Distinct dacryoadenitides autoadoptively transferred to rabbits by different subpopulations of lymphocytes activated ex vivo

Abstract

Purpose: To test whether CD4+ T cells proliferate in mixed cell reactions with autologous lacrimal gland (LG) acinar cells and whether these cells can autoadoptively transfer disease.

Methods: Purified acinar cells were gamma irradiated and cocultured with peripheral blood lymphocytes. Activated CD4+ T cells were sorted by fluorescence-activated cell sorting (FACS). Unfractionated activated peripheral blood lymphocytes (UF), CD4+-enriched and CD4+-depleted T cells from an autologous mixed cell reaction were injected into the donor rabbit's remaining LG. After 4 weeks, ocular examinations were performed, and the rabbits were euthanized; LGs were removed for histopathology, immunohistochemistry, and real-time reverse transcription-polymerase chain reaction studies.

Results: CD4 T cells increased in the autologous mixed cell reaction from 20% to 80%. Tear production decreased in the induced disease/UF (ID/UF) group and declined even more in the ID/CD4+-enriched group. Tear breakup times decreased and rose bengal staining increased in all groups. All LGs exhibited significant histopathology and increased messenger RNAs for tumor necrosis factor α. The ID/UF group exhibited the largest increases of CD4+ and rabbit T-lymphocyte antigen-positive cells. The ID/CD4+-enriched group contained fewer infiltrating CD4 cells but more eosinophils, severely altered acinar morphology, and increased fibrosis. LG of the ID/CD4+-depleted group exhibited large increases of CD18, major histocompatibility complex II, and CD4+ cells. Messenger RNAs for interleukin 2, interleukin 4, and CD4+ increased in the ID/CD4+-enriched group compared with the CD4+-depleted group.

Conclusions: Autoreactive CD4+ effector cells activated ex vivo and autoadoptively transferred, caused what seems to be a distinct dacryoadenitis. The CD4+-depleted cell fraction also contained pathogenic effector cells capable of inducing disease.

Figure 1. Marked expansion of CD4+ T cells in autologous mixed cell reaction (AMCR)

(A) Mononuclear cells were stained with an anti-T cell PE- labeled antibody before (unstimulated) and after (stimulated) culture in an AMCR. (B) Same as for (A) except the cells were stained with an anti-CD4 PE-labeled antibody. (C) Same as for (A) except the cells were stained with an anti-CD8 FITC-labeled antibody.

Figure 1. Marked expansion of CD4+ T cells in autologous mixed cell reaction (AMCR)

(A) Mononuclear cells were stained with an anti-T cell PE- labeled antibody before (unstimulated) and after (stimulated) culture in an AMCR. (B) Same as for (A) except the cells were stained with an anti-CD4 PE-labeled antibody. (C) Same as for (A) except the cells were stained with an anti-CD8 FITC-labeled antibody.

Figure 1. Marked expansion of CD4+ T cells in autologous mixed cell reaction (AMCR)

(A) Mononuclear cells were stained with an anti-T cell PE- labeled antibody before (unstimulated) and after (stimulated) culture in an AMCR. (B) Same as for (A) except the cells were stained with an anti-CD4 PE-labeled antibody. (C) Same as for (A) except the cells were stained with an anti-CD8 FITC-labeled antibody.

Figure 2. Clinical Ocular Parameters

A comparison of basal tear production, tear break-up time and rose bengal staining for both the OD (injected LG) and OS (excised inferior LG) eyes challenged with ID, ID/CD4⁺-enriched or ID/CD4⁺- depleted cell preparations is presented 30 days post-injection. The procedure for these clinical examinations was described previously (21). Each group consisted of at least six animals and one group was normal animals. (A). Compared to the normal group basal tear production for the OD eyes of the ID/UF group was reduced by an average of 35%, the ID/CD4⁺-enriched group by 71%, and the ID/CD4⁺depleted group by 12%. Compared to normal tear production, the ID/UF group’s OS eyes declined by 26%, the ID/CD4⁺- enriched group by 35%, and the ID/CD4⁺depleted group by 6%. (B). Compared to the tear break-up time of normal animals, the OD eyes of ID/UF group declined by 46%, ID/CD4⁺-enriched by 66%, and the ID/CD4⁺-depleted group by 54%. Values for the OS eyes of the ID/UF group declined by 48%, ID/CD4⁺-enriched group by 64%, and the ID/CD4⁺depleted group by 67%. (C). OD rose bengal scores for the ID/UF group increased by 5.5 fold, ID/CD4⁺enriched group by 6.1 fold, and the ID/CD4⁺depleted group by 3.7 fold. The OS rose bengal scores increased five- to sixfold.

Figure 3. Histopathology with H&E

(A) Normal LG showed acinar cells (ac) with pale apical cytoplasm due to accumulated secretory vesicles and nuclei at the basal side of the cells; d represents ducts. (B) Section from an ID/UF group lacrimal gland 4 weeks post-injection contained large lymphocytic foci (black arrows) around venules and within the connective tissue around ducts. (C) Rabbits injected with CD4⁺ enriched preparations (ID/ CD4⁺ enriched) had multiple large foci of lymphocytic infiltrates (black arrows), morphologically altered acinar cells (aa), and ducts (d) and blood vessels were surrounded by expanded fibrous connective tissue (fct). In the inset of (C) the black arrowhead identifies an eosinophil within the lumen. In addition to the numerous eosinophils within the acinar lumens, it appears that activated macrophages (black arrow) are present, as well as other blood cells. (D) Rabbits injected with CD4⁺ depleted lymphocyte preparations ID/CD4⁺ depleted had lymphocytic infiltration similar to ID/UF rabbits and less severe infiltration and fibrosis than ID/CD4⁺ enriched rabbits.

Figure 4. Abundances of CD molecule and cytokine mRNAs

Abundances of target mRNAs relative to the abundance of GAPDH mRNA in each gland were determined by real time RT-PCR. Values presented are means ± sems for 5 normal glands, 3 glands from ID/UF animals, 8 glands from ID/CD4⁺ enriched animals, i.e., CD4(+), and 5 glands from ID/CD4⁺ depleted animals, i.e., CD4(−). Also presented are values for one additional gland each from the ID/CD4⁺ enriched group and the ID/CD4⁺ depleted group which were atypical with respect to several of the measured transcripts.