TWO SIDES OF A CELLULAR COIN: CD4 + CD3 - CELLS ORCHESTRATE

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1 TWO SIDES OF A CELLULAR COIN: CD4 + CD3 - CELLS ORCHESTRATE MEMORY ANTIBODY RESPONSES AND LYMPH NODE ORGANISATION Peter J.L. Lane 1 (Fabrina M.C. Gaspal) and Mi-Yeon Kim * MRC Centre for Immune Regulation, Birmingham Medical School, Vincent Drive, Birmingham B15 2TT, UK 1 Correspondence: should be addressed to Peter Lane Address: MRC Centre for Immune Regulation, Birmingham Medical School, Vincent Drive, Birmingham B15 2TT, UK Telephone FAX p.j.l.lane@bham.ac.uk LAB BIOGRAPHY Peter Lane s lab investigates the signals and cells regulating the survival of the CD4 + T cells that provide B-cell help. They use a combination of approaches: key cellular interactions are identified by tracking antigen-specific T cells during immune responses in mice; and functions for the co-stimulatory molecules expressed during cognate events are investigated using genetic techniques. They hope to understand the complexity of the adaptive B-cell response in humans by comparing the genotype and phenotype of the immune systems in mice, men and lower vertebrates, and to use this knowledge to illuminate the immunopathology of human disease.

2 PREFACE We propose that CD4 + CD3 - cells have two functions: a well-established role in organizing lymphoid tissue during development, and a novel role in supporting T-cell help for B cells, both during affinity maturation in germinal centres and for memory antibody responses. As these CD4 + CD3 - cells express the HIV coreceptors CD4 and CXCR4, we think that infection of these cells by virus and their subsequent destruction by the host immune system could help to explain the loss of memory antibody responses and the destruction of lymphoid architecture that accompany disease progression to AIDS. A key feature of the immune system of both birds and mammals is that infection with pathogens evokes, within weeks, class-switched antibodies of exquisite specificity. This process occurs in B-cell germinal centres (GCs), which are potent advertisements for the power of natural selection. The generation of millions of B-cell- receptor specificities in the rapidly proliferating B centroblast population is followed by their ruthless selection by antigen and follicular T cells in an iterative cycle. At the end of the process, the affinity of the remaining B cells can have increased up to a million fold. Also of importance, B-cell progeny survive as memory cells that can be stimulated by re-exposure to small amounts of antigen to make rapid memory responses that eliminate the pathogen without signs of reinfection. All of this depends on the collaboration of T cells; follicular T cells help and select B cells during the critical phase of affinity maturation in the GC, and memory T cells support memory B-cell antibody responses. This opinion article proposes a role for CD4 + CD3 - accessory cells in promoting the survival of T cells during both phases of the immune response. Furthermore, we suggest that these CD4 + CD3 - accessory cells are the adult equivalent of the inducer cells that organize the development of lymphoid tissue 1, and that these two functions organization of lymphoid tissue and the promotion of survival of T cells that help B cells are regulated by discrete sets of tumour-necrosis factor (TNF)-family members. By virtue of their expression of CD4 and CXCR4, these accessory cells are potential targets for HIV, and we speculate that the loss of these cells is responsible for the loss of antibody responses and the disorganization of lymphoid tissue that is observed with disease progression to AIDS. CD4 + CD3 - CELL INTERACTIONS WITH PRIMED CD4 + T CELLS. Digital confocal microscopy of spleen sections enabled the identification of interactions between T cells in GCs and a CD4 + CD3 - cell population with dendritic morphology but lacking the mouse dendritic-cell (DC) marker CD11c 2. CD4 + CD3 - cells could be found not only in the B-cell follicles and GCs, where they were interacting with follicular T cells, but they were also aligned at the B:T interface where the T-cell zone and B-cell follicle adjoin, which is a site of T:B collaboration in both primary and secondary antibody responses 3,4 (Figure 1). Priming of marked transgenic CD4 + T cells occurred first on the conventional DC population in the T- cell zone, following which some of the CD4 + T cells migrated to the outer T-cell zone and into B-cell follicles, where a substantial fraction was found to interact with CD4 + CD3 - cells 2. Due to technical difficulties in isolating CD4 + CD3 - cells from mice with an intact CD4 + T-cell repertoire, these cells were first isolated from T-cell-deficient mice. Two

3 CD4 + populations could be distinguished from CD11c + bright DCs. One expressed B220 (CD11c low) and was identified as the plasmacytoid DC population 5 ; the other was CD4 + CD3 - CD11c - B220 - IL-7R +. Humans also have a distinct CD4 + CD3 - cell population within GCs 6. Although the human cells express CD11c, they express CD4 and their location is the same, so we regard them as equivalent to the mouse CD4 + CD3 - population. WHICH SIGNALS DO PRIMED T CELLS RECEIVE FROM CD4 + CD3 - CELLS? Because of their close association with primed T cells, both inside B-cell follicles (follicular T cells) (Figure 2) and at the interface between the B- and T-cell areas (newly primed and recirculating memory T cells) (Figure 3), it seemed plausible that CD4 + CD3 - cells provide co-stimulatory signals to T cells. Unlike conventional DCs, which express and can be activated through CD40, CD4 + CD3 - cells lack significant CD40 expression 2. Furthermore, their expression of the conventional DC-associated costimulatory molecules such as CD80 and CD86 is low 2. However, two T-cell costimulatory molecules are expressed at high levels by CD4 + CD3 - cells: OX40-ligand (OX40L; TNFSF4) and CD30-ligand (CD30L; TNFSF8). These are both members of the TNF family, whose receptors (OX40 and CD30) are expressed by primed but not naïve T cells. In the context of the GC environment, OX40L can also be expressed by activated B cells 7. Uniquely, however, the expression of both OX40L and CD30L by CD4 + CD3 - cells is high and constitutive, is independent of antigen activation and is not modified by cytokines, particularly IL-4 8. Also, it has been shown from the histology that there is membrane contact between OX40 hi CD4 + T cells and OX40L + CD4 + CD3 - cells that would enable delivery of an OX40 signal to the primed T cells 2. FUNCTION OF OX40 AND CD30 SIGNALS DELIVERED BY CD4 + CD3 - CELLS. OX40 and CD30 (the receptors for OX40L and CD30L expressed by CD4 + CD3 - cells) are genetically linked in a TNF-receptor (TNFR) cluster containing 7 members, located on mouse chromosome 4 and human chromosome 1. Like many members of the TNFR family, they share common signaling pathways: both bind to members of the TNF-related adaptor factor (TRAF) family (TRAF1, TRAF2, TRAF3 and TRAF5) 9,10, and OX40 signals have been shown to upregulate expression of antiapoptotic Bcl-2-family members that promote survival 11. Given that activated T cells can express both receptors, and that CD4 + CD3 - cells express both ligands, it is expected that there should be partial redundancy between OX40 and CD30 signals, as shown by the results from single- and double-deficient mice. Th2-cell survival in vitro. CD4 + T cells activated in vitro upregulate expression of both OX40 and CD30, but their expression is particularly marked on Th2-differentiated cells (cultured in the presence of IL-4) 2. Although many studies have linked OX40 and CD30 signals with preferential Th2-cell development in vitro, CD4 + T cells deficient in OX40 and/or CD30 can still proliferate and differentiate into Th2 cells 2,12. The most important difference that these signals make is to the survival of Th2, but not Th1 cells. Coculture of normal Th2-differentiated cells with CD4 + CD3 - cells shows independent additive effects of OX40 and CD30 signals on Th2-cell survival 12.

4 In keeping with the survival effects of OX40 and CD30 signals in vitro, in vivo analysis of the immune response of mice deficient in both OX40 and CD30 showed independent and additive effects of these genes on two aspects of T-cell help for B cells 12 : affinity maturation and memory T-cell help for secondary antibody responses. Affinity maturation supported by follicular T cells. The survival of the follicular T cells that orchestrate affinity maturation of GC B cells depends on additive signals through CD30 and OX Transgenic T cells deficient in both CD30 and OX40 signals initially proliferated normally, but like their in vitro counterparts, they failed to survive. As a consequence, although T- cell help for primary antibody responses was normal, and GC development was initiated, GCs involuted after 7 days. GC failure led to impaired affinity maturation of antibody responses. OX40- and CD30-deficient CD4 + T cells in vivo had restricted survival in B-cell follicles, indicating that OX40 and CD30 have overlapping roles in the maintenance of follicular T cells. These data support the in vitro observation that the role of OX40 and CD30 signals from CD4 + CD3 - cells, which express OX40L and CD30L, is to keep follicular T cells alive. It is not difficult to see why follicular T-cell survival is important for the affinity maturation of antibodies in GCs. As affinity maturation progresses, the antigen driving the reaction becomes scarce (antigen is removed by phagocytes and masked by secreted antibodies). It is precisely at this time that follicular T cells are needed to select rare B-cell mutants with high-affinity receptors. Rapidly proliferating GC B centroblasts exit the cell cycle and differentiate into centrocytes that compete for antigen fragments trapped on follicular dendritic cells. Successful B-cell mutants bearing high-affinity receptors are able to take up antigen and successfully present peptide fragments to follicular T cells, which then provide selective CD40Ldependent rescue signals. The constitutive expression of OX40L and CD30L by CD4 + CD3 - cells, irrespective of antigen concentration, equips them to provide antigen-independent survival signals to follicular T cells so that they can continue to select B cells (Figure 2). T cell help for memory antibody responses. Memory antibody responses in OX40 and CD30 double-deficient mice are markedly reduced 12. By contrast, defects in memory antibody responses in mice deficient in either OX40 or CD30 were sufficiently mild to be ignored by many investigators who studied OX or CD30 17,18 single-deficient mice. Memory T cells that provide help for B-cell responses recirculate through lymphoid organs 19, ensuring that the production of memory antibody responses does not depend on the site of initial immunization. The effects of OX40 and CD30 deficiency on memory antibody responses indicated that CD4 + CD3 - cells might specifically influence the survival of these memory T cells, as well as the follicular T cells involved in affinity maturation. Evidence from mouse studies indicates that recirculating memory T cells do not arise from the CXCR5-expressing follicular T- cell population responsible for affinity maturation in GCs, which does not recirculate 20. A subset of memory T cells that home to lymph nodes express both CXCR5 and CCR7 21. Co-expression by B cells of CCR7 (the ligand for which produced by stromal cells in the T-cell zone 22 ) and CXCR5 (the ligand for which is produced by stromal cells in the B-cell areas 23 ) results in the alignment of B cells at

5 the B:T interface 24, and this seems also to be the case for the CXCR5 + CCR7 + memory T cells in mice (Reinhold Forster, personal communication). In addition to being located in B-cell follicles adjacent to follicular T cells, CD4 + CD3 - cells are also found at the B:T interface. Although mouse reagents are not available to check for surface CCR7 expression, there is evidence that CCR7 mrna is expressed by the CD4 + CD3 - cells (Kim, M. and Lane, P., unpublished observations). So, similar to their B- and T-cell counterparts, there are probably two subsets of CD4 + CD3 - cells; one CXCR5 + subset localized in B-cell follicles that interacts with CXCR5 + follicular T cells, and the other subset at the B:T interface that co-expresses CXCR5 and CCR7 and interacts with CXCR5 + CCR7 + recirculating memory T cells. Recirculating memory T cells do not normally express either OX40 or CD30, but can be induced to re-express OX40 12 when exposed to the common gamma-chain signaling cytokine, IL-7, which has been implicated in the maintenance of memory CD4 + T cells We think that memory T cells, co-expressing CXCR5 and CCR7, upregulate OX40 in response to IL-7 signals, perhaps from stromal cells or follicular dendritic cells 28, as they migrate through secondary lymphoid organs, allowing them to receive OX40-dependent survival signals from CD4 + CD3 - cells each time they passed through lymphoid tissue (Figure 3). T-cell-dependent memory B-cell responses are therefore also promoted by CD4 + CD3 - cell-mediated survival of memory T-cell responses. NEONATAL CD4 + CD3 - CELLS DO NOT EXPRESS OX40L OR CD30L. In contrast to adult mice, CD4 + CD3 - cells isolated from neonates lack expression of the molecules associated with T-cell memory, OX40L and CD30L, indicating that the expression of these molecules is developmentally regulated 29. Their absence is specific: the expression of TNF-family ligands associated with the development of lymph nodes is otherwise comparable in adult and neonatal CD4 + CD3 - cell populations 2. This may help to explain the observations of Medawar, who reported 50 years ago that immunization of neonatal rodents resulted in tolerance rather than immunity 30. We predict that T-cell help for B-cell responses would effectively be aborted in neonatal rodents as a consequence of the absence of T-cell survival signals through OX40 and CD30 from CD4 + CD3 - cells. This does not render the neonate immunodeficient because of protection from maternal antibodies at this time. By the time of weaning, expression levels of OX40L and CD30L on CD4 + CD3 - cells are normal 29 and mice become immunocompetent to respond. ARE CD4 + CD3 - CELLS RELATED TO THE CELLS THAT INDUCE LYMPH NODES? Detailed phenotyping of adult CD4 + CD3 - cells revealed that they are similar to a CD4 + population first described by Mebius to colonize neonatal lymph nodes 31 but also found in neonatal spleen 32 in mice. The name inducer was later coined for these cells because of their essential role in the induction of lymph nodes, Peyer s patches 1 and, more recently, isolated lymphoid follicles in the submucosa 33. The splice variant of the retinoic orphan receptor gamma (RORγt) is expressed by inducer cells and required for their function in the induction of lymph nodes 34,35. Using a green fluorescent protein (GFP) marker to identify RORγt-expressing cells specifically, Eberl and Littman 33 reported a phenotype for these cells similar to the CD4 + CD3 - cells that we have identified in adults 2. Evidence from adult 2 and neonatal 31-33,36 mice indicates that the shared phenotype between these CD4 + CD3 -

6 populations is: RORγt +, CD45 +, CD4 +, LTα +, LTβ +, TRANCE +, c-kit +, IL7-Rα +, IL2- Rα +, common cytokine receptor gamma chain +, CXCR5 +, CCR7 +, α4β7 +, Thy1.2 +, CD11b -, CD11c -, B220 -, NK1.1 -, CD8 - and CD3 -. We have found that RORγtspecific mrna is expressed by adult CD4 + CD3 - cell populations, albeit at lower levels than in neonates (Kim, M. and Lane, P., unpublished observations). The expression of TNF-family members linked with the development and organization of lymphoid tissue 1 lymphotoxin-α (LT-α; TNFSF1), LT-β (TNFSF3) and TRANCE (TNFSF11) is also common to both adult and neonatal CD4 + CD3 - cells. These data are consistent with adult CD4 + CD3 - cells being related to neonatal inducer cells. A difficulty with this argument is that RORγt-regulated GFPexpressing inducer cells are reported to be absent from adult secondary lymphoid organs (Dan Littman, personal communication). This may be because RORγt expression is attenuated in adult CD4 + CD3 - cells as it is in T cells, which express RORγt as double-positive thymocytes 37, but which lose expression as they mature 37. IMMUNE MEMORY AND HIGH AFFINITY ANTIBODY RESPONSES ANTEDATED LYMPH NODE DEVELOPMENT Assuming that adult CD4 + CD3 - cells and neonatal inducer cells are related, what was the original evolutionary function of cells of this phenotype? Birds and mammals evolved from a common reptilian ancestor ~300 million years ago 38. In terms of their immune responses, both form GCs when immunized, make high-affinity classswitched antibodies and have B- and T-cell memory. However, birds lack lymph nodes, unlike the most primitive mammals, the monotremes 39. This indicates that the genetic and cellular mechanisms for maintaining CD4 + memory T cells to support antibody production were established before birds and mammals went their separate evolutionary ways. The key advantage of mammalian immunity is that the major investment made during the GC reaction in terms of B-cell affinity maturation is not lost; the memory response is systemic, not local to the site of initial vaccination, because memory lymphocytes recirculate 19. Once local immune memory was established, one could envision a selective survival advantage for animals able to redistribute the memory response globally. Lymph nodes provide the infrastructure for a systemic response; the blood vessels and lymphatics supply the conduit. Genetic paleontology supports this as an evolutionary sequence. The chicken genome published in silico (ensembl.org/gallus_gallus/) contains orthologues of many genes that are essential for GC development (such as CD28 and CD40), and also orthologues of CD30 and OX40, which we have identified as important for T-cell memory. In contrast to these genes, the master gene for lymph-node development, LTβR, seems to be missing from the chicken genome. Therefore, a cell type to control lymph-node development (the neonatal inducer cell) may have arisen simply through the evolution of a new function for an old cell type: the adult CD4 + CD3 - cell that regulates the development and maintenance of memory antibody responses. CD4 + CD3 - CELL FUNCTION AND PUTATIVE ROLE IN THE EVOLUTION OF AIDS Our data therefore link CD4 + CD3 - cells with memory antibody responses and maintenance of the integrity of lymphoid architecture, both of which are lost as HIV infection progresses CD4 + CD3 - cells in mice express CD4 and the chemokine

7 receptor CXCR4 (Kim, M. and Lane, P., unpublished observations), which are coreceptors for viral entry into human cells 43. Although HIV + individuals are infected initially with the CCR5-tropic variant, which mainly targets CD4 + T-cell populations in the gut 44, mutation of the virus to the CXCR4-tropic form is associated with depletion of T cells in secondary lymphoid tissue and a poor prognosis 43. High levels of HIV are known to be trapped on follicular dendritic cells in B-cell follicles 45, where CD4 + CD3 - cells could be infected directly. The histopathology of HIV-infected lymph nodes is also consistent with the immunopathology of CD4 + CD3 - cells. Lymph nodes from HIV-infected individuals show hyperplastic GC formation initially, but then the GCs involute and the follicular architecture disappears 46. If CD4 + CD3 - cells became targets during the course of HIV infection, not only would it impair the capacity to make and maintain neutralizing antibody to the virus and other pathogens, but also, by destroying the cells that establish and maintain lymphoid architecture, undermine immune responses to the large number of normally nonpathogenic infections associated with AIDS. How might CD4 + CD3 - cells be destroyed? There are two possibilities: destruction by the virus or destruction by the host cytolytic CD8 + T-cell response against infected cells. There is some evidence for the latter in that invasion of B-cell follicles by CD8 + T cells is associated with follicular destruction 47. In humans, however, longterm survival with untreated HIV correlates with strong CD4 + and CD8 + T-cell responses against virally infected cells. This seems to argue against CD4 + CD3 - cell destruction by CD8 + T cells being relevant to AIDS pathogenesis. However, the opposite is true in the natural non-human primate hosts. Chimpanzees (SIV1) 48 and Sooty Mangabeys (SIV2) 49 show high levels of viraemia without progressive CD4 + T- cell deficiency or AIDS, and this is associated with preservation of the follicular architecture in chimpanzees 47. This is not because of resistant mutations within the chimpanzee CXCR4 gene, which is identical to humans 50, but rather due to evidence of a hypoplastic CD8 + T-cell response against the virus 47,51, possibly allowing the survival of a CD4 + CD3 - cell population. As HIV maintains latency in memory CD4 + T cells and is therefore impossible to eradicate 52, an ignorant CD8 + T-cell response may have a selective survival advantage in the long-run, provided that the virus is not inherently lethal. By preserving CD4 + CD3 - cell function, the likelihood of making and maintaining neutralizing antibody responses is increased, which limits the spread of virus, at the same time preserving immune responses to normally non-pathogenic organisms. Why then does this not seem to be the case for long-term human survivors of HIV? Although there is excellent evidence that SIV/HIV mutates to evade recognition by individual MHC molecules 53, the large number of MHC class I molecules in humans may make it difficult for the virus to evade the CD8 + T-cell response entirely. Anything but an all out assault on virus-infected cells, which apparently is achieved by HIV survivors through the strength of their CD8 + T-cell response, allows persistent viraemia, the infection of CD4 + CD3 - cells, and the subsequent attrition of the lymphoid architecture that we propose leads to AIDS. SUMMARY In this opinion article, we have highlighted the importance of CD4 + CD3 - cells in organizing the development and perhaps maintenance of secondary lymphoid organs, as well as in the support of adaptive memory antibody responses. Expression of CD4 and CXCR4 by these cells in mice indicates that they may be targets for HIV in

8 humans, and that their destruction by the host CD8+ T-cell response could account for many of the features associated with progressive disease. Pinpointing their identity in humans will help to establish whether they are infected by HIV and to confirm or refute this hypothesis. The result is relevant to the control of the HIV pandemic. We predict that in individuals that make suboptimal responses those that progress to AIDS strategies that evoke CD8 + T-cell responses 54 may aggravate disease, consequent on CD4 + CD3 - cell destruction, whereas vaccine strategies designed to evoke neutralizing antibody responses 55 are likely to preserve CD4 + CD3 - cells and lymphoid architecture and to improve prognosis.

9 LEGENDS Each legend shows a photograph with accompanying schematic diagram. LEGEND TO FIGURE 1. Digitally processed image of an immunostained section from mouse spleen to show the location of CD4 + CD3 - CD11c - cells (red) in relation to CD11c + DCs (green) and IgM + B cell areas (gray). CD4 + CD3 - cells are mainly located at the B:T interface and in B-cell follicle adjacent to follicular T cells whereas DCs are located in T cell area and red pulp. Plasma cells are located in the red pulp that surrounds the white pulp areas that contain lymphocytes. T=T cell area, B=B follicle, MZ=marginal zone. DC=dendritic cell. LEGEND TO FIGURE 2. Antigen-specific T cells are first primed on DCs in T cell area. T cells that upregulate CXCR5 and migrate into B follicles. The white arrow indicates CD4 T cell migration from T cell area to B follicle. In the follicle, follicular T cells (T f ) drive the proliferation of B cells that differentiate into centroblasts undergoing somatic mutation within immunoglobulin variable genes. Centroblasts proliferate rapidly and differentiate into centrocytes that compete for antigen fragments trapped on follicular dendritic cells (FDCs). B-cell mutants bearing high-affinity receptors take up antigen and present peptide fragments to T f, which then provide selective CD40L-dependent rescue signals. When antigen is scarce, T f are sustained by OX40 and CD30 signals from CD4 + CD3 - cells, which express OX40L and CD30L constitutively so that that they can continue to select B cells. Centrocytes that are positively selected leave the GC and differentiate into memory B cells or mature into long-lived plasma cells. Inset white shows a T cell and a DC (T:DC) interaction in T cell area (TZ). Inset red shows interaction between a T f and a CD4 + CD3 - cell within a B cell GC and the left shows this at high magnification. LEGEND TO FIGURE 3. Re-activation mechanism of memory T cells (T m ) by CD4 + CD3 - cells. Antigenspecific T m that co-express CXCR5 + CCR7 +, transmigrate through high endothelial venules (HEVs) in lymph nodes via their expression of ligands for peripheral lymph node addressin (PNAd). IL-7 produced by putative stromal cells or follicular dendritic cells (FDCs) within lymphoid tissue upregulates expression of OX40 on T m. T m then interact with interface CD4 + CD3 - cells, which express OX40L constitutively at the B:T border. The interaction of CD4 + CD3 - cells and T m cells provide survival signals to T m through OX40 by upregulation of BCL-2 and BCL-XL. Inset shows high magnification of this interaction.

10 ACKNOWLEDGMENTS This work is supported by the Wellcome Trust. We thank I. MacLennan, F. McConnell and G. Anderson for reading the manuscript and making many helpful comments. We also thank C. Raykundalia, who organized us all and made sure everything in the lab worked.

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15 MZ B T Red Pulp Plasma cells ADULT B follicle DC CD4 + CD3 - Fig 1A. Fig. 1 T=T cell area, B=B follicle, MZ=Marginal zone B

16 Hypermutation and selection in mature germinal centres Memory B cell Apoptotic B cell GC T cell CD4 + CD3 - Plasma cell Centrocyte FDC Centroblast OX40/CD30 B follicle T f GC TZ T:DC 1B. Fig. 2

17 Fig. 3