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. 2009 Mar;85(3):382-90.
doi: 10.1189/jlb.0808491. Epub 2008 Dec 30.

Differential lymphopenia-induced homeostatic proliferation for CD4+ and CD8+ T cells following septic injury

Jacqueline Unsinger  1 , Hirotaka Kazama, Jacquelyn S McDonough, Richard S Hotchkiss, Thomas A Ferguson
Affiliations

Differential lymphopenia-induced homeostatic proliferation for CD4+ and CD8+ T cells following septic injury

Jacqueline Unsinger et al. J Leukoc Biol. 2009 Mar.

Abstract

Sepsis is a severe, life-threatening infection and a leading cause of death in hospitals. A hallmark of sepsis is the profound apoptosis-induced depletion of lymphocytes generating a lymphopenic environment. As lymphopenia can induce nonantigen-driven homeostatic proliferation (HP), we examined this process during sepsis. CD4(+) and CD8(+) T cells, which were depleted within 24 h of sepsis induction, remained at significantly reduced levels until Day 21 when normal numbers were detected. When HP was examined, naïve CD8(+) T cells proliferated between Day 7 and Day 21 post-cecal ligation and puncture, developing into memory cells with relatively few cells expressing an activation phenotype. Conversely, naïve CD4(+) T cells did not undergo HP, but proportionally higher numbers expressed activation markers. Adoptive transfer studies revealed that T cells from mice that had recovered from sepsis were not protective when transferred to naïve mice undergoing sepsis. In addition, the TCR repertoire was not skewed toward any specific Vbeta type but resembled the repertoire found in normal mice, suggesting that T cells were not primed to antigens resulting from the infection. Interestingly, depletion of endogenous CD8(+) but not CD4(+) T cells restored the ability of naive CD4(+) T cells to undergo HP, increasing the number of CD4(+) T cells with memory but not activation markers. We conclude that homeostatic control in the postseptic environment permits recovery of the T cell repertoire to normal levels without generating antigen-specific memory or aberrant T cell specificities. Restoration of homeostatic control mechanisms might be a rational therapy for this disorder.

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Figures

Fig. 1.
Fig. 1.
T cell depletion in the spleen and thymus. Mice underwent a sham operation or CLP. At various times postsurgery, spleen and thymus were harvested and the absolute number of T cell subsets enumerated. (A) CD4+ and CD8+ splenic T cells; (B) Double-positive (DP; CD4+CD8+ T cells), single-positive (SP; CD4+ or CD8+ T cells), and double-negative (DN; CD4–CD8– T cells) thymic T cells. Values are expressed as total counts per spleen or thymus. Each value represents the average of three to five mice. Significance: ***, P < 0.001; **, P < 0.01; *, P < 0.05.
Fig. 2.
Fig. 2.
HP of CD4+ and CD8+ T cells during CLP. Recipient mice underwent a sham operation or CLP. Seven days later, mice received 1 × 106 CFSE-labeled: (A) OT-I CD8+ T cells; (B) naïve (wt) CD8+ T cells; (C) OT-II CD4+ T cells; (D) naïve CD4+ T cells. On Day 21 postsurgery, spleens were harvested, and CFSE content was assessed by flow cytometry. Data shown represent one of three sham mice and two of five CLP animals examined. (E) Mice received 600 R γ-irradiation, and 1 day later, they were infused with 1 × 106 CFSE-labeled, naïve CD4+ or CD8+ T cells. CFSE content in splenic cells was assessed 7 days later by flow cytometry. Three of five representative mice are shown for CD4+ and CD8+ T cells.
Fig. 3.
Fig. 3.
Proliferation of naïve CD4+ T cells to IL-7 and cognate antigen. (A) Mice underwent a sham operation or CLP. Seven days later, they were given 1 × 106 naïve, CFSE-labeled CD4+ T cells. Each mouse received 5 μg rIL-7 via i.p. injection every other day for a total of 14 days. Mice were then killed on Day 21 postsurgery and proliferation determined by CFSE content. (B) Mice underwent a sham operation or CLP. Seven days later, they were given 1 × 106 naïve, CFSE-labeled OT-II T cells. Twenty-four hours later, mice received 100 μg Ova peptide (OVA323–339) i.v., and the proliferation of OT-II CD4+ T cells was evaluated 3 days later. Two of five representative mice are shown for sham and CLP.
Fig. 4.
Fig. 4.
Activated and memory cells following CLP. Mice underwent a sham operation or CLP. Twenty-one days later, spleens were harvested and analyzed by flow cytometry for activation (CD69+) and memory (CD44hiCD62Llow) T cells in the (A) CD8+ T cell population and (B) CD4+ T cell population. (C) CD45.2-expressing C57BL/6 mice underwent a sham operation or CLP. At Day 7 post-CLP, mice received 2 × 106 naïve, congenic (CD45.1-expressing), CFSE-labeled CD4+ T cells. Twenty-one days postsurgery, spleens were harvested, and the percentage of activation and memory endogenous (CD45.2+) and transferred (CD45.1+) CD4+ T cells was evaluated by flow cytometry. Each value represents the average of eight to 10 mice over two separate experiments.
Fig. 5.
Fig. 5.
Vβ analysis and immunologic memory following recovery from sepsis. (A) On Day 21 following a sham or CLP operation, spleens were harvested, and the expression of Vβ TCR epitopes was assessed by flow cytometry. Each value represents the average of five mice. (B) Mice received sham or CLP surgery. At Day 21 postsurgery, these mice were used as donors for the adoptive transfer of 50 × 106 whole spleen cells to naïve mice. Recipient mice then underwent sham or CLP surgery 24 h later. Survival was monitored for 7 days. Each group consisted of 12 mice.
Fig. 6.
Fig. 6.
Adoptive transfer of cells or serum from septic mice, which received sham or CLP surgery. At Day 7 postsurgery, these mice were used as donors for the adoptive transfer of whole spleen cells and serum, which were transferred to mice that had received 600 R irradiation and 2 × 106 naïve, CFSE-labeled CD4+ T cells 24 h earlier. Proliferation was assessed by CFSE content 7 days later. One of two sham and one of three CLP mice are shown for each group.
Fig. 7.
Fig. 7.
In vivo depletion of CD4+ and CD8+ T cells. Mice underwent a sham or CLP operation. (A) Depletion of CD8+ T cells was achieved by three daily, 100 μg doses of αCD8 (mAb 2.43) beginning 3 days prior to infusion of CFSE-labeled, naïve CD4+ T cells on Day 7. (B) CD4+ T cells were depleted by three daily i.v. doses of 100 μg GK1.5 on Days 1–3 postsurgery. Seven days postsurgery, 1 × 106 CFSE-labeled, naïve CD4+ T cells were adoptively transferred i.v. HP was assessed on Day 21 by flow cytometry. (C) On Day 21 post-CLP, the numbers of activated (CD69+) and memory (CD44hiCD62low) CD4+ T cells in CD8-depleted mice were assessed by flow cytometry.
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