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Eur J Immunol. Author manuscript; available in PMC 2017 July 01. Published in final edited form as: Eur J Immunol. 2016 July ; 46(7): 1587–1591. doi:10.1002/eji.201646500.
CD28 Days Later: Resurrecting Costimulation for CD8+ Memory T Cells Verena van der Heide1,2 and Dirk Homann1,2 1Diabetes,
Obesity and Metabolism Institute, Icahn School of Medicine at Mount Sinai, New York,
NY
Author Manuscript
2Immunology
Institute, Icahn School of Medicine at Mount Sinai, New York, NY
Abstract Rapid activation and proliferative expansion of specific CD8+ memory T cells (CD8+TM) upon antigen re-encounter is a critical component of the adaptive immune response that confers enhanced immune protection. In this context, however, the requirements for costimulation in general and CD28 signaling in particular remain incompletely defined. In the current issue of the European Journal of Immunology, Fröhlich et al. now provide definitive evidence that optimal elaboration of CD8+TM recall responses is indeed contingent on CD8+TM-expressed CD28. Here, we discuss the “CD28 costimulation paradigm” in its historical context and highlight some of the unresolved complexities pertaining to CD28-dependent interactions that shape CD8+T cell phenotypes, functionalities and recall reactivity.
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A superficial reading of Thomas Kuhn’s “The Structure of Scientific Revolutions” (1962) [1] might suggest that relevant scientific progress occurs in leaps and bounds, through revolutions small and large, and with abrupt course corrections that topple conventional wisdom in a fashion that in contemporary parlance has degenerated into the concept of “disruption”. But that would distort Kuhn’s ultimately more balanced assessment of the dynamics of scientific knowledge production and disregard the influence of his acknowledged predecessor Ludwik Fleck [2] who emphasized the gradual, discursive nature of scientific practice within and amongst different “thought collectives”. In that sense, Fleck’s 1935 monograph [3], provocatively entitled “Genesis and Development of a Scientific Fact”, might less controversially be labeled “The Structure of Scientific Evolutions”. And so it has come to pass that in the world of T cell costimulation, a “paradigm shift”, two decades in the making, has taken hold: recall responses of CD8+ memory T cells (CD8+TM), once considered “costimulation-independent”, in fact rely on CD28-mediated interactions for their optimal development. The work of Fröhlich et al. in the current issue of the European Journal of Immunology makes this case in a
Correspondence: Dirk Homann, MD, Diabetes, Obesity and Metabolism & Immunology Institutes, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place - Box 1152, New York, NY 10029, ph: 212 241-1935, fax: 212 241-2485, [email protected]. Commentary on Fröhlich et al., “Interrupting CD28 costimulation before antigen re-challenge affects CD8+T-cell expansion and effector functions during secondary response in mice” The authors declare that they do not have any conflict of interest.
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straightforward and compelling manner and thus offers, the provisional nature of all scientific conclusions notwithstanding, some “closure” to an ongoing discussion [4].
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The concept of T cell costimulation is rooted in the 1970 Bretscher-Cohn two-signal model for B cell tolerance [5], its subsequent extension and refinement by Lafferty and Cunningham in 1975 [6], and eventual application to the induction of T cell immunity and tolerance by 1980 [7, 8]. With the discoveries of CD28 and its ligands made over the ensuing decade, the phenomenon of T cell costimulation got its first molecular correlate [9 —11], and a rich tapestry of intricately interwoven insights was established soon after [12— 14]. The notion that secondary, in contrast to primary, T cell responses are CD28independent is largely a product of the late 1990s and its ensuing promotion to the status of a “paradigm” [15] is in hindsight perhaps better characterized as the dissemination of a “widely held belief” owing to the comparatively few studies it was originally based upon (reviewed in refs. [16, 17]). Indeed, differential costimulation requirements for naïve and memory T cells were originally conceived in relative rather than absolute terms [18], and reports emerging from late 1990s onwards began to slowly build momentum, accelerated over the past 10 years, in support of CD28 costimulation as a non-redundant pathway for the effective elaboration of secondary T cell responses ([16, 17]; also summarized for antiviral T cells in ref. [19]). Nearly a dozen studies, including the first decisive report on the importance of CD28 for CD8+TM recall responses by P. Katsikis’ group [20], reached this conclusion by focusing their interrogations on the specific context of secondary T cell immunity using adoptive transfers of in vivo generated wild-type CD8+TM into CD80/86deficient recipients and/or interference with costimulation (CTLA-4 Ig treatment or CD28 blockade) restricted to the recall setting. As with all experimentation, however, the inevitable constraints and limitations of particular model systems had to somewhat temper any urges to proclaim a more general relevance of the observed phenomena. The introduction of mice with a floxed Cd28 gene in 2010 [21] therefore constituted a practical foundation for contemporary models of conditional or induced CD28 deletion that eschew potential artifacts arising from the employment of constitutively immunodeficient mice, adoptive transfers and/or antibody-based costimulation blockade. Though tantalizing clues about CD28-dependent CD8+TM responses were initially offered [21], subsequent investigations were devoted to the regulation of various CD4+T cell responses by CD28. Accordingly, CD28 ablation prior to a re-challenge was shown to limit CD4+TH2-dependent allergic airway disease [22] and to compromise protective CD4+TM responses in a model of parasite infection [23]. In addition, CD28 expression by CD4+T cells was not only required for initial control of bacterial burden (C. rodentium) and effective primary expansion of TH1 and TFH subsets in response to an influenza infection, but also for the subsequent maintenance of TFH [24]; similarly, sustained CD28 expression was found to be critical for CD4+TREG survival and function [25]. Which brings us to the report by Fröhlich et al. [4]. Combining an established infectious disease model (primary and secondary challenge with low- and high-dose, respectively, of recombinant L. monocytogenes expressing ovalbumin [LM-OVA]) with an inducible CD28deficient strain (iCD28ko mice), the authors demonstrate that global CD28 deletion just prior to a re-challenge resulted in a ~4-fold reduction of the OVA-specific CD8+TM recall response (Fig.1A). Complementary experiments in which systemic CD28 blockade was Eur J Immunol. Author manuscript; available in PMC 2017 July 01.
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applied to LM-OVA-immune wild-type (wt) mice only in the context of a secondary LMOVA infection yielded essentially the same results (Fig.1B) and confirm similar observations made earlier for antiviral CD8+TM recall responses [19, 20]; a practical advantage of the present work is the use of a murine non-depleting CD28 antibody that will facilitate future studies considering prolonged CD28 blockade. The most compelling evidence comes from the employment of wt mice seeded with a small number of iCD28ko OT-I cells (TCR transgenic CD8+T cells specific for the OVA257-264 determinant) before primary LM-OVA infection. The subsequent targeting of CD28 deletion in a precise temporal fashion (only before secondary challenge) and cell-specific manner (only OT-I CD8+TM with their fixed TCR specificity/affinity) provides incontrovertible evidence that optimal proliferative expansion of secondary CD8+ effector T cells (CD8+TE) is contingent on available CD28 signaling (Fig.1C).
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The impact of CD28 ablation on other aspects of the specific CD8+TE response is apparently more modest and also more difficult to interpret. Under conditions of systemic CD28 inhibition prior to reinfection (iCD28ko mice or CD28 blockade), secondary CD8+TE exhibited a somewhat reduced capacity for inducible degranulation and IFNγ synthesis (Fig. 1A/B). Yet when CD28 deletion was targeted exclusively to CD8+TM populations in the iCD28ko OT-I system, secondary OT-I CD8+TE featured a trend towards enhanced degranulation and IFNγ production (Fig.1C). The differential outcomes reported by Fröhlich et al. point towards a scenario where multiple T cell populations rely on effective CD28 signaling and thus in concert shape the evolution of optimal secondary CD8+TE responses. For example, consistent with the notion that CD4+TREGs restrain the CD8+TM recall response in the LM-OVA model, CD4 depletion before re-challenge enhanced secondary CD8+TE expansions, and while the increased magnitude of the recall response remained susceptible to effective reduction by concomitant CD28 blockade, the interventions also impaired the elaboration of secondary CD8+TE functions (degranulation/IFNγ production) [4]. To reconcile these findings with the development of slightly improved CD8+TE functionalities in the context of CD28 ablation restricted to OT-I CD8+TM, future experiments will have to carefully differentiate between the specific contributions of CD4+TM and TREG subsets, and may consider the possibility that CD8+T cell populations other than the specific CD8+TM responders exert “helper functions” [26] in a CD28dependent fashion. To complicate the situation even further, targeted deletion of CD28 from OT-I cells before primary challenge also precipitated CD8+TE differentiation at the level of facilitated effector functionalities but “paradoxically” resulted in the generation of more CD127+/KLRG1− “memory precursor effector cells” [4]. These findings are reminiscent of the greater CD62L and CD127 expression by antiviral CD8+TE primed in CD28- or CD80/86-deficient mice and confirm an at least partial uncoupling of primary CD8+TE differentiation at the levels of phenotype, function and numerical expansion in the absence of CD28 costimulation (summarized in ref. [19]). Lastly, Fröhlich et al. found that CD28-deletion in the iCD28ko OT-I model right after the primary response affected neither OT-I CD8+TM maintenance nor recall capacity (though secondary CD8+TE functionalities were again slightly enhanced). The authors plausibly speculate that the CD28-deficient OT-I CD8+TM may have adapted to more effectively harness other costimultory pathways which in the context of a recall response may Eur J Immunol. Author manuscript; available in PMC 2017 July 01.
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compensate for the lack of CD28 [4]. But it is also important to note that the latter experiments were conducted ~3 months after primary challenge rather than the ~1 month interval employed for all other experiments (Fig.1). In fact, the precise timing for the evaluation of recall responses appears to be an underappreciated variable [16, 17] and our own preliminary data indicate that the relative dependence of secondary antiviral CD8+TE reactivity on CD28 costimulation progressively increases with time elapsed after original CD8+TM generation (D.H., unpublished). Reconciling all these observations and resolving seeming inconsistencies will require experiments that will assess the broader molecular, phenotypic and functional properties of the various CD8+T cell populations, rigorously control for CD8+TM numbers and defined recall settings (precise nature, dosage, route of administration and timing of re-challenge protocols), include relevant clinical readouts (e.g., altered immune protection), and consistently explore the many possible permutations of targeted CD28 deletion. And the iCD28ko mouse now offers a suitable model system to do just that.
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Over the past half century, the concept of costimulation has evolved in a spectacular manner. “Signal 2”, initially a theoretical abstraction, now comprises probably more than 4,000 stimulatory and inhibitory cell surface signaling molecules [27], and the integration of multiple signals decisively shapes the development of T cell immunity in general and the CD8+TM recall response in particular [28, 29]. Furthermore, a growing awareness about the contextual importance of costimulation, vividly illustrated for the CD28 pathway in a recent report by Welten et al. [30], underscores the need to conduct complementary investigations with a wide variety of model systems that cover specific T cell immunity in different infectious, neoplastic, autoimmune and allergic diseases as well as various transplantation settings. Unmoored now from the burden of perceived dogma [16, 17], the recognition that CD28 costimulation is essential for the optimal development of secondary CD8+TE immunity will undoubtedly foster the generation of many other important insights but if the timeline sketched out above for knowledge formation about CD28 costimulation is any indication, progress will be gradual, incremental, protracted and occasionally punctuated by “paradigm shifts”. In the meantime, however, an emerging consensus about the contextual CD28-dependence of memory T cell responses, no matter its evolving nature, will have to be considered for an improved design of relevant clinical treatment modalities [17, 31, 32].
Acknowledgments This work was supported by NIH grant R01 AI093637 (D.H.)
REFERENCES Author Manuscript
1. Kuhn, TS. The Structure of Scientific Revolutions. Chicago, IL: University of Chicago Press; 1962. 2. Kuhn, TS. Foreword to "Ludwik Fleck: Genesis and Development of a Scientific Fact". Chicago, IL: University of Chicago Press; 1979. p. xii-xi. 3. Fleck, L. Entstehung und Entwicklung einer wissenschaftlichen Tatsache: Einführung in die Lehre vom Denkstil und Denkkollektiv. Basel, Switzerland: B. Schwabe und Co. Verlagsbuchhandlung; 1935. 4. Frohlich M, Gogishvili T, Langenhorst D, Luhder F, Hunig T. Interrupting CD28 costimulation before antigen re-challenge affects CD8 T-cell expansion and effector functions during secondary response in mice. Eur J Immunol. 2016
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5. Bretscher P, Cohn M. A theory of self-nonself discrimination. Science. 1970; 169:1042–1049. [PubMed: 4194660] 6. Lafferty KJ, Cunningham AJ. A new analysis of allogeneic interactions. Aust J Exp Biol Med Sci. 1975; 53:27–42. [PubMed: 238498] 7. Cunningham AJ, Lafferty KJ. A simple conservative explanation of the H-2 restriction of interactions between lymphocytes. Scand J Immunol. 1977; 6:1–6. [PubMed: 300494] 8. Lafferty KJ, Andrus L, Prowse SJ. Role of lymphokine and antigen in the control of specific T cell responses. Immunol Rev. 1980; 51:279–314. [PubMed: 7000674] 9. Gmunder H, Lesslauer W. A 45-kDa human T-cell membrane glycoprotein functions in the regulation of cell proliferative responses. Eur J Biochem. 1984; 142:153–160. [PubMed: 6235110] 10. Aruffo A, Seed B. Molecular cloning of a CD28 cDNA by a high-efficiency COS cell expression system. Proc Natl Acad Sci U S A. 1987; 84:8573–8577. [PubMed: 2825196] 11. Linsley PS, Clark EA, Ledbetter JA. T-cell antigen CD28 mediates adhesion with B cells by interacting with activation antigen B7/BB-1. Proc Natl Acad Sci U S A. 1990; 87:5031–5035. [PubMed: 2164219] 12. Lenschow DJ, Walunas TL, Bluestone JA. CD28/B7 system of T cell costimulation. Annu Rev Immunol. 1996; 14:233–258. [PubMed: 8717514] 13. Sharpe A, Freeman GJ. The B7-CD28 superfamily. Nat Reviews Immunology. 2002; 2:116–126. 14. Chambers CA, Allison JP. Co-stimulation in T cell responses. Curr Opin Immunol. 1997; 9:396– 404. [PubMed: 9203422] 15. Sprent J, Surh CD. T cell memory. Annu Rev Immunol. 2002; 20:551–579. [PubMed: 11861612] 16. Boesteanu AC, Katsikis PD. Memory T cells need CD28 costimulation to remember. Semin Immunol. 2009; 21:69–77. [PubMed: 19268606] 17. Ville S, Poirier N, Blancho G, Vanhove B. Co-Stimulatory Blockade of the CD28/CD80-86/ CTLA-4 Balance in Transplantation: Impact on Memory T Cells? Front Immunol. 2015; 6:411. [PubMed: 26322044] 18. Croft M. Activation of naive, memory and effector T cells. Curr Opin Immunol. 1994; 6:431–437. [PubMed: 7917111] 19. Eberlein J, Davenport B, Nguyen TT, Victorino F, Sparwasser T, Homann D. Multiple Layers of CD80/86-Dependent Costimulatory Activity Regulate Primary, Memory, and Secondary Lymphocytic Choriomeningitis Virus-Specific T Cell Immunity. J Virol. 2012; 86:1955–1970. [PubMed: 22156513] 20. Borowski AB, Boesteanu AC, Mueller YM, Carafides C, Topham DJ, Altman JD, Jennings SR, Katsikis PD. Memory CD8+ T cells require CD28 costimulation. J Immunol. 2007; 179:6494– 6503. [PubMed: 17982038] 21. Hunig T, Luhder F, Elflein K, Gogishvili T, Frohlich M, Guler R, Cutler A, Brombacher F. CD28 and IL-4: two heavyweights controlling the balance between immunity and inflammation. Med Microbiol Immunol. 2010; 199:239–246. [PubMed: 20390297] 22. Gogishvili T, Luhder F, Kirstein F, Nieuwenhuizen NE, Goebbels S, Beer-Hammer S, Pfeffer K, Reuter S, Taube C, Brombacher F, Hunig T. Interruption of CD28-mediated costimulation during allergen challenge protects mice from allergic airway disease. J Allergy Clin Immunol. 2012; 130:1394–1403. e1394. [PubMed: 23102920] 23. Ndlovu H, Darby M, Froelich M, Horsnell W, Luhder F, Hunig T, Brombacher F. Inducible deletion of CD28 prior to secondary nippostrongylus brasiliensis infection impairs worm expulsion and recall of protective memory CD4(+) T cell responses. PLoS Pathog. 2014; 10:e1003906. [PubMed: 24516382] 24. Linterman MA, Denton AE, Divekar DP, Zvetkova I, Kane L, Ferreira C, Veldhoen M, Clare S, Dougan G, Espeli M, Smith KG. CD28 expression is required after T cell priming for helper T cell responses and protective immunity to infection. Elife. 2014; 3 25. Gogishvili T, Luhder F, Goebbels S, Beer-Hammer S, Pfeffer K, Hunig T. Cell-intrinsic and extrinsic control of Treg-cell homeostasis and function revealed by induced CD28 deletion. Eur J Immunol. 2013; 43:188–193. [PubMed: 23065717]
Eur J Immunol. Author manuscript; available in PMC 2017 July 01.
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26. Frentsch M, Stark R, Matzmohr N, Meier S, Durlanik S, Schulz AR, Stervbo U, Jurchott K, Gebhardt F, Heine G, Reuter MA, Betts MR, Busch D, Thiel A. CD40L expression permits CD8+ T cells to execute immunologic helper functions. Blood. 2013; 122:405–412. [PubMed: 23719298] 27. Zhu Y, Yao S, Chen L. Cell surface signaling molecules in the control of immune responses: a tide model. Immunity. 2011; 34:466–478. [PubMed: 21511182] 28. Duttagupta PA, Boesteanu AC, Katsikis PD. Costimulation signals for memory CD8+ T cells during viral infections. Crit Rev Immunol. 2009; 29:469–486. [PubMed: 20121696] 29. Chen L, Flies DB. Molecular mechanisms of T cell co-stimulation and co-inhibition. Nat Rev Immunol. 2013; 13:227–242. [PubMed: 23470321] 30. Welten SP, Redeker A, Franken KL, Oduro JD, Ossendorp F, Cicin-Sain L, Melief CJ, Aichele P, Arens R. The viral context instructs the redundancy of costimulatory pathways in driving CD8(+) T cell expansion. Elife. 2015; 4 31. Bour-Jordan H, Esensten JH, Martinez-Llordella M, Penaranda C, Stumpf M, Bluestone JA. Intrinsic and extrinsic control of peripheral T-cell tolerance by costimulatory molecules of the CD28/B7 family. Immunol Rev. 2011; 241:180–205. [PubMed: 21488898] 32. Beyersdorf N, Kerkau T, Hünig T. CD28 co-stimulation in T-cell homeostasis: a recent perspective. ImmunoTargets and Therapy. 2015; 2015(4):111–122.
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Figure 1. Strategies for timed CD28 ablation in an infectious disease model
As developed by Fröhlich et al. [4], iCD28ko mice are heterozygous for floxed Cd28 and ERT-driven Cre (Cd28−/flCre+/−) allowing for tamoxifen- (TM-) mediated CD28 deletion at various stages of the immune response (despite somewhat lower CD28 expression levels, heterozygous CD28+/− mice generate pathogen-specific primary and secondary CD8+T cell responses comparable to wt mice; not shown). A., iCD28ko mice are infected with low-dose LM-OVA (recombinant L. monocytogenes expressing ovalbumin), allowed to establish CD8+T cell memory, treated with TM to ablate CD28, and re-challenged with high-dose
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LM-OVA. This intervention reduces the proliferative expansion of OVA-specific secondary CD8+TE populations in comparison to a control group treated with vehicle (oil) only. B., LM-OVA-immune wt mice are treated with a CD28-blocking antibody (clone E18, mIgG2b) or PBS before a high-dose LM-OVA re-challenge. C., wt mice are seeded with iCD28ko OTI cells (TCR transgenic CD8+T cells specific for the OVA257-264 determinant) before a primary LM-OVA infection; TM treatment just prior to the secondary challenge permits elimination of CD28 exclusively from the OT-I CD8+TM.
Author Manuscript Author Manuscript Author Manuscript Eur J Immunol. Author manuscript; available in PMC 2017 July 01.
Eur J Immunol. Author manuscript; available in PMC 2017 July 01. Published in final edited form as: Eur J Immunol. 2016 July ; 46(7): 1587–1591. doi:10.1002/eji.201646500.
CD28 Days Later: Resurrecting Costimulation for CD8+ Memory T Cells Verena van der Heide1,2 and Dirk Homann1,2 1Diabetes,
Obesity and Metabolism Institute, Icahn School of Medicine at Mount Sinai, New York,
NY
Author Manuscript
2Immunology
Institute, Icahn School of Medicine at Mount Sinai, New York, NY
Abstract Rapid activation and proliferative expansion of specific CD8+ memory T cells (CD8+TM) upon antigen re-encounter is a critical component of the adaptive immune response that confers enhanced immune protection. In this context, however, the requirements for costimulation in general and CD28 signaling in particular remain incompletely defined. In the current issue of the European Journal of Immunology, Fröhlich et al. now provide definitive evidence that optimal elaboration of CD8+TM recall responses is indeed contingent on CD8+TM-expressed CD28. Here, we discuss the “CD28 costimulation paradigm” in its historical context and highlight some of the unresolved complexities pertaining to CD28-dependent interactions that shape CD8+T cell phenotypes, functionalities and recall reactivity.
Author Manuscript Author Manuscript
A superficial reading of Thomas Kuhn’s “The Structure of Scientific Revolutions” (1962) [1] might suggest that relevant scientific progress occurs in leaps and bounds, through revolutions small and large, and with abrupt course corrections that topple conventional wisdom in a fashion that in contemporary parlance has degenerated into the concept of “disruption”. But that would distort Kuhn’s ultimately more balanced assessment of the dynamics of scientific knowledge production and disregard the influence of his acknowledged predecessor Ludwik Fleck [2] who emphasized the gradual, discursive nature of scientific practice within and amongst different “thought collectives”. In that sense, Fleck’s 1935 monograph [3], provocatively entitled “Genesis and Development of a Scientific Fact”, might less controversially be labeled “The Structure of Scientific Evolutions”. And so it has come to pass that in the world of T cell costimulation, a “paradigm shift”, two decades in the making, has taken hold: recall responses of CD8+ memory T cells (CD8+TM), once considered “costimulation-independent”, in fact rely on CD28-mediated interactions for their optimal development. The work of Fröhlich et al. in the current issue of the European Journal of Immunology makes this case in a
Correspondence: Dirk Homann, MD, Diabetes, Obesity and Metabolism & Immunology Institutes, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place - Box 1152, New York, NY 10029, ph: 212 241-1935, fax: 212 241-2485, [email protected]. Commentary on Fröhlich et al., “Interrupting CD28 costimulation before antigen re-challenge affects CD8+T-cell expansion and effector functions during secondary response in mice” The authors declare that they do not have any conflict of interest.
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straightforward and compelling manner and thus offers, the provisional nature of all scientific conclusions notwithstanding, some “closure” to an ongoing discussion [4].
Author Manuscript Author Manuscript Author Manuscript
The concept of T cell costimulation is rooted in the 1970 Bretscher-Cohn two-signal model for B cell tolerance [5], its subsequent extension and refinement by Lafferty and Cunningham in 1975 [6], and eventual application to the induction of T cell immunity and tolerance by 1980 [7, 8]. With the discoveries of CD28 and its ligands made over the ensuing decade, the phenomenon of T cell costimulation got its first molecular correlate [9 —11], and a rich tapestry of intricately interwoven insights was established soon after [12— 14]. The notion that secondary, in contrast to primary, T cell responses are CD28independent is largely a product of the late 1990s and its ensuing promotion to the status of a “paradigm” [15] is in hindsight perhaps better characterized as the dissemination of a “widely held belief” owing to the comparatively few studies it was originally based upon (reviewed in refs. [16, 17]). Indeed, differential costimulation requirements for naïve and memory T cells were originally conceived in relative rather than absolute terms [18], and reports emerging from late 1990s onwards began to slowly build momentum, accelerated over the past 10 years, in support of CD28 costimulation as a non-redundant pathway for the effective elaboration of secondary T cell responses ([16, 17]; also summarized for antiviral T cells in ref. [19]). Nearly a dozen studies, including the first decisive report on the importance of CD28 for CD8+TM recall responses by P. Katsikis’ group [20], reached this conclusion by focusing their interrogations on the specific context of secondary T cell immunity using adoptive transfers of in vivo generated wild-type CD8+TM into CD80/86deficient recipients and/or interference with costimulation (CTLA-4 Ig treatment or CD28 blockade) restricted to the recall setting. As with all experimentation, however, the inevitable constraints and limitations of particular model systems had to somewhat temper any urges to proclaim a more general relevance of the observed phenomena. The introduction of mice with a floxed Cd28 gene in 2010 [21] therefore constituted a practical foundation for contemporary models of conditional or induced CD28 deletion that eschew potential artifacts arising from the employment of constitutively immunodeficient mice, adoptive transfers and/or antibody-based costimulation blockade. Though tantalizing clues about CD28-dependent CD8+TM responses were initially offered [21], subsequent investigations were devoted to the regulation of various CD4+T cell responses by CD28. Accordingly, CD28 ablation prior to a re-challenge was shown to limit CD4+TH2-dependent allergic airway disease [22] and to compromise protective CD4+TM responses in a model of parasite infection [23]. In addition, CD28 expression by CD4+T cells was not only required for initial control of bacterial burden (C. rodentium) and effective primary expansion of TH1 and TFH subsets in response to an influenza infection, but also for the subsequent maintenance of TFH [24]; similarly, sustained CD28 expression was found to be critical for CD4+TREG survival and function [25]. Which brings us to the report by Fröhlich et al. [4]. Combining an established infectious disease model (primary and secondary challenge with low- and high-dose, respectively, of recombinant L. monocytogenes expressing ovalbumin [LM-OVA]) with an inducible CD28deficient strain (iCD28ko mice), the authors demonstrate that global CD28 deletion just prior to a re-challenge resulted in a ~4-fold reduction of the OVA-specific CD8+TM recall response (Fig.1A). Complementary experiments in which systemic CD28 blockade was Eur J Immunol. Author manuscript; available in PMC 2017 July 01.
van der Heide and Homann
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applied to LM-OVA-immune wild-type (wt) mice only in the context of a secondary LMOVA infection yielded essentially the same results (Fig.1B) and confirm similar observations made earlier for antiviral CD8+TM recall responses [19, 20]; a practical advantage of the present work is the use of a murine non-depleting CD28 antibody that will facilitate future studies considering prolonged CD28 blockade. The most compelling evidence comes from the employment of wt mice seeded with a small number of iCD28ko OT-I cells (TCR transgenic CD8+T cells specific for the OVA257-264 determinant) before primary LM-OVA infection. The subsequent targeting of CD28 deletion in a precise temporal fashion (only before secondary challenge) and cell-specific manner (only OT-I CD8+TM with their fixed TCR specificity/affinity) provides incontrovertible evidence that optimal proliferative expansion of secondary CD8+ effector T cells (CD8+TE) is contingent on available CD28 signaling (Fig.1C).
Author Manuscript Author Manuscript Author Manuscript
The impact of CD28 ablation on other aspects of the specific CD8+TE response is apparently more modest and also more difficult to interpret. Under conditions of systemic CD28 inhibition prior to reinfection (iCD28ko mice or CD28 blockade), secondary CD8+TE exhibited a somewhat reduced capacity for inducible degranulation and IFNγ synthesis (Fig. 1A/B). Yet when CD28 deletion was targeted exclusively to CD8+TM populations in the iCD28ko OT-I system, secondary OT-I CD8+TE featured a trend towards enhanced degranulation and IFNγ production (Fig.1C). The differential outcomes reported by Fröhlich et al. point towards a scenario where multiple T cell populations rely on effective CD28 signaling and thus in concert shape the evolution of optimal secondary CD8+TE responses. For example, consistent with the notion that CD4+TREGs restrain the CD8+TM recall response in the LM-OVA model, CD4 depletion before re-challenge enhanced secondary CD8+TE expansions, and while the increased magnitude of the recall response remained susceptible to effective reduction by concomitant CD28 blockade, the interventions also impaired the elaboration of secondary CD8+TE functions (degranulation/IFNγ production) [4]. To reconcile these findings with the development of slightly improved CD8+TE functionalities in the context of CD28 ablation restricted to OT-I CD8+TM, future experiments will have to carefully differentiate between the specific contributions of CD4+TM and TREG subsets, and may consider the possibility that CD8+T cell populations other than the specific CD8+TM responders exert “helper functions” [26] in a CD28dependent fashion. To complicate the situation even further, targeted deletion of CD28 from OT-I cells before primary challenge also precipitated CD8+TE differentiation at the level of facilitated effector functionalities but “paradoxically” resulted in the generation of more CD127+/KLRG1− “memory precursor effector cells” [4]. These findings are reminiscent of the greater CD62L and CD127 expression by antiviral CD8+TE primed in CD28- or CD80/86-deficient mice and confirm an at least partial uncoupling of primary CD8+TE differentiation at the levels of phenotype, function and numerical expansion in the absence of CD28 costimulation (summarized in ref. [19]). Lastly, Fröhlich et al. found that CD28-deletion in the iCD28ko OT-I model right after the primary response affected neither OT-I CD8+TM maintenance nor recall capacity (though secondary CD8+TE functionalities were again slightly enhanced). The authors plausibly speculate that the CD28-deficient OT-I CD8+TM may have adapted to more effectively harness other costimultory pathways which in the context of a recall response may Eur J Immunol. Author manuscript; available in PMC 2017 July 01.
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compensate for the lack of CD28 [4]. But it is also important to note that the latter experiments were conducted ~3 months after primary challenge rather than the ~1 month interval employed for all other experiments (Fig.1). In fact, the precise timing for the evaluation of recall responses appears to be an underappreciated variable [16, 17] and our own preliminary data indicate that the relative dependence of secondary antiviral CD8+TE reactivity on CD28 costimulation progressively increases with time elapsed after original CD8+TM generation (D.H., unpublished). Reconciling all these observations and resolving seeming inconsistencies will require experiments that will assess the broader molecular, phenotypic and functional properties of the various CD8+T cell populations, rigorously control for CD8+TM numbers and defined recall settings (precise nature, dosage, route of administration and timing of re-challenge protocols), include relevant clinical readouts (e.g., altered immune protection), and consistently explore the many possible permutations of targeted CD28 deletion. And the iCD28ko mouse now offers a suitable model system to do just that.
Author Manuscript
Over the past half century, the concept of costimulation has evolved in a spectacular manner. “Signal 2”, initially a theoretical abstraction, now comprises probably more than 4,000 stimulatory and inhibitory cell surface signaling molecules [27], and the integration of multiple signals decisively shapes the development of T cell immunity in general and the CD8+TM recall response in particular [28, 29]. Furthermore, a growing awareness about the contextual importance of costimulation, vividly illustrated for the CD28 pathway in a recent report by Welten et al. [30], underscores the need to conduct complementary investigations with a wide variety of model systems that cover specific T cell immunity in different infectious, neoplastic, autoimmune and allergic diseases as well as various transplantation settings. Unmoored now from the burden of perceived dogma [16, 17], the recognition that CD28 costimulation is essential for the optimal development of secondary CD8+TE immunity will undoubtedly foster the generation of many other important insights but if the timeline sketched out above for knowledge formation about CD28 costimulation is any indication, progress will be gradual, incremental, protracted and occasionally punctuated by “paradigm shifts”. In the meantime, however, an emerging consensus about the contextual CD28-dependence of memory T cell responses, no matter its evolving nature, will have to be considered for an improved design of relevant clinical treatment modalities [17, 31, 32].
Acknowledgments This work was supported by NIH grant R01 AI093637 (D.H.)
REFERENCES Author Manuscript
1. Kuhn, TS. The Structure of Scientific Revolutions. Chicago, IL: University of Chicago Press; 1962. 2. Kuhn, TS. Foreword to "Ludwik Fleck: Genesis and Development of a Scientific Fact". Chicago, IL: University of Chicago Press; 1979. p. xii-xi. 3. Fleck, L. Entstehung und Entwicklung einer wissenschaftlichen Tatsache: Einführung in die Lehre vom Denkstil und Denkkollektiv. Basel, Switzerland: B. Schwabe und Co. Verlagsbuchhandlung; 1935. 4. Frohlich M, Gogishvili T, Langenhorst D, Luhder F, Hunig T. Interrupting CD28 costimulation before antigen re-challenge affects CD8 T-cell expansion and effector functions during secondary response in mice. Eur J Immunol. 2016
Eur J Immunol. Author manuscript; available in PMC 2017 July 01.
van der Heide and Homann
Page 5
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
5. Bretscher P, Cohn M. A theory of self-nonself discrimination. Science. 1970; 169:1042–1049. [PubMed: 4194660] 6. Lafferty KJ, Cunningham AJ. A new analysis of allogeneic interactions. Aust J Exp Biol Med Sci. 1975; 53:27–42. [PubMed: 238498] 7. Cunningham AJ, Lafferty KJ. A simple conservative explanation of the H-2 restriction of interactions between lymphocytes. Scand J Immunol. 1977; 6:1–6. [PubMed: 300494] 8. Lafferty KJ, Andrus L, Prowse SJ. Role of lymphokine and antigen in the control of specific T cell responses. Immunol Rev. 1980; 51:279–314. [PubMed: 7000674] 9. Gmunder H, Lesslauer W. A 45-kDa human T-cell membrane glycoprotein functions in the regulation of cell proliferative responses. Eur J Biochem. 1984; 142:153–160. [PubMed: 6235110] 10. Aruffo A, Seed B. Molecular cloning of a CD28 cDNA by a high-efficiency COS cell expression system. Proc Natl Acad Sci U S A. 1987; 84:8573–8577. [PubMed: 2825196] 11. Linsley PS, Clark EA, Ledbetter JA. T-cell antigen CD28 mediates adhesion with B cells by interacting with activation antigen B7/BB-1. Proc Natl Acad Sci U S A. 1990; 87:5031–5035. [PubMed: 2164219] 12. Lenschow DJ, Walunas TL, Bluestone JA. CD28/B7 system of T cell costimulation. Annu Rev Immunol. 1996; 14:233–258. [PubMed: 8717514] 13. Sharpe A, Freeman GJ. The B7-CD28 superfamily. Nat Reviews Immunology. 2002; 2:116–126. 14. Chambers CA, Allison JP. Co-stimulation in T cell responses. Curr Opin Immunol. 1997; 9:396– 404. [PubMed: 9203422] 15. Sprent J, Surh CD. T cell memory. Annu Rev Immunol. 2002; 20:551–579. [PubMed: 11861612] 16. Boesteanu AC, Katsikis PD. Memory T cells need CD28 costimulation to remember. Semin Immunol. 2009; 21:69–77. [PubMed: 19268606] 17. Ville S, Poirier N, Blancho G, Vanhove B. Co-Stimulatory Blockade of the CD28/CD80-86/ CTLA-4 Balance in Transplantation: Impact on Memory T Cells? Front Immunol. 2015; 6:411. [PubMed: 26322044] 18. Croft M. Activation of naive, memory and effector T cells. Curr Opin Immunol. 1994; 6:431–437. [PubMed: 7917111] 19. Eberlein J, Davenport B, Nguyen TT, Victorino F, Sparwasser T, Homann D. Multiple Layers of CD80/86-Dependent Costimulatory Activity Regulate Primary, Memory, and Secondary Lymphocytic Choriomeningitis Virus-Specific T Cell Immunity. J Virol. 2012; 86:1955–1970. [PubMed: 22156513] 20. Borowski AB, Boesteanu AC, Mueller YM, Carafides C, Topham DJ, Altman JD, Jennings SR, Katsikis PD. Memory CD8+ T cells require CD28 costimulation. J Immunol. 2007; 179:6494– 6503. [PubMed: 17982038] 21. Hunig T, Luhder F, Elflein K, Gogishvili T, Frohlich M, Guler R, Cutler A, Brombacher F. CD28 and IL-4: two heavyweights controlling the balance between immunity and inflammation. Med Microbiol Immunol. 2010; 199:239–246. [PubMed: 20390297] 22. Gogishvili T, Luhder F, Kirstein F, Nieuwenhuizen NE, Goebbels S, Beer-Hammer S, Pfeffer K, Reuter S, Taube C, Brombacher F, Hunig T. Interruption of CD28-mediated costimulation during allergen challenge protects mice from allergic airway disease. J Allergy Clin Immunol. 2012; 130:1394–1403. e1394. [PubMed: 23102920] 23. Ndlovu H, Darby M, Froelich M, Horsnell W, Luhder F, Hunig T, Brombacher F. Inducible deletion of CD28 prior to secondary nippostrongylus brasiliensis infection impairs worm expulsion and recall of protective memory CD4(+) T cell responses. PLoS Pathog. 2014; 10:e1003906. [PubMed: 24516382] 24. Linterman MA, Denton AE, Divekar DP, Zvetkova I, Kane L, Ferreira C, Veldhoen M, Clare S, Dougan G, Espeli M, Smith KG. CD28 expression is required after T cell priming for helper T cell responses and protective immunity to infection. Elife. 2014; 3 25. Gogishvili T, Luhder F, Goebbels S, Beer-Hammer S, Pfeffer K, Hunig T. Cell-intrinsic and extrinsic control of Treg-cell homeostasis and function revealed by induced CD28 deletion. Eur J Immunol. 2013; 43:188–193. [PubMed: 23065717]
Eur J Immunol. Author manuscript; available in PMC 2017 July 01.
van der Heide and Homann
Page 6
Author Manuscript Author Manuscript
26. Frentsch M, Stark R, Matzmohr N, Meier S, Durlanik S, Schulz AR, Stervbo U, Jurchott K, Gebhardt F, Heine G, Reuter MA, Betts MR, Busch D, Thiel A. CD40L expression permits CD8+ T cells to execute immunologic helper functions. Blood. 2013; 122:405–412. [PubMed: 23719298] 27. Zhu Y, Yao S, Chen L. Cell surface signaling molecules in the control of immune responses: a tide model. Immunity. 2011; 34:466–478. [PubMed: 21511182] 28. Duttagupta PA, Boesteanu AC, Katsikis PD. Costimulation signals for memory CD8+ T cells during viral infections. Crit Rev Immunol. 2009; 29:469–486. [PubMed: 20121696] 29. Chen L, Flies DB. Molecular mechanisms of T cell co-stimulation and co-inhibition. Nat Rev Immunol. 2013; 13:227–242. [PubMed: 23470321] 30. Welten SP, Redeker A, Franken KL, Oduro JD, Ossendorp F, Cicin-Sain L, Melief CJ, Aichele P, Arens R. The viral context instructs the redundancy of costimulatory pathways in driving CD8(+) T cell expansion. Elife. 2015; 4 31. Bour-Jordan H, Esensten JH, Martinez-Llordella M, Penaranda C, Stumpf M, Bluestone JA. Intrinsic and extrinsic control of peripheral T-cell tolerance by costimulatory molecules of the CD28/B7 family. Immunol Rev. 2011; 241:180–205. [PubMed: 21488898] 32. Beyersdorf N, Kerkau T, Hünig T. CD28 co-stimulation in T-cell homeostasis: a recent perspective. ImmunoTargets and Therapy. 2015; 2015(4):111–122.
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Figure 1. Strategies for timed CD28 ablation in an infectious disease model
As developed by Fröhlich et al. [4], iCD28ko mice are heterozygous for floxed Cd28 and ERT-driven Cre (Cd28−/flCre+/−) allowing for tamoxifen- (TM-) mediated CD28 deletion at various stages of the immune response (despite somewhat lower CD28 expression levels, heterozygous CD28+/− mice generate pathogen-specific primary and secondary CD8+T cell responses comparable to wt mice; not shown). A., iCD28ko mice are infected with low-dose LM-OVA (recombinant L. monocytogenes expressing ovalbumin), allowed to establish CD8+T cell memory, treated with TM to ablate CD28, and re-challenged with high-dose
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LM-OVA. This intervention reduces the proliferative expansion of OVA-specific secondary CD8+TE populations in comparison to a control group treated with vehicle (oil) only. B., LM-OVA-immune wt mice are treated with a CD28-blocking antibody (clone E18, mIgG2b) or PBS before a high-dose LM-OVA re-challenge. C., wt mice are seeded with iCD28ko OTI cells (TCR transgenic CD8+T cells specific for the OVA257-264 determinant) before a primary LM-OVA infection; TM treatment just prior to the secondary challenge permits elimination of CD28 exclusively from the OT-I CD8+TM.
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