Lymphopenia-induced Proliferation Research Articles

Lymphopenia-Induced Homeostatic Proliferation of CD8+ T Cells Is a Mechanism for Effective Allogeneic Skin Graft Rejection following Burn Injury

Burn patients are immunocompromised yet paradoxically are able to effectively reject allogeneic skin grafts. Failure to close a massive burn wound leads to sepsis and multiple system organ failure. Immune suppression early (3 days) after burn injury is associated with glucocorticoid-mediated T cell apoptosis and anti-inflammatory cytokine responses. Using a mouse model of burn injury, we show CD8+ T cell hyperresponsiveness late (14 days) after burn injury. This is associated with a CD8+ T cell pro- and anti-inflammatory cytokine secretion profile, peripheral lymphopenia, and accumulation of a rapidly cycling, hyperresponsive memory-like CD8+CD44+ IL-7R- T cells which do not require costimulation for effective Ag response. Adoptive transfer of allospecific CD8+ T cells purified 14 days postburn results in enhanced allogeneic skin graft rejection in unburned recipient mice. Chemical blockade of glucocorticoid-induced lymphocyte apoptosis early after burn injury abolishes both the late homeostatic accumulation of CD8+ memory-like T cells and the associated enhanced proinflammatory CD8+ T cell response, but not the late enhanced CD8+ anti-inflammatory response. These data suggest a mechanism for the dynamic CD8+ T cell response following injury involving an interaction between activation, apoptosis, and cellular regeneration with broad clinical implications for allogeneic skin grafting and sepsis.

The generation of protective memory-like CD8+ T cells during homeostatic proliferation requires CD4+ T cells

Antigen-specific memory T cells are a critical component of protective immunity because of their increased frequency and enhanced reactivity after restimulation. However, it is unclear whether 'memory-like' T cells generated during lymphopenia-induced homeostatic proliferation can also offer protection against pathogens. Here we show that homeostatic proliferation-induced memory (HP-memory) CD8(+) T cells controlled bacterial infection as effectively as 'true' memory CD8(+) T cells, but their protective capacity required the presence of CD4(+) T cells during homeostatic proliferation. The necessity for CD4 help was overcome, however, if the HP-memory CD8(+) T cells lacked expression of TRAIL (tumor necrosis factor-related apoptosis-inducing ligand; also called Apo-2L). Thus, like conventional CD8(+) memory T cells, the protective function of HP-memory CD8(+) T cells shows dependence on CD4(+) T cell help.

Accelerated Memory Cell Homeostasis during T Cell Depletion and Approaches to Overcome It

Partial T cell depletion is used in solid organ transplantation as a valuable strategy of peritransplant induction immunosuppression. Using a murine cardiac allograft model, we recently demonstrated that this led to lymphopenia-induced (homeostatic) proliferation among the residual nondepleted lymphocytes. Rather than promoting tolerance, peritransplant T cell-depleting Abs actually resulted in resistance to tolerance induction by costimulatory blockade. In this study we show that memory T cells predominate shortly after subtotal lymphodepletion due to two distinct mechanisms: relative resistance to depletion and enhanced homeostatic proliferation. In contrast, regulatory cells (CD4+ CD25+ Foxp3+) are depleted as efficiently as nonregulatory cells and exhibit reduced homeostatic expansion compared with memory cells. The resistance to tolerance induction seen with subtotal T cell depletion can be overcome in two different ways: first, by the adoptive transfer of additional unprimed regulatory cells at the time of transplant, and second, by the adjunctive use of nondepleting anti-CD4 and anti-CD8 mAbs, which effectively block homeostatic expansion. We conclude that the resistance to tolerance induction seen after subtotal lymphocyte depletion can be attributed to alterations in the balance of naive, memory, and regulatory T cells. These data have clinically relevant implications related to the development of novel strategies to overcome resistance to tolerance.

The Death Receptor Fas (CD95/APO-1) Mediates the Deletion of T Lymphocytes Undergoing Homeostatic Proliferation

Murine T cells adoptively transferred into syngeneic lymphopenic recipients undergo proliferation. Despite continued cell division, this lymphopenia-induced or homeostatic proliferation of a limited number of transferred T cells does not fill the T cell compartment. The continued expansion of the transferred T cells, even after stable T cell numbers have been reached, suggests that active cell death prevents further increase in T cell number. In this study, we show that wild-type T cells undergoing homeostatic proliferation are sensitive to Fas-mediated cell death. In the absence of Fas, T cells accumulate to significantly higher levels after transfer into lymphopenic recipients. Fas is, thus, a principal regulator of the expansion of peripheral T cells in response to self-peptide/MHC during T cell homeostasis. As Fas-deficient lpr mice manifest no significant abnormalities in thymic negative selection or in foreign Ag-induced peripheral T cell deletion, their lymphadenopathy may result from unrestrained homeostatic proliferation.

Effects of Increasing IL-7 Availability on Lymphocytes during and after Lymphopenia-Induced Proliferation

IL-7 is critically involved in regulating peripheral T cell homeostasis. To investigate the role of IL-7 on lymphopenia-induced proliferation of polyclonal lymphocytes, we have transferred CFSE-labeled cells into a novel T-lymphopenic, IL-7-transgenic mouse line. Results obtained indicate that T and B cells do not respond in the same way to IL-7-homeostatic signals. Overexpression of IL-7 enhances proliferation of both CD4(+) and CD8(+) T cells but with distinctly temporal effects. Expansion of naturally arising CD4(+)-regulatory T cells was like that of conventional CD4(+) T cells. IL-7 had no effect on B cell proliferation. By immunohistology, transferred T cells homed to T cell areas of spleen lymphoid follicles. Increasing IL-7 availability enhanced T cell recovery by promoting cell proliferation and reducing apoptosis during early stages of lymphopenia-induced proliferation. Taken together, these results provide new insights into the pleiotropic effects of IL-7 on lymphopenia-induced T cell proliferation.

A Major Role for Memory CD4 T Cells in the Control of Lymphopenia-Induced Proliferation of Naive CD4 T Cells

In a state of lymphopenia, naive and memory CD4 T cells compete with each other for expansion at the expense of naive T cells. This competition prevents the proliferation as well as the phenotypic and functional conversion of naive T cells to "memory-like" T cells and may consequently prevent immune pathology frequently associated with lymphopenia-induced proliferation of naive cells. However, in T cell replete mice, memory T cells do not compete with naive T cells, indicating independent homeostatic control of naive and memory CD4 T cells in conditions that do not involve profound lymphopenia. Moreover, within the memory compartment, subsequent generation of new memory T cells precludes the survival of memory-like T cells. Thus, memory T cells have a major role in the control of lymphopenia-induced proliferation of naive cells because they inhibit both the generation of memory-like T cells and their persistence within the memory compartment.

The molecular program induced in T cells undergoing homeostatic proliferation

Naïve T cells proliferate independently of cognate antigen when introduced into lymphopenic hosts. Lymphopenia-induced proliferation depends on low-affinity MHC/self-peptide complexes and on IL-7. To elucidate the intracellular signals mediating this proliferation, we analyzed changes in gene expression in naive CD8+ T cells at different times after their transfer into a lymphopenic environment. The genes induced in response to lymphopenia were largely an attenuated subset of those turned up by full antigenic stimulation, including genes related to cell cycling, whereas excluding genes specifically associated with effector activity. After the initial phase of proliferation in an empty compartment, the naive T cells adopted a stable pattern of gene expression similar to that of antigen-experienced memory cells. Thus, T cells proliferating in lymphopenic hosts do not exhibit a unique gene-expression profile, instead relying on "traditional" signals for this antigen-independent proliferation; this process ultimately results in differentiation to "authentic" memory cells.

Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation

The function of the Src-family kinases (SFKs) Lck and Fyn in T cells has been intensively studied over the past 15 years. Animal models and cell line studies both indicate a critical role for Lck and Fyn in proximal T-cell antigen receptor (TCR) signal transduction. Recruited SFKs phosphorylate TCR ITAMs (immunoreceptor tyrosine-based activation motifs) in the CD3 and zeta chains, which then serve as docking sites for Syk-family kinases. SFKs then phosphorylate and activate the recruited Syk-family kinase. Lck and Fyn are spatially segregated in cell membranes due to differential lipid raft localization, and may undergo sequential activation. In addition to the CD4 and CD8 coreceptors, a recently described adaptor, Unc119, may link SFKs to the TCR. CD45 and Csk provide positive and negative regulatory control of SFK functions, respectively, and Csk is constitutively bound to the transmembrane adapter protein, PAG/Cbp. TCR-based signaling is required at several stages of T-cell development, including at least pre-TCR signaling, positive selection, peripheral maintenance of naive T cells, and lymphopenia-induced proliferation. SFKs are required for each of these TCR-based signals, and Lck seems to be the major contributor.

Enhanced lymphocyte proliferation responses in pediatric patients early after myelosuppressive chemotherapy.

It has long been known that patients both after myelosuppressive chemotherapy (ChTh) and after myeloablative bone marrow transplantation (BMT) show a long lasting impairment of cellular immune functions. However, recent reports have revealed that early after BMT a passing state of augmented immune responsiveness exists. Adoptive T cell therapy in this period of lymphopenia-induced (homeostatic) proliferation has shown better results than in steady state in murine studies. To determine whether also early after myelosuppressive ChTh enhanced immune responses can be found, we have determined proliferation of peripheral blood lymphocytes and calcium influx and performed immunophenotyping in pediatric patients recovering from myelosuppressive ChTh in comparison to immunoreconstituted patients late after BMT. The lymphocytes of the ChTh patients were found to proliferate vigorously in response to stimulation with a variety of antibodies and mitogens, while in the BMT patients any stimulation was severely reduced. The increase of intracellular calcium after stimulation was similar in both patient groups. ChTh patients showed an expansion of an activated "naive" phenotype (CD45RO- HLA-DR+) in both the CD4 and CD8 subsets. In contrast, BMT patients showed a prominent expansion of "memory type" T lymphocytes (CD45RO+ HLA-DR+). Early after ChTh, a period of immunoaugmentation seems to exist. Whether this observation can be used clinically to increase cure rates remains to be elucidated.

Cutting edge: IL-7-dependent homeostatic proliferation of CD8+ T cells in neonatal mice allows the generation of long-lived natural memory T cells.

Healthy, nonimmunized C57BL/6 (B6) mice contain memory phenotype CD8+ T cells, which are assumed to be generated in response to environmental Ags. Since neonatal mice are functionally lymphopenic within the first days after birth, we investigated the alternative possibility that the memory CD8+ T cells of untreated B6 mice are the result of lymphopenia-induced proliferation during neonatal life. We show here that adoptively transferred CD8+ T cells proliferate in neonatal B6 mice, rapidly produce IFN-gamma, and develop into memory cells which are maintained until adulthood. In contrast to CD4+ T cells, neonatal lymphopenia-induced proliferation of CD8+ T cells was IL-7 dependent. Thus, neonatal lymphopenia seems to allow CD8+ thymic emigrants to undergo lymphopenia-induced proliferation during early neonatal life to equip the immune system with a set of preactivated CD8+ T cells before any infection, which might contribute to the rapid initiation of immune responses in the adult.

On the role of MHC class II molecules in the survival and lymphopenia-induced proliferation of peripheral CD4+ T cells.

CD4(+) T cells expand after transfer into lymphopenic H-2(b) A(beta)-/- mice (I-A(beta)-, I-E(alpha)-deficient mice) but not after transfer into lymphopenic MHC II(Delta/Delta) mice (I-A(alpha)-, I-A(beta)-, I-E(alpha)-, and I-E(beta)-deficient mice), implying that in H-2(b) A(beta)-/- mice, A(alpha) chain and E(beta) chain associate to form a hybrid A(alpha)E(beta) MHC class II molecule. In light of this unexpected result, we reexamined the MHC class II requirement in the survival and lymphopenia-induced proliferation of CD4(+) T cells. Here we show that expansion, but not short-term survival, of CD4(+) T cells depends on interactions with MHC class II molecules in lymphopenic mice. Nevertheless, interactions with classical MHC class II molecules are required for CD4(+) T cells to survive in CD8(+) T-cell-containing mice.

Phenotypic changes with immunosuppression in human recipients

The differentiation of lymphocyte subsets plays an important role in transplant medicine. The review describes alterations in the expression of surface molecules on circulating T cells associated with immunosuppressive therapy. Phenotypic changes are reflecting the various stages of T cell development, the degree of thymic involution, and disease- and treatment-associated perturbations. The most pronounced effects on T cell subsets are mediated by depleting therapies with mono- or polyclonal antibodies. The lymphopenia-induced homeostasis-driven proliferation leads to a novel homeostasis characterized by the expansion of cell subsets that characteristically show a phenotype of terminally differentiated cells such as CD8+CD57+CD28- T cells. Cells with these markers have long been linked with suppressor functions and recent data suggest their specific regulatory role. In addition cell phenotypes seen in transplant patients show striking similarities with changes in immune cells associated with aging. These phenotypic changes suggest an accelerated aging of the immune system in transplantation.

Neonates Support Lymphopenia-Induced Proliferation

T cells expand without intentional antigen stimulation when transferred into adult lymphopenic environments. In this study, we show that the physiologic lymphopenic environment existing in neonatal mice also supports CD4 T cell proliferation. Strikingly, naive CD4 T cells that proliferate within neonates acquire the phenotypic and functional characteristics of memory cells. Such proliferation is inhibited by the presence of both memory and naive CD4 T cells, is enhanced by 3-day thymectomy, is independent of IL-7, and requires a class II MHC-TCR interaction and a CD28-mediated signal. CD44 bright CD4 T cells in neonates have a wide repertoire as judged by the distribution of Vβ expression. Thus, lymphopenia-induced T cell proliferation is a physiologic process that occurs during the early postnatal period.

Cutting Edge: Depletion of CD4+CD25+ Regulatory T Cells Is Necessary, But Not Sufficient, for Induction of Organ-Specific Autoimmune Disease

Thymectomy of BALB/c mice on day 3 of life results in the development of autoimmune gastritis (AIG) due to the absence of CD4(+)CD25(+) regulatory T cells. However, depletion of CD4(+)CD25(+) T cells by treatment with anti-CD25 rarely resulted in AIG. Depletion was efficient, as transfer of splenocytes from depleted mice induced AIG in nu/nu mice. One explanation for this result is that CD4(+)CD25(-) T cells upon transfer to nude recipients undergo lymphopenia-induced proliferation, providing a signal for T cell activation. Cotransfer of CD25(+) T cells did not inhibit initial proliferation but did suppress AIG. Surprisingly, immunization with the AIG target Ag, H/K ATPase, in IFA failed to induce disease in normal animals but induced severe AIG in CD25-depleted mice. These results demonstrate that second signals (nonspecific proliferation, TCR activation, or inflammation) are needed for induction of autoimmunity in the absence of CD25(+) regulatory T cells.

T cell proliferation, differentiation, and restoration in lymphopenic individuals: for contributed volumes.

When a small number of naive T cells are adoptively transferred into syngeneic lymphopenic hosts, such as mice deficient in recombination activating gene-1 (RAG-/-) or sublethally irradiated normal mice, the transferred T cells proliferate, a phenomenon referred to as homeostasis-driven or lymphopenia-induced proliferation. Although deliberate antigen stimulation is not required, homeostatic T cell proliferation requires i) the engagement of T cell receptor (TCR) with self peptide-major histocompatibility complexes (pepMHC) and ii) the presence of lymphopenia (or “space”) (Tanchot et al.,, 1997, Ernst et al., 1999, Viret et al., 1999, Goldrath and Bevan, 1999, Bender et al., 1999). We have shown that following homeostatic proliferation naive CD8 T cells acquire the phenotype and functional properties characteristic of antigen-induced memory T cells. We have also developed an in vitro system for studying homeostatic T cell proliferation. These findings and their implications for memory T cell development and the stable maintenance of naive and memory T cell compartments are discussed.