individuals with PML.

<p>PBMC from all studied MS patients were stimulated with SEB. Background-subtracted frequency of memory CD4 T cells producing IL-10 is shown. <i>P</i> value from month 0 to month 1 is the result of Wilcoxon matched-pairs rank sum test. Panel A shows longitudinal samples from MS patients treated with natalizumab who did not have PML, and Panel B shows samples from 4 individuals with natalizumab-associated PML.</p

T cell responses to JC virus target each viral protein.

<p>PBMC from individuals with MS treated with natalalizumab who did not have PML were stimulated with JCV peptide pools or costimulatory molecules alone (negative control) for 6 hours. Panel A shows memory CD4 T cells from 3 samples; Panel C shows memory CD8 T cells from 3 samples. The fluorescence intensity of IFNγ and TNF are shown on the X and Y-axes, respectively. Panels B and D show baseline pre-treatment responses from all eight longitudinal subjects with MS, with the background-subtracted magnitude of the response to each JCV protein depicted by colored bars. Responses were measured by production of any combination of IFNγ, TNF and IL-2, using Boolean gates and then background subtracting from each Boolean population.</p

Magnitude and functional profile of JCV and CMV-specific T cells do not change upon treatment.

<p>PBMC from individuals with MS treated with natalalizumab who did not have PML were stimulated with JCV peptide pools, CMV pp65 peptide pool or costimulatory molecules alone (negative control) for 6 hours. A response was considered positive if the frequency of memory T cells producing IFNγ, TNF or IL-2 was higher in the peptide-stimulated cells than in those stimulated with costimulatory molecules alone. Response size was calculated by measuring the frequency of cells producing each Boolean combination of cytokines, and subtracting the frequency of these cells in the negative control. Summing the background-subtracted Boolean subsets gave the total frequency of cytokine-producing memory T cells specific for the peptide pool. The total response to JCV (Panel A) was calculated by summing the frequency of cells specific for each of the 5 JCV peptide pools. The functional profile of the response is shown in the pie charts above, with the blue slice representing the proportion of responding cells that produce all 3 cytokines, the red slices representing the proportion of cells that produce a combination of 2 cytokines, and the green slices representing the proportion of cells that produce only 1 cytokine. Panel B shows the frequency of CD4 (left) and CD8 (right) memory T cells responding to the CMV pp65 peptide pool.</p

JCV-specific T cell responses in subjects with PML.

<p>Panel A shows the total response to JCV. For each of 4 subjects with PML, the summed frequency of memory CD4 (left) and CD8 (right) T cells producing IFNγ, TNF, IL-2 or IL-10 (indicated by colors) in response to all JCV peptides is shown. Red bars indicate frequency of cells producing IFNγ, including those that produce any combination of IL-2 and TNF in addition to IFNγ. The responses for subjects PML-1 and PML-2 are not significantly above background in these samples. Panel B shows the JCV-specific IL-10 response in subjects PML-3 and PML-4. Subjects PML-1, 2, 3 and 4 were sampled 2 weeks, 2 months, 4 months and 5 years, respectively, after diagnosis with PML.</p