Multicenter Analysis of Immune Biomarkers and Heart Transplant Outcomes: Results of the Clinical Trials in Organ Transplantation-05 Study.

Identification of biomarkers that assess posttransplant risk is needed to improve long-term outcomes following heart transplantation. The Clinical Trials in Organ Transplantation (CTOT)-05 protocol was an observational, multicenter, cohort study of 200 heart transplant recipients followed for the first posttransplant year. The primary endpoint was a composite of death, graft loss/retransplantation, biopsy-proven acute rejection (BPAR), and cardiac allograft vasculopathy (CAV) as defined by intravascular ultrasound (IVUS). We serially measured anti-HLA- and auto-antibodies, angiogenic proteins, peripheral blood allo-reactivity, and peripheral blood gene expression patterns. We correlated assay results and clinical characteristics with the composite endpoint and its components. The composite endpoint was associated with older donor allografts (p < 0.03) and with recipient anti-HLA antibody (p < 0.04). Recipient CMV-negativity (regardless of donor status) was associated with BPAR (p < 0.001), and increases in plasma vascular endothelial growth factor-C (OR 20; 95%CI:1.9-218) combined with decreases in endothelin-1 (OR 0.14; 95%CI:0.02-0.97) associated with CAV. The remaining biomarkers showed no relationships with the study endpoints. While suboptimal endpoint definitions and lower than anticipated event rates were identified as potential study limitations, the results of this multicenter study do not yet support routine use of the selected assays as noninvasive approaches to detect BPAR and/or CAV following heart transplantation.

Peripherally circulating CD4⁺ FOXP3⁺ CXCR3⁺ T regulatory cells correlate with renal allograft function.

Peripheral immunoregulation depends on T regulatory cell trafficking into the allograft to modulate the local alloresponse. Little is known about the relevance of trafficking receptors for Tregs after solid organ transplantation in humans. In this study, expression of the peripheral chemokine receptors CXCR3 and CCR5 on CD4⁺ FOXP3⁺ Treg cells was analysed and correlated with allograft function in renal transplant recipients. Flow cytometry analysis of peripheral blood mononuclear cells of 54 renal transplant recipients receiving a calcineurin inhibitor-based immunosuppression was performed for CD4, CD25, FOXP3, CXCR3 and CCR5 within the first 18 months post-transplantation. Correlation analysis of chemokine receptor expression and glomerular filtration rate as calculated by MDRD (eGFR) was performed. Expression of the peripheral homing receptors CXCR3 (r = 0.44, P < 0.05) and CCR5 (r = 0.45, P < 0.05) on FOXP3⁺ Tregs correlated with renal allograft function (eGFR) in patients receiving tacrolimus (n = 28), but not cyclosporine A (CsA) (n = 26). CsA but not tacrolimus reduced surface expression of CXCR3 on FOXP3⁺ Tregs in renal transplant recipients as correlated to trough levels (r = -0.42, P < 0.05). In contrast to CD4⁺ CXCR3⁺ CD25(lo) T cells, flow-sorted CD4⁺ CXCR3⁺ CD25(hi) Tregs isolated from healthy individuals did not produce IFNγ or IL-17 ex vivo and expressed high levels of GARP mRNA both at baseline as well as after TCR activation indicating functional regulatory activity. Expression of the peripheral trafficking receptors CXCR3 and CCR5 on FOXP3⁺ Tregs is associated with renal allograft function. These results suggest that Treg trafficking may also depend on the interaction of CXCR3 or CCR5 and their respective ligands.

Endothelial cells modify the costimulatory capacity of transmigrating leukocytes and promote CD28-mediated CD4(+) T cell alloactivation.

Activated vascular endothelial cells (ECs) express major histocompatibility complex (MHC) class II molecules in vitro and in vivo in acute and chronic allograft rejection. However, human ECs may be limited in their ability to effectively activate CD4(+) T cells, because they do not express members of the B7 family (CD80 and CD86) of costimulatory molecules. In this study, we show that ECs promote the full activation of CD4(+) T cells via trans-costimulatory interactions. By reverse transcriptase polymerase chain reaction, Western blot, and FACS((R)) analysis, we could not detect the expression of CD80 and CD86 on activated ECs and found minimal expression on purified CD4(+) T cells. In contrast, both CD80 and CD86 were expressed in allogeneic CD4(+) T cell-EC cocultures. Expression of CD86 peaked at early times between 12 and 24 h after coculture, whereas CD80 was not expressed until 72 h. Addition of anti-CD86 but not anti-CD80 monoclonal antibodies to cocultures inhibited IL-2 production and the proliferation of CD4(+) T cells to allogeneic donor human umbilical vein ECs (HUVECs), as well as to skin and lung microvascular ECs. Furthermore, we found that interferon gamma-activated ECs but not untreated ECs induced mRNA and cell surface expression of CD80 and CD86 on CD4(+) T cells, and these T cells were functional to provide a trans-costimulatory signal to autologous CD4(+) T cells. Blockade of MHC class II and lymphocyte function-associated antigen 3 but not other EC cell surface molecules on IFN-gamma-activated ECs inhibited the induction of CD86 on CD4(+) T cells. Transmigration of purified populations of monocytes across EC monolayers similarly resulted in the induction of functional CD86, but also induced the de novo expression of the cytokines interleukin (IL)-1alpha and IL-12. In addition, EC-modified monocytes supported enhanced proliferation of allogeneic and autologous CD4(+) T cells. Taken together, these data define the ability of the endothelium to modify CD4(+) T cells and monocytes for trans-costimulatory events. This unique function of the endothelium in alloimmune T cell activation has functional consequences for the direct and the indirect pathways of allorecognition.

mTOR-understanding the clinical effects.

The target of rapamycin (TOR) is a highly conserved serine/threonine kinase that controls cell growth and metabolism in response to nutrients, growth factors, cellular energy, and stress. The TOR kinase, which was originally discovered in yeast, is also expressed in human cells as mammalian TOR (mTOR). In this review, we focus on how mTOR-inducible signals function in cell protection and cell survival of effector and regulatory T cells as well as its role in endothelial cell biology. We evaluate how signaling is important for vascular endothelial cell growth, survival, and proliferation; and we consider how the function of mTOR in endothelial cells may be clinically important in the rejection process. Understanding the biology of mTOR allows clinicians to use mTOR inhibitors optimally as therapeutics following solid organ transplantation.

Interactions between T lymphocytes and endothelial cells in allograft rejection.

Vascular endothelial cells participate in the process of allograft rejection by promoting both the recruitment and the activation of alloreactive T cells. There have been three major recent advances in the field of interactions between T cells and endothelial cells that are of direct relevance to the process of cell-mediated responses to allografts: first, endothelial cells mediate selective recruitment of CD4+ T cell subsets, including naive and memory T cells and T cell subsets of the Th1 and Th2 phenotypes; second, endothelial cells co-stimulate the production of effector cytokines by helper T cells; and third, endothelial cells regulate T cell apoptosis.

Expression of the chemokine receptor CXCR3 and its ligand IP-10 during human cardiac allograft rejection.

BACKGROUND: Chemokines play an essential role in regulating the infiltration of leukocytes into allografts in experimental models. Little is known of their expression or function after human cardiac transplantation. METHODS AND RESULTS: We analyzed 169 sequential human endomyocardial biopsies by immunocytochemistry for infiltration by CD3(+) T cells and the expression of the chemokine receptors CCR1, CCR3, CCR5, and CXCR3. In both cross-sectional and longitudinal analyses, the expression of each of the chemokine receptors correlated with the degree of CD3(+) T-cell infiltration. In particular, the expression of CXCR3 was temporally and spatially associated with CD3(+) T-cell infiltrates and correlated with the histopathological diagnosis of acute rejection (OR, 11.73 and 4.05, respectively; P<0.001). Of 7 patients followed up longitudinally for 1 year, 4 with consecutive biopsies developed intimal thickening by intravascular ultrasound. In these patients, there was a trend for persistent expression of CD3- and CXCR3-expressing infiltrates in the later part of the first posttransplant year. The chemokines eotaxin, IP-10, lymphotactin, MCP-1, Mig, RANTES, and SDF-1 were examined in an additional 35 biopsies by RT-PCR. Eotaxin, lymphotactin, MCP-1, Mig, and SDF-1 were present in both normal and rejecting biopsies. However, the CXCR3 ligand IP-10, which was rarely expressed in normal biopsies, was markedly induced in acute rejection (OR, 19.43; P=0.01). CONCLUSIONS: The presence of CXCR3(+) T cells and the CXCR3 ligand IP-10 within endomyocardial biopsies is strongly associated with acute rejection. The CXCR3-IP-10 interaction warrants consideration as a therapeutic target in the management of cardiac allograft recipients.

Predictive value of inducible endothelial cell adhesion molecule expression for acute rejection of human cardiac allografts.

We conducted a prospective longitudinal study to determine the clinical significance of endothelial adhesion molecule expression in endomyocardial biopsies from human cardiac allografts. Ten to 18 (mean 13) consecutive allograft biopsies were obtained from 20 serial human transplant recipients over a one-year period. A total of 267 biopsies was examined. The expression of endothelial adhesion molecules intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, as well as the presence of CD3+ T cell infiltrates was assessed by immunocytochemical staining of frozen sections. Separate specimens taken at the same time were analyzed histologically for ischemic injury or rejection. ICAM-1--and, to a lesser extent VCAM-1--was expressed at low levels in normal biopsies. E-selectin was only expressed in 15% of histologically normal biopsy specimens. Ischemic injury noted in the immediate posttransplant period was associated with increased expression of all three adhesion molecules. VCAM-1 expression increased both with the degree of CD3+ T cell infiltrates (P < 0.001) and with the degree of rejection (P < 0.05). ICAM-1 increased over constitutive levels in association with diffuse CD3+ infiltrates (P < 0.001) and with rejection (P < 0.05). E-selectin was increased on occasional vessels in association with CD3+ infiltrates (P < 0.001), but was not associated with active rejection. Increases in E-selectin were most likely to occur in biopsies just prior to rejection episodes (odds ratio 3.3), and were least likely to occur in biopsies following rejection (odds ratio 0.3). ICAM-1, but not VCAM-1, was also elevated in prerejection specimens. VCAM-1 and ICAM-1 declined in postrejection specimens. These data suggest a dynamic pattern in the expression of endothelial cell adhesion molecules during the course of cardiac allograft rejection. This study also suggests that endothelial E-selectin expression may be a useful clinical marker of impending rejection. Finally, inducible VCAM-1 expression may be a helpful adjunct in the diagnosis of ongoing acute rejection, and decreases in its expression may be indicative of successful antirejection therapy.

The allogeneic response to cultured human skin equivalent in the hu-PBL-SCID mouse model of skin rejection.

BACKGROUND: Engineered tissues have been proposed for the treatment of a variety of conditions including the partial or complete replacement of human organs. To determine the basis for the rejection of these tissues, we analyzed the immune response to allogeneic human skin equivalent (HSE, also called Apligraf) in the humanized SCID mouse (hu-PBL-SCID). METHODS: Two models of hu-PBL-SCID were used for these studies. In one model, human skin or HSE was transplanted onto humanized mice so that graft survival could be analyzed. In the other model, skin grafts were allowed to heal on naive mice before humanization. This model was used to analyze the immunologic response to the vascularized skin allograft. Humanization was performed by adoptive transfer of human PBL into SCID mice by i.p. injection. RESULTS: Both human foreskin and HSE successfully engrafted onto naive SCID mice and remained stable for more than 6 months. In contrast, human foreskin was rejected by 21 days posttransplant in hu-PBL-SCID, whereas HSE consistently engrafted for more than 28 days. Treatment of HSE grafts with interferon-y for 5 days to induce maximal MHC class II molecule expression before grafting failed to induce rejection. HSE also engrafted onto hu-PBL-SCID mice that were exposed to alloantigen by prior injection with interferon-gamma-treated keratinocytes identical to those used to generate the HSE. In addition, we determined that humanization of SCID mice following engraftment and vascularization of human foreskin resulted in marked CD3+ T cell infiltrates and a lymphocyte-induced vasculitis. In contrast, the response in vascularized HSE was associated with minimal CD3+ T cell infiltration in the absence of vasculitis or morphological features of rejection. CONCLUSION: These results support the use of HSE and other allogeneic engineered tissues in humans provided that such tissues are limited in their antigen presenting capabilities. In addition, our findings suggest a critical function for the donor endothelial cell in rejection.

Angiogenesis in the huPBL-SCID model of human transplant rejection.

BACKGROUND: Angiogenesis is characteristic of chronic inflammatory reactions. The process of angiogenesis is reported to be proinflammatory in part due to enhanced adhesion events and in part due to increased perfusion and permeability to sites of inflammation. However, little is known about the association between angiogenesis and rejection. METHODS: Severe combined immune deficient mice are permissive for the growth of human skin allografts and human peripheral blood mononuclear cells (PBMC). Human PBMC were injected into mice by intravenous or intraperitoneal injection. The infiltration of cells and the associated angiogenesis reactions in the skin allografts were analyzed temporally by videomicroscopy and spatially by immunohistochemistry. RESULTS: Human alloreactive mononuclear cells migrated to human skin but not mouse skin within hours after the intravenous infusion of PBMC. Within 3 days, areas of angiogenesis were observed in the skin grafts at the sites of infiltrates. The vessel densities in skin grafts were 24+/-6 vessels per calibrated grid at baseline on the day of the infusion and increased to 55+/-16 vessels per calibrated field by day 10. Skin grafts harvested from humanized severe combined immune deficient mice 7-14 days after the intraperitoneal infusion of human PBMC showed a similar increased density of vessels that were spatially associated with mononuclear cell infiltrates. CONCLUSIONS: A significant angiogenesis response was associated with the cell infiltrates in the human skin allografts. The onset of angiogenesis appeared after the initial development of localized infiltrates and preceded the development of microvascular destruction. These findings suggest that alloreactive T cells and/or monocytes mediate the angiogenesis response in skin allografts.

CD40 and CD40 ligand (CD154) are coexpressed on microvessels in vivo in human cardiac allograft rejection.

BACKGROUND: CD40 is expressed by a wide variety of cells in the immune system, including endothelial cells. It binds to CD40 ligand ([CD40L] CD154), which was originally reported to be restricted in its expression to early-activated T cells. We report here the expression of CD40 and CD40L in human cardiac allografts. METHODS: A total of 123 consecutive biopsies from 11 human cardiac allograft recipients were analyzed by immunohistochemistry for the expression of CD40 and CD40L. The expression of CD40L was also examined in vitro in homogeneous cultures of umbilical vein endothelial cells by reverse transcriptase-polymerase chain reaction and by flow cytometry. RESULTS: CD40 was expressed at low levels, and CD40L was minimal or absent in histologically normal biopsies in the absence of CD3+ T-cell infiltrates. In rejection, the expression of CD40 increased on vascular endothelial cells and on graft-infiltrating leukocytes throughout biopsy specimens. Induced expression of CD40 was strongly associated with the presence of CD3+ T-cell infiltrates, acute rejection, and ischemic injury (P<0.05). CD40L was expressed in biopsies with rejection and was prominent on a subset of infiltrating leukocytes as well as on microvascular endothelial cells. In contrast to CD40, staining of endothelial CD40L was focal in most biopsies. Overall, the expression of CD40L correlated with the presence of CD3+ T-cell infiltrates and rejection (P<0.05), but not ischemic injury (P=0.9). To confirm that the endothelium can synthesize CD40L, we also evaluated the expression of endothelial CD40L in vitro. Cultured endothelial cells were found to express little constitutive CD40L that markedly increased after 24 hr of treatment with supernatants from phytohemagglutinin-activated peripheral blood mononuclear cells or by the cytokines tumor necrosis factor-alpha, interleukin-1a, interleukin-4, or interferon-gamma. CONCLUSION: Both CD40 and CD40L are expressed in vivo on infiltrating leukocytes and on microvascular endothelium in human cardiac allograft rejection. We suggest that endothelial cell CD40 and CD40L play a role in human cell-mediated immune responses such as cardiac allograft rejection.

Risk factors for mortality in infants and young children on dialysis.

The factors associated with a greater mortality risk in infants and young children undergoing dialysis have not been clearly determined. We report the results of a North American Pediatric Renal Transplant Cooperative Study designed to assess risk factors in patients aged younger than 6 years at initiation of dialysis therapy. Sixty-four nonsurvivors were matched with 110 survivors for age at dialysis initiation, primary renal disease, and year of entry onto the database. Questionnaires on 137 patients (51 nonsurvivors, 86 survivors) were completed by participating centers. Seventy-five percent (103 of 137 patients) of the patients were aged younger than 2 years at dialysis initiation; 42% (58 of 137 patients) had renal aplasia, dysplasia, and/or hypoplasia or obstructive uropathy; 62% were boys; and 62% were white. One-year patient survival rates were 83% in infants beginning dialysis at younger than 3 months of age, 89% in 3- to 23-month-olds, and 95% in 2- to 5-year-olds (P = 0.001). Comorbid nonrenal disease occurred in 37 of 51 nonsurvivors (74%) versus 46 of 84 survivors (55%; P = 0.027). Nonsurvivors had pulmonary disease and/or hypoplasia more often (14 of 37 nonsurvivors; 37.8% versus 8 of 46 survivors; 17.4%; P = 0.04). Oliguria or anuria was present in 23 of 33 nonsurvivors (70%) aged younger than 2 years versus 26 of 64 survivors (41%; P = 0.007). Infection accounted for 15 of 51 deaths (29.4%). In summary, these results suggest that age at dialysis initiation; presence of nonrenal disease, particularly pulmonary disease and/or hypoplasia; and oliguria or anuria in children aged younger than 2 years are identifiable as risk factors for mortality in these young patients.

Challenges after pediatric transplantation.

In this review we address the current challenges facing the pediatric transplant caregiver. We focus most of our discussion on recent developments that have resulted in improved graft survival in renal allograft recipients. We discuss the issue of growth failure posttransplant and the realization that it is time to reassess strategies that optimize the immunosuppression and growth after pediatric transplantation. Lastly, we discuss some recent findings suggesting that these issues are also of critical importance for success after pediatric liver transplantation.

Immunologic targets for currently available immunosuppressive agents: what is the optimal approach for children?

In this review, the authors discuss immunologic targets and events in T cells that are dysregulated by commonly used immunosuppressive agents. These include a description of glucocortcoid receptors as well as targets of glucocorticoids, targets of cyclosporine and FK506, and the mammalian target of rapamycin. In addition, novel antibody-based targets on T cells and antigen-presenting cells including the IL-2 receptor and costimulatory molecules are described. Finally, the authors provide a rationale for an optimal approach to immunosuppression in pediatrics. Because many of the newer immunosuppressive agents are currently in clinical trials, the "optimal" immunosuppressive strategy for the next decade is forthcoming.

P-selectin expression in myocardium of children undergoing cardiopulmonary bypass.

Cardiopulmonary bypass is a planned support technique that results in a period of myocardial ischemia and reperfusion. In addition, it is associated with an inflammatory response likely involving endothelial cell activation. In previous studies, we showed that E-selectin and intercellular adhesion molecule-1 (ICAM-1) messenger ribonucleic acid (mRNA) are increased in human myocardium after cardiopulmonary bypass. We have now examined the expression of P-selectin mRNA by ribonuclease protection in paired atrial biopsy specimens from 12 patients before and after cardiopulmonary bypass. By means of immunocytochemistry, we have also examined the endothelial cell surface expression of P-selectin protein, as well as that of E-selectin and ICAM-1 in three additional patients. Patient ages ranged from 1 day to 8.5 years (median 12 months), and cardiopulmonary bypass times ranged from 46 to 196 minutes (median 144 minutes). By ribonuclease protection, there was marked variability in the expression of P-selectin in biopsy specimens before bypass. However, when compared with prebypass levels, P-selectin mRNA decreased modestly in 10 of 12 patients after bypass (median decrease 1.5-fold, p = 0.016). As seen with immunocytochemistry, P-selectin protein was distributed diffusely through the vascular bed on large vessels and small vessels before bypass but was virtually absent on capillaries in specimens taken after bypass. E-selectin, which was absent in prebypass biopsy specimens, was induced in one of the three specimens after bypass, but no change in ICAM-1 protein expression above baseline was noted. We also find that cultured human endothelial cells treated with tumor necrosis factor-alpha in doses which induce ICAM-1 mRNA simultaneously decrease their expression of P-selectin mRNA as compared with untreated cells. These observations suggest that endothelial P-selectin is transcriptionally downregulated after cardiopulmonary bypass at times when E-selectin and ICAM-1 are induced. Furthermore, we find that E-selectin and ICAM-1 are expressed at times and at sites where P-selectin is absent. Although it is possible that P-selectin may have been induced and lost at early times before reperfusion, these data suggest that endothelial P-selectin plays a limited role in the inflammatory response that ensues after cardiopulmonary bypass.

Differential regulation of leucocyte L-selectin (CD62L) expression in normal lymphoid and inflamed extralymphoid tissues.

AIMS: To study tissue expression of L-selectin, a leucocyte cell surface molecule that is considered to be involved in adhesion to certain endothelia, particularly in peripheral lymph nodes and during inflammation, and is shed upon leucocyte activation. METHODS: Leucocytes were examined by immunohistochemistry and double immunofluorescence staining in various lymphoid sites and normal and inflamed extralymphoid tissues. RESULTS: L-selectin was present on mantle zone B lymphocytes in different lymphoid sites, including in intestinal lymphoid tissue, but was absent on germinal centre B cells. Splenic white pulp B cells also expressed L-selectin. The proportion of T lymphocytes expressing L-selectin depended on the site under study, being greatest in peripheral lymph nodes (mean 48% of T cells positive), and lower in mucosal lymphoid sites and spleen (9 and 11% positive, respectively). Non-lymphocytic L-selectin staining was observed on follicular dendritic cells in tonsils and on macrophages in thymus. L-selectin positive leucocytes were rare in normal extralymphoid tissues, and relatively few were seen in most inflammatory settings. However, in rejecting renal transplants, a higher proportion (30%) of leucocytes expressed L-selectin. CONCLUSIONS: Overall, the results indicate how the degree of L-selectin expression by leucocytes in particular tissues may reflect a requirement for L-selectin expression for entry into those tissues and the activation state of leucocytes once localised there.

Role of leukocyte-endothelial cell adhesion molecules in renal inflammation: in vitro and in vivo studies.

An increasing body of evidence suggests that endothelial cells as well as parenchymal cells within the kidney express multiple cytokine-inducible leukocyte adhesion molecules. This paper reviews evidence from our own laboratory as well as others on the in vitro induction and function of adhesion molecules in the kidney. We review current data from the literature on the possible role of endothelial cell adhesion molecules in mediating leukocyte infiltration and renal injury in experimental and human transplant rejection and glomerulonephritis. These early studies suggest that leukocyte-endothelial adhesive interactions result in a complex cascade of biological and pathological processes leading to renal injury. Further analysis of such interactions in the kidney will elucidate their specific roles.

Effects of tumor necrosis factor, lipopolysaccharide, and IL-4 on the expression of vascular cell adhesion molecule-1 in vivo. Correlation with CD3+ T cell infiltration.

We have injected human TNF, LPS, and IL-4 into the skin of baboons to examine regulation of endothelial leukocyte adhesion molecules (ELAM) in vivo and to determine which endothelial adhesion molecules correlate temporally and spatially with cytokine-induced T cell infiltration. The expression of adhesion molecules ELAM-1 (E-selectin), VCAM-1, and ICAM-1 (CD54) were quantified by immunocytochemical staining of frozen sections obtained from skin biopsies; T cell infiltration was measured by immunocytochemical staining of CD3+ T cells in serial sections. We found that injection of TNF causes late (24 to 48 h) T cell infiltration whereas injection of LPS, in doses that do not cause tissue necrosis, does not. The ability of TNF (but not LPS) to recruit T cells correlates with the ability of TNF to cause sustained endothelial cell adhesion molecule expression. Expression of VCAM-1 on post-capillary venules showed the highest degree of spatial localization with infiltrates. IL-4, although not proinflammatory by itself, can cause T cell infiltration in combination with an ineffective dose of TNF. The ability of IL-4 to augment TNF-induced inflammation best correlates with the ability of the combination of IL-4 and TNF to increase endothelial VCAM-1 expression. In contrast, IL-4 does not promote T cell infiltration or endothelial VCAM-1 expression in combination with LPS. In cytokine-injected tissues, VCAM-1 is also expressed on connective tissue cells other than endothelium, including smooth muscle and perineural cells, where it is induced by cytokines in parallel with endothelial VCAM-1. Overall, our data support the hypothesis that endothelial VCAM-1 expression contributes to T cell extravasation at sites of inflammation. Furthermore, we find that IL-4, a product a Ag-activated T cells, can interact with TNF to selectively promote VCAM-1 expression and the development of T cell-rich infiltrates, characteristic of Ag-induced inflammatory reactions.