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The Journal of Immunology, 2007, 179, 80 -88
Copyright © 2007 by The American Association of Immunologists, Inc.
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Disparate Primary and Secondary Allospecific CD8+ T Cell Cytolytic Effector Function in the Presence or Absence of Host CD4+ T Cells1

Phillip H. Horne{dagger}, Mitchel A. Koester*, Kartika Jayashankar*, Keri E. Lunsford{dagger}, Heather L. Dziema* and Ginny L. Bumgardner2,*,{dagger}

* Department of Surgery, Comprehensive Transplant Center, Ohio State University Medical Center, Columbus, OH 43210; and {dagger} Integrated Biomedical Science Graduate Program, College of Medicine, Ohio State University, Columbus, OH 43210


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The role of CD4+ T cells in promoting CD8+ T cell effector activity in response to transplant Ags in vivo has not been reported. We used a hepatocellular allograft model known to initiate both CD4-dependent and CD4-independent rejection responses to investigate the contribution of CD4+ T cells to the development, function, and persistence of allospecific CD8+ T cell effectors in vivo. Complete MHC-mismatched hepatocellular allografts were transplanted into C57BL/6 (CD4-sufficient) or CD4 knockout (CD4-deficient) hosts. The development of in vivo allospecific cytotoxicity was determined by clearance of CFSE-labeled target cells. CD8+ T cell cytotoxic effector activity was enhanced in response to allogeneic hepatocellular grafts with a greater magnitude of allocytotoxicity and a prolonged persistence of CTL effector activity in CD4-sufficient hosts compared with CD4-deficient hosts. Cytolytic activity was mediated by CD8+ T cells in both recipient groups. In response to a second hepatocyte transplant, rejection kinetics were enhanced in both CD4-sufficient and CD4-deficient hepatocyte recipients. However, only CD4-sufficient hosts developed recall CTL responses with an augmented magnitude and persistence of allocytotoxicity in comparison with primary CTL responses. These studies show important functional differences between alloreactive CD8+ T cell cytolytic effectors that mature in vivo in the presence or absence of CD4+ T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Activation and maturation of CD8+ effector T cells are conventionally considered to require CD4+ T cell help. However, it is recognized that this requirement for CD4+ T cell help is conditional, such that CD8+ CTL maturation in the absence of CD4+ T cells occurs when APC activation is directly stimulated by adjuvants, infectious agents, or experimental agents (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). Furthermore, effective CD8+ CTL-mediated clearance of pathogens can occur in the absence of CD4+ T cells (11). More recently it has been recognized that the absence of CD4+ T cells significantly impairs CD8+ T cell memory or recall responses to Ag (11, 12, 13, 14, 15) (reviewed in Ref. 16). CD4+ T cells have been reported to contribute to the development of enhanced secondary or memory CD8+ CTL responses through epigenetic modification or programming (11, 17, 18, 19). Others have reported that CD4+ T cells are important for the maintenance (but not programming) of CD8+ memory T cells (13). In contrast to the critical influence of CD4+ T cells on development of efficient CD8+ T cell memory responses, the absence of CD4+ T cells during priming does not impair primary CD8+ T cell cytokine production, proliferation, or effector function in nontransplant models (17, 20).

In addition to the recognized roles of CD8+ T cells in clearance of infectious pathogens and tumor cells, CD8+ T cells are also known effectors of allograft rejection (21, 22, 23, 24, 25). Allograft rejection by conventional pathways is a CD4+ T cell-dependent process (reviewed in Ref. 26) that can be suppressed by targeting CD4+ T cells alone. This implies that allospecific CD8+ CTLs that develop concurrently with allospecific CD4+ T cells are CD4-dependent CD8+ CTLs. Several allograft tissues, including hepatocytes (26, 27), skin (28, 29), intestine (30, 31), and cardiac allografts in some models (32), have been reported to activate CD4-independent, CD8+ T cell rejection responses. These unconventional CD4+ T cell-independent immune pathways are associated with different costimulatory requirements (31, 33, 34, 35), skewed immune repertoire in the host (32), and resistance to immunotherapies that readily control CD4+ T cell-initiated rejection responses (27, 31, 32, 36, 37, 38, 39, 40, 41, 42). Furthermore, CD4-independent CD8+ T cell rejection responses contribute to costimulation blockade-resistant rejection and interfere with immune tolerance strategies (39, 41, 43, 44). Collectively, these studies underscore the importance of understanding the development and function of alloreactive CD8+ T cells that are activated independently of CD4+ T cells.

Few experimental transplantation studies have examined the in vivo development of alloreactive CD8+ CTL effector function in the presence or absence of CD4+ T cells. One study of cardiac allografts reported that host CD4+ T cells are necessary to license dendritic cells for CD8+ T cell activation, CTL function, and CD4-dependent graft rejection (45). However, the development of CTL effectors under CD4-independent conditions in response to allograft tissues such as skin, intestine, and hepatocytes has not been investigated, except for reports by our group showing that the rejection of allogeneic liver parenchymal cells in the absence of CD4+ T cells is accompanied by in vivo cytotoxic effector function (46, 47).

The purpose of the current study was to examine CD8+ CTL maturation, magnitude, and productive memory development in response to allogeneic hepatocytes in the presence or absence of CD4+ T cells. Experimental advantages of the hepatocellular allograft model to study these immune parameters include the relative homogeneity of the alloantigen conferred by transplantation of a purified cell population and a standard cell number or graft size. Furthermore, CD4-independent, CD8+ T cell immune responses to allogeneic hepatocytes have been studied using three experimental models, including recipient CD4+ T cell depletion (anti-CD4 mAb, GK1.5), recipient genetic deficiency (CD4 knockout (KO)),3 or reconstitution of immunoincompetent SCID (or Rag1–/–) recipients with CD8+ T cells. All of these models produce comparable results and are available to explore the consequence of alloreactive CD8+ T cell maturation in the presence or absence of recipient CD4+ T cells (27, 36, 48). Our studies show that CD8+ CTL responses are generated by hepatocellular allografts both in the presence and absence of CD4+ T cells, but the kinetics, magnitude of effector responses, and second set responses are strikingly different.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Experimental animals

FVB/N (H-2q; Taconic Farms), C57BL/6 (H-2b; Taconic Farms), CD4 KO (H-2b, C57BL/6-CD4tm1Mak; The Jackson Laboratory), DesTCR (H-2k transgenic mice with CD8+ T cells expressing TCR specific for H-2Kb; a gift from A. Mellor, Medical College of Georgia, Augusta, GA), and B10.BR (H-2k; The Jackson Laboratory) mouse strains were used in this study. Transgenic FVB/N and C57BL/6 mice expressing human {alpha}-1 anti-trypsin (hA1AT-FVB/N, H-2q; hA1AT-C57BL/6, H-2b) were the source of donor hepatocytes. These strains were created, bred, and maintained at the Biotechnology Center and Transgenic Animal Facility (Ohio State University) (27, 48). Mice that were 6–9 wk of age were used in experiments. All animals received humane care according to the criteria outlined in the "Guide for the Care and Use of Laboratory Animals" (National Academy Press, revised 1996).

Hepatocyte isolation and purification

Hepatocyte isolation and purification were performed, as described previously (27, 48). Briefly, the liver was perfused with 0.09% EGTA-containing calcium-free salt solution, followed by a 0.05% collagenase solution (Sigma-Aldrich; type IV) in 1% albumin. Liver tissue was minced, filtered, and washed in RPMI 1640 with 10% FBS. Hepatocytes were purified on a 50% Percoll gradient (Pharmacia Biotech). Hepatocyte viability and purity were consistently >99%, respectively.

Hepatocyte transplantation and monitoring of hepatocyte graft function

Donor hepatocytes were retrieved from transgenic mice expressing hA1AT under control of the liver-specific hA1AT promoter and transplanted into recipients by intrasplenic injection with circulation of donor hepatocytes to the host liver, as previously described (48). C57BL/6 (H-2b) and CD4 KO (H-2b) recipients were transplanted with 2 x 106 purified allogeneic hA1AT-FVB/N (H-2q) hepatocytes. DesTCR+CD8+ T cell-reconstituted B10.BR (DesrB10BR) recipients (H-2k) were transplanted with 2 x 106 allogeneic hA1AT-C57BL/6 (H-2b) hepatocytes. Graft function was determined by detection of the secreted transgenic reporter product, hA1AT, in serial recipient serum samples by sandwich ELISA (27, 48). Graft survival was determined by sustained serum hA1AT levels, and graft loss was determined as the time point at which host serum hA1AT was <0.5 µg/ml.

Abs used for T cell subset depletion in hepatocyte recipients

Recipients were depleted of circulating CD4+, CD8+, or NK cells using mAbs. Anti-CD4 (GK1.5; American Type Culture Collection) and anti-CD8 (53-6.72; American Type Culture Collection) mAbs were obtained from Bioexpress Cell Culture Services. Anti-NK1.1 (PK136; American Type Culture Collection) mAb was purified from pristane-primed mice. CD4+, CD8+, or NK cells were depleted by i.p. treatment with 250 µg of anti-CD4, or 300 µg of anti-CD8 or anti-NK1.1 mAbs on day –2 and day –1 relative to the in vivo cytotoxicity assay in hepatocyte recipients (as well as in control naive mice). Depletion was confirmed through flow cytometric analysis of recipient splenocytes, as previously described (36).

In vivo cytotoxicity assay

Detection of in vivo cytolytic T cell function through clearance of CFSE-stained allogeneic and syngeneic target cells was modified from published methods (49, 50), and has been previously described (47). Syngeneic target splenocytes from C57BL/6 mice were stained with 0.2 µM CFSE (CFSElow) (Vybrant CFDA SE Cell Tracer Kit; Molecular Probes). Allogeneic target splenocytes from FVB/N mice were stained with 2.0 µM CFSE (CFSEhigh). Equal numbers of CFSE-labeled syngeneic and allogeneic target splenocytes (20 x 106 each, mixed 1:1) were injected into the tail veins of allograft recipient and control naive mice. Eighteen hours after CFSE-labeled target cell injection, splenocytes from hepatocyte recipients were retrieved and analyzed by flow cytometry, gating on CFSE-positive splenocytes. Percentage of allospecific cytotoxicity was calculated using the following formula, in which #CFSEhigh represents the number of allogeneic target cells and #CFSElow represents the number of syngeneic target cells recovered from either naive or experimental mice:

Formula

In vivo proliferation assay

Preparation of Des+CD8+ T cells. Splenocytes from DesTCR mice were column purified for CD8+ T cells, according to manufacturer’s instructions (Mouse CD8 Subset Column Kit; R&D Systems), and phenotyped through immunolabeling with anti-Des hybridoma (mouse anti-mouse Des) supernatant (100 µl neat), washing twice in cold PBS-FACS buffer (5% goat serum and .001% sodium azide), then staining with anti-CD8 FITC (53-6.7, 1:200; BD Pharmingen), anti-CD3 PE (145-2C11, 1:200; BD Pharmingen), and goat anti-mouse IgG Alexa Fluor 647-R-PE (1:200; Molecular Probes), in cold PBS-FACS buffer. The cells were washed twice and then analyzed by flow cytometry. Column-purified splenocytes were ~50% Des+CD8+CD3+.

Adoptive transfer of Des+CD8+ T cells into B10.BR hepatocyte recipients. Des+CD8+ T cells were stained with 5 µM CFSE in PBS at 37°C for 8 min, then washed in warm DMEM with 10% FBS (37°C) for 5 min. The 5–8 x 106 column-purified splenocytes (~2–4 x 106 Des+CD8+CD3+) were adoptively transferred through tail vein injection in 0.5 ml of DMEM into syngeneic B10.BR hosts (day –1), some of which were previously depleted of CD4+ T cells (anti-CD4 mAb, days –4 and –2). Transgenic hA1AT-C57BL/6 hepatocytes were transplanted on day 0 through intrasplenic injection in some of the Des+CD8+-reconstituted B10.BR (DesrB10BR) hosts, and later host liver and spleen lymphocytes were retrieved (day 3 after hepatocyte transplant; day 4 after adoptive transfer of Des+CD8+ T cells) for analysis of CFSE dilution in Des+CD8+ T cells by flow cytometry.

Retrieval of graft-infiltrating cells (GICs) for phenotypic analysis

Hepatocytes transplanted by intrasplenic injection rapidly circulate to and engraft in the recipient liver (reviewed in Ref. 26) and stimulate the accumulation of GICs in the recipient liver (47). To retrieve GICs, recipient mice were euthanized for excision of the liver. The livers were digested in a solution containing 0.05% collagenase (Sigma-Aldrich; type IV) in 1% albumin for 10 min and cells were separated through mechanical dispersion. Erythrocytes were lysed using hypotonic solution, and lymphocytes were enriched using 60% gradient (Histodenz; Sigma-Aldrich) density centrifugation. Lymphocytes were enumerated and phenotyped by immunolabeling with anti-CD8 FITC (53-6.7, 1:200; BD Pharmingen), anti-CD43 PE (1B11, 1:200; BD Pharmingen), anti-CD62L PE (MEL-14, 1:200; BD Pharmingen), anti-CD44 PE-Cy5 (IM7, 1:200; BD Pharmingen), and flow cytometry, gating on CD8+ T cells.

Statistical analysis

Graft survival between experimental groups was compared using Kaplan Meier survival curves and log-rank statistics (SPSS version 14 for Windows). Other statistical calculations were performed using Student’s t test to analyze differences between experimental groups. Total cytotoxicity in CD4-sufficient (C57BL/6) and CD4-deficient (CD4 KO) recipient groups was compared through calculation of the total area under the allocytotoxicity curves divided by the summed SD of all individual measurements. Value of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Allospecific CD8+ CTL effector function develops in CD4-sufficient (C57BL/6) and CD4-deficient (CD4 KO) hepatocyte recipients

We have previously reported that allogeneic liver parenchymal cells initiate distinct rejection pathways in C57BL/6 (CD4+ and CD8+ T cell sufficient, H-2b) and CD4 KO (CD4 deficient, CD8+ T sufficient, H-2b) hosts (27, 36). Rejection in C57BL/6 recipients is dependent on both CD4+ and CD8+ T cells (36). Rejection in CD4 KO recipients is CD8+ T cell dependent (27). In the current study, C57BL/6 (H-2b) and CD4 KO (H-2b) mice were transplanted with hA1AT-FVB/N (H-2q) hepatocytes and had median survival time (MST) of 10 and 14 days, respectively (p = NS) (Fig. 1a). Hepatocyte recipients were assessed on day 7 posttransplant for in vivo allospecific cytotoxicity by measurement of CFSE-labeled allogeneic vs syngeneic target cell clearance. Both CD4-sufficient and CD4-deficient hepatocyte recipients demonstrated detectable in vivo allospecific cytotoxicity (Fig. 1a); however, the magnitude of allospecific cytotoxicity was greater in CD4-sufficient than in CD4-deficient recipient groups (p < 0.01). To determine whether CD8+ T cells were responsible for the observed allocytotoxicity, separate groups of C57BL/6 and CD4 KO recipients were depleted of CD8+ T cells (anti-CD8 mAb, 53-6.72) 2 days before the cytotoxicity assay. Depletion of CD8+ T cells abrogated allocytotoxicity in CD4-deficient recipients and significantly decreased allocytotoxicity in CD4-sufficient recipients (Fig. 1b). Furthermore, NK cells do not contribute to cytotoxicity in CD4-sufficient or CD4-deficient recipient groups, because depletion of NK cells (anti-NK1.1 mAb, PK136) 48 h before the in vivo cytotoxicity assay did not perturb cytotoxicity in comparison with NK replete hosts (Fig. 1b). The allospecificity of the CD8+ T cell-mediated cytotoxic effector function was assessed in separate groups of recipients that were transplanted, followed by assessment of in vivo cytotoxicity assay using syngeneic and third-party (B10.BR, H-2k) target cells. No enhanced clearance of third-party target cells was observed in hepatocyte allograft recipients (Fig. 1c). These studies demonstrate that allospecific CD8+ CTLs develop in both CD4-sufficient and CD4-deficient hepatocyte recipients.


Figure 1
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  		FIGURE 1. Allospecific CD8+ CTL activity develops in CD4-sufficient and CD4-deficient hosts. Untreated C57BL/6 (H-2b) and CD4 KO (H-2b) recipients were transplanted with allogeneic (FVB/N, H-2q) hepatocytes, followed by assessment for in vivo cytotoxicity on day 7 following hepatocellular transplantation. As shown, CD4-sufficient and CD4-deficient hosts reject hepatocyte allografts with MST of 10 and 14 days, respectively (left) (p = NS). Allospecific target cell clearance was detected in both recipient groups, as seen by the increased clearance of allogeneic (FVB/N, H-2q) CFSEhigh relative to syngeneic (C57BL/6, H-2b) CFSElow target cells; no allogeneic target cell clearance occurred in untransplanted controls. Greater allocytotoxicity was observed in C57BL/6 hosts (n = 5) in comparison with CD4 KO hosts (n = 5) (right) (p < 0.01) (a). Depletion of recipient CD8+ T cells 48 h before the cytotoxicity assay on day 7 posttransplant in CD4-sufficient hosts (n = 5) and on day 5 posttransplant in CD4 KO hosts (n = 3) significantly decreased allocytotoxicity in both recipient groups (p < 0.001, p < 0.01, respectively), whereas depletion of host NK cells did not impair allocytotoxicity in CD4-sufficient or CD4 KO hosts (n = 4) at comparable times posttransplant (p = NS) (b). Untreated C57BL/6 (n = 3) and CD4 KO (n = 3) hepatocyte allograft recipients were assessed for specificity of in vivo cytotoxicity on day 10 (CD4-sufficient hosts) or day 5 (CD4-deficient hosts) posttransplant using third-party (B10.BR, H-2k) CFSEhigh targets and syngeneic CFSElow targets. No clearance of third-party targets relative to syngeneic targets was observed in C57BL/6 or CD4 KO hepatocyte recipients (c).

 
In vivo allospecific CD8+ T cell cytolytic effector function is greater in magnitude and persists longer in CD4-sufficient recipients than in CD4-deficient recipients

To determine whether or not the difference in magnitude of in vivo allocytotoxicity observed on day 7 posttransplant in CD4-sufficient compared with CD4-deficient recipients was sustained over time or was limited to that time point, we performed a kinetic analysis of CD8+ CTL maturation under these disparate conditions. Groups of CD4-sufficient (C57BL/6, H-2b) and CD4-deficient (CD4 KO, H-2b) recipients were transplanted with hepatocellular allografts (hA1AT-FVB/N, H-2q) and assessed for allospecific in vivo cytotoxicity at serial time points posttransplant (Fig. 2). In CD4-sufficient recipients, CD8-mediated cytotoxic effector function is first observed 3 days posttransplant and rises to its peak magnitude on day 7 posttransplant. CD8-mediated allospecific cytotoxic effector function decreases after rejection (day 10 posttransplant), and slowly returns to near baseline by day 21 following transplant. In CD4-deficient recipients, CD8-mediated allocytotoxicity is also detected on day 3 posttransplant, but does not continue to increase in magnitude, as observed in CD4-sufficient recipients. Instead, CD4-independent CD8+ CTL activity peaks on day 3 posttransplant and decreases rapidly to baseline levels by the time of graft rejection (day 10–14 posttransplant). Peak cytotoxicity in CD4-sufficient recipients was significantly greater than in CD4-deficient hepatocyte recipients (78 vs 29%; p < 0.01), peaked later (day 7 vs day 3), and persisted longer posttransplant (day 21 vs day 10). Consequently, the total cytotoxic activity generated by CD4-dependent CD8+ CTLs (as reflected by the area under the curve) was much greater than that for CD4-independent CD8+ CTLs over the same time period (day 3 to day 35 posttransplant) (Fig. 2) (p < 0.001).


Figure 2
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  		FIGURE 2. Kinetic analysis of in vivo allospecific cytotoxicity in CD4-sufficient and CD4-deficient hepatocyte allograft recipients. C57BL/6 (H-2b)- and CD4 KO (H-2b)-untreated hosts were transplanted with allogeneic (FVB/N, H-2q) hepatocytes, and the presence of in vivo allocytotoxicity was measured at serial timepoints following transplantation. An increased magnitude of allospecific cytolytic activity was observed on days 5, 7, 10, 14, and 28 following hepatocyte transplant in CD4-sufficient (C57BL/6) compared with CD4-deficient (CD4 KO) hepatocyte recipients (p < 0.05; n = 4–6 animals for each timepoint for both groups). The total allospecific cytotoxicity (area under the curve from day 3 to day 35 posttransplant) occurring in response to hepatocyte transplantation in CD4-sufficient hosts was significantly greater than in CD4-deficient hosts (p < 0.001).

 
The differences observed in the CD4-sufficient and CD4-deficient recipients raised the issue of whether CD4+ T cells might directly contribute to cytotoxic effector function in CD4-sufficient recipients. To investigate this possibility, C57BL/6 mice were transplanted with hA1AT-FVB/N hepatocytes and were then depleted of CD4+ T cells (anti-CD4 mAb, i.p., days 6 and 7 posthepatocyte transplant) 48 h before assessment of in vivo allocytotoxicity near the peak of the cytotoxic response (day 8 posttransplant). CD4-depleted C57BL/6 hepatocyte rejectors demonstrated in vivo allospecific cytotoxicity that was comparable in magnitude to that observed in control C57BL/6 hepatocyte rejectors (p = NS) (Fig. 3). These collective results demonstrate that although recipient CD4+ T cells appear to significantly influence the maturation of CD8+ allo-CTLs, they do not directly contribute to in vivo alloreactive CD8+ CTL effector function.


Figure 3
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  		FIGURE 3. CD4+ T cells do not contribute to the effector phase of in vivo allospecific cytotoxicity in CD4-sufficient C57BL/6 hosts. C57BL/6 (H-2b) recipients (n = 6) were transplanted with allogeneic (FVB/N, H-2q) hepatocytes, followed by CD4+ T cell depletion (anti-CD4 mAb, i.p., days 6 and 7 posthepatocyte transplant) 48 h before the assay for in vivo allospecific cytolytic activity (day 8 posttransplant). The depletion of CD4+ T cells did not significantly decrease in vivo allocytotoxicity (allogeneic target cell clearance) relative to C57BL/6 control hosts (n = 5) (p = NS).

 
In vivo proliferation of allospecific TCR transgenic CD8+ T cells is equivalent in CD4-sufficient and CD4-deficient hepatocyte recipients

CD4+ T cells are a main source of the T cell growth factor IL-2 and are important for optimal CD8+ T cell proliferation (51). To determine whether the enhanced magnitude of CD8+ CTL activity observed in CD4-sufficient recipients was due to enhanced proliferation resulting in an increased number of CD8+ CTL effectors, we investigated the in vivo proliferation of allospecific TCR transgenic CD8+ T cells in response to hepatocyte transplant in the presence or absence of CD4+ T cells.

DesTCR CD8+ T cells (H-2k) express a TCR with specificity for H-2Kb MHC class I alloantigen. Column-purified Des+CD8+ T cells (~2–4 x 106) were stained with CFSE (5 µM) and adoptively transferred (day –1) into syngeneic B10.BR (H-2k) recipients. These hosts were either CD4 sufficient or CD4 depleted (anti-CD4 mAb, GK1.5, days –4 and –2) and received allogeneic hepatocyte transplants (hA1AT-C57BL/6, H-2b) on day 0. CD4-sufficient and CD4-depleted Des-reconstituted B10.BR recipients (DesrB10.BR) without hepatocyte transplant served as controls to detect background and/or homeostatic proliferation. Three days after hepatocyte transplant, recipient splenocytes and graft-infiltrating lymphocytes (liver-infiltrating lymphocytes) were retrieved and Des+CD8+ T cells were assessed for in vivo proliferation by flow cytometric analysis of CFSE dilutional fluorescence. Proliferation of allospecific Des+CD8+ T cells was observed in both splenic (data not shown) and graft-infiltrating lymphocyte populations in both CD4-sufficient and CD4-deficient hepatocyte recipients, but not in untransplanted controls (Fig. 4). In both groups of recipients, Des+CD8+ T cells underwent more rounds of cell division at the graft site (liver) than in the host spleen (data not shown). Proliferation of allospecific CD8+ T cells was similar in CD4-sufficient and CD4-deficient hepatocyte allograft recipients, as reflected by comparison of the percentage of Des+CD8+ T cells remaining undivided, or which underwent 1–3 divisions, 4–6 divisions, or >7 cell divisions (p = NS). This suggests that given an equal number of alloreactive CD8+ T cells from the outset, these cells proliferate to the same degree in response to allogeneic hepatocytes in the presence or absence of CD4+ T cell help.


Figure 4
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  		FIGURE 4. Allospecific graft-infiltrating TCR transgenic CD8+ T cells proliferate equally in CD4-sufficient or CD4-deficient recipients. A total of 2–4 x 106 TCR transgenic CD8+ T cells allospecific for H-2Kb (Des+TCR+CD8+CD3+ T cells) was stained with CFSE dye (5 µM) and adoptively transferred into syngeneic B10.BR (H-2k) hosts day –1 relative to hepatocyte transplantation. Some hosts were depleted of CD4+ T cells before hepatocyte transplant (anti-CD4 mAb, GK1.5, i.p., days –4 and –2). Allogeneic (C57BL/6, H-2b) hepatocytes were transplanted into B10.BR recipients, and 3 days posttransplant, host GICs and splenocytes (data not shown) were retrieved, enumerated, and immunolabeled for DesTCR+ and CD8+. Allospecific DesTCR+CD8+ T cells were assessed for in vivo proliferation by analysis of dilutional fluorescence and flow cytometry. The percentages of DesTCR+CD8+ T cells remaining undivided, dividing 1–3 times, 4–6 times, or 7+ times were compared between CD4-sufficient (n = 4) and CD4-deficient (n = 4) Des+TCR+CD8+ T cell-reconstituted (DesrB10.BR) recipient groups. The data shown are representative of four separate experiments.

 
However, given the potential of TCR transgenic cells to bypass the requirement for CD4+ T cell help due to high frequency or Ag avidity (52, 53), we determined both the number of activated CD8+ CTLs and the ultimate size of the nontransgenic CTL effector population occurring in CD4-dependent vs CD4-independent allograft rejection. To investigate the size of the CD8+ CTL effector population in CD4-sufficient (C57BL/6) and CD4-deficient (CD4 KO) hepatocyte allograft recipients, activated CD8+ T cells were retrieved from the liver (CD8+ GICs) and enumerated 1 wk posttransplant. The percentages and absolute numbers of CD8+ T cells with an activated phenotype (CD8+CD62LlowCD44high) were equivalent between recipient groups (p = NS). The number of recipient CD8+ GICs demonstrating a cytotoxic effector phenotype (CD8+CD43highCD44high) was also similar between CD4-suffficient and CD4-deficient recipient groups (p = NS) (Fig. 5). Collectively, these data support the conclusion that differences in CD8+ effector T cell population size, due to a higher frequency of alloreactive cells or through enhanced proliferation, are not responsible for the increased magnitude of CD8+ cytolytic effector function observed in CD4-sufficient recipients.


Figure 5
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  		FIGURE 5. CD4-sufficient and CD4-deficient hepatocyte recipients have equivalent numbers of CD8+ GICs with an activated and cytotoxic phenotype. FVB/N (H-2q) allogeneic hepatocytes were transplanted into untreated C57BL/6 (H-2b, n = 5) and CD4 KO (H-2b, n = 5) hosts. Day 7 posttransplant, recipient graft (liver)-infiltrating cells and splenocytes (data not shown) were retrieved, enumerated, and immunolabeled for flow cytometric analysis to quantify CD8+ T cells with an activated (CD62LlowCD44high) or cytotoxic (CD43highCD44high) phenotype. Results were compared with liver-infiltrating cells and splenocytes from naive controls and confirmed the expansion of effector populations in response to hepatocyte transplant (data not shown). Comparison between CD4-sufficient and CD4-deficient recipient groups showed no significant difference in percentage or absolute number of CD8+ T cells in GICs or splenocytes with an activated or cytotoxic phenotype developing in response to hepatocyte allografts (p = NS, respectively, for both cell populations).

 
Potent secondary CD8-mediated CTL activity develops in CD4-sufficient, but not CD4-deficient recipients

CD4+ T cell help in response to viral and other infectious stimuli or nominal Ags has been reported to critically influence CD8+ T cell memory and recall responses (3, 11, 12, 13, 14, 15, 16, 17, 18, 19). We hypothesized that recipient CD4+ T cells also critically influence secondary alloreactive CD8+ T cell cytotoxic effector function after hepatocyte transplant. To test this hypothesis, CD4-sufficient (C57BL/6) and CD4-deficient (CD4 KO) recipients were transplanted with allogeneic hepatocytes (hA1AT-FVB/N, H-2q). Following rejection of the first hepatocyte graft, recipients underwent a second donor-matched hepatocyte transplant (hA1AT-FVB/N, H-2q) on day 21–35 posttransplant (coinciding with return of in vivo CTL activity to baseline levels). Hepatocellular allograft survival and CTL activity were assessed in these primed recipients. Rejection of the second graft was faster than rejection of the primary graft in both CD4-sufficient (MST = 7 vs 10 days, p = 0.05) and CD4-deficient (MST = 10 vs 14 days, p < 0.05) recipient groups, consistent with a recall response. At 3 days following the second transplant, CD4-sufficient hosts showed a higher magnitude of allocytotoxicity in comparison with CTL activity at the comparable timepoint following primary transplant (95 vs 33%, respectively, p < 0.001). In contrast, there was no increase in CTL activity following the second transplant in CD4-deficient hosts (Fig. 6). Additionally, 2 wk after rejection of the second hepatocyte transplant, CD4-sufficient recipients still manifested heightened CTL effector function, whereas CTL activity in CD4-deficient recipients had returned to baseline (Fig. 6). These results support the conclusion that recipient CD4+ T cells facilitate development of an early augmented and sustained allospecific CD8+ CTL memory response beyond clearance of the alloantigenic stimulus. In contrast, in CD4-deficient recipients, secondary CTL responses are no different from the primary CTL response and are significantly decreased in magnitude and extinguished more rapidly than in CD4-sufficient recipients (Fig. 6).


Figure 6
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  		FIGURE 6. CD4-sufficient (but not CD4-deficient) hepatocyte transplant recipients develop recall responses with enhanced magnitude and persistence of allospecific cytotoxicity. FVB/N (H-2q) allogeneic hepatocytes were transplanted into C57BL/6 (H-2b) and CD4 KO (H-2b) hosts. Following rejection of hepatocytes and coinciding with loss of detectable in vivo allocytotoxicity, a second allogeneic FVB/N hepatocyte transplant was performed (~21–35 days following the initial hepatocyte transplant). The presence of in vivo allospecific cytolytic activity was assessed in both CD4-sufficient and CD4-deficient recipient groups on days 3 (n = 5, n = 6, respectively) and 21 (n = 5, n = 6, respectively) following the second hepatocyte transplant. Allospecific cytotoxicity was significantly increased in magnitude (p < 0.001 for day 3) and demonstrated prolonged persistence in CD4-sufficient recipients following secondary vs primary hepatocyte transplant (p < 0.001 for day 21). In contrast, allospecific cytotoxicity did not differ significantly in CD4-deficient recipients following primary or secondary hepatocyte transplant (p = NS for days 3 and 21 posttransplant, respectively).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The role of CD4+ T cell help on primary and secondary CD8+ T cell cytolytic responses has been extensively investigated in vitro (54, 55, 56, 57, 58, 59). The requirement for, and consequences of CD4+ T cell help on maturation of Ag-specific CD8+ CTLs in vivo have been examined primarily in response to infectious pathogens (viral and other microbial stimuli) and in response to immunization with nominal Ags (reviewed in Refs. 16, 18, 19 , and 60). To our knowledge, this is the first study to investigate the development of primary and secondary in vivo alloreactive CD8+ CTL activity in CD4-sufficient and CD4-deficient transplant recipients.

We found that allogeneic hepatocytes stimulate the development of in vivo allospecific cytotoxicity in both CD4-sufficient (C57BL/6) and CD4-deficient (CD4 KO) hepatocyte recipients (Fig. 1). In both recipient groups, this in vivo cytotoxicity was mediated by CD8+ CTL effectors (Fig. 1b) and was allospecific (Fig. 1c). Although NK cells have been reported to initiate and affect cytolytic activity in CD4-independent conditions in the setting of antitumor immunity (61) and costimulation blockade-resistant bone marrow allograft rejection (62), we found that NK cells do not participate in CTL effector function in hepatocyte rejectors (Fig. 1b). These studies expand upon our previous observations that CD8+ GICs in hepatocyte rejectors express a CTL phenotype (CD8+CD43+) (47) and that the detection of in vivo allocytotoxicity correlates with graft survival status (46, 47).

Experimental models that investigate CD8+ CTL responses to viral infection or to nominal Ag have reported that CD4+ T cell help does not significantly influence primary CD8+ T cell responses, but critically influences CD8+ T cell recall and memory responses (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 63). Of note, in these nontransplant models, CD8+ T cell cytokine production, proliferation, and CTL effector function during primary Ag-specific activation were determined to be comparable whether or not recipient CD4+ T cells were present (17, 20). In contrast, we found that CD4+ T cells significantly influence the development of alloreactive CD8+ CTLs during primary alloantigen exposure as reflected by the differences in kinetics and magnitude of in vivo allospecific cytotoxicity in CD4-sufficient vs CD4-deficient hepatocyte recipients (Fig. 2). These results agree with studies in which CD4-sufficient hosts developed higher CTL activity in response to allogeneic tumor cells than CD4-deficient hosts (64).

Heightened CTL activity occurring in the presence of CD4+ T cells has several possible explanations. First, CD4+ T cells could directly mediate in vivo allospecific cytotoxicity; however, it was observed that CD4+ T cell depletion before the in vivo cytotoxicity assay in CD4-sufficient hepatocyte recipients did not significantly inhibit clearance of allogeneic target cells (Fig. 3). Second, CD4+ T cell signals could drive enhanced CD8+ T cell effector proliferation. We have noted in other studies that a small percentage of CD8+ GICs in CD4 KO hepatocyte rejectors expresses the IL-2R{alpha} (CD25), implicating a role for local IL-2 in (CD4-independent) CD8+ T cell alloimmunity (46, 47). To address the possibility of enhanced CD8+ T cell proliferation in the presence of CD4+ T cells, we analyzed in vivo proliferation of allospecific TCR transgenic Des+CD8+ T cells in CD4-sufficient and CD4-deficient conditions. As shown in Fig. 4, proliferation of allospecific CD8+ T cells was similar in the presence or absence of CD4+ T cells. This suggests that either alloreactive CD8+ T cells provide self help through autocrine production of IL-2 and perhaps other cytokines or receive help from other non-CD4+ immune cells, or that a combination of these factors stimulates proliferation and maturation of alloreactive CD8+ T cells in CD4-deficient conditions (65). One caveat is that the in vivo proliferation of TCR transgenic CD8+ T cells may not reflect the proliferative requirements of wild-type CD8+ T cells. This could occur because TCR transgenic T cells could have decreased threshold for activation and proliferation. It could also occur due to the relatively high number of transgenic allospecific cells adoptively transferred (~2–4 x 106), which could circumvent the need for CD4+ T cell help in proliferation (52). The number of Des+CD8+ T cells transferred in this study was determined from titration studies designed to provide the minimum number of cells necessary to provide a sufficient yield of liver-infiltrating cells for flow cytometric analysis. Despite this caveat, our results agree with a published report using fewer CD8+ TCR transgenic cells (1–2 x 106 OT-I cells) for adoptive transfer and that also reported that proliferation of alloreactive CD8+ T cells occurs independent of CD4+ T cell help (45). Our results demonstrate that although the presence of CD4+ T cells significantly influences the magnitude and longevity of in vivo allocytotoxicity, this does not appear to occur due to enhanced proliferation of CD8+ CTLs.

A third possibility is that more alloreactive CD8+ T cell precursors are present in C57BL/6 vs CD4 KO hosts, resulting in a greater number of CD8+ CTL effectors. To analyze this, we measured the number of activated and cytotoxic CD8+ T cells arising in both immune conditions following hepatocyte transplantation. We observed that CD4-sufficient and CD4-deficient recipients had similarly sized effector CD8+ T cell populations; enumeration of CD8+ T cells from the graft site (liver) and in the spleen showed similar numbers of activated (CD62LlowCD44high) and cytotoxic (CD43highCD44high) CD8+ T cells (Fig. 5). These results are consistent with the in vivo proliferation experiments using TCR transgenic Des+CD8+ T cells, demonstrating equivalent proliferation of allospecific CD8+ T cells in response to allogeneic hepatocytes in the presence or absence of host CD4+ T cells. Therefore, regardless of any potential differences in the number of alloreactive CD8+ T cell precursors or their in vivo proliferation in C57BL/6 vs CD4 KO hosts, we find that there is no significant difference in the number of activated or cytotoxic CD8+ effector cells.

A fourth possibility is that alloreactive CD8+ T cells develop along a continuum in response to local activation signals such that in the presence of CD4+ T cell help, CD8+ allo-CTLs reach their maximum differentiation state and cytolytic effector potential. In contrast, in CD4-deficient conditions, CD8+ CTLs may only reach intermediate differentiation and cytolytic effector function. In this scenario, it is possible that the increased allocytotoxicity observed in CD4-sufficient hepatocyte recipients could result from the development of more efficient cytolytic mechanisms in the presence of CD4+ T cell help. The perforin/granzyme CTL lytic mechanism has been reported to mediate more efficient killing than Fas ligand (FasL)-mediated cytotoxicity (66, 67) and has been shown to require stronger activation signals than are required to activate the FasL cytotoxicity pathway (68). Consistent with this idea, CD8+ CTL activation in response to hepatocellular allografts in CD4-sufficient conditions could develop both efficient perforin/granzyme- and FasL-killing mechanisms, whereas CD8+ CTLs that mature in CD4-deficient conditions may only develop the less efficient FasL cytotoxic mechanism. Studies to define the mechanisms of CD8+-mediated cytotoxicity in the presence or absence of CD4+ T cells are ongoing.

Alloreactive CD8+ T cells maturing in a CD4-sufficient immune environment exhibit peak cytolytic activity on day 7 posttransplant, which declines to near-baseline levels on day 21 posttransplant (1–2 wk after hepatocyte rejection). In contrast, alloreactive (CD4-independent) CD8+ CTLs exhibit a distinct effector phase profile in which peak cytolytic activity occurs early posttransplant (day 3) and rapidly declines to baseline by the time of hepatocyte rejection (day 10–14) (Fig. 2). This kinetic profile of a rapid peak effector response that resolves rapidly is reminiscent of the kinetics of innate immune cell responses. It is intriguing to consider the possibility that CD4-independent, CD8+ allo-CTLs are novel, intermediate (less vigorous) cytolytic effectors bridging innate and adaptive immune pathways.

Whether or not CD4-independent CD8+ allo-CTLs represent differential maturation of the same precursor CD8+ T cells or whether they represent maturation of a unique subset of CD8+ T cells remains to be determined in future studies. One approach to this question would be to determine the functional fate and gene expression profile of a labeled population of in vivo activated CD8+ T cells (retrieved from CD4 KO hepatocyte recipients) that are subsequently adoptively transferred into CD4-sufficient vs control CD4-deficient hosts. If CD8+ allo-CTLs develop along a continuum in response to local stimuli including signals from CD4+ T cells, one would expect that CD8+CD43highCD44high T cells transferred into CD4-sufficient (C57BL/6) hepatocyte recipients would acquire the characteristics of CD8+ T cells developing de novo in CD4-sufficient hosts. In contrast, if CD4-independent CD8+CD43highCD44high T cells are terminally differentiated, there should be no difference in intracellular cytokine expression, expression of FasL vs perforin/granzyme molecules, gene expression profile, or in vivo CTL effector function between the populations when transferred into CD4-sufficient vs CD4-deficient hepatocyte recipients.

In nontransplant experimental models, CD4+ T cells clearly contribute to the development of enhanced secondary or memory CD8+ CTL and cytokine effector responses, although the mechanisms by which this occurs may vary (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 63, 69, 70). In the current study, we found that the absence of CD4+ T cells significantly impairs the magnitude and persistence of alloreactive CD8+ CTL recall responses, consistent with observations made in other experimental systems (Fig. 6). CD4-sufficient secondary hepatocyte transplant recipients demonstrated an enhanced magnitude and persistence of CTL activity, whereas CD4-deficient recipients displayed secondary in vivo cytolytic effector responses that were similar in magnitude and kinetics to the primary CD8-mediated CTL response. Despite the impaired development of enhanced secondary CTL responses in CD4-deficient hosts, both CD4-sufficient and CD4-deficient recipients demonstrated enhanced rejection kinetics in response to a second hepatocyte transplant. The accelerated kinetics of rejection, but not CTL activity, during the secondary response in CD4-deficient recipients could be explained by a reduced threshold for CTL activation without any enhancement in cytolytic differentiation, effector pool expansion, or CTL survival.

These studies used a hepatocellular allograft model to assess the influence of CD4+ T cells on the development of allospecific CD8+ CTL responses in vivo. The results support the concept that CD4-independent activation of alloreactive CD8+ T cells produces unique cytotoxic functional activity that is efficient in responding to and clearing donor Ag, but is not as potent in primary or memory in vivo cytolytic effector function when compared with CD8+ allo-CTLs, which mature in the presence of CD4+ T cells. A better understanding of alloreactive CD8+ T cells maturing in CD4-sufficient or CD4-deficient conditions is important for prevention of allograft rejection by these pathways as well as for design of immune tolerance strategies.


   Acknowledgments
 
We acknowledge the assistance of Cassidy Schwab, who provided excellent animal care and assistance in the laboratory. We also thank Dr. Gregg Hadley for critical review of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by grants from the American Society of Transplantation Basic Science Physician Scientist Award (to P.H.H.), Roche Organ Transplantation Research Foundation (to G.L.B.), the American Society of Transplant Surgeons (to G.L.B.), and National Institutes of Health DK072262 (to G.L.B.). Back

2 Address correspondence and reprint requests to Dr. Ginny L. Bumgardner, Ohio State University Medical Center, Department of Surgery, Division of Transplant, 1654 Upham Drive, 373 Means Hall, Columbus, OH 43210-1250. E-mail address: ginny.bumgardner{at}osumc.edu Back

3 Abbreviations used in this paper: KO, knockout; FasL, Fas ligand; GIC, graft-infiltrating cell; hA1AT, human {alpha}-1 antitrypsin; MST, median survival time. Back

Received for publication April 3, 2007. Accepted for publication April 16, 2007.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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