Spleen cell suspensions from overtly diabetic female NOD mice were prepared in Hank's balanced salt solution and injected i.v. into 18 disease free male NOD recipients 2- 4 months old previously given 650 rads from a cobalt source. The non-depleting anti-CD4 monoclonal antibody (Nd -YTS 177.1) was of the IgG2a subclass and 2mg was given i.v, i.p, i.p. on consecutive days and thereafter i.p. three times weekly. Groups of 4-5 mice either received no treatment after diabetic spleen cell transfer (group 1) or Nd anti-CD4 (non-depleting anti-CD4) starting at day 12 (group 2) or received no treatment after transfer but were sacrificed at day 12 (group 3). Animals in groups 1 and 2 were sacrificed 4 weeks after transfer for histological examination of pancreata by double immunofluorescent staining for insulin and 0X 6 (detects MHC class H positive cells) or anti-CD3 (detects T cells) (Figure 6 .1 A/B). At week 4 50% of the animals were diabetic in group 1 (1 animal had already died of diabetes ) but none of those receiving M.Ab. therapy were hyperglycemic. Results presented in the previous chapter have established that by 10-14 days after transfer macrophages and lymphocytes had infiltrated the pancreas and some intra-islet infiltration had occurred. These observations were confirmed in this study, as the control animals sacrificed at 2 weeks receiving no Ab. treatment had >10 class II positive cells at peri-islet sites in 40% of intact islets. Thus peri-islet infiltration was already extensive and 23% of the total islets analysed were residual and devoid of 6 cells (Table 6.2A).
In order to determine whether Nd CD4 could arrest the destructive process, mice in group 1 sacrificed at week 4 after transfer were analysed. All islets examined demonstrated severe peri-islet infiltration of MHC class II positive cells and 83% of the remaining intact islets showed severe intra-islet infiltration with 70% of the total islets devoid of 6 cells. Pancreata from animals given non-depleting anti-CD4 fi*om day 12 onwards and killed at 4 weeks after transfer, were indistinguishable from the group analysed at two weeks after transfer having 19% islet remnants, and 36% and 7% of islets demonstrated severe peri- and intra-islet infiltration of MHC class II positive cells respectively. Similarly analysis of pancreatic sections from all 3 groups stained for insulin and T cells revealed a similar pattern of infiltration to that obtained by staining for class II
MHC antigen'’’ cells. Table 6.2B shows that although there was considerable peri-islet infiltration in the anti-CD4 treated group, 49% of islets had >10 T cells at peri-islet locations compared to 77% for untreated controls at week 4. Severe intra-islet infiltration was also reduced from 59% of islets (control group at week 4) to 4% for those treated with Nd CD4 which is comparable to that observed in control animals analysed at week 2 (2%). Thus Nd.anti- CD4 can both independently reduce peri and prevent intra-islet infiltration by class n-h cells and T cells in diabetic spleen cell recipients and thereby prevent diabetes.
6 .2 .3 In vivo treatm ent o f diabetic spleen cell recipients with depleting anti-CD8 protects against disease induction.
The diabetic spleen cell transfer was carried out as indicated in chapter 4 on a cohort of 21, 9 week old male NOD mice. The rat monoclonal antibody to the mouse cell surface antigen Lyt-2 (anti-CD8-YTS 169.4) was diluted in PBS such that each animal received 400 M g in a final volume of 200 m1 i.v. on the first day of treatment and the same amount intraperitoneaUy on the 2 days following. Control mice were similarly treated with YTH3.2.6, an isotype-matched rat monoclonal antibody to human CD7. Fluorescence activated cell sorter (FACS) analysis showed that spleen ce lls from Ab untreated
m ice had 11.8% CD8’’’ T cells compared with 0% in spleen cells from animals
at week two after transfer
treated with YTS 169.4 (anti-CD8)^ Recipient mice of diabetic spleen cells were treated with anti-CD8, beginning on day 1 (3 mice), day 8 (5 mice), day 15 (4 mice), day 22 (4 mice) or untreated (5 mice). In another experiment a group of 5 diabetic spleen cell transfer recipients were treated with anti-CD8 commencing at day 12 after transfer (Table 6.2A/B). The pancreata were examined histologically 5 weeks after transfer. Pancreatic sections from all experimental animals were prepared and stained by immunoperoxidase^reviously for CD4 and CD8 or by immunofluorescence for insulin, and the macrophage markers Mac-1 and F4/80.
It can be seen from Table 6.3A that mice were afforded significant protection with anti-CD8 as 80% of untreated mice were diabetic at week 5 whereas none, 20%, 75% and 25% of mice treated with anti-CD8 on the next day, week 1, week 2 and week 3 respectively were diabetic (Figure 62). Some protection w as observed in mice treated
CDS from day 12 onwards, although not overtly diabetic, displayed evidence of considerably more 6 cell destmction (51% islet remnants) (Table 6.2A). Severe peri-islet infiltration by MHC class n + cells was observed in 60% of islets, although intra-islet infiltration was the same as in the non-depleting anti-CD4 treated animals in the same experiment (Table 6.2A, B). Histological examination at week 5 of the first experiment described showed a much reduced infiltrate in mice treated with anti-CDS immediately after transfer. The pancreata of these mice had no severe intra-islet infiltration of either CD4"*^ or
CDS’*" T cell populations and 25-45% of islets demonstrated no peri-islet infiltration compared to untreated controls which had 33% of islets demonstrating severe intra-islet infiltration by CD4‘‘‘ and CDS'*' T cells. The later the anti-CDS was administered after diabetic spleen cell transfer the less protection was afforded as administration at 1 week resulted in only 10% of islets without detectable 6 cells (“residual islets”) whereas delaying treatment with anti-CDS for 2 or 3 weeks resulted in 15% and 31% residual islets respectively (Table 6.3A). Additionally treatment with anti-CDS prevented severe macrophage infiltration as detected by Mac-1 and F4/S0 staining if administered on day 1 after transfer compared to later time points (Table 6.3B). Thus anti-CDS treatment can arrest disease if administered in time to prevent total destruction of the p cells .
6.2.4. In vivo treatment of diabetic spleen cell transfer recipients