Superantigen-presentation by rat major histocompatibility complex class II molecules RT1.Bl and RT1.Dl

Abstract

Rat major histocompatibility complex (MHC) class II molecules RT1.B(l) (DQ-like) and RT1.D(l) (DR-like) were cloned from the LEW strain using reverse transcription-polymerase chain reaction and expressed in mouse L929 cells. The transduced lines bound MHC class II-specific monoclonal antibodies in an MHC-isotype-specific manner and presented peptide antigens and superantigens to T-cell hybridomas. The T-cell-hybridomas responded well to all superantigens presented by human MHC class II, whereas the response varied considerably with rat MHC class II-transduced lines as presenters. The T-cell hybridomas responded to the pyrogenic superantigens Staphylococcus enterotoxin B (SEB), SEC1, SEC2 and SEC3 only at high concentrations with RT1.B(l)-transduced and RT1.D(l)-transduced cells as presenters. The same was true for streptococcal pyrogenic exotoxin A (SPEA), but this was presented only by RT1.B(l) and not by RT1.D(l). SPEC was recognized only if presented by human MHC class II. Presentation of Yersinia pseudotuberculosis superantigen (YPM) showed no MHC isotype preference, while Mycoplasma arthritidis superantigen (MAS or MAM) was presented by RT1.D(l) but not by RT1.B(l). Interestingly, and in contrast to RT1.B(l), the RT1.D(l) completely failed to present SEA and toxic shock syndrome toxin 1 even after transduction of invariant chain (CD74) or expression in other cell types such as the surface MHC class II-negative mouse B-cell lymphoma (M12.4.1.C3). We discuss the idea that a lack of SEA presentation may not be a general feature of RT1.D molecules but could be a consequence of RT1.D(l)beta-chain allele-specific substitutions (arginine 80 to lysine, asparagine 82 to aspartic acid) in the extremely conserved region flanking the Zn(2+)-binding histidine 81, which is crucial for high-affinity SEA-binding.

Figure 1

Flow cytometry of RT1.B^l-transduced (a) or RT1.D^l-transduced (b) L929 cells and of LEW splenocytes (c and d). Staining with phycoerythrin-conjugated OX6 (anti-RT1.B) and 14-4-4S (anti-RT1.D) is depicted as open histograms. Staining with the isotype-matched control antibody is depicted as filled histogram.

Figure 2

Interleukin-2 (IL-2) release of T-cell hybridoma 53/4 (a) or T-cell hybridoma 19 (b) after 40 hr culture with indicated amounts of guinea-pig myelin basic protein (gpMBP) (a) or casein (b): LEW rat thymocytes (square), L929 untransduced (circle), L929 RT1.B^l-transduced (triangle), L929 RT1.D^l-transduced (inverted triangle).

Figure 3

Interleukin-2 (IL-2) release by T-cell hybridoma 53/4 after 24–40 hr culture with the indicated amount of superantigen (a) staphylococcal enterotoxin B (SEB), (b) SEB, (c) SEC1, (d) SEC1, (e) SEC3, (f) staphylococcal pyrogenic exotoxin A (SPEA), (g) Yersinia pseudotuberculosis mitogen (YPM), (h) Mycoplasma arthritidis superantigen (MAS). (b) Depicts data from the SEB stimulation shown in (a) without the respective data generated with DR1-transduced presenting cells. (d) Depicts data from the SEC1 stimulation shown in (c) without the respective data generated with DR1-transduced presenting cells. Superantigen (SAg) presenting cells were: DR1 transductant P3/2 CD80 (square), L929 untransduced (circle), L929 RT1.B^l-transduced (triangle), L929 RT1.D^l-transduced (inverted triangle). Results were normalized for IL-2 production in the presence of DR1 transductants and the highest concentration of the respective superantigens. The average of three to five experiments is shown. Error bars indicate standard deviation.

Figure 5

Interleukin-2 (IL-2) release by T-cell hybridoma 3DO54.8 stimulated with staphylococcal enterotoxin A (SEA) or T-cell hybridoma FRN2.7 stimulated with toxic shock syndrome toxin 1 (TSST-1) with rat CD74-negative (left) or rat CD74-positive cells (right) as presenters (see Fig. 4). The left panel shows data from two experiments with L929 transductants, which are indicated by full lines (experiment 1, squares indicate DR1 transductants) or dotted lines (experiment 2, squares indicate RAJI cells). The other cells were L929 untransduced (circle), L929 RT1.B^l-transduced (triangle), L929 RT1.D^l-transduced (inverted triangle). The right panel shows data from two experiments with a culture period of 40 hr (full line experiment 1 and dotted line experiment 2) with CD74-transduced L929 rat MHC class II transductants and the DR1 transductant P3/2 CD80 (squares). The other cells are L929CD74 untransduced (circle), L929CD74 RT1.B^l-transduced (triangle), L929CD74 RT1.D^l-transduced (inverted triangle).

Figure 4

Major histocompatibility complex (MHC) class II expression before and after CD74 transduction. The left histograms indicate fluorescence of the enhanced green fluorescent protein (EGFP) reporter before (thin line) or after (bold line) transduction of the indicated L929 transductants with a bicistronic ratCD74-EGFP construct. In the right panel the same cells as in the left panel were tested for RT1.B^l expression (OX6), respectively, RT1.D^l expression (14-4-S) before (thin line) or after (bold line) transduction with the rat CD74-EGFP construct.

Figure 6

Major histocompatibility complex (MHC) class II expression and superantigen presentation of native or rat MHC class transduced M12.4.1.C3 cells: staining was performed with isotype-matched antibodies (filled histogram) or indicated MHC class II-specific antibodies (open histogram). The bold open histogram of M12.4.1.C3.RT1.D respresents staining with 14-4-S monoclonal antibody (RT1.Dα/H2-Aα chain) and the thin line staining with OX17 monoclonal antibody. The lower panel shows interleukin-2 (IL-2) production by the reactive T-cell hybridoma with indicated superantigens. Presenting cells were: untransduced M12.4.1.C3 cells; circle, RT1.B^l-transduced M12.4.C3 cells; triangle, RT1.D^l-transduced M12.4.C3 cells; inverted triangle, RAJI; squares. Data for staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin 1 (TSST-1) are mean values of three experiments with standard deviation. Data for streptococca pyrogenic exotoxin C (SPEC) are from two experiments distinguished by full and dotted lines.