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Keywords:

  • Alloantigens;
  • allorecognition;
  • lymphocytes;
  • T-cell reactivity;
  • T helper cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Alloreactive memory T cells can significantly impact graft survival due to their enhanced functional capacities, diverse tissue distribution and resistance to tolerance induction and depletional strategies. However, their role in allograft rejection is not well understood primarily due to the lack of suitable in vivo models. In this study, we use a novel approach to generate long-lived polyclonal alloreactive memory CD4 T cells from adoptive transfer of alloantigen-activated precursors into mouse hosts. We demonstrate that CD25 upregulation is a marker for precursors to alloantigen-specific memory and have created a new mouse model that features an expanded population of polyclonal alloreactive memory T cells that is distinguishable from the naive T-cell population. Furthermore, we show that alloreactive memory T cells exhibit rapid recall effector responses with predominant IFN-γ and IL-2 production, and mediate vigorous allograft rejection. Interestingly, while we found a heterogeneous distribution of allomemory T cells in lymphoid and nonlymphoid tissues, they were all predominantly of the effector-memory (CD62Llo) phenotype. Our results present a unique model for the generation and tracking of polyclonal allospecific memory CD4 T cells in vivo and reveal insights into the distinct and robust nature of alloreactive T-cell memory.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Immunological memory forms the basis for protective immunity against previously encountered antigens and is mediated by memory T cells that exhibit enhanced functional properties and activation kinetics (1). In transplantation, the presence of alloreactive memory T cells can be detrimental to a recipient of an allograft, and has been correlated with an increased risk of acute rejection and a decrease in posttransplant renal function (2,3). Alloreactive memory T cells can arise in individuals primed by exposure to blood transfusions, pregnancy, prior organ transplants or cross-reactivity to pathogens (4–9). The dramatic increase in memory T cells from 1–5% of total CD4 T cells in the newborn to 60% by age 30 (10), and the rising age of transplant recipients make it essential to understand the mechanisms by which alloreactive memory T cells impact long-term graft survival.

Memory T cells are a heterogeneous population comprising central-memory (CD62Lhi/CCR7+) and effector-memory (CD62Llo/CCR7) subsets that circulate in lymphoid and/or nonlymphoid tissues (11,12). They possess unique characteristics, which impart functional and survival advantages over naive T cells, such as reduced activation, costimulation and survival requirements (13–17) and the ability to mediate a secondary response that is faster in kinetics and greater in magnitude than the primary naive T-cell response (18,19). These enhanced features may account for their lack of susceptibility to tolerance induction and immunosuppressive strategies that inhibit naive T-cell activation. For example, costimulation blockade using anti-CD40L antibody and donor-specific transfusion can establish tolerance to cardiac allografts in naive mice but fails to prolong graft survival in the presence of alloreactive memory T cells (20–22). Moreover, in renal transplant patients receiving T-cell depletional therapy, memory CD4 T cells persist, while naive CD4 and total CD8 T cells are dramatically reduced (23). Therefore, it is essential to characterize the nature of alloreactive memory T cells in order to develop strategies that inhibit their activation and survival.

The role of memory CD4 T cells in allograft rejection has not been well defined primarily due to a lack of appropriate in vivo models. Alloantigen-specific T-cell models have been developed, which use TCR-transgenic systems (24, 25); however, they do not accurately recapitulate the polyclonal alloimmune response in graft rejection. Furthermore, the majority of studies on polyclonal alloreactive memory have been conducted in normal hosts directly primed with alloantigen (20, 21). A limitation of these studies is the inability to differentiate between memory and naive responses in vivo, and there is a great need for models that address the polyclonal nature of alloreactive memory and enable tracking and analysis of alloantigen-specific memory T cells.

In this study, we present a novel approach for generating polyclonal alloreactive memory CD4 T cells by adoptive transfer of in vitro-primed alloantigen-specific CD4 T cells into RAG2−/− or BALB/c hosts. We demonstrate that the activation marker CD25 can be used to select for alloactivated precursors to memory T cells and have generated a memory mouse with a trackable population of polyclonal allospecific memory T cells. Furthermore, we show that our allomemory T-cell population exhibits the characteristics of a memory response, including rapid proliferation and production of effector cytokines, and the ability to mediate accelerated allograft rejection. We also demonstrate that while allomemory T cells are distributed in both lymphoid and nonlymphoid tissues, they are predominantly of the effector-memory phenotype. These results reveal distinct characteristics of alloantigen-specific memory and a new in vivo strategy for generating and tracking an enriched population of polyclonal alloreactive memory T cells.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Mice

BALB/c (H-2d, Thy-1.2+) mice were purchased from Charles River Laboratories (Wilmington, MA), and C57BL/6 (H-2b) and B10.BR (H-2k) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). RAG2−/− mice on BALB/c and C57BL/6 backgrounds (Taconic, Germantown, NY) and BALB/c (Thy-1.1+) mice (University of North Carolina, Chapel Hill, NC) were bred as homozygotes and maintained in the Animal Facility at the University of Maryland, Baltimore under specific pathogen-free conditions and used at 8–12 weeks of age. All studies involving animal subjects were approved by the Institutional Animal Care and Use Committee at the University of Maryland, Baltimore.

Antibodies

The following antibodies were purchased from BioExpress (West Lebanon, NH): anti-CD8 (TIB105), anti-CD4 (GK1.5), anti-I-Ad (212.A1) and anti-Thy-1 (TIB238). PE-conjugated anti-CD45RB, anti-CD44, anti-CD62L, anti-CD25, anti-IFN-γ and anti-IgG1; FITC-conjugated anti-CD25 and anti-Thy-1.2; PerCP-conjugated anti-CD4 and anti-Thy-1.1; APC-conjugated anti-CD62L and anti-CD4; anti-Ly-6G and -Ly6C (Gr-1); anti-FcRγ (2.4G2) and anti-IL-2, anti-TNF-α, anti-IL-5 and anti-IL-10 enzyme-linked immunosorbent spot (ELISPOT) antibodies were purchased from BD Pharmingen (San Diego, CA). Anti-IFN-γ and anti-IL-4 ELISPOT antibodies were purchased from Mabtech (Mariemont, OH). Cells were analyzed on FACSCalibur using CellQuest software (Becton Dickinson, San Jose, CA).

Generation of effector and memory CD4 T cells

To generate alloantigen-specific effector CD4 T cells, CD4 cells were purified from spleens of BALB/c (Thy-1.2+) mice (26) and cultured (2 × 106/mL) with mitomycin C-treated APC (6 × 106/mL) prepared from C57BL/6 splenocytes (27) in supplemented Clicks media (Irvine Scientific, Irvine, CA) (28) for 3–4 days at 37°C and 5% CO2. In some cases, CD4 T cells were depleted of CD4+CD25+ regulatory T cells (Tregs) (29) prior to in vitro priming using FITC-conjugated anti-CD25 and anti-FITC magnetic microbeads (Miltenyi Biotec, Auburn, CA), and sorting for the CD25 fraction using automated magnetic separation (AutoMACS, Miltenyi Biotec). Effectors were recovered, purified by density centrifugation (LSM, MP Biomedicals, Aurora, OH), and sorted into CD25+ and CD25 fractions using AutoMACS. CD25+, CD25 and unstimulated BALB/c (naive control) CD4 T cells were injected (2.5 ×106/mouse) into the tail veins of syngeneic (H-2d) RAG2−/− or BALB/c (Thy-1.1+) hosts (Figure 1). Memory CD4 T cells were recovered from the spleen, lung and mesenteric lymph nodes ≥8 weeks posttransfer and purified by immunomagnetic depletion (28,30).

image

Figure 1. Schematic diagram of the method for generating polyclonal alloantigen-specific memory CD4 T cells. CD4 T cells isolated from BALB/c (Thy-1.2+) splenocytes were cultured with C57BL/6 APC in a 3–4-day MLR. The resultant alloantigen-activated CD4 T cells were sorted into CD25+ (activated) and CD25 (unactivated) subsets and adoptively transferred into syngeneic RAG2−/− or BALB/c (Thy-1.1+) mice. For controls, unstimulated BALB/c (naive control) CD4 T cells were transferred in parallel. Memory T cells were recovered and analyzed ≥8 weeks after transfer.

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Proliferation assays

CD4 T cells isolated from RAG2−/− hosts of CD25+, CD25 and naive control precursors, and freshly isolated BALB/c CD4 T cells were labeled with 3-μM CFSE (Molecular Probes, Eugene, OR) (31), and cultured (1–1.5 × 106/mL) with syngeneic BALB/c, allogeneic C57BL/6 and third-party B10.BR APC (3–4.5 × 106/mL) for 88 h. Proliferation was assessed by flow cytometry.

Cytokine assays

The frequency of cytokine-secreting cells was determined using ELISPOT and intracellular cytokine staining (ICS) (32,33). CD4 T cells (3 × 104− 1 × 105/well) were cultured in triplicate for 24 h with syngeneic BALB/c, allogeneic C57BL/6 and third-party B10.BR APC (9 × 104− 3 × 105/well) in 96-well MultiScreen-IP plates (Millipore, Billerica, MA). ELISPOTs were developed (32,33) and quantitated using the Immunospot reader (Cellular Technology, Cleveland, OH). For ICS, monensin (BD Pharmingen) was added 6 h prior to recovery, and cells were permeabilized and stained intracellularly with anti-IFN-γ or isotype control antibodies (32,33).

Assay for graft rejection

To assess naive and memory T-cell-mediated graft rejection, donor tail skin from C57BL/6 and B10.BR mice were transplanted on the lateral thoracic walls of RAG2−/− recipients (34). CD4 T cells from RAG2−/− recipients of CD25+, CD25 and naive control precursors, freshly isolated BALB/c, and CD4+CD25+ Treg-depleted BALB/c CD4 T cells were transferred into hosts (2.5 × 105/mouse) 1 day after grafting. Grafts were monitored daily for rejection.

Statistical methods

Kaplan-Meier survival analysis was used to evaluate differences between survival curves. A p-value of <0.05 was considered significant. All analyses were performed using MedCalc version 8.0.1.1 (Mariakerke, Belgium).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

A novel method for generating alloantigen-specific memory CD4 T cells

To generate alloreactive memory CD4 T cells, we used in vitro priming followed by adoptive transfer into mouse hosts based on systems well characterized in the laboratory for generating antigen-specific memory from TCR-transgenic CD4 T cells (Figure 1) (28, 30, 32, 33). In these systems, peptide antigen-activated precursors to memory T cells exhibited uniform upregulation of IL-2Rα (CD25) (30,35), and we hypothesized that alloantigen-specific precursors to memory may likewise be distinguished by CD25 upregulation. We activated purified BALB/c CD4 T cells with mitomycin C-treated allogeneic C57BL/6 APC and sorted effectors for CD25 expression after 4 days. As shown in Figure 2A, CD4 T cells which had upregulated CD25 were predominately large in size, CD44hi, and CD62Llo, consistent with an activated/effector phenotype, whereas the CD25 fraction was primarily small in size, CD44lo, and CD62Lhi, characteristic of an unactivated phenotype. The phenotypic distinctions between CD25+ and CD25 populations are graphically depicted based on mean fluorescence intensity and forward scatter (Figure 2A, right). These results indicate that sorting for CD25 expression separates alloantigen-activated from unactivated cells.

image

Figure 2. CD25 sorting separates alloantigen-specific effector cells. Mixed lymphocyte cultures of BALB/c CD4 T cells and C57BL/6 APC were recovered after 4 days, sorted into CD25+ and CD25 populations, and analyzed phenotypically and functionally. (A) Left: Representative flow cytometry analysis of cell size, CD25, CD44 and CD62L expression by CD25 and CD25+-sorted populations. Right: Graphical depictions of CD25, CD62L, CD44 as mean fluorescence intensity, and cell size as mean forward scatter. (B) Sorted CD25+ and CD25 cells were cultured in triplicate with syngeneic BALB/c and allogeneic C57BL/6 APC, and without APC for 24 h and IFN-γ production assessed by ELISPOT. Graph shows mean spot numbers ± standard deviation. Results are representative of four experiments.

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To confirm that the alloantigen-activated CD25+ population comprised functionally differentiated effector cells, we assessed IFN-γ production upon restimulation with allogeneic APC. Activated CD25+CD4+ T cells demonstrated alloantigen-specific IFN-γ production in response to C57BL/6-derived APC, in contrast to CD25 cells that produced negligible IFN-γ when stimulated (Figure 2B). These results demonstrate that the activated CD25+ population contains effector cells primed against the H-2b alloantigen.

We transferred primed CD25+ cells into RAG2−/− hosts to determine whether this subset contained precursors to memory development (Figure 1). As a control for the nonspecific effects of homeostatic expansion in lymphocyte-deficient hosts (36,37), we also transferred CD25 and unmanipulated BALB/c CD4 T cells into RAG2−/− hosts (naive control). Cells were recovered after 2–7 months, and analyzed functionally and phenotypically. We found that only alloactivated CD25+ cells developed into true alloreactive memory (see below). To assess any interference of CD4+CD25+ Tregs on memory generation, we also performed parallel assays using memory cells generated from CD25+-depleted naive CD4 T cells.

We asked whether the alloantigen-primed memory population exhibited hallmarks of a memory response, including enhanced recall proliferation and rapid production of effector cytokines. CD4 T cells from RAG2−/− recipients of CD25+, CD25 and naive control cells were isolated, CFSE labeled, and analyzed for their proliferative responses to allogeneic C57BL/6, third-party B10.BR and syngeneic BALB/c APC. As an additional control, we assessed the de novo alloreactive proliferative responses of CD4 T cells freshly isolated from unmanipulated BALB/c mice. Memory T cells derived from CD25+ precursors exhibited substantial proliferation when recalled with the C57BL/6 alloantigen but only low-level proliferation to syngeneic and third-party APC (Figure 3A). In contrast, CD4 T cells from CD25 and naive control precursors, and freshly isolated BALB/c CD4 T cells exhibited only low-level proliferation to C57BL/6 and third-party APC. These results demonstrate alloantigen-specific recall of CD25+-derived memory T cells, and a lack of spontaneous allospecific memory generation by naive and unactivated (CD25) precursors in RAG2−/− hosts.

image

Figure 3. Alloantigen-specific recall of memory CD4 T cells derived from CD25+ precursors in RAG2−/− hosts. (A) Spleen-derived CD4 T cells were isolated from unmanipulated BALB/c mice and RAG2−/ adoptive hosts 2–7 months posttransfer of CD25+, CD25 and naive control CD4 T cells. Cells were labeled with CFSE, cultured with syngeneic BALB/c, allogeneic C57BL/6 or B10.BR APC, and proliferation was assessed using flow cytometry after 88 h and is shown as percentage of dividing cells. Results are representative of three experiments. (B) Splenic CD4 T cells from RAG2−/− recipients of CD25+ and CD25 precursors 5 months after adoptive transfer, and freshly isolated BALB/c CD4 T cells were restimulated with C57BL/6 APC for 18–90 h and IFN-γ production assessed by ICS. Percentage of total CD4 T cells producing IFN-γ is shown. (C) IFN-γ production by H-2b-specific memory CD4 T cells after restimulation with syngeneic BALB/c, allogeneic C57BL/6 and third-party B10.BR APC in a 24-h ELISPOT. (D) ELISPOT analysis of H-2d-specific memory CD4 T cells isolated 2 months after adoptive transfer, restimulated with syngeneic C57BL/6 and allogeneic BALB/c APC, and without APC for 24 h. Graphs show mean spot numbers ± standard deviation of triplicate cultures.

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Memory CD4 T cells derived from alloactivated CD25+ precursors also exhibited rapid effector cytokine production upon recall with the priming alloantigen. CD25+-derived memory T cells produced IFN-γ within 24 h, which continued to increase after 2 days of activation, while CD25-derived and freshly isolated BALB/c CD4 T cells produced negligible IFN-γ at all time points (Figure 3B). To more precisely quantify early IFN-γ production, we analyzed cytokine production by ELISPOT after 24 h. Figure 3C demonstrates alloantigen-specific production of IFN-γ by CD25+-derived memory T cells for C57BL/6 APC that was not seen with syngeneic and third-party APC.

We generated CD25+ cells using a C57BL/6 anti-BALB/c reverse MLR and transferred these cells into RAG2−/− hosts to determine if our method of memory generation was applicable to other alloantigens. Alloantigen-specific recall (in this case to H-2d) was present only in memory cells derived from CD25+ precursors (200–500 IFN-γ spots/20 000 CD4 T cells; Figure 3D and data not shown). Negligible IFN-γ production was observed with syngeneic APC and by CD25-derived cells. Additionally, we used naive CD4 T cells depleted of endogenous Tregs before in vitro priming and found that removing this fraction did not affect the yield or development of memory in vivo (data not shown). These results demonstrate that in vitro priming and CD25+-sorting isolates precursors to long-lived alloreactive memory CD4 T cells exhibiting robust recall responses.

Generation of alloantigen-specific memory CD4 T cells in BALB/c hosts

To determine whether generation of alloreactive memory from CD25+ precursors could also occur in lymphocyte-replete hosts, we transferred alloantigen-activated CD25+Thy-1.2+CD4+ T cells into BALB/c (Thy-1.1+) hosts. Thy-1.2+CD4+ T cells derived from CD25+ precursors comprised an enriched population representing 9–10% of splenic and lung CD4 T cells compared to 1–3% of those derived from naive control and CD25 precursors (Figure 4A). As in RAG2−/− hosts, a large proportion of CD25+-derived memory in BALB/c hosts produced IFN-γ when stimulated with alloantigen, compared to the low frequency found in the CD25-derived and naive control groups (Figure 4B). These results demonstrate that transfer of alloactivated CD25+ effectors into lymphocyte-replete hosts yields an enriched population of alloantigen-specific memory CD4 T cells with robust recall function, compared to a negligible level of endogenous alloantigen-specific memory in unmanipulated BALB/c hosts.

image

Figure 4. Alloantigen-specific recall of memory CD4 T cells derived from CD25+ precursors in BALB/c hosts. (A) Spleen-, lung- and lymph node-derived CD4 T cells were isolated from BALB/c (Thy-1.1+) hosts 1 month after adoptive transfer of CD25+, CD25 and naive control Thy-1.2+CD4+ T cells. Percentage of Thy-1.2+CD4+ cells of total CD4 T cells is shown. (B) Spleen-derived CD4 T cells were isolated from BALB/c adoptive hosts as in (A) and assayed for IFN-γ production by ELISPOT in response to stimulation with syngeneic BALB/c, allogeneic C57BL/6 and third-party B10.BR APC for 24 h. Graph shows mean spot numbers ± standard deviation of triplicate cultures.

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Alloantigen-specific memory CD4 T cells are predominantly effector-memory

Given our finding that alloreactive memory T cells exhibit heterogeneity in tissue distribution, we asked whether they also exhibited heterogeneity in phenotype and function. We found that alloantigen-specific memory CD4 T cells recovered from the spleen, lung and lymph nodes of RAG2−/− and BALB/c hosts were small in size and exhibited a memory phenotype (CD25lo/CD44hi/CD45RBlo) (data not shown). Interestingly, these allomemory CD4 T cells isolated from lymphoid and nonlymphoid tissues of lymphocyte-deficient and lymphocyte-replete hosts were all predominantly CD62Llo, with CD62Lhi memory T cells representing a minority population (2–34%, 8–18% in BALB/c lung; Figure 5 and data not shown). This preponderance of effector-memory T cells was also observed when we depleted the naive population of CD4+CD25+ Tregs (data not shown), indicating that effector-memory generation is an inherent property of alloantigen priming. The difference in CD62Lhi expression between BALB/c and RAG2−/− lymph node-derived T cells may be due to the altered lymph node architecture of RAG2−/− mice and/or homeostatic proliferation. This predominant CD62Llo phenotype may account for the reduced frequency of memory CD4 T cells in the lymph node relative to spleen and lung in lymphocyte-replete hosts (Figure 4A).

image

Figure 5. CD62L expression of alloantigen-specific memory CD4 T cells in lymphoid and nonlymphoid tissues. CD62L expression of memory CD4 T cells recovered from the spleen, lung and lymph nodes of RAG2−/− and BALB/c (Thy-1.1+) hosts recovered 2–3 months after adoptive transfer of CD25+Thy-1.2+CD4+ T cells. Percentages in plots indicate proportion of CD62Lhi CD4 T cells. Results are representative of FACS analysis from 12 mice.

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We assessed the functional profiles of alloantigen-specific memory CD4 T cells and found that spleen-, lymph node- and lung-derived memory subsets produced predominantly IFN-γ, IL-2 and TNF-α in response to the priming alloantigen, with negligible production of IL-4, IL-5 and IL-10 (Figure 6 and data not shown). Overall, IL-2 production by lymph node-derived allomemory T cells was comparable or slightly less than spleen- and lung-derived cells (Figure 6 and data not shown). These results demonstrate that lymphoid and nonlymphoid alloreactive memory CD4 T-cell subsets exhibit a Th1-like profile with similar capacities for effector cytokine production.

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Figure 6. Cytokine profile of lymphoid and nonlymphoid alloantigen-specific memory CD4 T-cell subsets. Spleen, lung and lymph node-derived CD4 T cells were isolated from RAG2−/− recipients of CD25+ and CD25 precursors 2 months after transfer, cultured with syngeneic BALB/c, allogeneic C57BL/6 and third-party B10.BR APC for 24 h, and IFN-γ and IL-2 production were measured by ELISPOT. Graphs show mean spot numbers ± standard deviation from triplicate cultures.

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Alloantigen-specific memory CD4 T cells mediate robust allograft rejection

The results above demonstrate generation of alloantigen-specific memory T cells in lymphoid and nonlymphoid tissues with robust recall capacity in vitro. To evaluate the in vivo functional capacities of alloantigen-specific memory T cells, we transferred equal numbers of CD25+-derived memory, CD25-derived, naive control, and freshly isolated BALB/c CD4 T cells into RAG2−/− hosts grafted with allogeneic C57BL/6 and third-party B10.BR skin, and assessed graft rejection. We used lymphocyte-deficient hosts to study memory CD4 T-cell responses in isolation due to the robust alloreactive responses mediated by endogenous CD4 and CD8 T cells in lymphocyte-replete hosts. In this system, rejection is completely dependent on the transferred T-cell population, as indefinite graft survival occurs in unmanipulated RAG2−/− hosts (Figure 7A and (38)). We found that allomemory T cells mediated accelerated rejection of C57BL/6 skin grafts compared to naive control (Figure 7A), CD25-derived, and freshly isolated BALB/c CD4 T cells (Figure 7B). Earliest rejection by allospecific memory CD4 T cells occurred at day 9 with rejection of all grafts by day 15, while rejection by naive or unactivated control cells was delayed until day 12 and remained incomplete 4 weeks later (Figure 7A).

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Figure 7. In vivo rejection capacity of lymphoid and nonlymphoid allospecific memory CD4 T cells in RAG2−/− hosts. (A) Kaplan-Meier survival curve of C57BL/6 skin grafts showing accelerated rejection by splenic CD25+-derived allomemory cells (n = 11) versus naive control (n = 7) and no cells (n = 5). *p < 0.005 CD25+-derived versus naive control and no cells. (B) Rejection of C57BL/6 skin grafts by spleen CD25+-derived allomemory cells versus CD25-derived (n = 6) and freshly isolated BALB/c CD4 T cells (n = 7). *p < 0.005 CD25+-derived versus CD25-derived and freshly isolated BALB/c CD4. (C) C57BL/6 graft survival curve comparing the rejection capacities of spleen, lung and lymph node CD25+-derived allomemory cells (n = 11 for each group). Mean skin graft survival times ± standard deviation for spleen, lung and lymph node CD25+-derived memory T cells are as follows: 11.1 ± 1.7, 12.9 ± 3.7 and 11.8 ± 0.8 days, respectively (p = NS). Survival of third-party B10.BR skin grafts on hosts receiving CD25+-derived memory T cells (n = 33) is shown to demonstrate allospecificity.

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We compared the rejection capacities of spleen-, lung- and lymph node-derived memory CD4 T cells to analyze potential functional differences between allomemory T-cell subsets. All subsets mediated rapid rejection of C57BL/6 skin grafts with comparable kinetics (Figure 7C). Rejection of B10.BR grafts on hosts receiving CD25+-derived allomemory cells occurred at a slower rate, with 33% of recipients showing long-term graft survival. These results demonstrate the vigorous rejection capacities of lymphoid and nonlymphoid alloantigen-specific memory CD4 T cells that are specific for the priming alloantigen.

To distinguish between the possibilities that accelerated rejection mediated by CD25+-derived memory T cells may be due to a higher frequency of alloreactive cells rather than to the memory response, titrated numbers of CD25+-derived allomemory cells were transferred into grafted RAG2−/− mice. We found no differences in rejection kinetics even when the total number of memory cells transferred was reduced to one-fifth (Table 1). Hosts receiving 1 × 105 memory cells rejected allogeneic grafts faster than those receiving 2.5 times as many naive BALB/c cells. We also transferred CD4+CD25+ Treg-depleted BALB/c CD4 T cells to assess the influence of Tregs on graft rejection and found no differences in rejection capacity when compared to BALB/c CD4 T cells containing endogenous Tregs (Table 1). These results demonstrate the vigorous rejection capacity of our allomemory T cells at low frequencies and the negligible impact of endogenous Tregs on graft rejection in our system.

Table 1.  Rejection of C57BL/6 skin grafts by splenic CD25+-derived memory CD4 T cells in RAG2−/− hosts
 No. of CD4 cells transferredRejection time (days)Mean survival time ± SD (days)p-Value1
  1. 1Kaplan-Meier survival analysis.

CD25+-derived memory5 × 10410, 11, 1311.3 ± 1.50.3 vs. other memory groups
CD25+-derived memory1 × 10510, 11, 1110.7 ± 0.60.03 vs. both BALB/c groups
CD25+-derived memory2.5 × 1059, 10, 109.7 ± 0.60.03 vs. both BALB/c groups
 
Fresh BALB/c2.5 × 10512, 13, 1613.7 ± 2.1 
 
CD4+CD25+ Treg-depleted fresh BALB/c2.5 × 10512, 12, 1312.3 ± 0.60.3 vs. fresh BALB/c

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We present here a novel approach for generating polyclonal alloantigen-specific memory CD4 T cells by in vitro priming and transfer of alloactivated CD25+CD4+ T cells into adoptive hosts. We demonstrate that CD25+ effectors contain the precursors for an alloantigen-specific memory T-cell population that exhibits the hallmarks of a memory response, including rapid proliferation and production of effector cytokines with remarkable specificity for the priming alloantigen, and the ability to mediate accelerated rejection of allogeneic grafts in vivo. We used this system to explore the heterogeneous nature of alloreactive memory, and found that allospecific memory CD4 T cells are predominantly of the effector-memory phenotype in both lymphoid and nonlymphoid tissues, and that both lymphoid and nonlymphoid memory subsets exhibit comparable rapid recall effector function and robust capacity for allograft rejection in vivo. These results reveal important insights regarding the nature of alloreactive memory T cells that should be considered when designing strategies to target these potent cells.

In this study, we make novel use of CD25 upregulation as a selection marker for precursors of an enriched population of polyclonal alloantigen-specific memory T cells. CD25 is an early activation marker for T cells (39), but can remain upregulated in the presence of antigenic stimulus for up to 5 days on TCR-transgenic CD4 T cells (26). In polyclonal CD4 T cells activated with alloantigen in vitro, we observed a gradual upregulation of CD25 expression from 11% at 24 h to 50% after 4 days (data not shown). Memory generation from CD25+CD4+ T cells was maximized when derived from precursors activated for 3–4 days (data not shown), suggesting that a certain level of differentiation optimized the potential for memory generation in vivo. This method for sorting on CD25 expression to isolate precursors to memory can be applied to polyclonal nominal antigen systems.

The system presented here for generation of allospecific memory in RAG2−/− and normal hosts offers several advantages to direct priming in vivo (20,21). First, adoptive transfer of alloactivated CD25+ effectors into lymphocyte-deficient hosts allows expansion of an alloreactive memory population that usually constitutes only a small percentage of total T cells in the normal host. Second, it enables study of alloreactive memory without the interference of other lymphocytes. Finally, transfer of CD25+ cells into normal hosts bearing Thy-1 allelic differences results in a “mosaic-memory” mouse in which polyclonal alloreactive memory CD4 T cells can be distinguished from the endogenous T cells (Figure 4A), and allows the tracking of the secondary memory response (mediated by transferred alloreactive memory T cells) separately from the primary immune response (mediated by naive T cells).

Using our system to analyze the characteristics of alloantigen-specific memory, we found two distinguishing aspects of alloantigen-specific memory CD4 T cells compared to memory T cells generated against peptide antigens or viruses. First, we observed that activation with alloantigen biases the generation of effector-memory T cells, resulting in allospecific memory CD4 T cells bearing a predominant CD62Llo phenotype in lymphoid and nonlymphoid tissues in both lymphocyte-deficient and normal hosts. This phenotypic profile contrasts the comparable distribution of CD62Lhi and CD62Llo subsets among memory CD4 T cells specific for influenza hemagglutinin (HA) or ovalbumin (13, 28, 33), and in memory CD8 T cells specific for multiple viruses (40, 41). The reason for this disparity is uncertain, but may be due to differences in the manner of antigen recognition (direct with alloantigen vs. indirect with peptide antigen) and/or the avidity for or density of allogeneic MHC compared to antigenic peptide-MHC complexes on the surface of APC.

The second major difference between alloreactive memory CD4 T cells compared to antigen- or virus-specific memory, is the comparable robust recall effector function of both lymphoid and nonlymphoid subsets when tested in vitro and in vivo. Other studies found that lymphoid memory CD4 and CD8 T cells specific for peptide antigens or viruses, respectively, exhibited less effector function than nonlymphoid counterparts (42,43). However, for lymphocytic choriomeningitis virus-specific memory CD8 and HA-specific memory CD4 T cells, similar effector function is found in both compartments (33,41). These results suggest that the effector capacity of memory T cells in different tissue sites may depend on the priming antigen, and that direct priming with alloantigen generates functionally robust effector-memory T cells in peripheral and lymphoid tissues.

Current interest in CD4+CD25+ Tregs rests in their ability to modulate immune responsiveness to self and nonself antigens and their role in transplantation tolerance (44–47). We show that the generation of alloreactive memory T cells with a predominately effector-memory phenotype is independent of the presence or absence of Tregs. Additionally, endogenous Tregs did not delay nor prevent rejection of allogeneic skin grafts in RAGZ−/− hosts used here, similar to results in T-cell-depleted hosts in which naive and Treg-depleted CD4 T cells showed comparable skin graft rejection kinetics (48).

It has become increasingly clear that memory T cells present a significant impediment to long-term allograft survival in transplant recipients. While it is well known that presensitized patients with high panel-reactive antibody titers experience significantly shorter graft survival (22,49,50), the presence of alloreactive memory CD4 T cells has also been correlated with increased episodes of acute rejection and decreased graft function (2,3). Moreover, Kirk and colleagues have shown that effector-memory CD4 T cells persist in patients treated with antithymocyte globulin or anti-CD52 antibody (Campath-1H) (23), which may increase the risk for early rejection. The susceptibility of memory CD4 T cells, particularly the effector-memory subset, to conventional immunosuppression in vivo has not been studied, and is a critical issue not only for designing new targeted strategies for transplantation tolerance, but also for adapting existing protocols to patients with preexisting alloreactive memory. Our system for the generation and analysis of polyclonal alloreactive memory CD4 T cells in vivo can be an invaluable tool for addressing these issues as long-term graft survival may depend on our ability to suppress these resilient memory T cells.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We wish to thank Dr. Stephen T. Bartlett, Dr. Deepa Patke, Modesta Ndejembi and Smita Chandran for critical reading of this manuscript, and Wendy Lai for mouse colony maintenance. This study is supported by NIH AI50632 and Harry and Jeanette Weinberg Foundation Grant.

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  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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