CD4 T cells guarantee optimal competitive fitness of CD8 memory T cells

Eur. J. Immunol. 2004. 34: 91–97

CD4 T cells guarantee optimal competitive fitness of CD8 memory T cells

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CD4 T cells guarantee optimal competitive fitness of CD8 memory T cells Pål Johansen1,2, Panagiota Stamou2, Ricardo E. Tascon1, Douglas B. Lowrie1 and Brigitta Stockinger2 1 2

Division of Mycobacterial Research, National Institute for Medical Research, London, GB Division of Molecular Immunology, National Institute for Medical Research, London, GB

We studied the contribution of CD4 T cell help to survival and competitive fitness of CD8 memory T cells specific for influenza virus nucleoprotein. In agreement with recent studies, the optimal generation of functional memory CD8 T cells required CD4 help, although longterm maintenance of resting CD8 memory T cells did not absolutely depend on the presence of CD4 T cells. Nonetheless, CD4 T cells were essential during differentiation of CD8 memory T cells to imprint on them the capacity to compete effectively with other memory T cells. CD8 memory cells generated with help survived better in secondary polyclonal hosts, and co-transfer into lymphopenic hosts together with “un-helped” CD8 memory cells showed improved homeostatic expansion of CD8 memory cells that had been generated with CD4 help. Therefore, the requirement for CD4 help in CD8 T cell memory extends to homeostatic parameters that ensure the maintenance and competitive fitness of memory clones. Key words: CTL / T lymphocytes / CD4 help / Memory / Vaccination

1 Introduction Numerous reports on the susceptibility of T cell- or MHC-deficient mice to a number of pathological diseases suggest that CD4 and CD8 T cells are pivotal in generating protective immunity [1–3]. Nonetheless, questions concerning immunological memory continue to occupy investigators, and paradigms on memory have been constantly shifting [4–7]. It is a general notion that memory CD8 T cells are much less dependent on MHC class I for their survival than are naive CD8 T cells. They divide in the periphery even without evidence of persisting antigen. Memory CD8 T cells specific for lymphocytic choriomeningitis virus (LCMV) survive indefinitely in MHC class I-deficient hosts [8], and cells expressing the TCR transgene for the male antigen HY, survived and proliferated without a preference for the restricting allele [9]. Generally, it is accepted that naive CD8 T cells rarely divide, whereas memory cells divide on a weekly basis [10], even in an MHC class I-deficient host [8]. This division is at least in part, driven by the cytokine IL-15. IL-15 binds CD122 (IL-2R g ), which is selectively expressed on CD44hi CD8 T cells, and causes proliferation of these, but not of CD44hi CD4 T cells. IL-2, on the other hand, has an

Received Revised Accepted

4/6/03 6/10/03 12/11/03

inhibitory effect on the background turnover of CD8 memory cells [11], a process assumed to be regulated by CD25-positive CD4 T cells [12]. CD4 T cells have been described to influence CD8 responses in a number of ways. Initially it was proposed that their function consisted of “licensing” antigenpresenting cells to provide costimulatory signals through CD40 expression to naive CD8 T cells [13–15]. Recent findings indicate that CD8 T cells express CD40 molecules themselves upon activation, and can therefore receive direct help from CD4 T cells expressing CD40L [16]. CD4 T cells may also supply the necessary bystander cytokines and chemokines for survival of activated CD8 T cells [17, 18]. Moreover, it has been reported that memory CD8 T cells generated in the absence of CD4 T cell have weak proliferating and cytokine-secreting properties upon restimulation with antigen [19–22]. Here, we use the influenza A virus as a model and confirm that the generation of memory cells is favored by the presence of CD4 help, however, the survival of such CD8 memory cells is not dependent on constitutive CD4 help. Finally, we show an additional function of CD4 help that imprints on differentiating CD8 memory T cells the capacity for homeostatic expansion and effective competition with other memory clones.

[DOI 10.1002/eji.200324231]

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2 Results 2.1 The generation of memory, but not effector CD8 T cells, is improved by CD4 help To analyze the role of CD4 T cells in the generation of effector and memory CD8 T cells, TCR transgenic Rag1–/– mice (F5) were immunized with the influenza virus A/NT/60/68 in the presence or absence of 106 adoptively transferred naive wild-type CD4 T cells; the injection of CD4 T cells alone did not cause activation of F5 cells (Fig. 1). Seven days after immunization, the numbers of CD8 T cells in spleens from mice immunized in the absence or presence of CD4 T cells did not differ (Fig. 1a). The activation status of the cells was similar as determined by the level of CD44 expression (Fig. 1b) and by the frequency of IFN- + -producing cells (Fig. 1c). Similar results were obtained in mesenteric lymph nodes (LN) and when cells were stimulated with the NP366–374 peptide instead of PdBu for IFN- + analysis (data not shown). In

Fig. 1. Absolute numbers (means ± SE) of F5 cells (a, d), F5 cells expressing high levels of CD44 (b, e) or F5 cells staining positive for intracellular IFN- + after stimulation in vitro with PdBu (c, f) 7 (a–c) and 28 (d–f) days after priming of F5 mice with 106 PFU of the influenza virus A/NT/60/68 in the presence or absence of 106 CD4 T cells. As a negative control, one group of mice received CD4 T cells and no virus. Representative results in spleens from two (a–c) to three (d–f) experiments with comparable results are shown (n=3–4). The differences between the group receiving virus and CD4 T cells and the group that received virus only were statistically significant only after 28 days (*: p X 0.05; **: p X 0.01). © 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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contrast, 4 weeks after immunization, significantly higher total numbers of F5 cells (Fig. 1d), as well as CD44hi and IFN- + -producing F5 cells (Fig. 1e, f) were found in mice immunized in the presence of CD4 T cells. Hence, the generation of memory, but not of effector F5 cells was improved in the presence of CD4 T cell help.

2.2 The presence of CD4 T cells is not essential for the survival of CD8 memory cells GFP-F5 memory cells generated in the absence or presence of CD4 T cells were transferred into polyclonal hosts containing CD4 T cells (B10) or not (MHC II ko). This allowed us to study the role of CD4 T cells in maintaining signals for survival of CD8 memory cells, an issue previously not studied. As shown in Fig. 2a, the survival of memory GFP-F5 cells generated without T cell help was not impaired in the absence of CD4 T cells in the host animal (MHC II ko). On the contrary, consistently higher numbers of GFP-F5 memory cells could be recovered from MHC II ko mice than from wild-type B10 mice as analyzed in spleen and mesenteric, inguinal, and axillary LN 12–20 weeks after the adoptive transfer (Fig. 2a). On the other hand, GFP-F5 memory cells generated in the presence of CD4 T cells survived equally well in the two strains of recipient mice (Fig. 2b), and the F5 memory cells in B10 profited from receiving CD4 help during priming (p=0.0945; Fig. 2a vs. b). There was no significant difference in the numbers of surviving un-helped F5 memory cells in MHC II ko mice as compared to helped cells in B10 (p=0.136) or in MHC II ko (p=0.119). The transfer of GFP-F5 memory into B10 hosts primed or not four weeks earlier with A/NT/60/68 showed that the antigen-specificity of the CD4 T helper cells had no significant effect on the survival of GFP-F5 memory cells (data not shown). The distribution of both types of mem-

Fig. 2. Absolute numbers of CD44hi GFP-F5 memory cells surviving in B10 and MHC II ko hosts after transfer of memory cells obtained from hosts primed with A/NT/60/68 in the absence (a) or presence (b) of CD4 T cells. GFP-F5 cells were recovered from spleen and LN 18–20 weeks after the transfer (**: p X 0.01). The data are representative of three experiments with comparable results. www.eji.de

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ory cells was different in the two hosts, inasmuch as they distributed evenly between spleen and LN in B10 hosts, whereas in MHC II ko mice, they preferentially homed to LN ( G 75%) (data not shown). Finally, the functional properties of the memory cells were confirmed by direct staining for intracellular IFN- + or by analyzing in vivo proliferation and intracellular IFN- + 7 days after an intravenous injection of the immunizing influenza virus. There was a higher frequency of IFN- + -producing GFP-F5 cells in helped than in un-helped memory cells (results not shown).

2.3 Generation of memory CD8 in the absence of CD4 help compromises their ability to compete with other, normally generated CD8 T cells The finding that un-helped CD8 memory cells survived better in MHC II ko than in B10 hosts, suggests that there may be differences in their potential to compete

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with endogenous CD8 T cells. It is conceivable that endogenous CD8 memory T cells in the MHC II ko mice were of similar “weakness” as the transferred un-helped F5 memory cells. On the other hand, endogenous CD8 memory cells in the B10 hosts might be more robust and therefore more likely to out-compete the transferred F5 memory cells, which differentiated without CD4 help. In order to directly compare the expansion potential for the two types of F5 memory cells in the same host, we cotransferred memory cells generated in the absence (F5) and in the presence (GFP-F5) of CD4 help into lymphopenic Rag1–/– hosts. The expansion of either type of cells was monitored over 24 days. Seven days after transfer both memory populations are represented at a similar level in the spleen, whereas after 24 days, memory cells that were generated in the presence of CD4 help, i.e. GFP-F5 cells had expanded more than their un-helped F5 counterparts (Fig. 3, dot blots). The total pool of CD8 memory T cells recovered from spleen and LN was significantly skewed towards GFP-F5 memory T cells that had been generated in the presence of CD4 help (Fig. 3, histograms).

3 Discussion The extent to which CD8 T cell responses depend on help from CD4 T cells varies from mostly independent [23–25], strictly dependent [19, 26, 27] or conditional [28–30]. In the absence of CD4 T cell help, cytotoxic CD8 T cells are reported to be deleted [17], tolerized [31], rendered lethargic [19] or persisting un-perturbed [24]. To some extent these differences are due to differences in experimental systems and the antigens used. Here, we used intravenous immunization of mice with the influenza type A/NT/60/68 virus and analyzed whether transgenic CD8 T cells, specific for a H-2Db CD8 epitope, depended on CD4 help for their generation and longterm maintenance.

Fig. 3. Generation of memory CD8 in the absence of CD4 help compromises their homeostatic expansion in Rag1–/– hosts. Equal numbers of F5 and GFP-F5 generated in the absence and presence, respectively, of CD4 T cells were transferred into Rag1–/– mice. Left panel: the dot plot shows the relative frequency of CD44hi F5 and GFP-F5 T cells in percent as represented in one mouse 7 days after transfer. The histogram shows the average absolute numbers of CD44hi F5 and GFP-F5 cells as determined in spleens and LN of three mice. Right panel: dot plot and histogram of relative frequency and absolute numbers of GFP-F5 and F5 T cells 24 days after transfer. Cells in the dot blots were gated on viable and CD8-positive lymphocytes. © 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

CD4 help is transmitted via co-stimulatory CD40/CD40L interactions. CD4 and CD8 T cells types are brought together by an antigen-loaded dendritic cell that displays antigens to both [14, 15], or the dendritic cells are conditioned by stimulation from antigen-specific CD4 T cells, whereby they subsequently mature and activate CD8 T cells [13]. Recently, it was shown that generation of memory CD8 T cells depended on CD4 T cell help, but not on CD40 expression by the antigen-presenting cells [16]. Furthermore, whereas the frequency of effector cells were similar, memory cells derived from mice immunized with CD4 help were significantly higher in numbers [16, 19], a finding more recently confirmed by others [20–22]. www.eji.de

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In contrast to Liu et al. [32], who found that CD4 T-helper cells are required in the early phase of the immune response in order to promote the initial phase of clonal expansion and differentiation into CD8 effectors, but in agreement with Bourgeois et al. [16, 19] and others [20–22], we found that the differentiation of CD8 T cells into memory cells, but not effector cells was controlled by the presence of CD4 T cell help during priming of CD8 T cells. Others have shown that T-helper cells were required for functional reactivation of memory CTL specific for a tumor antigen, but effector CTL did not need T help to kill tumors [33]. Furthermore, generation of longlived protective CTL memory was associated with stimulation of the innate immune system and immunization with peptides induced short-lived CTL unless the peptide also provided T-helper epitopes in addition to CD8 T cell epitopes [31]. The induction of functional CTL and the clearance of viral infections vary, depending on the nature of a virus, and lymphocytic choriomeningitis, vaccinia and influenza A viruses were reported to elicit CD4independent CD8 T cell responses [34]. Finally, CTL responses could be converted from CD4 T cell dependency to independency in a cross-presentation model, by increasing the frequency of precursor ctl [29]. This finding might be explained by the fact that CD8 T cells express CD40 upon activation [16], and with increased precursor frequency, they might simply provide their own costimulatory CD40-CD40L interactions. Generally, the different role of CD4 help in the above mentioned systems might reflect the different costimulatory and proinflammatory signals elicited by the antigenic triggers utilized. For instance do viral and bacterial vectors typically up-regulate costimulatory molecules such as CD80, CD86 and CD40 on antigen-presenting cells, partly through Toll-like receptors [35]. They also represent a more serious pathological danger [36], thereby increasing the number and amounts of other proinflammatory mediators, which will probably also affect the absolute function of CD4 T cells in eliciting CTL. The requirement for CD4 T cells for long-term survival of CD8 memory T cells has not been addressed previously. We show that although CD4 help has an impact on the number of memory CD8 T cells generated, the long-term survival of these is not hampered by the removal of CD4 cells and transfer to a MHC II ko host. Four months after transfer, and 5 months after vaccination, comparable numbers of memory CD8 T cells survived in hosts with (B10) and without (MHC II ko) endogenous CD4 T cells, provided the CD8 T cells were generated in the presence of CD4 help. This suggests that CD4 T cells do not have a crucial bystander effect for the survival of resting CD8 memory T cells and is in accordance with the finding that CD40 is not required for maintenance of CD8 memory [37]. Helped CD8 memory cells are functionally superior © 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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to un-helped memory cells [19–22]. The triggering, division and acquisition of CD8 effector functions could occur in the absence of help, but was not sufficient to generate memory cells able to mount efficient secondary responses. When CD8 T cells were activated in the absence of CD4 T cells, their survival in a CD4-empty host (MHC II ko) was even better than in the wild-type B10 host (Fig. 2a). This could be an effect of homeostatic proliferation of naive CD8 T cells which were cotransferred with the memory cells, if available space in the host mice. However, CFSU-labeled naive F5 cells did not divide upon transfer in MHC II or B10 mice (results not shown), excluding such an explanation. Another explanation could be the inhibitory effect of CD25 positive regulatory CD4 T cells [12]. Again, no such memoryregulatory mechanism was evident here. Survival of resting CD8 memory T cells generated in the presence of CD4 cells was similar in B10 and MHC II ko mice, despite the presence of CD25+ CD4 T cells in B10. A third explanation for the improved survival of un-helped F5 memory cells in MHC II ko mice could be that the resident host CD8 memory cells with which they would have to compete also differentiated in the absence of help and therefore would be equally disadvantaged. In contrast, CD8 memory cells of the B10 hosts would have higher competitive fitness and a greater expansion potential the F5 memory cells because they had the benefit of CD4 help during their differentiation. This is an important feature and likely to have an impact on CD8 T cell attrition, a parameter that affects the numbers of CD8 memory cells in consecutive infections [38]. Co-transfer of the two kinds of memory populations into syngeneic Rag1–/– hosts directly assessed their relative capacity for expansion and indeed verified that CD8 memory T cells generated without CD4 help were disadvantaged and, therefore, compromised by the presence of robust CD8 memory T cells that had been generated in the presence of CD4 help. Reduced competitive fitness due to defective CD4 T cell help during generation of CD8 T cell memory might play a role in HIV pathology and could have consequences for generation of CD8 memory by therapeutic vaccination of individuals compromised at the level of CD4 T cells during HIV infection [39–41]. A recent report on supervised treatment interruptions (STI) in HIV therapy [42] indicated that the immune activity generated by a healthy person in response to an HIV vaccine will be qualitatively distinct from that of HIV-infected persons, especially one whose immune system is already damaged by years of infection. In this case, increases in virus levels after treatment interruption were probably associated with preferential infection of HIV-specific CD4 T cells [43]. CTL induced at a time point where CD4 T cells counts were low would be short-lived due to their inability to compete with memory www.eji.de

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CD8 T cells with other specificities generated before the onset of pathology and death of CD4 T cells. Taken together, this study showed that CD4 T cells not only favors the generation of CD8 memory T cells but that help also ensure competitive fitness of CD8 memory T cell probably by making them more capable of utilizing homeostatic parameters.

4 Materials and methods 4.1 Mice F5 Rag1–/– (F5) and F5 expressing GFP under control of the human CD2 promoter in all T cells (GFP-F5) are TCRtransgenic mice recognizing the CD8 epitope 366–374 (ASNENMDAM) from the influenza A virus A/NT60/68 nucleoprotein in the context of H-2Db [44, 45]. C57BL/10 (B10) mice, MHC class II–/– mice (MHC II ko) on a B10 background, or Rag1–/– mice were used as recipients for F5 or GFP-F5 cells. All mice were bred and kept under standard UK Home Office approved conditions at the National Institute for Medical Research.

4.2 Immunization and adoptive transfer of memory cells To study the role of CD4 T cells in the generation of memory CD8 T cells, F5 mice were primed intravenously with 106 PFU of the influenza virus A/NT/60/68 in the presence or absence of 106 CD4 T cells purified from wild-type B10 mice; the CD4 T cells were isolated from spleen and LN using magnetic beads (AutoMACS®, Miltenyi Biotech) according to the manufacturer’s protocol (94–96% purity) and injected into F5 mice together with the virus. The maintenance of CD8 memory was assessed after adoptive transfer of GFP-F5 memory CD8 T cells. These were generated in the presence or the absence of CD4 T cells, and CD8positive T cells were selected by AutoMACS®. The transfer inoculum, containing also naive CD8 T cells, was adjusted to 106 CD44hi CD8 T cells, which were intravenously transferred to B10 or MHC II ko mice; there was no homeostatic proliferation of naive F5 cells in either host (results not shown).

4.3 Flow cytometry analysis Single-cell suspension of lymphocytes recovered from spleen, LN, peritoneum or heparinized blood, were stained with specific mAb on ice followed by acquisition on a FACS®Calibur (BD BioSciences) and data analysis with a CellQuest® software. Staining was preceded by incubating cells with an § Fc receptor Ab (clone 2.4.G2) to block nonspecific staining of secondary Ab via Fc-receptor binding. © 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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All Ab were purchased from BD PharMingen, and the stainings were performed in 2% PBS/FCS.

4.4 FACS staining for IFN- q secreting cells Intracellular staining for IFN- + in F5 cells was performed following stimulation of cells from spleens or LN with PdBu, ionomycin and brefeldin A at 37°C for 4 h. After the stimulation period, the cells were incubated with § Fc receptor Ab, surface stained for CD8, CD44 and TCR (H57) expression, fixed in PBS/paraformaldehyde 1% for 10 min, and permeabilized in PBS/NP40 0.1% for 3 min, before stained intracellularly with an anti-IFN- + Ab. All incubation steps were performed on ice and intercepted by a washing step in 2% PBS/FCS. Since fluorescence intensity of GFP on GFP-F5 cells is diminished by fixation and permeabilization, cytokine secretion from GFP-F5 cells in B10 and in MHC II ko mice was measured using a IFN- + secretion kit as recommended by the provider (Miltenyi Biotech). Briefly, 5×106 cells were stimulated with 1 ? M NP366–374 peptide at 37°C for 4 h in 1 ml AIM-V medium (Gibco) supplemented with 2- g mercaptoethanol. After washing, the cells were exposed to bi-specific anti-CD45 Ab conjugated to an anti-IFN- + Ab for 5 min on ice and then, at 37°C and for 45 min while lightly agitated. After washing, the cells were stained with a PEconjugated secondary anti-IFN- + Ab and with other Ab for CD8, CD44 and TCR expression. Acquisition and analysis was done as described above.

4.5 Competitive fitness of CD8 memory T cells To study the fitness of CD8 memory T cells, generated in the absence of CD4 help, to compete with normally generated memory cells, the following experiment was performed. F5 or GFP-F5 cells (2×106) were transferred intravenously into sex-matched Rag1–/– mice along with 106 PFU A/NT/60/68 ± 106 B10 naive CD4 T cells (purified with AutoMACS). After 4 weeks, CD8-positive T cells were selected from LN and spleens and purified using AutoMACS. Finally, 2.5×106 F5 memory cells and 2.5×106 GFP-F5 memory cells were cotransferred into a sex-matched Rag1–/– host and the relative difference in expansion of F5 and GFP-F5 memory cells in spleen and LN was determined over 24 days.

Acknowledgements: This study was in part supported with grants to P. J. from the Swiss National Science Foundation, Berne, and the Royal Society, London, GB. We thank George Kassiotis and Christine Bourgeois for critical discussions, Dimitris Kioussis for the F5 and GFP-F5 CD8 transgenic mice and Trisha Norton for excellent animal husbandry.

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Correspondence: Pål Johansen, Department of Dermatology, University Hospital of Zurich, Gloriastrasse 31, CH8091 Zurich, Switzerland Fax: +41-1-255-4418 e-mail: pal.johansen — usz.ch

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