Decreased intrabulbar CD8 T cells or decreased peribulbar CD4/CD8 T-cells ratio :- which one is more specific and positive prognostic marker of hair re-growth in alopecia areata patients under treatment with topical squaric acid dibutyl ester (SADBE) ? – An analysis by histopathology and immunohistochemistry. Dr.Anup Kumar Tiwary JRA III

OBJECTIVE To specify and reiterate the role of intrabulbar CD8 T cells in the causation of alopecia areata. To clear out the conundrums in understanding the pathogenesis of alopecia areata by interpreting the distribution of T cell subsets at intrabulbar and peribulbar location before and after therapy.

MATERIAL & METHOD Four cases (all are male, age group 18-50 yrs) of patchy alopecia areata (acute stage) fulfilling the following criteria were enrolled in this study after taking written informed consent & treated with topical SADBE on weekly basis as per the standard protocol.

INCLUSION CRITERIA : Immunocompetent patients Patients > 12 years of age Acute stage of current episode of hair loss (≤ 3 months) No local/systemic therapy in last 6 months EXCLUSION CRITERIA : Pregnancy or lactation Serious intercurrent medical illness Significant cardiovascular disease Localized scalp infections / inflammation Immunosuppressed patients History of local/systemic therapy within 6 months

At T0 (before initiating the treatment) & T3 (after 3 months of treatment) Biopsy samples were taken with 4 mm punch of all the 4 patients and sent for histopathologic examination (HPE). Biopsy samples of two patients with grade 3 hair regrowth were also analyzed by immunohistochemistry (IHC). Statistical analysis was done using Chi-square test considering the level of significance (P) value < 0.05 to be as significant.

OBSERVATION AND RESULTS McDonald Hull and Norris grading AT T3: Clinical observation Two patients showed grade 3 and other two had grade 1 & 2 hair re-growth (Mcdonald Hull and Norris grading). McDonald Hull and Norris grading Grade 1:- regrowth of vellus hair Grade 2:- regrowth of sparse pigmented terminal hairs( <50% increment in %SALT score according to NAAF guidelines) Grade 3:- regrowth of terminal hair with patches of alopecia(50-75% ↑ in % SALT score) Grade 4:- regrowth of terminal hair on the whole alopecic patch(>75% ↑ in % SALT score)

Histopathological Examination . Histopathological Examination All the four biopsy samples revealed decrease in peri- and intrabulbar lymphocytic infiltrate. GRADE 3 HAIR REGROWTH PATIENT 1 PATIENT 2 T0 T3

Analysis by Immunohistochemistry in 2 patients with grade 3 hair re-growth Decrease in immunoreactivity for CD4+ T cells in peribulbar infiltrates from 4+ to 3+ and 3+ to 2+. PERIBULBAR CD 4 T - CELLS T0 T3

P VALUE WAS CALCULATED AS 0.04 BY CHI – SQUARE TEST Immunoreactivity for peribulbar CD8 T cells either increased or remained constant. PERIBULBAR CD 8 T- CELLS Intrabulbar CD8 T cells infiltrate markedly reduced to 1/4th & 1/5th of the initial load. 1+ 4+ INTRABULBAR CD 8 T CELLS P VALUE WAS CALCULATED AS 0.04 BY CHI – SQUARE TEST (SPSS V22 SOFTWARE)

DISCUSSION AA is a chronic inflammatory, tissue-specific autoimmune disease involving hairs and sometimes nails, mediated by autoreactive CD4+ and CD8+ T Cells in genetically predisposed individuals. The pathogenesis of AA is still an enigma, but now there are many evidences signifying the importance of CD4+ & CD8+ T cells and cytokines like IL-1α,IL-1β,IL-2,IL-10,IL-17,IFN-γ and TNF-α. Pathogenesis is initially driven by CD4 T Cells and hallmark of HPE is peribulbar collection of lymphocytes, chiefly CD4 T-cells in “swarm of bees” appearance. Loss of hair in AA is more causally related to intrabulbar CD8 T cells rather than peribulbar CD 4 T cells or elevated CD4/CD8 T Cells ratio. Increase in peribulbar CD8 T Cells can be attributed to the chemotactic movement of CD8 T Cells from intrabulbar to peribulbar location.

CONCLUSIONS SOURCE OF SUPPORT : NIL CONFLICT OF INTEREST : NONE Intrabulbar infiltrates of CD8 T cells is a more specific prognostic marker for the development of non scarring hair loss in AA. Assessment of intrabulbar CD8 T cells on HPE would have better prognostic significance. SOURCE OF SUPPORT : NIL CONFLICT OF INTEREST : NONE