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Narrowed TCR repertoire and viral escape as a consequence of heterologous immunity - PubMed

. 2006 May;116(5):1443-56.

doi: 10.1172/JCI27804. Epub 2006 Apr 13.

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Narrowed TCR repertoire and viral escape as a consequence of heterologous immunity

Markus Cornberg et al. J Clin Invest. 2006 May.

Erratum in

  • J Clin Invest. 2007 May;117(5):1450

Abstract

Why some virus-specific CD8 TCR repertoires are diverse and others restricted or "oligoclonal" has been unknown. We show here that oligoclonality and extreme clonal dominance can be a consequence of T cell cross-reactivity. Lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PV) encode NP(205-212) epitopes that induce different but highly cross-reactive diverse TCR repertoires. Homologous viral challenge of immune mice only slightly skewed the repertoire and enriched for predictable TCR motifs. However, heterologous viral challenge resulted in a narrow oligoclonal repertoire with dominant clones with unpredictable TCR sequences. This shift in clonal dominance varied with the private, i.e., unique, specificity of the host's TCR repertoire and was simulated using affinity-based computer models. The skewing differences in TCR repertoire following homologous versus heterologous challenge were observed within the same private immune system in mice adoptively reconstituted with memory CD8 T cell pools from the same donor. Conditions driving oligoclonality resulted in an LCMV epitope escape variant in vivo resembling the natural Lassa virus sequence. Thus, T cell oligoclonality, including extremes in clonal dominance, may be a consequence of heterologous immunity and lead to viral escape. This has implications for the design of peptide-based vaccines, which might unintentionally prime for skewed TCR responses to cross-reactive epitopes.

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Figures

Figure 1
Figure 1. CD8 T cells specific to LCMV NP205 and PV NP205 show widespread cross-reactivity at effector level.

(A) Intracellular IFN-γ assay. Splenocytes from day 8 LCMV- or PV-infected B6 mice were stimulated with the indicated peptides. Shown are gated CD8 cells stained for CD44 (x axis) and IFN-γ (y axis). Numbers in the upper-left corners represent the percentage of CD8-producing IFN-γ. The background IFN-γ response was less than 0.1%. This is representative of 3 experiments with 2–5 mice/group. (B) Double-tetramer staining. Splenocytes were stained with anti-CD8 and the indicated tetramers. Numbers represent the percentage of gated CD8 T cells binding tetramers. This is representative of 2 experiments with 3 mice/group. (C and D) Peptide titration. Production of IFN-γ by splenocytes (n = 3) in response to different concentrations of LCMV NP205 or PV NP205 peptide. (C) IFN-γ responses plotted as a percentage of the maximal response (% of max). IFN-γ responses without peptide stimulation were subtracted from each value. Differences between LCMV NP205 and PV NP205 were statistically significant at a peptide concentration of 10–11 M (P = 0.006). FACS plots of data for a representative mouse of 3 with stimulation of 10–6 and 10–11 M of the indicated peptide are shown in D. Numbers recorded in the upper-left corners represent the percentage of gated CD8 T cells producing IFN-γ.

Figure 2
Figure 2. Differences in the TCR Vβ repertoire of CD8 T cells specific to LCMV NP205 and PV NP205 .

(A) Splenocytes from day 8 LCMV- or PV-infected mice (n = 8) were stimulated with LCMV NP205, or PV NP205 peptides in an ICS assay and then stained with Vβ-specific mAbs. The percentage of Vβ usage was calculated after gating on the IFN-γ–positive CD8 T cell population. The percentage of other Vβ (Vβ15–18 were not included in the antibody pool) was calculated by subtracting the sum of Vβ2–14 from 100%. (B and C) TCR Vβ mRNA expression of LCMV NP205–specific CD8 T cells. LCMV NP205 tetramer–positive CD8 T cells were sorted from PBMCs 8 days after LCMV infection, and RNA was isolated. RT-PCR was performed with specific primers for Vβ1–18 (B). Spectratype analysis with specific primers for the indicated Vβ families (C). (D) The TCR Vβ16 repertoire of acute LCMV–infected mice is diverse. This shows the CDR3 amino acid sequence and the frequency of each unique Vβ16 LCMV NP205–sorted CD8 T cell clone represented in B. Clones with the same amino acid sequence that are plotted more than once have a different nucleic acid sequence. T cell clones with an XGGX-Jβ2.5 (QDTQY-F) motif dominate the response. (E) The TCR Vβ16 repertoire of acute PV–infected mice is diverse. (F) The TCR Vβ5.1 repertoire of acute PV–infected mice is diverse.

Figure 3
Figure 3. Variability and skewing of NP205 -specific T cells dependent on private specificity after heterologous infection.

(A) TCR Vβ repertoire of PV NP205 tetramer–positive cells of 2 PV-immune mice 8 days after LCMV challenge (PV+LCMV). (B and C) Double-tetramer staining and TCR Vβ5.1,5.2 analysis. (B) Splenocytes from day 8 PV-infected LCMV-immune mice (LCMV+PV) stained with CD8 and tetramers. Numbers in upper-right corners represent the percentage of gated CD8 T cells binding LCMV NP205 and PV NP205 tetramers; representative of 3 experiments. (C) Histograms of Vβ5.1,5.2-positive cells after gating on CD8 and double tetramer–positive cells. (D and E) Private specificity of NP205 response. Splenocytes from individual LCMV-immune donor mice were transferred into 3 congenic recipients. (D) Numbers in upper-right corners represent the percentage of gated CD8 T cells binding both tetramers 8 days after PV infection. (E) TCR Vβ repertoire of LCMV NP205-specific CD8 T cells in LCMV-immune mice before and 8 days after PV infection. Percentage of Vβ usage was calculated after gating on the IFN-γ–positive CD8 T cell population. The percentage for other Vβ was calculated by subtracting the sum of the indicated Vβ families from 100%. Numbers in lower bar graphs represent mean percent of Vβ5.1,5.2 usage (A versus B: P < 0.005; A versus C: P < 0.0002; B versus C: P < 0.03). In B and C, letters A–J represent individual mice. In D, letters A–C represent donor mice, and numbers 1–3 represent recipient mice receiving cells from the specified donor.

Figure 4
Figure 4. Narrowing of the private LCMV NP205 –specific CD8 T cell Vβ repertoire after heterologous PV infection.

(A) PBMCs were isolated from 10 LCMV-immune mice (white bars), and the LCMV NP205-specific CD8 T cells were analyzed either by ICS or by tetramer staining and costaining with Vβ-specific antibodies. The same mice were infected with PV (black bars), and 8 days after infection PBMCs were isolated, and the TCR Vβ repertoire of LCMV NP205 tetramer–positive cells was analyzed. (B and C) The cross-reactive NP205-specific TCR repertoire is oligoclonal after heterologous virus infection. (B) The FACS dot plots show the dominant Vβ usage of the NP205-specific CD8 T cells from 2 representative LCMV+PV–infected mice (mouse 1 [M1] and M8). (C) The LCMV NP205–specific CD8 T cells from M1 and M8 were sorted, and the dominant Vβ family was subcloned and sequenced.

Figure 5
Figure 5. A subset of LCMV NP205 –specific clones is expanded after heterologous PV infection.

PBMCs were from 2 LCMV-immune mice before and 8 days after PV infection. (A and C) LCMV NP205 tetramer–positive CD8 T cells were sorted, and Vβ mRNA expression was analyzed by RT-PCR with specific primers for Vβ1–18. (B, D, and E) The PCR products from dominant Vβ16 (M1, A and B; M2, D) and from Vβ 5.1 (M2, D) were subcloned, and 13–32 clones were sequenced per group.

Figure 6
Figure 6. The LCMV NP205 –specific CD8 T cell repertoire is not skewed after homologous LCMV virus infection.

PBMCs were from LCMV-immune mice before and 8 days after LCMV infection. (A) LCMV NP205 tetramer–positive CD8 T cells were sorted, and Vβ mRNA expression was analyzed by RT-PCR with specific primers for Vβ1–18. (B) The PCR product from dominant Vβ16 was subcloned, and 12–13 clones were sequenced per group. This is a representative experiment of 2.

Figure 7
Figure 7. Homologous versus heterologous infection of a private TCR repertoire.

Splenocytes from an LCMV-immune donor mouse were transferred into 2 congenic recipients. One recipient was infected with LCMV and the other was infected with PV. (A) TCR Vβ repertoire of NP205-specific CD8 T cells. Splenocytes from the LCMV-immune donor and recipient mice 8 days after LCMV or PV infection were stimulated with LCMV NP205 peptide. The percentage of Vβ usage was calculated on the gated IFN-γ–positive CD8 T cell population. Vβ17 served as a negative control. The percentage for other Vβ was calculated by subtracting the sum of the indicated Vβ families from 100%. C57BL/6-A, C57BL/6 donor A. (B) TCR Vβ 5.1,5.2 of LCMV NP205 tetramer–positive CD8 T cells before and after infection. Staining with LCMV NP205 tetramer and Vβ5.1,5.2 antibody was performed on the CD8 LCMV-immune or congenic donor CD8 T cells (LCMV+LCMV, LCMV+PV). The numbers shown above the gates represent the percentage of cells in the gate. The number in the upper-right quadrant (gray box) represents the percentage of LCMV NP205–specific CD8 T cells positive for Vβ5.1,5.2. This NP205-specific Vβ5.1,5.2 frequency was similar in the ICS assay. Data are from 1 of 5 experiments, where Vβ5.1,5.2 proliferated after PV infection. (C) Vβ clonotypes after PV or LCMV infection of mice harboring the same memory pool. Splenocytes from LCMV-immune mice were adoptively transferred into 2 recipient mice, which were then infected with LCMV or PV. This experiment used mice different from those represented in A and B.

Figure 8
Figure 8. Computer simulation of homologous versus heterologous virus challenge.

(A) Left: Clonal distribution of the memory population before (white bars) and after (black bars) a homologous challenge. Right: The same population before (white bars) and after (gray bars) a heterologous challenge. The x axis shows the absolute number of CD8 T cells, while y axis labels indicate hexadecimal representation of each clone involved in the response. Number in brackets on the left of the clone labels represents the place occupied by a particular clone in the immune hierarchy at the end of the primary response. Clones generated after the primary response are ordered according to decreasing cell number. This order is upset when there is a change of hierarchy occurring after the second challenge. rn, random number. (B) Computer simulation of effects of affinity on heterologous virus challenge–induced skewing of the T cell repertoire. Graphs show 2 examples of heterologous challenge in mice selected to possess a memory population with a defined percentage of high/low-affinity cells specific for the challenging virus (10% high/90% low in the left graph, 50% high/50% low in the right graph). White bars represent the memory population after the primary response and before the challenge, while gray bars represent the same population after the heterologous challenge. The x axis shows the size of each clone in terms of absolute cell number. On the y axis labels indicate the affinity of each clone for the heterologous virus: H, high affinity; M, medium affinity; L, low affinity; N, non–cross-reacting (see Methods).

Figure 9
Figure 9. Characterization of the NP205 (V→A) variant.

(A) NP205 epitope sequences from Old World arenaviruses. (B) Peptide MHC stabilization assay. RMA-S cells were incubated with 100 μM of the indicated peptides overnight. The y axis indicates the mean fluorescence intensity (MFI) of the anti-Kb mAb staining. Data are representative of 2 experiments. (C) Peptide titration. IFN-γ production of splenocytes from PV-immune + LCMV mice (n = 3) in response to serial dilutions of LCMV NP205 wild-type and LCMV NP205 (V→A) variant peptides. A representative FACS plot with stimulation of 5,000 nM and 0.5 nM of peptides is shown. Numbers in the upper-left corners represent the percentage of CD44hi T cells producing IFN-γ. IFN-γ responses are plotted as percentage of the maximal response to each peptide stimulation. Differences between LCMV NP205 wild-type and LCMV NP205 (V→A) were statistically significant (P = 0.0045) at 0.5 nM. (D) Virus-induced CD8 T cells. Splenocytes from mice inoculated with either LCMV (n = 2) or LCMV NP205 (V→A) variant virus (n = 3) 8 days after infection were stimulated with the indicated peptides in an intracellular IFN-γ assay. Numbers in the upper-left quadrants represent the percentage of CD8 T cells producing IFN-γ.

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