Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Jun;28(6):847-58.
doi: 10.1016/j.immuni.2008.04.018.

Selective CD4+ T cell help for antibody responses to a large viral pathogen: deterministic linkage of specificities

Alessandro Sette  1 , Magdalini Moutaftsi, Juan Moyron-Quiroz, Megan M McCausland, D Huw Davies, Robert J Johnston, Bjoern Peters, Mohammed Rafii-El-Idrissi Benhnia, Julia Hoffmann, Hua-Poo Su, Kavita Singh, David N Garboczi, Steven Head, Howard Grey, Philip L Felgner, Shane Crotty
Affiliations

Selective CD4+ T cell help for antibody responses to a large viral pathogen: deterministic linkage of specificities

Alessandro Sette et al. Immunity. 2008 Jun.

Abstract

Antibody responses are critical components of protective immune responses to many pathogens, but parameters determining which proteins are targeted remain unclear. Vaccination with individual MHC-II-restricted vaccinia virus (VACV, smallpox vaccine) epitopes revealed that CD4(+) T cell help to B cells was surprisingly nontransferable to other virion protein specificities. Many VACV CD4(+) T cell responses identified in an unbiased screen targeted antibody virion protein targets, consistent with deterministic linkage between specificities. We tested the deterministic linkage model by efficiently predicting new vaccinia MHC II epitopes (830% improved efficiency). Finally, we showed CD4(+) T cell help was limiting for neutralizing antibody development and protective immunity in vivo. In contrast to the standard model, these data indicate individual proteins are the unit of B cell-T cell recognition for a large virus. Therefore, MHC restriction is a key selective event for the antiviral antibody response and is probably important for vaccine development to large pathogens.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Vaccinia Virus CD4+ helper T cells and helper T cell dependent antibodies
(A) Quantitative ELISA of anti-VACV IgG (µg/ml), day 30 post-infection. Anti-VACV IgG is absent in MHCII−/− mice. “WT”, wildtype B6 mice. (B) Splenocytes from day 10 VACVWR infected mice were incubated with CD11c+ dendritic cells (DCs) pulsed with VACV peptides. Cells were incubated for 6 hrs and then stained for flow cytometry. Gated CD4+ CD62Llo lymphocytes are shown, stained for intracellular IFNγ and CD40L. DCs infected with VACVWR (MOI=5) for 2h prior addition of splenocytes were used to identify the total anti-VACV CD4+ T cell response (bottom right, “VV+ DCs”). Background levels were determined using uninfected DCs (“neg”). Low frequency A28-specific response was only detectable by ELISPOT (not shown).
Figure 2
Figure 2. Selective protein-specific CD4 T cell help to B cells after VACV infection
Mice peptide vaccinated with VACV I121–35 MHCII epitope were then infected with vaccinia virus. (A) Vaccinia-specific IgG responses in VACV infected mice primed with adjuvant alone (“mock prime”, open circles), I1-primed mice subsequently infected with VACV (squares), I1-primed only mice (X symbol), and uninfected control mice (+ symbol) measured by ELISA. n = 4/group. Error bars ± SEM. (B) Virus neutralizing antibody titers (PRNT50) were unimproved in I1-primed mice (P ≫ 0.05). I121–35 MHCII peptide primed mice (“+”, n = 4) and mock primed mice (“—”, adjuvant only prime. n = 4), tested after VACVWR infection. (C–D) Sets of VACV proteins were synthesized and printed in microarray format to generate VACV proteome arrays that could be probed with serum samples (see Methods). VACV proteome microarrays were probed with serum day 7 post-VACV infection from representative mice (C) primed with I121–35 or (D) mock primed. Star indicates anti-I1 signal. Error bar indicates range of replicates. (E–H) Antibody responses to individual vaccinia virus protein determinants after VACVWR infection in I121–35 MHCII peptide primed mice (“+”, n = 4) and mock primed mice (“—”, adjuvant only prime. n = 4). (E) Quantitation of anti-I1 IgG revealed 19.3-fold increase in primed mice (P < 0.0004). (F) Anti-A10 IgG (P ≫ 0.05), (G) Anti-D8 IgG (P ≫ 0.05), and (H) Anti-H3 IgG (P ≫ 0.05) levels were unchanged. (I) IgG responses in I1 primed and not primed mice at day 30 after VACV infection. Anti-I1 IgG was selectively increased (P < 0.02), while anti-A10 and anti-D8 responses were unchanged (P ≫ 0.05). *, P < 0.05. ** P < 0.01. ***, P < 0.001. Data is representative of multiple independent experiments.
Figure 3
Figure 3. CD4 T cell dependent MHCII restricted help for VACV antibody response
(A) CD4 T cell adoptive transfers, experiment schematic. Mice were immunized with a VACV MHCII-binding peptide, I121–35. CD4 T cells were purified from donor mice and transferred to unimmunized mice. Recipient mice were then infected with VACV. (B–C) Quantitative analysis of IgG antibody responses to individual VACV proteins in representative mice that (B) received I121–35 primed CD4 T cells, or (C) did not received primed CD4 T cells. Star indicates anti-I1 IgG. (D) Anti-I1 IgG response was increased 1210% in I121–35 MHCII peptide primed CD4 T cell recipient mice (“+”. P < 0.0001, n = 3. Control mice “—”, n = 4), while the (E) anti-A10 and (F) anti-D8 IgG responses were unimproved. Data are representative of two experiments.
Figure 4
Figure 4. Highly selective CD4 T cell help to B cells specific for VACV virion components
Antiviral antibody responses in mice immunized with vaccinia virus H3272–286, D8238–252, or L4176–190 peptide MHC II epitopes and then infected with VACV. (A) Vaccinia-specific IgG responses, measured by ELISA, in VACV infected mice primed with adjuvant alone (“mock primed,” open circles), or H3 primed (closed circles), or D8 primed (closed squares), or L4 primed (closed diamonds) mice subsequently infected with VACV. Peptide primed only mice (X symbol) and untreated uninfected mice (+ symbol) served as controls. n = 4/group. Error bars ± SEM. (B) Left panel: Anti-H3 IgG response after VACV infection was increased 48-fold in H3272–286 MHCII peptide primed mice (“+”. P < 0.0001, n = 11. Adjuvant only “mock primed” mice, “—”. n = 12. Composite data from three independent experiments). Right panel: The anti-A10 response was unchanged (P ≫ 0.05). (C) Virus neutralizing antibody titers (PRNT50) were increased in H3-primed mice. (D) Anti-D8 IgG response was unchanged in H3272–286 primed mice (P ≫ 0.05). (E) Anti-D8 IgG response was increased 2.2-fold in D8238–252 MHCII peptide primed mice (primed, “+”. P < 0.04, n = 4. Adjuvant only “mock primed” mice, “—”. n = 4), while (right panel) the anti-A10 response was unchanged (P ≫ 0.05). (F) Anti-L4 IgG response was increased 3.2-fold in L4176–190 MHCII peptide primed mice (primed, “+”, n = 4. Adjuvant only “mock primed” mice, “—”. n = 4), while (right panel) the anti-A10 response was unchanged. (G) Vaccinia-specific IgM responses, measured by ELISA in VACV infected mice (open circles), or H3 primed (closed circles) or I1 primed (closed squares) or D8 primed (closed triangles) or L4 primed (closed diamonds) mice subsequently infected with VACV. CFA primed only mice (+ symbol) served as controls. n = 4/group. (H) Anti-H3 IgM response (left panel) was increased in H3272–286 primed mice (P < 0.0019), while (right panel) the anti-A10 IgM response was unchanged (P ≫ 0.05). *, P < 0.05. ** P < 0.01. ***, P < 0.001. Data is representative of multiple independent experiments.
Figure 5
Figure 5. Interrelationship between anti-VACV CD4 T cell and antibody responses in virus infected mice
(A–C) Quantitative analysis of IgG antibody responses to individual VACV proteins in two representative VACV-infected B6 mice (A–B) and one uninfected mouse (C), as measured by proteomic microarray (RU, relative fluorescence units). Stringent limit of detection is indicated by dashed line. Representative of > 20 animals. (D) Representative immunofluorescence microarray scan of VACV protein microarray probed with sera from an uninfected mouse versus a VACV infected mouse. Each VACV protein is present as duplicate spots. (E) Tabulation of interrelationship between the antiviral CD4 T cell targets (columns) and antibody targets (rows). Matched CD4 T cell and antibody specificities are indicated in red. Specificities are ranked roughly in descending order based on immunodominance / strength of response (T cell targets, left to right. Antibody targets, top to bottom). 17 IgG targets were identified in the majority of infected mice (shown), and variable IgG responses were also seen to minor antigens F9, I3, A56, A17, A13, and WR149 in some infected mice (not shown, but included in the statistical analysis to be conservative). B cell specificities subsequently selected for prediction of CD4 T cell responses are highlighted in yellow.
Figure 6
Figure 6. Utilization of antibody specificities to predict new vaccinia virus protein targets of CD4 T cell responses, and test of protective immunity induction in vivo
(A) IFNγ and CD40L intracellular staining for VACV B246–60, B546–60, A33116–130, and A466–80 -specific CD4 T cells at day 10 after VACV infection. Gated on CD4+ CD62Llo lymphocytes. “Neg” = no peptide control. As negative controls, two virion proteins that are not IgG targets were also selected, A9 and D3. No CD4 T cell responses were detected to control ORFs A9 and D3 (data not shown). (B) Tabulation of the full set of discovered interrelationships between the antiviral CD4 T cell targets (columns) and antibody targets (rows). 11 of 18 CD4 T cell responses (highlighted in pink) are matched by IgG responses to the same smallpox vaccine virus protein. Specificities are ranked and marked as described above. (C) Antibody responses to B5 (left) and VACV MV (right) proteins after VACVWR infection of B546–60 MHCII peptide primed mice (“+”) and mock primed mice (“—”, adjuvant only prime). (D) Weight loss in B6 mice infected intranasally with VACVWR. Groups were primed to generate B5-specific CD4 T cells (closed circles, n = 8), H3- specific CD4 T cells (squares, n = 8), or primed with CFA adjuvant alone (n = 8) prior to challenge. (E) Weight loss in B cell deficient mice (µMT) infected intranasally with VACVWR. Experiment done concurrently with that of panel D. N = 4 per group. (F) Survival curves in B5 CD4 T cell primed (left) and H3 CD4 T cell primed (right) C57BL/6 (“WT”) and B cell deficient mice after VACVWR challenge.
Figure 6
Figure 6. Utilization of antibody specificities to predict new vaccinia virus protein targets of CD4 T cell responses, and test of protective immunity induction in vivo
(A) IFNγ and CD40L intracellular staining for VACV B246–60, B546–60, A33116–130, and A466–80 -specific CD4 T cells at day 10 after VACV infection. Gated on CD4+ CD62Llo lymphocytes. “Neg” = no peptide control. As negative controls, two virion proteins that are not IgG targets were also selected, A9 and D3. No CD4 T cell responses were detected to control ORFs A9 and D3 (data not shown). (B) Tabulation of the full set of discovered interrelationships between the antiviral CD4 T cell targets (columns) and antibody targets (rows). 11 of 18 CD4 T cell responses (highlighted in pink) are matched by IgG responses to the same smallpox vaccine virus protein. Specificities are ranked and marked as described above. (C) Antibody responses to B5 (left) and VACV MV (right) proteins after VACVWR infection of B546–60 MHCII peptide primed mice (“+”) and mock primed mice (“—”, adjuvant only prime). (D) Weight loss in B6 mice infected intranasally with VACVWR. Groups were primed to generate B5-specific CD4 T cells (closed circles, n = 8), H3- specific CD4 T cells (squares, n = 8), or primed with CFA adjuvant alone (n = 8) prior to challenge. (E) Weight loss in B cell deficient mice (µMT) infected intranasally with VACVWR. Experiment done concurrently with that of panel D. N = 4 per group. (F) Survival curves in B5 CD4 T cell primed (left) and H3 CD4 T cell primed (right) C57BL/6 (“WT”) and B cell deficient mice after VACVWR challenge.
Figure 6
Figure 6. Utilization of antibody specificities to predict new vaccinia virus protein targets of CD4 T cell responses, and test of protective immunity induction in vivo
(A) IFNγ and CD40L intracellular staining for VACV B246–60, B546–60, A33116–130, and A466–80 -specific CD4 T cells at day 10 after VACV infection. Gated on CD4+ CD62Llo lymphocytes. “Neg” = no peptide control. As negative controls, two virion proteins that are not IgG targets were also selected, A9 and D3. No CD4 T cell responses were detected to control ORFs A9 and D3 (data not shown). (B) Tabulation of the full set of discovered interrelationships between the antiviral CD4 T cell targets (columns) and antibody targets (rows). 11 of 18 CD4 T cell responses (highlighted in pink) are matched by IgG responses to the same smallpox vaccine virus protein. Specificities are ranked and marked as described above. (C) Antibody responses to B5 (left) and VACV MV (right) proteins after VACVWR infection of B546–60 MHCII peptide primed mice (“+”) and mock primed mice (“—”, adjuvant only prime). (D) Weight loss in B6 mice infected intranasally with VACVWR. Groups were primed to generate B5-specific CD4 T cells (closed circles, n = 8), H3- specific CD4 T cells (squares, n = 8), or primed with CFA adjuvant alone (n = 8) prior to challenge. (E) Weight loss in B cell deficient mice (µMT) infected intranasally with VACVWR. Experiment done concurrently with that of panel D. N = 4 per group. (F) Survival curves in B5 CD4 T cell primed (left) and H3 CD4 T cell primed (right) C57BL/6 (“WT”) and B cell deficient mice after VACVWR challenge.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Amanna IJ, Slifka MK, Crotty S. Immunity and immunological memory following smallpox vaccination. Immunol Rev. 2006;211:320–337. - PubMed
    1. Bachmann MF, Zinkernagel RM. Neutralizing antiviral B cell responses. Annu Rev Immunol. 1997;15:235–270. - PubMed
    1. Balazs M, Martin F, Zhou T, Kearney J. Blood dendritic cells interact with splenic marginal zone B cells to initiate T-independent immune responses. Immunity. 2002;17:341–352. - PubMed
    1. Batista FD, Iber D, Neuberger MS. B cells acquire antigen from target cells after synapse formation. Nature. 2001;411:489–494. - PubMed
    1. Belyakov IM, Earl P, Dzutsev A, Kuznetsov VA, Lemon M, Wyatt LS, Snyder JT, Ahlers JD, Franchini G, Moss B, Berzofsky JA. Shared modes of protection against poxvirus infection by attenuated and conventional smallpox vaccine viruses. Proc Natl Acad Sci U S A. 2003;100:9458–9463. - PMC - PubMed

Publication types

MeSH terms

-