Development of virus-specific CD4⁺ T cells during primary cytomegalovirus infection

Although virus-specific CD4⁺ T cells have been characterized extensively in latently infected individuals, it is unclear how these protective T-cell responses develop during primary virus infection in humans. Here, we analyzed the kinetics and characteristics of cytomegalovirus-specific (CMV-specific) CD4⁺ T cells in the course of primary CMV infection in kidney transplant recipients. Our data reveal that, as the first sign of specific immunity, circulating CMV-specific CD4⁺ T cells become detectable with a median of 7 days after first appearance of CMV-DNA in peripheral blood. These cells produce the T helper 1 type (Th1) cytokines IFNγ and TNFα, but not the T helper 2 type (Th2) cytokine IL4. In primary CMV infection, the vast majority of these circulating virus-specific T cells have features of recently activated naive T cells in that they coexpress CD45RA and CD45R0 and appear to be in the cell cycle. In contrast, in people who have recovered from CMV infection earlier in life, virus-specific T cells do not cycle and express surface markers characteristic of memory T cells. After the initial rise, circulating virus-specific CD4⁺ T cells decline rapidly. During this phase, a strong rise in IgM and IgG anti-CMV antibody titers occurs, concomitant with the reduction of CMV-DNA in the circulation.

In immune competent individuals, primary infection with herpes viruses results in lifelong viral persistence without serious clinical manifestations. A cease-fire is being negotiated in which, on one hand, viral gene products that interfere with immune recognition mask latently or persistently infected cells from the immune system, and, on the other hand, immune effector mechanisms repress abundant virus replication (1). The crucial role of the immune system in maintaining the symbiotic relation with herpes viruses is emphasized by the fact that in congenital or acquired immune deficiency states, herpes virus reactivation may give rise to serious complications.

Virus-specific CD4⁺ T cells are of prime importance in initiating and maintaining immunity against most viruses, including herpes viruses. Recent findings have shown that antigen-specific CD4⁺ T cells activate antigen-presenting cells after interaction of CD154 on the CD4⁺ T cell with CD40 on the antigen-presenting cell. CD8⁺ T cells specific for class I peptides from the same antigen, cross-presented on this activated antigen-presenting cell, are then primed to become cytolytic effector cells (2). Importantly, not only in the acute phase but also in the latent stage, CD4⁺ T cells are critical for maintaining functionality and diversity of cytotoxic T-lymphocyte responses (3). Apart from inducing class I–restricted viral-peptide–specific cytolytic T cells, CD4⁺ T lymphocytes are indispensable in guiding naive B cells to become plasma cells that secrete high-affinity virus-specific antibodies, a process in which again the interaction between CD154 on T cells and CD40 on B cells appears to be the major regulatory event (4–8). In line with these diverse effects of CD4⁺ T cells on effector pathways of the specific immune system, depletion and/or dysfunction of CD4⁺ T cells can result in reactivation of persistent viruses such as cytomegalovirus (CMV) and Epstein Barr virus (EBV) (9–12).

Acute and latent virus-specific CD4⁺ T-cell responses have been well studied in experimental animals in which infectious dose, mode, and timing can be standardized. Because of advances in analyzing virus-specific CD4⁺ T cells, the frequency and functional properties of these cells during latency and reactivation of persistent infections, e.g., CMV, have been assessed recently in humans (13, 14). In contrast, development of specific cellular immune responses from a naive state in humans has remained unresolved so far. Investigation of this subject is hampered by the fact that timing infection is generally impossible. We circumvented this problem by studying CMV-seronegative renal transplant recipients of seropositive organ donors. In these patients, the exact time point of infection is known because the donated kidney contains the virus and will induce primary CMV infection in about 60% of the recipients (15, 16). We investigated patients on basic immunosuppressive therapy, consisting of prednisolone and cyclosporin. These patients generally develop a protective immune response against CMV, characterized by a CD8⁺ T-cell lymphocytosis in peripheral blood, an inversion of CD4/CD8 ratio, generation of CMV-specific antibodies, and, generally, clearance of the virus from the blood (17). We collected longitudinal blood samples and determined the temporal relationship between appearance of CMV-DNA, CMV-specific CD4⁺ T cells, and anti-CMV antibodies. Next, the functional distinctions between virus-specific T cells during acute and latent viral infection were defined. Our data reveal, we believe for the first time, the kinetics and properties of a primary, protective CD4⁺ T-cell immune response to herpes viruses in humans. Insight into this process is essential in understanding disturbed immunity to herpes viruses in different clinical conditions.

Detection of CMV-specific CD4⁺ T cells in renal transplant recipients. We first validated the method of analyzing CMV-specific CD4⁺ T-cell frequency in immunosuppressed patients receiving standard immunosuppressive therapy. In agreement with other published data (13), CMV-specific CD4⁺ T-cell frequencies were detectable in healthy CMV-seropositive individuals (group II) with a median of 0.15% of CD4⁺ T cells (range 0.05–1.63%, n = 6), whereas no CMV-specific CD4⁺ T cells were found in CMV-seronegative individuals (group I). Also, in PBMC of seropositive renal transplant recipients (group IV), a median CMV-specific CD4⁺ T-cell frequency of 0.39% (range 0.05–1.24%, n = 11) was found, whereas no CMV-specific CD4⁺ T cells were detectable in CMV-seronegative renal transplant recipients (group III; Figures 1, a–c, and 2a). These data demonstrate that under conditions of standard immunosuppression CMV-specific CD4⁺ T-cell frequencies can be measured in renal transplant recipients. Moreover, when CD69 upregulation and IFNγ production to SEB stimulation were determined by flow cytometric analysis, frequencies of SEB-specific CD4⁺ T cells from renal transplant recipients were similar to those of SEB-specific CD4⁺ T cells from healthy individuals (Figure 2a).

Figure 1

Flow cytometric determination of CMV-specific CD4⁺ T cells. Dot plots of anti–IFNγ-PE fluorescence (X-axis; arbitrary units, log scale) versus CD69-FITC fluorescence (Y-axis; arbitrary units, log scale). Depicted are lymphocytes positive for CD4-APC fluorescence. Left panels: cells incubated with control antigen; middle panels: cells incubated with CMV antigen; and right panels: cells incubated with SEB. Cells from (a) a CMV-seropositive healthy individual (group II) (b) a CMV-seronegative renal transplant recipient (group III) (c) a CMV-seropositive renal transplant recipient (group IV) (d) a CMV-seronegative renal transplant recipient of CMV-seropositive organ (group V, patient 2), after CMV-DNA was detected, but before seroconversion.

Figure 2

(a) CMV- and SEB-specific CD4⁺ T-cell frequencies. Each dot represents 1 individual. Left 4 scattergrams (left Y-axis): CMV-specific CD4⁺ T-cell frequencies (percent of CD4⁺ T cells) in healthy individuals and renal transplant recipients. CMV-HI, CMV-seronegative healthy individuals (group I); CMV-RTx, CMV-seronegative renal transplant recipients on basic immunosuppressive therapy (group III); CMV+HI, CMV-seropositive healthy individuals (group II); CMV+RTx, CMV-seropositive renal transplant recipients on basic immunosuppressive therapy (group IV). Right 2 scattergrams (right Y-axis): SEB-specific CD4⁺ T-cell frequencies (in percent of CD4⁺ T cells); HI, healthy individuals; RTx, renal transplant recipients on basic immunosuppressive therapy; ns, not significant (P > 0.05, Mann-Whitney). (b) Development of CMV-specific CD4⁺ T-cell frequencies in peripheral blood in relation to the number of CMV-DNA copies per milliliter blood. One representative seronegative renal transplant recipient of a kidney from a seropositive organ donor (patient 2, group V) is shown. Time (days after transplantation) versus frequency of CMV-specific CD4⁺ T cells (percent of CD4⁺ cells) and CMV-DNA copies (copies per milliliter blood). Open circles, CMV-DNA copies; filled circles, CMV-specific CD4⁺ T cells.

Longitudinal analysis of seronegative renal transplant recipients of organs from seropositive donors. In 5 of the 7 renal transplant recipients enrolled in the follow-up study of seronegative recipients of a kidney from seropositive organ donors, a primary CMV infection was diagnosed by positive CMV culture or seroconversion. During the first 18 days after transplantation, neither CMV-DNA nor CMV-specific CD4⁺ T cells were detectable in any of the patients from group V or VI. In 5 out of 7 patients, viral DNA was detected at a median of 25 days (range 18–29 days) after transplantation. These patients were assigned to group V. One of these 5 patients was treated with ganciclovir because of persistent fever with positive CMV buffy coat culture and a pulmonary infiltrate (patient 1). In all 5 patients from group V, CMV-specific CD4⁺ T cells were detected by flow cytometry at a median of 7 days after first detection of CMV-DNA (range 4–14 days; for a representative experiment see Figures 1d and 2b). Peak frequencies ranged from 0.46 to 2.50% of peripheral blood CD4⁺ T cells (Table 2). In all patients, CMV-specific CD4⁺ T-cell frequencies precipitously increased after detection of the virus, followed by a rapid decrease (Figure 2b). In the 2 renal transplant recipients without evidence of CMV infection (remaining negative in CMV-PCR, CMV culture of urine and blood, and anti-CMV antibodies; group VI) no CMV-specific CD4⁺ T cells were detected during follow-up for up to 100 days after transplantation (not shown).

Table 2

Appearance of CMV-DNA, CMV-specific CD4⁺ T cells, and CMV-specific antibodies in CMV-seronegative renal transplant recipients of kidneys from CMV-seropositive donors (group V)

Functional and phenotypic properties of CMV-specific CD4⁺ T cells shortly after infection. When peak frequencies were expected in patients from group V, CMV-specific T cells were further analyzed. The CMV-specific CD4⁺ T cells were CD38⁺ during primary infection, contrasting profoundly with their phenotype during viral latency, when CMV-specific CD4⁺ T cells were exclusively CD38^– (Figure 3a). CMV-specific T cells in healthy CMV-seropositive individuals were predominantly CD27^–, whereas shortly after primary infection, a substantial proportion of the CMV-specific T cells had not yet lost CD27 expression (Figure 3b). Furthermore, CMV-specific CD4⁺ T cells were CD49d(VLA-4)⁺, CD62L^–, and CD11a^high, irrespective of whether they were obtained from an acutely or latently infected person (not shown). Next to this phenotype, which strikingly resembles the phenotype of recently activated T cells, CMV-specific CD4⁺ T cells were in cell cycle during acute primary infection, as evidenced by Ki67 expression. In healthy seropositive individuals, these cells were typically nonproliferating in peripheral blood (Figure 3c).

Figure 3

Phenotypic characteristics of CMV-specific CD4⁺ T cells. Left column: a representative patient (patient 4) analyzed at the peak of CMV-specific CD4⁺ T cells. Right column: a CMV-seropositive healthy individual (group II). (a–c) During the acute phase, CMV-specific CD4⁺ T cells display a recently activated, proliferating phenotype. Dot plots of anti-IFNγ fluorescence (X-axis; arbitrary units) versus CD38 (a), CD27 (b), Ki67-fluorescence (c), respectively (Y-axis; arbitrary units) within a CD4⁺CD69⁺ lymphocyte gate. (d and e) CMV-specific CD4⁺ T cells display the CD45RA⁺ CD45R0high phenotype. Histograms of CD45RA fluorescence (d) or CD45R0 fluorescence (e). Filled histograms represent CMV-specific CD4⁺ T cells only, i.e., lymphocytes gated on positive CD4, intracellular IFNγ, and CD69 expression. Open histogram overlays represent all CD4⁺ T cells. Histogram overlays were generated using the “each histogram” option for clarity. Therefore, cell number of filled histograms and open histograms do not have the same Y-axis values.

During viral latency, CMV-specific CD4⁺ T cells displayed a typical memory phenotype, i.e., CD45RA^– and CD45RO⁺. In contrast, during acute CMV infection, the CMV-specific CD4⁺ T cells expressed both the naive T-cell marker CD45RA and the memory marker CD45R0 (Figure 3, d and e). Interestingly, the expression of CD45R0 on these CMV-specific T cells was higher when compared with the bulk memory CD4⁺ population (Figure 3e, left panel).

Around the time of peak frequency, the CMV-specific CD4⁺ T cells displayed typical Th1 cytokine production profiles, IFNγ-producing cells showing the highest frequency. Figure 4 shows that all TNFα-producing cells also produced IFNγ. Few cells produced IL2, whereas virtually no IL4-producing cells were detected. In contrast, considerable frequencies of IL2-producing cells and, to a lesser extent IL4 producing cells, could be detected using SEB-stimulated cultures (not shown). Finally, after incubation with CMV antigen in the presence of brefeldin A, very few peripheral blood CD4⁺ T cells displayed cell surface expression of CD154. However, if CD154 staining was performed after fixation and permeabilization, the molecule was found to be selectively expressed intracellularly in the CMV-specific CD4⁺ T cells (not shown).

Figure 4

CMV-specific CD4⁺ T cells display a Th1 cytokine secretion pattern (arbitrary units for all axes). Dot plots of lymphocytes from patient 4, gated on positive CD4-PerCP and CD69-APC fluorescence. (a) Anti–IFNγ-PE fluorescence (X-axis) versus anti TNFα-FITC fluorescence (Y-axis). (b) Anti–IL4-PE fluorescence (X-axis) versus anti–IL2-FITC fluorescence (Y-axis).

CMV-specific antibody responses in relation to CMV-specific CD4⁺ T cells. Figure 5 shows the relation of CMV-specific CD4⁺ T-cell frequencies with CMV-specific antibody production in 1 representative patient. In all patients (except for patient 1), the appearance of CMV-specific CD4⁺ T cells preceded the formation of CMV-specific antibodies.

Figure 5

Relation of CMV-specific CD4⁺ T cells with CMV-specific antibody production. Time (days after transplantation) versus frequency of CMV-specific CD4⁺ T cells (percent of CD4⁺ T cells) and antibody concentration (IgM, ratio to a standard serum; IgG, antibody units per milliliter serum). One representative patient (patient 2) is shown with respect to anti-CMV IgM levels and anti-CMV IgG levels. Filled circles, CMV-specific CD4⁺ T-cell frequency; open squares, anti-CMV IgM levels; open diamond, anti-CMV IgG levels.

The present study shows that in naive individuals infection of CMV from donated kidneys needs between 2.5 to 4 weeks to arrive at a detectable level of viral replication and spread. Recently, a mean in vivo doubling time of human CMV was calculated to be around 1 day (21). Therefore, in our patient group the virus needed at least 17 doubling times to emerge at a detectable level, assuming that no pathways for viral clearance are available before generation of the specific immune response and that viral replication commences immediately after transplantation. At a median of 7 days after first detection of CMV-DNA, CMV-specific CD4⁺ T cells appeared in the circulation. The time between transplantation and appearance of CMV-specific CD4⁺ T cells probably reflects a series of events: generation of immunogenic viral particles, uptake of antigen by antigen-presenting cells, migration of these cells to secondary lymphoid organs, presentation of antigenic peptides to the naive CMV-specific helper T cells, activation, initial proliferation and naïve-to-memory transition of these T cells. After this process, seeding of the CMV-specific CD4⁺ T cells to the peripheral blood apparently ensued.

We chose to study renal transplant recipients for the apparent reason that if one wants to define the first steps in the generation of an immune response to a live virus in humans, the exact time of infection should be known. To this aim, these patients were routinely screened for CMV serology before transplantation and followed longitudinally after transplantation with an organ from a CMV-seropositive donor. The study of antigen-specific T-cell dynamics in these patients might be complicated by the administration of immunosuppressive therapy. However, despite immunosuppressive drug therapy consisting of cyclosporin and low-dose prednisolone, renal transplant recipients are known to develop an adequate immune response against the virus. First, CMV-specific antibodies are being produced and, generally, primary CMV infection in these patients does not lead to serious clinical manifestations. Without antiviral therapy, CMV appears to be largely neutralized in most patients. Finally, after patients have recovered from primary infection, they seldom develop clinical signs of either persistent or secondary CMV infection (16). We therefore conclude that CMV-seronegative renal transplant recipients, treated with cyclosporin and low-dose prednisolone, develop a protective response to primary CMV infection and can be used to characterize features of the primary immune response to CMV in humans. In agreement, we found similar frequencies of CMV-specific T cells in CMV-seropositive renal transplant recipients as compared with CMV-seropositive healthy individuals. Also, frequencies of IFNγ producing CD4⁺ T cells after SEB stimulation did not differ between renal transplant recipients and healthy individuals. It cannot be formally excluded, however, that the basic immunosuppressive drug therapy administered to the patients in this study potentially affects T-helper cell differentiation, proliferation, and cytokine production, both in vivo as well as in the assay used here (22), and for this reason the frequencies reported here may still underestimate the magnitude of the CD4 response.

After the initial peak, at least up to 10 weeks after the acute primary infection, CMV-specific CD4⁺ T cell frequencies were low. This low magnitude of the antigen-specific CD4 response shown here fits with the empirical fact that the peripheral blood CD4 pool does not emphatically expand, whereas the CD8 pool does. It is in accordance with the notion that CD4⁺ T cells exert their functions mainly within lymphoid organs, whereas cytotoxic effector T cells are designed to exert their function throughout the body, especially in generalized viral infections, necessitating their massive transport through the peripheral blood compartment. In HIV infection, memory CD4⁺ T cells were shown to be preferentially contained within the peripheral lymphoid organs, whereas they emigrate toward the blood soon after viral replication is blocked (23). The observation that in the primary CMV-infected patients CMV-DNA levels are still readily detectable after mean follow-up periods of about 100 days would be in accordance with the idea that at that time point virus replication still takes place and that therefore the majority of CMV-specific T cells are actively retained within lymph nodes and spleen.

The detailed follow-up of CMV-naive renal transplant recipients allowed, for the first time, to our knowledge, documentation of changes in T-helper cell phenotype in response to a pathogen in vivo. The phenotype of early CMV-specific T cells strikingly confirms predictions, based on both ex vivo and in vitro studies. Human lymph nodes have been reported to contain T cells highly expressing both CD45RA and CD45R0. This phenotype is also found relatively early after in vitro activation of sorted naive (CD45RA^single+) T cells, suggesting that the CD45RA^highCD45R0^high phenotype is one of the earliest stages of antigen-primed, formally naive T cells (24). In addition, in healthy individuals the peripheral blood contains small numbers of these CD45RA^highCD45R0^high helper T cells, which are enriched for cycling cells and primarily produce IFNγ (25, 26). The first antigen-specific CD4⁺ T cells appearing in peripheral blood after primary CMV infection specifically displayed this CD45RA^highCD45R0^high phenotype and were actively cycling based on Ki67 expression. Thus, the small numbers of circulating CD45RA^highCD45R0^high cells may represent T cells that, after initial activation by neoantigens, emigrate from lymph nodes to other sites of the body. The reason for this early migration is unclear at this moment, but it could be speculated that spreading of antigen-specific helper cells contributes to an efficient, specific immune response that eventually takes place in most secondary lymphoid organs.

We postulated previously that the expression of CD27 on CD4⁺ T cells is downregulated after persistent stimulation of T cells in vivo (26, 27). In fact, the CD27^– phenotype of CMV-specific CD4⁺ T cells that we found in CMV carriers is consistent with repetitive in vivo stimulation and suggests also that during viral latency, continuous or intermittent activation of CMV-specific helper T cells by viral products takes place to control overt virus replication.

Antigen-specific T cells need several rounds of cell division to obtain the capacity to produce IL4, whereas IFNγ may be produced in low quantities without previous cell division (28). The lack of IL4-producing cells found during primary infection may therefore be due to the early time point of analysis of cytokine phenotype. However, long after establishment of antiviral memory, we and others found low frequencies of IL4-producing CMV-specific cells, which suggest that CMV elicits a prototype Th1 response (13, 29).

In conclusion, to our knowledge this is the first study that reveals the developing immune response to primary CMV infection in vivo in humans. These data may serve as a reference to which patients with more profound states of immunodeficiency may be compared. The test can be used to monitor primary CMV infection in other groups of patients and may be helpful in decisions regarding antiviral prophylaxis or treatment.

The authors thank the patients and healthy subjects for their participation, S. Surachno for patient inclusion, A.J. Kolk for initial reagent supply, and Yvette Gerrits for accurate performance of the CMV-PCR. We thank D. Hamann, F. Bemelman, and P. Schellekens for critically reading the manuscript. Grants from the Dutch Kidney Foundation support R.J. Rentenaar and F.N.J. van Diepen (C95-1455) and L.E. Gamadia (C98-1724).

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