Selective Depletion of Nonspecific T Cells During the Early Stage of Immune Responses to Infection

Mice, bacteria, viruses, and infection

C57BL/6 mice (Thy1.2) were purchased from the National Cancer Institute (Frederick, MD). C57BL/6 (Thy1.1), P14, and OT-I TCR transgenic mice (14, 15) on B6.Thy1.1, B6.Thy1.2, and B6.Thy1.1/Thy1.2 backgrounds were bred in-house. All mice were maintained in institutional animal care and use committee-approved facilities at the University of Pennsylvania School of Medicine (Philadelphia, PA). Mice were age- and sex-matched within each experiment. Recombinant LM strains, rLM-gp33 and rLM-OVA, which express the H-2D^b-restricted gp33–41 epitope of lymphocytic choriomeningitis virus (LCMV) and the H-2D^b-restricted OVA epitope, OVA_323–339, respectively, were constructed by chromosomal integration into wild-type strain 10403s (16, 17). Mice were infected i.v. with ∼5 × 10⁴ CFU LM or i.p. with ∼5 × 10⁶ PFU of LCMV Armstrong strain (18).

Adoptive transfers

Splenocytes were isolated from P14 or OT-I mice and labeled with 5 μM CFSE (Molecular Probes, Eugene, OR) in PBS, then quenched with 100% FCS. Cells were then washed with RPMI 1640/10% FCS and resuspended in PBS. P14 and OT-I T cells were purified by MACS using CD8a Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and were transferred i.v. into recipient mice (2–5 × 10⁶ cells/mouse, or 1–2 × 10⁷ cells/mouse for confocal microscopy). For cotransfer experiments, equal numbers of purified CFSE-labeled P14 and OT-I cells were mixed and transferred. One day after transfer, recipients were infected with LM or LCMV.

Flow cytometry

Single-cell suspensions of splenocytes were resuspended in PBS/1% BSA/0.1% NaN₃ at a concentration of 1–5 × 10⁷ cells/ml, stained for cell surface markers using mAbs from BD PharMingen (San Diego, CA), and fixed with 2% paraformaldehyde/PBS. Flow cytometry was performed with a FACSCalibur (BD Biosciences, San Jose, CA), and data were analyzed using FlowJo software (Tree Star, San Carlos, CA).

In situ detection of apoptotic cells in spleens using TUNEL

Frozen 5-μm-thick spleen sections were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. TUNEL-positive cells were detected using the In Situ Cell Death Detection Kit/TMR Red (Roche, Indianapolis, IN). Sections were viewed and photographed using a laser-scanning confocal fluorescence microscope system (1024-ES; Bio-Rad, Hercules, CA).

Kinetics of T cell depletion and expansion following LM infection

To examine the early events of T cell response, we infected C57BL/6 mice with LM and followed the kinetics of activation and expansion of CD4 and CD8 T cells (Fig. 1⇓). As reported by others (1, 4, 5, 6, 7), substantial depletion of T cells was evident by day 3 post-LM infection. The CD4 T cell population decreased from ∼8.5 × 10⁶/spleen (∼23% of splenocytes) on day 0 to ∼2.6 × 10⁶/spleen (∼11% of splenocytes) on day 3 after infection. Similarly, the CD8 T cell population decreased from ∼5 × 10⁶/spleen (∼15% of splenocytes) on day 0 to ∼1.8 × 10⁶/spleen (∼7% of splenocytes) on day 3. Beginning from day 3, however, both CD4 and CD8 T cell populations rebounded, and by day 7, CD4 and CD8 T cells reached ∼8.1 × 10⁶/spleen (∼21% of splenocytes) and ∼8.5 × 10⁶/spleen (∼32% of splenocytes), respectively. The increase in CD4 and CD8 T cells from days 3–7 was exclusively due to accumulation of activated (CD44^high) cells. CD44^high cells made up only ∼13% of CD8 cells on day 3, but increased to ∼81% on day 7 after infection (Fig. 1⇓B), and a similar increase in the CD44^high population was observed for CD4 T cells (data not shown). Thus, T cell compartments were repopulated from days 3–7 by activated T cells, which made up the majority of the T cell population at the peak of response on day 7.

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FIGURE 1.

Kinetics of T cell depletion and expansion during the early stage of LM infection. C57BL/6 mice were infected with LM, and on days 0, 3, 5, and 7 splenocytes were stained for CD4, CD8, and CD44 and analyzed by FACS. A, Percentages of splenocytes that are CD4⁺ or CD8⁺. B, Percentages of CD8⁺ cells that are CD44^high or CD44^low. C, The number of CD4 and CD8 T cells per spleen. FACS plots in A and B are from representative mice in each group, and data in C are the mean ± SD of three to five mice per group. Similar results were observed in two independent experiments.

Selective depletion of nonspecific T cells during LM infection

To determine whether most of the activated T cells are specific to LM Ags or there is a substantial bystander activation of nonspecific T cells, we employed an adoptive transfer model using P14 TCR transgenic cells that are specific to the H-2D^b-restricted gp33–41 epitope of LCMV. Before transfer, P14 cells were labeled with CFSE, a vital fluorescent dye that is equally partitioned into daughter cells, thus allowing visualization of cell division. Recipient mice were infected with a recombinant LM strain (rLM-gp33) that expresses the gp33–41 epitope or, as a control, the parental wild-type strain of LM (wtLM). On day 3 postinfection we examined the proliferation of P14 cells by analyzing CFSE profiles and determined their activation status by staining for CD62L, a surface marker whose expression is down-regulated in activated T cells. As expected, the transferred P14 cells became activated and proliferated rapidly in response to rLM-gp33 infection, while few, if any, P14 cells divided or displayed an activation phenotype in PBS-treated or wtLM-infected mice (Fig. 2⇓A). Surprisingly, however, a substantial loss of P14 cells was noted in the wtLM-infected mice. Transferred P14 cells constituted 0.22% of the total splenocytes, which dropped to only 0.03% in wtLM-infected mice by day 3 postinfection. The total numbers of P14 cells per spleen were significantly lower in wtLM-infected (∼5 × 10³) compared with PBS-treated (∼1 × 10⁵) mice. Thus, >90% of transferred P14 cells were depleted in the spleens of wtLM-infected mice. In contrast, the P14 cells expanded >10-fold, to ∼2 × 10⁶/spleen by day 3 in rLM-gp33 infected mice. These results indicate that gp33-specific P14 cells proliferated rapidly in response to rLM-gp33 infection, but were depleted in the spleen of wtLM-infected mice.

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FIGURE 2.

Proliferation of Ag-specific T cells is accompanied by depletion of nonresponding T cells during the early stage of LM infection. A, CFSE-labeled gp33-specific P14 T cells (Thy1.2) were adoptively transferred into congenic Thy1.1 C57BL/6 mice. Recipient mice (three to five per group) were infected with wtLM or rLM-gp33. On day 3 postinfection, transferred P14 cells and host CD8 T cells in the spleen were identified by CD8⁺/Thy1.2⁺ and CD8⁺/Thy1.2⁻ staining, respectively (upper panel; numbers indicate the percentages of total splenocytes). Proliferation and activation of transferred P14 cells were visualized by loss of CFSE and down-regulation of CD62L (lower panel; gated on Thy1.2⁺/CD8⁺ cells). Absolute numbers of transferred P14 cells in spleens were determined (bar graph). Similar results were obtained in five separate experiments. B, Equal numbers of CFSE-labeled, MACS-enriched OT-I cells (Thy1.1⁺/Vβ5⁺) and P14 cells (Thy1.1⁺/Vβ8⁺) were cotransferred into congenic Thy1.2 C57BL/6 mice. Recipient mice (three per group) were infected with rLM-gp33 or rLM-OVA. Proliferation of transferred OT-I and P14 cells in spleens was examined 3 days postinfection (dot plots are gated on Thy1.1⁺ cells, and OT-I cells are distinguished from P14 cells by Vβ5⁺ staining). Total numbers of OT-I and P14 cells in spleens were determined (bar graph). Similar results were obtained in three independent experiments.

Depletion of gp33-specific P14 cells in wtLM-infected, but not rLM-gp33-infected, mice could be due to 1) genetic alterations in rLM-gp33 that render this strain less virulent and/or unable to cause T cell depletion, or 2) expression of the gp33 epitope in rLM-gp33 that provides antigenic stimulation and rescues P14 cells from depletion. We examined the first possibility by assessing the virulence of rLM-gp33 and its ability to induce depletion of polyclonal T cells. The 50% lethal dose and in vivo growth kinetics were similar between rLM-gp33 and wtLM, and infection with rLM-gp33 and wtLM induced similar levels of depletion of polyclonal T cells in normal mice (data not shown). In the adoptive transfer mice (Fig. 2⇑A), the host CD8 T cells (CD8⁺/Thy1.2⁻) decreased from 8.26% in PBS-treated to 3.30 and 3.91% in wtLM- and rLM-gp33 infected mice, respectively. These results demonstrate that genetic alterations in rLM-gp33 did not render this strain less virulent or unable to cause T cell depletion. We then considered the second possibility, that T cell depletion during the early stage of LM infection is limited to nonspecific T cells, while Ag-specific cells are activated to expand rapidly. To investigate this possibility, we transferred a mix of two populations of CD8 T cells with defined specificities: P14 cells specific to the gp33 epitope and OT-I cells specific to OVA. Recipient mice were then infected with isogenic strains of rLM-gp33 or rLM-OVA (16). By day 3 postinfection OVA-specific OT-I cells were depleted, while gp33-specific P14 cells proliferated in mice infected with rLM-gp33 (Fig. 2⇑B). Conversely, OT-I cells proliferated and increased in number, while P14 cells were depleted in rLM-OVA-infected mice. These results provide internal controls within the same mice and clearly demonstrate that nonspecific CD8 T cells in the spleen are depleted while Ag-specific CD8 T cells are activated to expand during LM infection.

It is possible that depletion of Ag-specific cells also occurs but is accompanied or followed by Ag-driven expansion, resulting in an increase in total P14 cells despite their depletion. We thus took a quantitative approach to assess possible depletion of P14 cells in rLM-gp33 infected mice. We closely followed the proliferation of transferred, CFSE-labeled P14 cells at different time points after infection (Fig. 3⇓). In wtLM-infected mice, there was no sign of P14 cells dividing before their depletion, which occurred between 36–48 h, resulting in >90% reduction of P14 cells by 48 h (Fig. 3⇓, A and B). In rLM-gp33 infected mice there was no loss of P14 cells before their recruitment into division (0–36 h). By 48 h more P14 cells were present as a result of their proliferation, which gave rise to many daughter cells in successive divisions, as evident by their distinct peaks of CFSE fluorescence (Fig. 3⇓A). To determine whether there was loss of P14 cells during proliferation, we deduced the number of original P14 cells (precursors) required to generate these daughter cells as previously described (19). If some P14 cells were lost during proliferation, the sum of the deduced number of precursors should be fewer than the number of P14 cells in PBS-treated control mice. However, our calculations revealed that the number of deduced P14 precursors in rLM-gp33 infected mice was not significantly different from that in PBS-treated control mice (Fig. 3⇓C). Therefore, there was marginal loss of gp33-specific CD8 T cells in rLM-gp33-infected mice before and during the early phase of their expansion.

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FIGURE 3.

Ag-specific CD8 T cells are not depleted before and during the initial expansion following LM infection. CFSE-labeled, MACS-enriched P14 cells (Thy1.1) were transferred into congenic Thy1.2 C57BL/6 mice, and recipient mice (three to five per group) were infected with wtLM or rLM-gp33. At 0, 24, 36, and 48 h splenocytes were analyzed by FACS to visualize the proliferation of transferred P14 cells (A; gated on Thy1.1⁺/CD8⁺), and the total numbers of P14 cells per spleen were determined (B). To examine whether there was loss of P14 cells during expansion, the numbers of precursors required to generate the daughter cells present at 48 h in PBS-treated, wtLM-infected, and rLM-gp33-infected mice were compared (C). At 48 h many P14 cells in rLM-gp33-infected mice had divided, giving rise to distinct peaks of CFSE fluorescence that represent populations of daughter cells resulting from successive divisions (n). The percentages of P14 cells under each peak of CFSE fluorescence were experimentally determined, and the numbers of P14 cells in each division were then calculated by multiplying the percentages by the total number of P14 cells recovered. The number of original P14 cells (precursors) required for generating these daughter cells was then deduced by dividing the number of daughter cells in n divisions by 2ⁿ. The sum of the deduced precursor number in rLM-infected mice would have been less than the starting number of P14 cells present in PBS-treated mice if there had been loss of P14 cells during expansion. Similar results were obtained in two separate experiments.

Depletion of nonspecific T cells by apoptosis

Early T cell depletion in the spleen of LM-infected mice may be explained by two possible mechanisms: altered trafficking or cell death. Previous studies have examined the possibility of altered trafficking as a potential mechanism, but found no supporting evidence (1, 6, 7). Lymphocyte depletion during the early stage of LM infection was observed in many organs (1, 7), and no redistribution of radiolabeled T cells from spleens into other organs was evident following LM infection (4). In light of our new findings, it is possible that Ag-specific T cells might be selectively retained in the spleen to respond to antigenic stimulation, while nonspecific T cells migrate out of spleen into other organs. We examined total T cell numbers and the distribution of Ag-specific and nonspecific T cells in different compartments following LM infection. Consistent with the reported data (4, 7), we found that LM infection resulted in depletion of total T cells not only in the spleen, but also in other compartments, including blood, liver, bone marrow, and lymph nodes (data not shown). In all organs examined, the P14 population expanded while the numbers of OT-I cells decreased following rLM-gp33 infection. Conversely, the OT-I population increased while the number of P14 cells declined in rLM-OVA-infected mice. Thus, depletion of nonspecific cells following LM infection occurs, although to varying degrees, in all compartments examined. Furthermore, we did not observe any increase in total or nonspecific T cells in the blood preceding their depletion, which otherwise would indicate increased trafficking of these cells to other organs, nor did we observe the return of nonspecific T cells by day 7 when the spleen was repopulated (data not shown). Nonspecific T cells do not become fully activated during LM infection (Figs. 2⇑ and 5⇓), and the increase in T cell number from days 3–7 is mostly by accumulation of activated (CD44^high) T cells, which make up the majority (>80%) of the T cells by day 7 (Fig. 1⇑). Together these results indicate that the repopulation of T cell compartments is mostly by the proliferation of Ag-specific T cells. Although it is difficult to completely rule out the possibility that some T cells may have trafficked to unusual places that we have not examined, our results together with previous studies by several groups indicate that depletion of nonspecific T cells is unlikely to be due to redistribution of these cells into other organs.

The foci of LM infection are characterized by massive lymphocyte apoptosis, and this in situ cell death has been shown to be responsible for T cell depletion (7). We considered the possibility that apoptotic lymphocytes seen during the early stage of LM infection might be restricted to nonspecific T cells. We examined splenic sections using confocal microscopy to visualize transferred CFSE-labeled P14 cells and TUNEL-positive apoptotic cells (Fig. 4⇓). Upon transfer, CFSE-labeled P14 cells homed appropriately to the T cell zone, as shown in Fig. 4⇓A. In wtLM-infected mice, fewer CFSE-labeled P14 cells were detected compared with PBS-treated controls (Fig. 4⇓B), consistent with depletion of P14 cells observed by FACS analysis (Fig. 2⇑). In rLM-gp33-infected mice, P14 cells were increased in number, were enlarged in size, and had reduced CFSE fluorescence, consistent with their proliferation. TUNEL-positive cells were readily visible in splenic sections of wtLM- and rLM-gp33-infected mice, but not in PBS-treated controls. Most importantly, some TUNEL-positive cells colocalized with CSFE-labeled P14 cells in wtLM-infected, but not in rLM-gp33-infected, mice. When multiple sections from different mice were examined (>200 P14 cells counted for each group), ∼38% of P14 cells were TUNEL-positive in wtLM-infected mice, while <2% of P14 cells were TUNEL-positive in rLM-gp33-infected and PBS-treated mice. These results clearly demonstrate that P14 cells undergo apoptosis in wtLM-infected, but not in rLM-gp33-infected, mice. Thus, nonspecific T cells are depleted as a result of in situ death by apoptosis, while Ag-specific T cells are spared, providing conclusive evidence for selective depletion of nonspecific T cells during the early stage of LM infection.

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FIGURE 4.

Nonspecific CD8 T cells are depleted by apoptosis during the early stage of LM infection. CFSE-labeled and MACS-enriched P14 cells (2 × 10⁷; >95% Thy1.2⁺/CD8⁺) were transferred into C57BL/6 mice, which were then infected with wtLM or rLM-gp33. A, Spleen sections from PBS-treated control mice were stained with anti-B220 mAb (red) and examined by confocal microscopy (×200), showing proper localization of transferred CFSE-labeled P14 cells (green) to the T cell regions of the spleen. B, Sections of spleens harvested 48 h postinfection were stained for apoptosis by TUNEL staining and examined by confocal microscopy (×600). The single-color images show CFSE-labeled P14 cells in green, TUNEL-positive cells in red, and color overlay images show apoptotic P14 cells in yellow. Inset histograms show CFSE profiles of transferred P14 cells.

Partial activation of nonspecific T cells before depletion

Resting and activated lymphocytes differ in their sensitivities to various death signals and have dissimilar requirements for survival (20). We examined the activation phenotypes of nonspecific T cells before their depletion (Fig. 5⇓). Within 24 h following LM infection, most T cells in the spleen up-regulated their surface expression of the early activation marker CD69 (Fig. 5⇓A), and this included both Ag-specific and nonspecific T cells (Fig. 5⇓B). Ag-specific T cells (P14 in rLM-gp33-infected mice and OT-I in rLM-OVA-infected mice) were further activated to divide and display a full spectrum of late activation phenotypes (CD44^high, CD62L^low, and CD25^high). On the other hand, nonspecific T cells (P14 cells in rLM-OVA-infected mice and OT-I in rLM-gp33-infected mice) did not divide or display any late activation markers. These results indicate that nonspecific T cells are activated in the early stage of LM infection, but only to a state of partial activation before their depletion. T cells receiving incomplete and inadequate activation signals do not progress through the cell cycle, but instead undergo apoptosis (20). Thus, it is possible that partial activation may lead to apoptosis of nonspecific T cells during the early stage of LM infection. Ag-specific T cells, on the other hand, are rescued from apoptosis by TCR engagement, which stimulates them to become fully activated, divide, and differentiate into effector cells to participate in the control of infection.

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FIGURE 5.

Nonspecific T cells are partially activated before their depletion during LM infection. A, C57BL/6 mice were infected with wtLM and on days 0, 1, and 2, and surface expression of CD69, CD44, CD62L, and CD11a on CD8 T cells was examined (gated on CD8⁺ cells). B, OT-I and P14 cells were cotransferred into C57BL/6 mice, which were then infected with rLM-OVA or rLM-gp33 as described in Fig. 1⇑B. The expression of activation markers (CD69, CD44, CD62L, and CD25) on OT-I and P14 cells was examined on day 3 after infection. Solid line, OT-I cells; filled gray area, P14 cells.

Depletion of nonspecific T cells during LCMV infection

IFN-α/IFN-β are strongly induced in many viral and bacterial infections (21, 22) and are known to stimulate naive T cells to a state of partial activation with up-regulation of CD69 surface expression (23, 24). Products of viral and bacterial pathogens, such as poly I:C, LPS, and undermethylated CpG DNA, induce IFN-α/IFN-β-mediated partial activation of naive T cells (23, 24). Thus, we asked whether partial activation and selective depletion of nonspecific T cells also occur in the early stage of other infections. A previous study reported depletion of T cells during LCMV infection, but did not demonstrate whether the depletion is limited to nonspecific T cells (3). We cotransferred P14 and OT-I cells into recipient mice and examined these two populations on day 3 following LCMV infection (Fig. 6⇓). LCMV infection resulted in depletion of OT-I and the host polyclonal CD8 T cell population, but not P14 cells (Fig. 6⇓A). Most T cells, including both P14 and OT-I cells, expressed the early activation marker (CD69), while only P14 cells specific to the LCMV gp33 epitope displayed late activation markers (e.g., CD62L^low; Fig. 6⇓B). Confocal microscopy showed that a substantial number of OT-I cells (∼40% with ∼200 cells counted), but few P14 cells (<2% with ∼200 cells counted), were TUNEL-positive in LCMV-infected mice (Fig. 6⇓C). These results demonstrate that LCMV infection also induces partial activation of most T cells, which is followed by selective depletion of nonresponding T cells. Together, our results indicate that selective depletion of nonresponding T cells is probably a general phenomenon and an integral part of the T cell response to many infections.

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FIGURE 6.

Partial activation and selective depletion of nonspecific CD8 T cells occur during LCMV infection. MACS-purified OT-I (Thy1.1) and P14 (Thy1.1/Thy1.2) cells were cotransferred into C57BL/6 (Thy1.2) mice, which were then infected with LCMV Armstrong strain (three mice per group). Three days after LCMV infection, the numbers of total CD8 T cells and transferred OT-I and P14 cells in spleens were quantified (A), and the surface expression of CD69 and CD62L on OT-I and P14 cells was measured (B). To visualize OT-I and P14 cells by confocal microscopy (×600), one group of mice received a mix (1/1) of CFSE-labeled OT-I cells and unlabeled P14 cells, and the other group received a mix (1/1) of CFSE-labeled P14 cells and unlabeled OT-I cells. Frozen sections of spleens from PBS-treated and LCMV-infected mice were stained for apoptosis by TUNEL. The images in C show CFSE-labeled OT-I or P14 cells (green), TUNEL-positive cells (red), and colocalization of CFSE-labeled and TUNEL-positive cells (yellow). Inset histograms show CFSE profiles of transferred P14 and OT-I cells. Similar results were seen in three separate experiments.