Journal of Leukocyte Biology
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Originally published online as doi:10.1189/jlb.1104669 on May 17, 2005

Published online before print May 17, 2005
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(Journal of Leukocyte Biology. 2005;78:80-84.)
© 2005 by Society for Leukocyte Biology

Cooperation between platelet-derived CD154 and CD4+ T cells for enhanced germinal center formation

Bennett D. Elzey*, Julieann F. Grant{dagger},{ddagger}, Haley W. Sinn*, Bernhard Nieswandt§, Thomas J. Waldschmidt{dagger},{ddagger} and Timothy L. Ratliff*,{dagger},¶,{cjs0822}{cjs0822},**,{dagger}{dagger},1

* Departments of Urology,
{dagger} Pathology, Interdisciplinary Immunology Program, and
** Microbiology,
{ddagger} Carver College of Medicine, Medical Scientist Training Program,
Prostate Cancer Research Group, and
{dagger}{dagger} Holden Comprehensive Cancer Center, University of Iowa, Iowa City;
§ Rudolf Virchow Center for Experimental Biomedicine, University of Wurzburg, Germany; and
{cjs0822}{cjs0822} Iowa City Veterans Affairs Medical Center, Iowa City

1 Correspondence: University of Iowa, Department of Urology, 3206 MERF, 375 Newton Road, Iowa City, IA 52242. E-mail: tim-ratliff{at}uiowa.edu

ABSTRACT

It has been demonstrated previously that platelet-derived CD154 communicates with the adaptive immune compartment, enhancing B and T cell responses in CD154–/– mice. The presence of platelets was also shown to be necessary for optimal production of immunoglobulin G (IgG) in normal C57BL/6 mice. These data led us to hypothesize that platelets perform a sentinel function, quickly relaying activating signals to the adaptive immune compartment. Here, we report that platelet-derived CD154 increases serum IgG levels and germinal center formation under conditions where antigen-specific CD4+ T cell numbers are limiting. We propose that in the physiologic setting where antigen-specific B and T cells are rare, platelets function to enhance signals required for robust adaptive humoral immunity.

Key Words: B cells • humoral immunity • mouse • innate immunity • knockout

It is well established that CD4+ T cell CD154 is required for germinal center (GC) formation and production of appreciable levels of switched antibody isotypes (reviewed in ref. [1 ]). The current paradigm for antibody production proposes that T cells first expand in the T cell zone and then migrate toward the follicle border, where antigen-stimulated B cells have also localized. CD154-dependent communication then occurs between B and T cells, resulting in initial rounds of B cell proliferation. The resulting B cell blasts differentiate into antibody-forming plasma cells or coalesce in the follicle to initiate a mature GC response. Experiments by Garside and colleagues [2 ], who tracked antigen-specific T and B cells after immunization, best illustrated the paradigm described above. In these studies, 2.5 x 106 transgenic B cells and 5 x 106 transgenic CD4+ T cells were adoptively transferred intravenously (i.v.) to visualize interactions of antigen-specific lymphocytes in host lymph nodes. Although it is unknown how many actually localized to the draining nodes, the studies used a precursor frequency about four orders of magnitude higher than the in vivo-anticipated, physiologic number. The need for high numbers of transgenic lymphocytes to perform experiments of this type is of interest and underscores the efficiency at which rare antigen-specific T and B cells are able to interact and affect humoral immunity under normal conditions. For example, it has been postulated that there are maximally 25 antigen-specific T or B cells present before expansion [3 ]. Although stimulated T cells can have a doubling time as short as 6 h [4 , 5 ], it is remarkable that many follicles within the spleen contain sizable GCs detectable as early as 4 days post-immunization if B cells must wait to be "licensed" by T helper (Th) cells. Indeed, a two-phase model of B cell activation has been proposed to account for these considerations [3 ].

It is clear that in addition to mediating hemostasis, platelets exert a strong influence on innate immunity through modulation of inflammatory responses [6 ]. Additionally, their involvement in adaptive immunity has been reported and reviewed recently [7 8 9 ]. In mice, platelets can be present at over 5 x 108/ml blood, and in humans, up to one-third of the total in circulation is present in the spleen and can be a significant source of serum CD154 [10 , 11 ]. As a result of their extremely high numbers, omnipresence in circulation, and ability to release inflammatory mediators, platelets are uniquely poised to perform sentinel functions and can quickly communicate with the immune system. Given the limiting numbers of CD4+ T cells available during the initiation of an antibody response and the ready availability of platelet-derived CD154, we hypothesize that platelets provide early signals to the B cell compartment. Through CD154-CD40 interactions, platelets may prime antigen-specific B cells and thus facilitate their efficient differentiation into GC B cells upon stimulation by emerging Th cells.

Previous studies demonstrated that normal platelets adoptively transferred to CD154–/– mice enhanced the production of adenovirus-specific immunoglobulin G (IgG) [7 ]. However, the levels were transient, peaking at day 7 and disappearing by day 14, indicating that platelet-derived CD154 alone is not sufficient for the development of a mature GC response. We hypothesized, based on the proposed sentinel function of platelets, that early progression signals had been provided by platelets, but these were insufficient to drive GC formation in the absence of CD4+ T cells. To test this hypothesis, studies were initiated to determine whether normal platelets, in combination with normal CD4+ T cells, could enhance thymus-dependent GC responses when adoptively transferred into CD154–/– mice. As only small numbers of CD4+ T cells are required for a detectable GC response [12 ], the number of B6 CD4+ T cells was titrated into CD154–/– mice to determine a threshold number unable to stimulate GC formation but able to cooperate with platelets for a significant GC response (data not shown). Platelets and CD4+ T cells were then transferred into CD154–/– mice alone or in combination and followed by adenoviral challenge 4 h later. After 28 days, adenovirus-specific production of IgG was assessed by enzyme-linked immunosorbent assay (ELISA). Figure 1 shows that CD154–/– mice receiving normal platelets and T cells produced the highest levels of specific IgG compared with other experimental groups. The results were highly reproducible over four experiments of five mice per group. To confirm platelet-associated enhancement of GC formation, histologic examination of GCs was performed. Figure 2 clearly demonstrates that platelet-derived CD154, in the presence of normal CD4+ T cells, increased GC formation. In CD154–/– mice, GCs only form in the presence of normal CD4+ T cells, but when normal platelets are also present, there is a statistically significant increase in GC frequency. Normal platelets alone did not result in detectable GCs nor did any of the CD154–/– control groups. Next, we sought to determine if increasing the number of adoptively transferred normal platelets would further enhance GC formation. After injecting two and five times the number of platelets used in Figure 2 , Figure 3A clearly demonstrates that more platelets do not increase, but rather decrease, GC formation. The reasons for this are unclear but may relate to the fact that activated platelets can express Fas ligand (FasL) [14 ], which could induce apoptosis in B cells stimulated by CD40L [15 , 16 ] prior to B cell receptor ligation [17 , 18 ]. It is interesting that when sufficient numbers of CD4+ T cells were adoptively transferred into CD154–/– mice, the presence of normal platelets did not enhance GC formation (Fig. 3B) . These data suggest that when limiting numbers of CD154-bearing T cells are present, platelets can act as "costimulators" in B cell activation. From this, we predicted that in normal B6 mice, platelets would also be able to perform a costimulatory role when antigen dose was low, but this contribution would be diminished in the presence of high antigen levels. Figure 4 shows that this is indeed the case. When B6 mice were depleted of platelets, their ability to produce IgG in response to low doses of adenovirus was compromised severely, but at higher doses, IgG production was unaffected. Taken together, these data suggest that platelets may play a costimulatory role early in primary responses when T cell help is limiting. As large numbers of platelets are activated during early phases of infection and inflammation, it is intuitively attractive to speculate that the resulting pool of platelet-derived CD154 may engage CD40-bearing B cells and prime them for further signals.



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  		Figure 1. Normal B6 platelets (plt) enable limiting numbers of normal B6 CD4+ T cells to induce antiadenovirus IgG in CD154–/– mice, which were depleted of platelets with 10 µg p0p3/4 24 h prior to platelet and CD4+ T cell-adoptive transfer (a titrated dose of monoclonal antibody, which depletes host platelets but permits adoptive transfer of donor platelets; ref. [7 ]). B6 (108) or CD154–/– [knockout (KO)] platelets and 4 x 106 negatively selected, naïve B6 or KO CD4+ T cells were injected i.v. into appropriate mice. Four hours later, all groups were injected i.v. with 108 plaque-forming units of adenovirus. Twenty-eight days later, plasma was harvested, and antiadenovirus-specific IgG was quantitated by ELISA. Shown is one representative experiment of four, each with five mice per group. Normal C57BL/6 (B6) mice were purchased from the National Cancer Institute (Frederick, MD). CD154 gene KO mice (CD154–/–; B6 background) were purchased from Jackson Laboratories (Bar Harbor, ME) and bred at the University of Iowa (Iowa City) animal facility. All mice were used between the ages of 6 and 12 weeks. Murine platelets were isolated as described previously [7 , 13 ] and did not contain detectable lymphocytes (data not shown). Negative isolation of CD4+ T cells was performed by magnetic bead separation according to the manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). No B cells were detectable by flow cytometry in the T cell preparation (data not shown).

 


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  		Figure 2. Normal B6 platelets enable limiting numbers of normal B6 CD4+ T cells to induce GC reactions in CD154–/– mice, which were treated as in Figure 1 , except 10 days after adenovirus immunization, spleens were harvested and processed for histologic analysis of frozen sections. (A–C) Selected histologic images. (A) Normal B6 spleen containing multiple GC. (B) Lack of GC in CD154–/– mice adoptively transferred with CD154–/– platelets and CD154–/– CD4+ T cells. (C) Restoration of GC response in CD154–/– mice adoptively transferred with normal B6 platelets and B6 CD4+ T cells. (D) Table shows number of GCs formed in all experimental groups. GCs were visualized by histologic examination and reported as GC/PALS units (PALS unit=the follicles surrounding a T cell zone). Results are from two independent experiments each with five mice per group. Blue = T cell zone (anti-CD4 and CD8 staining); red = B cell follicles (anti-B220); green = peanut agglutinin (PNA; GC B cells stain PNAhi). GCs are visualized as B220 PNA double-positive (green-yellow) areas of cells.

 


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  		Figure 3. (A) Dose titration of normal platelets for GC induction with a fixed CD4+ T cell number. Mice were treated and evaluated as in Figure 2 , except additional numbers of platelets were transferred as indicated. Approximately 400 PALS units were examined for GCs per group. Results are combined from two separate experiments of five mice per group. (B) Sufficient numbers of CD4 T cells do not require platelet help for GC formation. Mice were treated as in Figure 2 , except that 107 CD4 T cells were adoptively transferred instead of 4 x 106. Histology was performed as in Figure 2 . Approximately 200 PALS units were evaluated per group. Results are combined from two separate experiments of five mice per group.

 


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  		Figure 4. B6 mice require platelets to produce IgG in response to low doses of adenovirus. B6 mice were depleted of platelets (over 99%, data not shown) by i.v. injection of 100 µg p0p3/4 and 24 h later, were immunized i.v. with the indicated dose of adenovirus particles. Twenty-one days later, plasma was harvested, and ELISAs were performed to measure levels of antiadenovirus-specific IgG. Shown is one representative experiment of two with four to five mice per group.

 
Although platelet activation is regulated tightly, platelets are among the first cells to respond to injury and inflammatory stimuli. Thus, platelets represent an early signaling compartment that releases highly inflammatory substances such as 12(S)-hydroxy-6,8,11,14-eicosatetraenoic acid, cationic proteins, histamine, macrophage-inflammatory protein-1{alpha}, and RANTES (regulated on activation, normal T expressed and secreted) upon activation [6 ]. Many of these components likely contribute to nonspecific inflammation via vasodilation and chemoattraction of inflammatory cells. Similarly, human platelet CD154 has been described to provoke release of interleukin-8 and monocyte chemoattractant protein from endothelial cells [19 ], but our work clearly defines a role for platelet CD154 at the level of adaptive immunity in the murine model [7 ] (Figs. 1 2 3 4) . Platelets also express the membrane-bound immunomodulatory proteins FasL and tumor necrosis factor-related apoptosis-inducing ligand [14 , 20 ], which are molecules that play key regulatory roles in the adaptive response. The mechanism by which platelets modulate antibody levels and increase GC formation is not known. Recently, it has been reported that human platelets can directly contact autologous B cells in vitro and cause them to proliferate and produce specific antibody in a CD154-dependent manner [21 ]. It is possible that signaling via direct B cell contact occurs in vivo, but it may be that the action is through the antigen-presenting cell. Indeed, platelets have been reported to activate dendritic cells [7 , 22 23 24 ]. Although the mode of action requires clarification, our unpublished, preliminary data indicate that microparticles released from activated platelets may deliver the B cell stimulus and not the intact platelets themselves. Identification of the mechanism of signal delivery is currently underway.

It is not surprising that platelet CD154 alone is insufficient to drive GC development. This observation is consistent with previous studies demonstrating the inability of an agonistic anti-CD40 antibody to promote GC formation [25 ]. In addition to CD154, Th cells must provide a range of other signals and cytokines required for full differentiation of B cells and the GC reaction. It is significant, however, that platelet CD154 can enhance GC formation when T cell numbers are limiting and increase specific IgG production to low antigen doses, suggesting a key role for platelets in B cell activation during primary responses. In summary, our studies demonstrate the previously unknown participation of platelets in antibody production and GC formation, suggesting that the current paradigm for antibody production may require modification.

ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants AI60924 and A096691. The authors thank Dr. Tom Griffith for critical reading of the manuscript and Mike Carlson for technical help with histology.

Received November 18, 2004; revised April 21, 2005; accepted April 22, 2005.

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